proteins

STRUCTURE O FUNCTION O BIOINFORMATICS

Conservation of structural fluctuations

in homologous protein kinases and its
implications on functional sites
Raju Kalaivani,1 Alexandre G. de Brevern,2,3,4,5 and Narayanaswamy Srinivasan1*
1 Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka 560012, India
2 INSERM, U 1134, DSIMB, Paris F-75739, France
3 Sorbonne Paris Cite, University of Paris Diderot, Paris F-75739, France
4 Institut National de la Transfusion Sanguine (INTS), Paris F-75739, France
5 Laboratoire d’Excellence GR-Ex, Paris F-75739, France

ABSTRACT

Our aim is to explore the similarities in structural fluctuations of homologous kinases. Gaussian Network Model based Normal
Mode Analysis was performed on 73 active conformation structures in Ser/Thr/Tyr kinase superfamily. Categories of kinases
with progressive evolutionary divergence, viz. (i) Same kinase with many crystal structures, (ii) Within-Subfamily, (iii) Within-
Family, (iv) Within-Group, and (v) Across-Group, were analyzed. We identified a flexibility signature conserved in all kinases
involving residues in and around the catalytic loop with consistent low-magnitude fluctuations. However, the overall structural
fluctuation profiles are conserved better in closely related kinases (Within-Subfamily and Within-family) than in distant ones
(Within-Group and Across-Group). A substantial 65.4% of variation in flexibility was not accounted by variation in sequences
or structures. Interestingly, we identified substructural residue-wise fluctuation patterns characteristic of kinases of different cat-
egories. Specifically, we recognized statistically significant fluctuations unique to families of protein kinase A, cyclin-dependent
kinases, and nonreceptor tyrosine kinases. These fluctuation signatures localized to sites known to participate in protein-protein
interactions typical of these kinase families. We report for the first time that residues characterized by fluctuations unique to the
group/family are involved in interactions specific to the group/family. As highlighted for Src family, local regions with differen-
tial fluctuations are proposed as attractive targets for drug design. Overall, our study underscores the importance of considera-
tion of fluctuations, over and above sequence and structural features, in understanding the roles of sites characteristic of kinases.

Proteins 2016; 84:957–978.

C 2016 Wiley Periodicals, Inc.
V

Key words: homologous proteins; conservation of protein flexibility; structural fluctuations; functional site identification;
family-specific interactions; STY kinases; protein kinases.

Additional Supporting Information may be found in the online version of this article.
Abbreviations: Abl, Abelson murine leukemia homolog; Ack, activated cdc42-associated tyrosine kinase; AGC, group containing PKA, PKG, PKC, and related
families; Akt, PKB or protein kinase B; CAMK, group containing calcium/calmodulin regulated kinases and related families; CDK, cyclin-dependent kinase;
CDKN3, cyclin-dependent kinase inhibitor 3; CK1, group containing casein kinase 1 and related families; CKS, cyclin-dependent kinase regulatory subunit;
CMGC, group containing CDK, MAPK, GSK3, CLK, and related families; DAPK, death-associated protein kinase; DMPK, myotonic dystrophy protein kinase;
EGFR, epidermal growth factor receptor; ENM, elastic network model; FS score, flexibility similarity score; GNM, Gaussian network model; GRK, G-protein
coupled receptor kinase; GSK, glycogen synthase 3 kinase; InsR, family containing insulin receptor and associated kinases; MAPK, mitogen-activated protein
kinase; MAST, microtubule-associated serine/threonine kinase; NDR, nuclear DBF2-related kinases; NMA, normal mode analysis; nRTK, nonreceptor tyrosine
kinase; PDK1, phosphoinositide-dependent protein kinase 1; PHK, phosphorylase kinase; PKA, protein kinase A or cAMP-dependent protein kinase; PKC,
protein kinase C; PKG, protein kinase G or cGMP-dependent protein kinase; PKN, protein kinase N; PTF, phototrophin and flippase kinases; RSK, ribo-
somal S6 kinase; RSKL, RSK-like kinases; RSKR, RSK-related kinases; RTK, receptor tyrosine kinase; SGK, family containing serum and glucocorticoid
responsive kinase and related kinases; SH2, Src homology 2; Src, family containing SrcA, SrcB, Frk, and related subfamilies; SRPK, SR protein kinase; phos-
phorylates serine/arginine rich splicing factors; STE, group containing MAP kinase cascade kinases; STE20, family containing MAP4K (MAP kinase kinase
kinase kinase) and related kinases; STY kinases, serine/threonine/tyrosine kinases; TK, tyrosine kinase; YANK, yet another novel kinase.

Grant sponsors: Indo-French Collaborative Grant CEFIPRA, Grant number: 5203–2 (to N.S. and A.dB.); Mathematical Biology Initiative, Department of Science and Technol-
ogy and Department of Biotechnology, Government of India, J. C. Bose National Fellowship (to N.S.), National Institute for Health and Medical Research (INSERM), University
Paris Diderot, Sorbonne Paris Cite, National Institute for Blood Transfusion (INTS), and laboratory of excellence GR-Ex (to A.dB.), and University Grants Commission, Gov-
ernment of India (to R.K.).
*Correspondence to: Narayanaswamy Srinivasan, Lab no. 103, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, Karnataka, India.
E-mail: ns@mbu.iisc.ernet.in
Received 20 August 2015; Revised 2 February 2016; Accepted 17 March 2016
Published online 30 March 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/prot.25044

C 2016 WILEY PERIODICALS, INC.

V PROTEINS 957
 R. Kalaivani et al.
INTRODUCTION
Consideration of homology has profoundly influenced
our understanding of functional relatedness between pro-
teins. However, traditional homology detection methods
using sequence,1–5 structure,6–10 and functional
motif11–14 similarities have shortcomings. A trait that is
tightly related to the protein’s function would be more
effective as a proxy to understand relatedness among
proteins. Recent studies have shown that dynamics of
proteins carry activity-specific information15 and are
coupled to their functions.16 But, studies that compre-
hensively analyse the relationship between dynamics and
functions of related proteins are sparse.17–20 In the cur-
rent work, an understanding on the mobility of proteins
was arrived at using the fluctuations derived from Gaus-
sian Network Model (GNM) based Normal Mode Analy-
sis (NMA). Variations in structural fluctuations among
homologous kinases are then viewed in the light of hier-
archical categories such as subfamily and family. In sum-
mary, we primarily aim to understand how the mobility
of homologous kinases vary, and how well this variation
is consistent with the hierarchical grouping of kinases.
Protein kinases, which target the hydroxyl group in
the side-chains of Ser, Thr, and Tyr residues, were chosen
as the model system of homologous proteins. They form
a large and well-studied protein superfamily with several
structures belonging to diverse families/subfamilies
already available. They catalyse phosphotransfer from a
nucleoside triphosphate to a Ser/Thr/Tyr residue on its
cognate protein substrate. For this reason, we refer them
as STY kinases. Various studies have analyzed their
sequence,21–23 structure,24,25 and function/mecha-
nism.26,27 Reflective of the diverse regulatory mecha-
nisms that tightly control the activity of different STY
kinases, their catalytic domains have a modest 20%
sequence identity across groups of families.28 On the
other hand, consistent with the common ancestry and
catalytic mechanism, all the members of the superfamily
have a conserved structural fold29 made of a smaller N-
terminal-lobe and a bigger C-terminal-lobe. While the C-
lobe is entirely made of helices, the N-lobe mainly con-
tains b sheets and a conserved helical region referred as
aC-helix. The aC-helix is a crucial structural motif
whose conformational variation has significant conse-
quence on the activity levels of the kinase.25,30 Besides
the aC-helix, ATP binding loop is the other functionally
important flexible motif in the N-lobe and it is involved
in binding of the nucleoside in the catalytic cleft. Con-
nected to the N-lobe through the hinge region is the
C-lobe, which contains the catalytic loop, with a com-
pletely conserved aspartate,31 followed by the activation
loop. Both these loops are known to play significant roles
in the switch of the kinases25 between two extreme func-
tional states: active and inactive. Through several struc-
tural studies,30 it has been shown that the STY kinases
958 PROTEINS
adopt highly similar conformations in their active states,
especially in the functional motif regions. Contrastingly,
the inactive state conformations of STY kinases are less
similar in general.
Although the proteins of STY kinase superfamily
broadly perform the same catalytic function, they are
involved in a myriad of signalling pathways;32 are regu-
lated by varied mechanisms33–38 and localize in differ-
ent cellular regions, resulting in distinct biochemical
effects.39–46 The sequence and structure features of STY
kinases alone cannot identify their cognate substrates;
specific regulatory mechanisms or interactions with other
proteins. We hypothesise that the structural fluctuations
or mobility of STY kinases is an important consideration
to understand the basis of their regulatory features, espe-
cially their specific recognition of other domains/pro-
teins. If this hypothesis is valid, one would expect the
structural fluctuations to be better conserved in closely
related STY kinases than distantly related ones. For
example, fluctuations would be conserved better within a
subfamily of kinases than across subfamilies within a
family. If the structural fluctuations of related STY
kinases are indeed better conserved, is it only a trivial
consequence of conservation of sequence and structure
information? Or, do the fluctuations truly contain infor-
mation that is not inherent in sequences and structures?
How do such conserved structural fluctuations, if pres-
ent, corroborate with the specific association of kinases
with other domains/proteins?
To answer these questions, we first grouped the STY
kinases into five categories: (i) Same kinase with more
than one crystal structure available, (ii) Within-
Subfamily, (iii) Within-Family, (iv) Within-Group, and
(v) Across-Group. These categories are derived from Kin-
Base21 (see Methods) classification scheme and corre-
spond to progressive evolutionary divergence of kinases.
STY kinases within a group have similar substrate site
specificity; those within a family have related regulatory
features and those within a subfamily have similar
sequence and regulatory features across phyla. GNM
based NMA was performed on 73 active conformation
structures of STY kinase catalytic domains (Table I)
using only the spatial positions of Ca atoms (Gaussian
network model based normal mode analysis section in
Methods). We characterized the resulting structural fluc-
tuations of STY kinases of different categories in the light
of their regulatory features such as specific association
with domains/proteins.
METHODS
KinBase classification of STY kinases
Hanks and Hunter originally devised a scheme for
classifying47 STY kinases based on the sequence similar-
ity between their catalytic domains. Based on the
 Structural Fluctuations in Protein Kinases

Table I
Data Set of 73 STY Kinases used in the Present Study

KinBase classification
Kinase domain
no. Group Family Subfamily UniProt ID PDB ID ATOM residues Res ()
1 AGC Akt P31751 1O6K_A 152–409 1.7
1O6L_A 152–409 1.6
2 AGC DMPK GEK Q09013 2VD5_A 71–339 2.8
3 AGC DMPK ROCK Q28021 2F2U_A 92–354 2.4
4 AGC PDK1 O15530 1H1W_A 82–342 2
5 AGC PKA P05132 1APM_E 43–297 2
1ATP_E 43–297 2.2
1FMO_E 43–297 2.2
1JBP_E 43–297 2.2
1L3R_E 43–297 2
1RDQ_E 43–297 1.26
2CPK_E 43–297 2.7
2ERZ_E 43–297 2.2
6 AGC PKA P36887 1CDK_A 43–297 2
7 AGC PKC PKCa P05771 2I0E_A 342–600 2.6
8 AGC PKC PKCd Q04759 2JED_A 380–634 2.32
9 AGC PKC PKCi P41743 1ZRZ_A 245–513 3
10 CAMK CAMKL CHK1 O14757 1IA8_A 9–265 1.7
11 CAMK DAPK DAPK P53355 1JKK_A 13–275 2.4
1IG1_A 13-275 1.8
12 CAMK MAPKAPK MK2 P49137 1NXK_A 64–325 2.7
13 CAMK PHK P00518 1PHK_A 19–287 2.2
2PHK_A 19–287 2.6
14 CAMK PIM P11309 1XR1_A 38–290 2.1
15 CK1 CK1 CK1-D Q06486 1CKJ_A 12–282 2.46
16 CMGC CDK CDK2 P24941 1FIN_A 4–286 2.3
1JST_A 4–286 2.6
1JSU_A 4–286 2.3
1QMZ_A 4–286 2.2
1W98_A 4–286 2.15
17 CMGC CDK CDK4 Q00534 1JOW_B 4–286 3.1
18 CMGC CDK CDK5 Q00535 1H4L_A 4–286 2.65
19 CMGC DYRK DYRK1 Q13627 2VX3_A 159–479 2.4
20 CMGC GSK P49841 1GNG_A 56–340 2.6
1O9U_A 56–340 2.4
21 CMGC MAPK ERK1 P63086 2ERK_A 23–311 2.4
22 CMGC MAPK p38 P53778 1CM8_A 27–311 2.4
23 CMGC MAPK p38 P47811 3PY3_A 24–308 2.1
24 CMGC SRPK Q96SB4 1WAK_A 80–653 1.73
1WBP_A 80–653 2.4
25 Other Aur O14965 1OL5_A 133–383 2.5
26 STE STE-Unique Q99558 4DN5_A 400–655 2.5
27 STE STE20 PAKA Q13153 1YHV_A 270–521 1.8
1YHW_A 270–521 1.8
3Q52_A 270–521 1.8
3Q53_A 270–521 2.09
28 STE STE20 TAO Q9JLS3 1U5Q_A 28–281 2.1
1U5R_A 28–281 2.1
29 TK Abl P00519 1OPL_A 261–512 3.42
2F4J_A 242–493 1.91
2G2I_A 242–493 3.12
2GQG_A 242–493 2.4
30 TK Ack Q07912 1U46_A 126–385 2
1U4D_A 126–385 2.1
1U54_A 126–385 2.8
31 TK Csk P32577 1K9A_A 195–445 2.5
32 TK EGFR P00533 1M14_A 688–955 2.6
2GS2_A 688–955 2.8
2ITP_A 712–979 2.74
33 TK InsR P06213 1IR3_A 996–1271 1.9
34 TK InsR P08069 1K3A_A 969–1244 2.1
35 TK PDGFR P10721 1PKG_A 589–927 2.9
36 TK Src SrcA P12931 1Y57_A 267–520 1.91

PROTEINS 959
 R. Kalaivani et al.

Table I
(Continued)

KinBase classification
Kinase domain
no. Group Family Subfamily UniProt ID PDB ID ATOM residues Res ()
1YOJ_A 269–522 1.95
1YOL_A 269–522 2.3
1YOM_A 269–522 2.9
37 TK Src SrcA P00523 3DQW_A 267–520 2.02
38 TK Src SrcB P06239 1QPC_A 245–498 1.6
1QPD_A 245–498 2
1QPE_A 245–498 2
1QPJ_A 245–498 2.2
3LCK_A 245–498 1.7
39 TK VEGFR P35968 1VR2_A 834–1162 2.4

A total of 73 STY kinase domain structures, accounting for 39 unique STY kinases, are listed along with hierarchical classification of KinBase. KinBase classifies STY
kinases broadly into groups. STY kinases within a group are further clustered into families. Additionally, some of the within-family members are sub-categorised into
subfamilies. UniProt ID, Protein Data Bank identities of the structures (PDB ID) and the subunit used are also enlisted. The region of ATOM residues (numbering
according to the PDB structure file) that form the kinase catalytic domain and the resolution of the X-ray crystal structure are also tabulated.

phylogeny of catalytic domains, they classified the then
known STY kinases into a few major groups and subdi-
vided them into families and subfamilies. This classifica-
tion resulted in clusters of kinases with related regulatory
features such as association with specific domains/pro-
teins and similar structural features. Nonetheless, with
the emergence of new kinomes and expanding knowledge
of the mechanistic and regulatory features of
kinases,48,49 the shortcoming of using only the catalytic
domain sequence became apparent and an empirical sys-
tem of classification was required.
In 2002, the complete protein kinome of humans was
mapped and classified into 9 major groups,21 subclassi-
fied into families and finely into subfamilies. This classi-
fication, called the KinBase classification, was an
augmented Hanks-Hunter system, where the primary
mode of classification was still based on the catalytic
domain sequence. Parameters such as sequence similarity
outside the catalytic domain, domain architecture, bio-
logical functions, association with cellular pathways,
localisation in the cell and orthology found in the other
kinomes were used to incorporate changes over and
above the Hanks-Hunter method. This scheme also
involves manual curation to attain a useful classification
scheme that substantiates experimental results and evolu-
tionary conservation within and across phyla. As a result,
kinases within a group may have broadly similar sub-
strate site specificity or similar strategies of regulation
such as binding of a second messenger and SH2 interac-
tion; those within a family are related by similar regula-
tory features and those within a subfamily have highly
conserved sequence and regulatory features across spe-
cies. With several kinomes now available, the KinBase
classification system is a widely accepted, well curated,
and revised database, providing classification of >3000
kinase sequences across phyla. Definitions of group, fam-
ily and subfamily of the STY kinases in the current study
are obtained from KinBase.
Definition of categories derived from
KinBase classification
We performed all possible pairwise comparisons of
structural fluctuations of the 73 STY kinases considered
in the study. These pairs were grouped into five catego-
ries based on the KinBase classification of the entities: (i)
Same-Protein, (ii) Within-Subfamily, (iii) Within-Family,
(iv) Within-Group, and (v) Across-Group. These catego-
ries represent progressive divergence in the evolution of
kinases, with Within-Subfamily corresponding to the
most closely related kinases and Across-Group corre-
sponding to the most divergent kinases. Same-Protein
category has 78 pairs, whose entities are different crystal
structures of the same STY kinase. For example, PDB
IDs 1Y57_A (KinBase classification—TK group: Src fam-
ily: SrcA subfamily; UniProt ID P12931) and 1YOJ_A
(KinBase classification—TK group: Src family: SrcA sub-
family; UniProt ID P12931) are a pair in the Same-
Protein category. Within-Subfamily category has 14 pairs,
whose entities are crystal structures of STY kinases of
identical group, family and subfamily denominations but
different UniProt IDs. For example, PDB IDs 1Y57_A
(KinBase classification—TK group: Src family: SrcA sub-
family; UniProt ID P12931) and 3DQW_A (KinBase
classification—TK group: Src family: SrcA subfamily;
UniProt ID P00523) form a pair in the Within-Subfamily
category. Within-Family category has 50 pairs, whose
entities are crystal structures of STY kinases of identical
group and family denominations but different subfamily
assertions. For example, PDB IDs 1Y57_A (KinBase clas-
sification—TK group: Src family: SrcA subfamily; Uni-
Prot ID P12931) and 1QPC_A (KinBase classification—
TK group: Src family: SrcB subfamily; UniProt ID
P06239) are a pair in the Within-Family category.
Within-Group category consists of 441 pairs, whose enti-
ties are crystal structures of STY kinases of identical
group but different family denominations. For example,

960 PROTEINS
 Structural Fluctuations in Protein Kinases
PDB IDs 1Y57_A (KinBase classification—TK group: Src
family: SrcA subfamily; UniProt ID P12931) and 1IR3_A
(KinBase classification—TK group: InsR family; UniProt
ID P06213) are a pair in the Within-Group category.
Across-Group category has 2045 pairs, whose entities are
crystal structures of STY kinases belonging to different
groups. For example, PDB IDs 1Y57_A (KinBase classifi-
cation—TK group: Src family: SrcA subfamily; UniProt
ID P12931) and 1U5R_A (KinBase classification—STE
group: STE20 family: TAO subfamily; UniProt ID
Q9JLS3) are a pair in the Across-Group category.
Gaussian network model based normal mode
analysis
Seventy three STY kinase structures in active confor-
mation (Table I) were all prepared by (i) retaining only a
single kinase catalytic domain structure, and not consid-
ering other domains/chains and (ii) deleting any bound
ligands, substrates or inhibitors. Thus prepared structures
were each modelled as a 3-dimensional mass-spring sys-
tem using coarse-grained Elastic Network Model
(ENM).50 The spatial coordinates of the back bone Ca
atoms were virtually modelled as masses and all the Ca-
Ca pairs with inter-Ca distance less than 7Å51 were
virtually connected by springs of identical spring con-
stants.52 Assuming that the crystal structure of the STY
kinase represents the equilibrium state, the topology of
harmonic potentials was solved for their vibrational
modes around this stable state.53 Further simplification
of the model was brought about by assuming isotropic
Gaussian fluctuations around the masses or Ca atoms.54
The resulting Gaussian Network Model (GNM) of the
STY kinases, determines direction-nonspecific relative
fluctuations at each mass or Ca atom. Despite the
above-stated simplifications and microsampling around
the equilibrium state alone, GNM-based NMA fluctua-
tions have been successful in identifying biologically rele-
vant and physiologically feasible motions of the
proteins.16
Construction of network topologies and calculation of
eigenvectors (normal modes) and eigenvalues (frequency)
of the structures were carried out as described in previ-
ous studies.15 Only the global mode or the mode of least
frequency was used in all the analyses in the study. This
is because the global mode represents the slowest motion
that usually involves contribution from a large fraction
of residues. As a result, the global mode usually denotes
conformational changes or biologically relevant motions
in the protein.55,56
Treatment of missing residues
Twenty four of the seventy three STY kinase structures
analyzed in the study have one or more missing Ca
atomic positions in the positional coordinate data files.
For the construction of network topology, only the Ca
atoms with resolved spatial coordinates were used.
GNM-based NMA was performed on the network thus
obtained and square fluctuations of the Ca atoms with
available positional coordinates were calculated. Square
fluctuations of two residues sequentially preceding the
missing residues and two residues sequentially succeeding
the missing residues were disregarded for all analyses.
For instance, if the Ca atoms of the 10th and 11th resi-
dues were missing in the crystal structure of the kinase
catalytic domain spanning residues 5 to 260, spatial coor-
dinates of Ca atoms 5 to 9 and 12 to 260 were used to
construct the virtual mass-spring topology. However, the
square fluctuations of only the residues 5 to 7 and 14 to
260 were used for all the analyses in the study. This is
done in order to remove any spurious fluctuations
caused by the virtue of missing residues in the immedi-
ate surroundings. For each of the 73 crystal structures
used in the study, the kinase catalytic domain, missing
regions, residues used for network construction and the
residues whose fluctuations were used in the study are
tabulated in Supporting Information Table S1.
It is arguable that the results obtained in the study are
an incidental effect of the missing residues in the struc-
tures, and the consequent erroneous network topology.
In order to address this concern, we performed two con-
trol experiments (“Missing residues control experiments”
section in Supporting Information). In the first experi-
ment, we used the 49 STY kinase structures containing
no missing Ca coordinates and replicated the primary
results of the study (Supporting Information Figs. S1–
S3). This experiment shows that there is no significant
change in the results of the study even if the structures
with missing residues are eliminated from the dataset. In
the second experiment, we modelled the missing residues
in 22 of the remaining 24 structures. We show in
detailed comparative analysis that the square fluctuations
of residues more than two amino acids away from the
missing region is unaltered in the modelled structures as
compared with the native structures with missing resi-
dues (Fig. S4 in Supporting Information). The above
results clearly advocate that the results of the study are
not a consequence of the missing regions in the crystal
structures.
Residue-wise square fluctuations
For every residue i in each STY kinase structure, the
square fluctuation in the global mode g is given by:
" #
T
3k B T ug u g
hDRi2 ig 5
g kg
ii
where hDRi2 ig is the square fluctuation of residue i in the
global mode g, kB is the Boltzmann constant, T is the
PROTEINS 961
 R. Kalaivani et al.
absolute temperature, g is the spring constant of all the
Ca-Ca interactions defined by the Kirchhoff matrix, kg
is the least eigenvalue or frequency, and ug is the eigen-
vector corresponding to the least frequency or the global
normal mode.
Normalized global mode square fluctuations
The residue-wise square fluctuations derived in the
global mode of a given structure was normalized by
dividing with the highest individual-residue square fluc-
tuation in the same mode of the same structure. The
fluctuation of residue i in the global mode g is normal-
ized as:
Normalized square f luctuations
hDRi2 ig
5hDRi2 inormalized 5 n o
max hDR 2 ig
n o
where max hDR 2 ig is a constant for every structure,
and is the square fluctuation of the most mobile residue
in the global mode of the structure.
Calculation of flexibility similarity (FS) score
FS score, or Flexibility Similarity score, is a quantita-
tive measure of similarity between the global mode struc-
tural fluctuations of any two given STY kinases. In this
study, FS scores were calculated using three different
measures.17,19,20
FS score using Spearman rank order correlation
Seventy-three STY kinases were all individually com-
pared with one another, giving rise to 2628 possible pair-
wise comparisons. For each of the 2628 pairwise
comparisons, structure-based sequence alignment of the
kinase catalytic domain was performed using TM-
Align57 to identify topologically equivalent residues in
the kinase pair. GNM-based NMA was individually car-
ried out for the kinase catalytic domains and the
residue-wise square fluctuations were determined for the
two constituent kinases in the pair. Spearman rank order
correlation coefficient was calculated between the square
fluctuations of the two kinases, taking into account only
those residues which have topological equivalence in
each other. It is to be noted that the number of aligned
residues, or residues with topological equivalence, will
vary depending on the kinases in comparison. This could
potentially introduce a bias in the calculated correlation
coefficient. To counter this bias, each correlation coeffi-
cient was calculated with a correction for the number of
data points (the length of aligned residues) using the
formula:
962 PROTEINS
 
  N21
FSSP 512 12 g2SP 3
N22
where FSSP is the FS score calculated using Spearman
rank order correlation, gSP is the Spearman rank order
correlation coefficient calculated between the square fluc-
tuations of the two kinases, taking into account only
those residues which have topological equivalence in
each other and N is the length of aligned residues. The
effect of length of aligned residues N on FSSP , sequence
identity and RMSD was investigated and found to be
insignificant (Supporting Information Fig. S5).
FS score using Pearson rank order correlation
This is calculated in a manner similar to the FS score
calculated using Spearman rank order correlation, with
the modification of using Pearson rank order correlation
instead of Spearman:
 
  N21
FSPS 512 12 g2PS 3
N22
where FSPS is the FS score calculated using Pearson rank
order correlation, gPS is the Pearson rank order correla-
tion coefficient calculated between the square fluctua-
tions of the two kinases, taking into account only those
residues which have topological equivalence in each
other, and N is the length of aligned residues.
FS score using overlap of eigenvectors
GNM-based NMA was carried out for the kinase cata-
lytic domains and the global mode eigenvector was
determined for the two constituent kinases in each of the
2628 pairs. Dot product or overlap between the global-
mode eigenvectors of the two kinases was calculated, tak-
ing into account only those residues which have topolog-
ical equivalence in each other. This corresponds to the
FS score calculated using overlap of eigenvectors:
d1  EV
FSOL 5jEV d2 j
where FSOL is the FS score calculated using the overlap
d1 is the unit vector in the direc-
of two eigenvectors, EV
tion of global mode eigenvector of the first constituent
d2 is the unit vector in the
kinase of the pair and EV
direction of global mode eigenvector of the second con-
stituent kinase of the pair.
Multivariate correlation analysis
Multivariate correlation analysis was carried out to
understand the extent of consequence of sequence and
structure information in the global mode structural fluc-
tuations. For each of the 2628 STY kinase pairs, sequence
 Structural Fluctuations in Protein Kinases
identity and RMSD were calculated using structure based
sequence alignment algorithm TM-Align.57 Further, the
similarity in the flexibility profiles of the constituent
kinases in each of the kinase pairs was calculated as FS
scores using Spearman rank order correlation (FSSP ), as
described above. The fraction of variance in FSSP scores
that can be accounted for by the variance in sequence
identity and RMSD was calculated as:
0sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi12
FSS2 1FSR2 2ð23FSS3FSR3SRÞA
Vboth 5@
12SR2
FSS5 gSP ðFSSP ; seqidÞ
FSR5 gSP ðFSSP ; RMSDÞ
SR5 gSP ðseqid; RMSDÞ
where Vboth is the fraction of variance in FSSP that can
be explained by the variance in sequence identity and
RMSD, gSP ðFSSP ; seqidÞ is the Spearman rank order cor-
relation coefficient between FSSP and sequence identity,
gSP ðFSSP ; RMSDÞ is the Spearman rank order correlation
coefficient between FSSP and RMSD, gSP ðseqid; RMSDÞ is
the Spearman rank order correlation coefficient between
sequence identity and RMSD and FSSP is the FS score
calculated using Spearman rank order correlation after
correcting for the number of aligned residues.
The fraction of variance in FSSP scores that can be
accounted for by the variance in sequence identity alone
is given by:
VS 5Vboth 2FSR2
The fraction of variance in FSSP scores that can be
accounted for by the variance in RMSD alone is given
by:
VR 5Vboth 2FSS2
Thus, the fraction of variance in FSSP that is explained
by both the sequence identity and RMSD inseparably is
given by:
VSR 5Vboth 2VS 2VR
Sequence and structure similarity across
73 kinases
Topological equivalence of residues across the 73 STY
kinases was derived from multiple structure superposi-
tion based multiple sequence alignment.58 For each
of the topologically aligned position, the sequence
divergence was calculated by Jensen-Shannon entropy59
and structure divergence was calculated as Ca RMSD.
Interaction density analysis from iPfam
structures
iPfam60 is a database of all domain-domain interac-
tions, validated through structure determination. We
queried iPfam for all the PDB structures that contained a
protein kinase domain and another domain of interest
(say, C-terminal, SH2, and so forth). All the resulting
PDB entries (say, n hits) were parsed to identify the num-
ber of interactions (inter-Ca distance of 6 Å or less) each
kinase residue had with the domain of interest. The
sequences of the kinase catalytic domains of the n struc-
tures were aligned using T-Coffee61 to identify the topo-
logically equivalent residues. At every aligned position, the
number of interactions of the kinase residue with the
domain of interest across the n structures was summed
and referred to as the interaction density. This interaction
density was mapped on the kinase fold for visualization.
Linear discriminant analysis
Linear discriminant analysis was performed using the
Statistics and Machine Learning toolbox of MATLAB62
to understand how well a simple classifier is able to
ascertain the group and family to an STY kinase based
solely on its global mode structural fluctuations. The lin-
ear classifier was trained and tested on a data set of 71
STY kinases (Supporting Information Fig. S6) for group
prediction. A leave-one-out approach was used, that is,
using 70 STY kinases as the training set and testing on
the remaining one, repeating 71 times. GNM perceived
global mode normalized square fluctuations of the resi-
dues that had topological equivalence in each of the 71
kinases were used for training and testing. This
accounted for normalized square fluctuations at 326
alignment positions. The accuracy with which the group
of the test kinase was predicted was calculated as an
average across the 71 structures. Similarly, the linear clas-
sifier was trained and tested on a dataset of 62 STY
kinases (Supporting Information Fig. S7) for family pre-
diction using the global mode fluctuations of the same
326 alignment positions in a leave one out method.
RESULTS
Seventy three STY kinase structures, solved in active
conformations at resolutions better than 3.4 Å by X-ray
crystallography, were investigated (Table I). The reason
for selecting active conformations is twofold: first, the
inactive conformations are varied30 and secondly, struc-
tural fluctuations of active conformations differ system-
atically from those of inactive conformations.15 The
dataset, comprising 39 unique kinases of 7 KinBase21
PROTEINS 963
 R. Kalaivani et al.
groups, has an average pairwise sequence identity of
29.7% 6 15.3 (mean 6 standard deviation) and RMSD of
2.27 Å 6 0.57. In each of the 73 structures, the spatial
positions of Ca atoms of the kinase catalytic domain
alone were used to construct the Kirchhoff matrix for
GNM based NMA (Gaussian network model based nor-
mal mode analysis section in Methods). All other atoms,
including bound ligands/peptides and other domains,
were omitted. GNM based NMA was performed on the
structures, thus processed, with identical spring constants
and standard cut-off value.54 Residue-wise square fluctu-
ations were calculated for each kinase from the mode of
the lowest frequency, or global mode (Residue-wise
square fluctuations section in Methods). For all analyses,
fluctuations of two residues sequentially preceding and
two residues sequentially succeeding the missing regions
in the structure, if any, were disregarded (Treatment of
missing residues section in Methods).
Fluctuations of closely related STY kinases
are correlated better than those of distant
homologues
Definition of relatedness from KinBase classification
In order to assess the similarity in fluctuation profiles
of STY kinases, we performed all possible 2628 pairwise
comparisons of residue-wise square fluctuations of the
73 STY kinases. Of these, based on KinBase classifica-
tion,21 the entities of 78 pairs correspond to identical
kinases in different crystal structures (Definition of cate-
gories derived from KinBase classification section in
Methods); 14 pairs correspond to kinases of the same
subfamily; 50 pairs to kinases of the same family but dif-
ferent subfamilies; 441 pairs to the same group but dif-
ferent families; and 2045 pairs to different groups. The
above are referred as (i) Same-Protein, (ii) Within-
Subfamily, (iii) Within-Family, (iv) Within-Group, and
(v) Across-Group categories respectively (Table S2 in
Supporting Information).
The similarity between the global mode residue-wise
square fluctuations of any two kinases, referred as Flexi-
bility Similarity score (FS score), was calculated using
three measures: (i) Spearman rank order correlation [Fig.
1(A–C)], (ii) Pearson rank order correlation [Fig. 1(D–
F)], and (iii) overlap of the two global mode eigenvectors
[Fig. 1(G–I)]. Figure 1(A) shows the histogram of FS
scores of all the 2628 STY kinase pairs, calculated using
Spearman rank order correlation (FSSP ) and segregated
into one of five different categories. The FSSP scores of
Same-Protein (red) and Within-Subfamily (blue) catego-
ries are accrued near 1.0; Within-Family (green) and
Within-Group (purple) categories are accumulated
around 0.95 and Across-Group (orange) category is dis-
persed over a range of 0.60 to 0.96. Cumulative fre-
quency plot of the FSSP scores [Fig. 1(B)] shows that
the Across-Group similarity (orange curve) scores are
964 PROTEINS
the lowest in magnitude and are distributed over a wide
range of values. Within-Group (purple curve), Within-
Family (green curve), Within-Subfamily (blue curve) and
Same-Protein (red curve) FSSP scores are shifted to the
right of the Across-Group scores in a sequential manner,
suggesting that the distributions of correlation coefficients
increase systematically and hierarchically. Kinase pairs that
are more closely related (Same-Protein and Within-Sub-
family) have higher FSSP scores than the less closely
related ones (Within-Family and Within-Group), which
in turn have higher scores than the Across-Group pairs.
The median of the FSSP scores in the Across-Group cat-
egory, denoted by q0, is 0.84 [dotted gray line in Fig.
1(A,B)]. Thus, the fraction of Across-Group pairs that
have FSSP scores greater than q0 , P ðFSSPAG > q0 Þ, is 0.5 by
construction. The fraction of Within-Group pairs that
have FSSP scores greater than q0 , P ðFSSPwG > q0 Þ, is 0.84.
Hence, the conservation of flexibility is relatively higher in
the Within-Group pairs than in the Across-Group pairs,
that is, P ðFSSPwG > q0 Þ > P ðFSSPAG > q0 Þ50:5 (binomial
test, P 5 4.94E-52). The fractions of Within-Family,
P ðFSSPwF > q0 Þ, Within-Subfamily, P ðFSSPWS > q0 Þ, and
Same-Protein, P ðFSSPSP > q0 Þ, pairs that have FSSP scores
greater than q0 are all 1.0. This indicates that all of the
Within-Family, Within-Subfamily, and Same-Protein pairs
have FS scores higher than an average Across-Group pair
(Binomial test, P  E-04). Further, unpaired t-test [upper-
right in Fig. 1(C)] and Kolmogorov-Smirnov test [lower-
left in Fig. 1(C)] also distinguish the FSSP scores of the five
categories significantly from one another, arguing that the
categories indeed have different scores. Corresponding
analyses done for FS scores calculated using Pearson rank
order correlation [FSPS , Fig. 1(D–F)] and overlap of global
mode eigenvectors [FSOL , 1(G–I)] also yield similar results.
Relationship between flexibility similarity scores and
sequence/structural similarity
We have demonstrated that Within-Subfamily kinase
pairs have better conserved flexibility profiles than
Across-Group kinase pairs. This was achieved upon con-
sideration of category definitions from KinBase.21 How-
ever, as will be applicable to other protein families that
are not manually classified, we wanted to understand the
conservation in flexibility profiles purely as a function of
sequence and structure similarity among kinases. To this
end, we calculated pairwise sequence identity and RMSD
between the kinase catalytic domains of all the 2628
pairs57 (Supporting Information Table S2). The mean
and standard error of mean of pairwise sequence identity
and RMSD are 0.992 6 0.001 and 0.60 Å 6 0.04 for
Same-Protein pairs; 0.938 6 0.026 and 0.67 Å 6 0.12 for
Within-Subfamily pairs; 0.575 6 0.014 and 1.33 Å 6 0.05
for Within-Family pairs; 0.385 6 0.003 and 1.77 Å 6 0.02
for Within-Group pairs and 0.240 6 0.001 and 2.48
Å 6 0.01 for Across-Group pairs. We plotted the FSSP
 Structural Fluctuations in Protein Kinases

Figure 1
Distribution of FS scores of different categories. Each of the 73 STY kinases’ global mode square fluctuations were compared with that of every
other kinase, resulting in Flexibility Similarity scores (FS scores) for 2628 kinase pairs. Based on the KinBase classification and UniProt IDs of the
constituent kinases, the pairs and the corresponding FS scores were divided into the following categories: Same-Protein (red), Within-Subfamily
(blue), Within-Family (green), Within-Group (purple), and Across-Group (orange). FS scores were calculated using Spearman rank order correla-
tion coefficient (FSSP , A–C), Pearson rank order correlation coefficient (FSPS , D–F) or overlap of global mode eigenvectors (FSOL , G–I). (A) Fre-
quency distribution of FSSP scores is plotted for all the five categories. The dotted gray line, labelled medianAG, represents the median value q0 of
the Across-Group similarity score (FSSPAG , orange) and corresponds to a Spearman rank order correlation coefficient of 0.84. (B) Cumulative fre-
quency distribution of FSSP scores is plotted for all the five categories. The medianAG or q0, corresponding to the median of FSSPAG (orange), is
plotted as a dotted gray line. (C) FSSP score distributions of each of the five categories were compared with that of every other category using
unpaired t-test (upper-right triangle) and Kolmogorov-Smirnov test (lower-left triangle) and the resulting P values are tabulated with color codes
in a brown-purple scheme. Predominance of brown color indicates that the FSSP score distributions of the five categories are different from each
other with statistical significance. (D–F) are similar to (A–C), respectively, but the FS scores are calculated using Pearson rank order correlation
coefficients (FSPS Þ. (G–I) are similar to (A–C), respectively, but the FS scores are calculated using overlap of the global mode eigenvectors (FSOL ).
[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

scores as a function of their sequence identity [Fig. 2(A)]
and structure similarity [Fig. 2(B)]. Only a weak correla-
tion between flexibility similarity (FSSP ) scores and the
corresponding sequence identity is seen [Fig. 2(A),
Spearman rank order correlation, cc 5 0.56, P 5 E-215].
Similarly, RMSD values also correlate weakly with con-
servation in flexibility [Fig. 2(B), Spearman rank order
correlation, cc 5 20.53, P 5 E-192], although the flexibil-
ity profiles are determined solely based on the three-
dimensional spatial coordinates of Ca atoms. As the cor-
relation of flexibility similarity with sequence identity
and RMSD is weak, it is arguable that the conservation
observed in flexibility is not a trivial consequence of
sequence/structure similarity. In this regard, we hypothe-
sise that the flexibility profiles contain more information
than can be explained by sequence/structure alone.
To understand the extent of similarity in flexibility
contributed by sequence and structure similarity, we con-
ducted a multivariate correlation analysis (Multivariate
correlation analysis section in Methods) on the FSSP
scores. Only 6.27% of the variance in FSSP scores could
be accounted by the variance in sequence identity alone,
3.41% by the variance in RMSD alone and 24.9% by the
variance of both sequence identity and RMSD insepara-
bly. There still remains a considerable 65.4% of variation
in FSSP scores, which is unexplained by sequence identity
or RMSD. This implies that the conservation in flexibility
profiles of STY kinases is not always accompanied by
conservation in sequence/structure. In this context, one
could speculate that the flexibility patterns important for
function/regulation/stability of a protein were evolutio-
narily selected against other variations that compromised
such flexibility patterns.
Fluctuations conserved in all protein kinases
Although a large and divergent superfamily, STY
kinases have a common catalytic mechanism. Therefore,

PROTEINS 965
 R. Kalaivani et al.

Figure 2
FS scores as a function of sequence and structure similarity. (A) FSSP scores of 2628 kinase pairs are plotted against their sequence identity, calcu-
lated from structure based sequence alignment using TM-align.57 The scatter points are colored according to which of the following categories the
kinase pair belongs to: Same-Protein (red), Within-Subfamily (blue), Within-Family (green), Within-Group (purple), and Across-Group (orange).
The FSSP scores, which are indicative of the similarity in fluctuation profiles, show a weak correlation (Spearman rank order correlation, cc 5 0.56,
P 5 2.6E-215) with sequence identity. (B) FSSP scores show a weak correlation with the corresponding RMSD (Spearman rank order correlation,
cc 5 20.53, P 5 5.2E-192). (C) The RMSD of the 2628 kinase pairs are plotted against their sequence identity, showing a strong correlation (Spear-
man rank order correlation, cc 5 20.73, P  0.0) between sequence and structure similarity. [Color figure can be viewed in the online issue, which
is available at wileyonlinelibrary.com.]

it is possible that a common flexibility trait, necessary
for the catalytic function, is present in all the STY
kinases. We sought to identify this flexibility pattern that
is conserved through evolution and is invariably present
in all the 73 STY kinases in our dataset.
After identifying the topologically equivalent residues
from sequence based alignment,61 normalized global
mode square fluctuations of the corresponding residues
in the 73 STY kinases were plotted [Fig. 3(A), orange
curves]. The variance in normalized square fluctuations
across kinases at each residue position is color coded in
a red-gray scheme below the zero ordinate, with red
indicating the least and gray indicating the highest var-
iance. Also plotted are the Z-scores of variance [Fig.
3(A), blue dots]. It is clearly seen that some residues
show lower variation in structural fluctuations across
kinases [Fig. 3(A), red, Z-scores < 21] than others [Fig.
3(A), gray]. Such residues also have a low mean magni-
tude of normalized square fluctuations. When the
residue-wise variance in structural fluctuations across
kinases is mapped on to the kinase catalytic fold [Fig.
3(B)], it is seen that the residues spatially proximal to
the catalytic loop in the STY kinase structure possess
highly conserved structural fluctuations. The residues
around the catalytic loop show remarkable conservation
of low magnitude structural fluctuations consistent
across all kinases. This signature flexibility profile is
arguably the essential dynamics for an STY kinase. This
is reasonable because the aspartate in the catalytic loop
acts as a hydrogen acceptor in the catalysis of phospho-
transfer, the single common function of all the STY
kinases. Previous studies on other families of proteins63
have shown that the catalytic residues have lower struc-
tural fluctuations than other functional residues.
As in the previous section, we examined whether the
observed essential flexibility profile of STY kinases is
reflected in their sequence and structure. To this end, we
structurally aligned the 73 kinase structures,58 calculated
the sequence conservation scores at every alignment
position using Jensen-Shannon divergence59 and
mapped them on the kinase fold [Fig. 3(C), red indicat-
ing the most and gray indicating the least conserved res-
idues]. Analogously, the RMSD at the aligned Ca
positions of the multiple structure alignment is mapped
onto the kinase fold in Figure 3(D) (red indicating the
least and gray indicating the highest RMSD values). We
observe barely any correlation of the variance in square
fluctuations [Fig. 3(B)] with sequence conservation [Fig.
3(C), Spearman rank order correlation, cc 5 0.32,
P 5 1.5E-13] or RMSD [Fig. 3(D), Spearman rank order
correlation, cc 5 20.09, P 5 0.03]. Thus, it is ascertained
that the flexibility profile found in all the 73 kinases in
the dataset, is not a trivial consequence of sequence or
structure information. Further, we probed to determine
whether residue-wise structural differences can explain
residue-wise fluctuation differences. To this end, we
measured Spearman rank order correlation between
residue-wise difference in square fluctuations and
residue-wise Ca-Ca displacement for each of the 2628
kinase pairs [Supporting Information Fig. S8(A–D)]. We
found that 92% of the 2628 pairs had a correlation coef-
ficient of less than 0.25. Additionally, we also observed
that residue-wise difference in Voronoi packing den-
sities64 correlated weakly (Spearman rank order correla-
tion, 98% of the 2628 pairs had cc  0.1) with residue-
wise difference in square fluctuations [Supporting Infor-
mation Fig. S8(A,E–G)]. Thus, we conclude that the var-
iation in structural fluctuations cannot be explained by

966 PROTEINS
 Structural Fluctuations in Protein Kinases

Figure 3
Fluctuations conserved in all STY kinases are proximal to the catalytic loop. (A) Normalized square fluctuations of 73 STY kinases listed in Table I
are plotted (orange lines) as a function of topologically aligned positions. At every alignment position, the variance in normalized square fluctua-
tions in calculated and color coded in a red-gray scheme below the zero ordinate. Also, the Z-score of variance in normalized square fluctuations at
every alignment position is plotted (blue dots). It can be seen that some residues from noncontiguous regions exhibit very low variance in struc-
tural fluctuations (marked red below the ordinate; Z-score 20.1) than others. (B) The red-gray color code on the sequence depicted in (A) is
mapped on to the kinase fold. All the noncontiguous regions with low variance are either in or proximal to the catalytic loop in the three-
dimensional space. (C) The catalytic domain structures of the 73 STY kinases were aligned using MUSTANG58 and the Jensen-Shannon59 conser-
vation score at every aligned position is mapped on the kinase fold in a red-gray scheme. The variance in normalized square fluctuations (B) is
weakly correlated to the measure of sequence divergence (C) with a Spearman rank order correlation coefficient of 0.32 (P 5 1.5E-13). (D) The cat-
alytic domain structures of the 73 STY kinases were aligned using MUSTANG58 and the Ca RMSD at every aligned position is mapped on the
kinase fold in a red-gray scheme. The variance in normalized square fluctuations (B) is not correlated with the measure of structure similarity (D)
(Spearman rank order correlation, cc 5 20.09, P 5 0.03). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.
com.]

trivial sequence divergence, residue-wise
difference or global RMSD.
Flexibility patterns characteristic of a kinase
group
Since the previous analyses of the study suggest that
there exists a characteristic low fluctuation profile
around the catalytic loop in all the STY kinases, we
explored if there also existed signature flexibility profiles
for the different subtypes of STY kinases. To this end, we
scrutinised two of the well-studied KinBase groups, AGC
and TK, to find such group-specific fluctuation profiles
and discuss their functional implications.
Case study: AGC group (with special reference to PKA)
AGC group encompasses diverse families of STY
kinases, viz. Akt, DMPK, GRK, MAST, NDR, PDK1,
PKA, PKC, PKG, PKN, RSK, RSKL, RSKR, SGK, YANK,
PTF. The member families participate in distinct signal
transduction pathways; are modulated by diversified
structural mechanisms; respond to assorted signalling molecules
and occur in concurrence with diverse domain architec-
tures. However, there are common features in most, if
not all, members of AGC group: (i) presence of a C-
terminal tail outside the kinase domain containing a
hydrophobic (HF) motif,65 (ii) repositioning of aC-helix
by binding of the C-terminal tail between the aC-helix
and b4-strand,66 (iii) activation loop phosphorylation
by PDK1,67 which is docked to the kinase by the HF
motif,68,69 and (iv) translocation of the kinase to the
membrane.70 From the above common attributes, it is
discernible that the C-terminal tail is the most distin-
guishing feature of all the AGC kinases. In support of
this argument, previous studies have reported remarkable
conservation of kinase catalytic residues that bind the C-
terminal tail and the resulting inactivity of the kinase
upon mutations/deletions of such residues.71
We queried iPfam60 for all crystal structures contain-
ing both the STY kinase domain (Pfam IDs: PF00069 or
PF07714) and the C-terminal tail (Pfam ID: PF00433).
All the 58 structure hits belong to AGC group of STY

PROTEINS 967
 R. Kalaivani et al.

Figure 4
Fluctuations specific to AGC group are seen in residues involved in AGC-specific protein-protein interactions. (A) An example AGC group STY
kinase structure (PDB ID: 2JED_A) is depicted. The C-terminal tail is represented in sticks (green). The catalytic domain itself is represented in
cartoon, with the residues colored red if they interacted (inter Ca distances of 6 Å or less) with the C-terminal tail and gray if they did not. (B)
The C-terminal interaction density at every residue position, calculated from 58 AGC structures retrieved from iPfam, is plotted on the kinase fold
in a red-gray scheme. The residues that interacted with the C-terminal tail the most number of times in all the 58 structures are colored red and
those that interacted the least are represented in gray. (C) The residues that show AGC-specific structural fluctuations are mapped (red) on the
kinase fold. The residues that showed statistically different (unpaired t-test, P < 0.005) structural fluctuations in the 17 AGC kinase structures,
when compared with the 56 non-AGC kinase structures in Table I are colored red. (D) Residue-wise mean of normalized square fluctuations of 17
AGC group kinase structures (green) and 56 non-AGC group kinase structures (purple) are plotted as a function of the aligned residue positions.
Those alignment positions that showed statistically different (unpaired t-test, P < 0.005) structural fluctuation distributions between the AGC and
the non-AGC groups are marked red below the ordinate. (E) Residue-wise mean of (i) normalized square fluctuation differences (red), (ii) Ca-Ca
displacements (blue), and (iii) Voronoi packing density differences (green) between all possible AGC-non-AGC kinase pairs are plotted as a func-
tion of the aligned residue positions. On an average, we observe only a weak correlation of fluctuation differences with Ca-Ca displacements
(Spearman rank order correlation, mean cc 5 0.37) and Voronoi packing density differences (Spearman rank order correlation, mean cc 5 0.23).
[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

kinases and form the dataset for interaction analysis
(Interaction density analysis from iPfam structures sec-
tion in Methods). An example structure (PDB ID
2JED_A) is shown in Figure 4(A), where the C-terminal
residues are colored green and the kinase domain resi-
dues are colored red or gray depending on whether they
interact with the C-terminal tail or not respectively.
Across the 58 structures, we counted the number of
times each residue in the kinase domain interacts with
the C-terminal tail. We mapped this interaction density
onto the kinase fold in Figure 4(B), with red indicating
maximum and gray indicating minimum interaction
density. It can be noted that the C-terminal tail wraps
around the kinase, predominantly anchoring at regions

968 PROTEINS
 Structural Fluctuations in Protein Kinases
near aC-helix, b1-strand, hinge and C-terminal end of
the kinase. Previous studies65,71 have recognized a frac-
tion of these residues to be indispensable for kinase func-
tioning and C-terminal tail binding. Since the C-terminal
tail binding is a unique feature of most families within
the AGC group, we examined if the involved residues
show differential dynamics, in the form of structural
fluctuations, in the AGC group of kinases as compared
with others. To this end, we compared the normalized
square fluctuations of AGC STY kinases against those of
non-AGC STY kinases in our dataset. In Figure 4(D),
residue-wise mean, across 17 AGC STY kinases (green
curve), of the normalized square fluctuations is plotted,
contrasting that of 56 non-AGC STY kinases (purple
curve). Every residue position, that exhibits statistically
different (unpaired t-test, P < 0.005) structural fluctua-
tions between the AGC and non-AGC groups is marked
red below the zero ordinate. Upon mapping onto the
kinase structure fold [Fig. 4(C), red], we note that these
residues are clustered around aC-helix, b1-strand, hinge,
and C-terminal end. We observe remarkable agreement
between residues in AGC kinases, as determined from
structure complexes, to be involved in group-specific reg-
ulatory interactions [Fig. 4(B)] and those that were
derived from structural fluctuation analysis [Fig. 4(C)].
We probed if the difference in flexibility profiles observed
between the AGC and non-AGC kinases could be
explained by structural parameters like Ca-Ca displace-
ments or Voronoi packing density differences. To this
end, we calculated the Ca-Ca displacements, difference
in Voronoi packing densities and difference in square
fluctuations at every alignment position between all pos-
sible AGC-nonAGC structure pairs. In Figure 4(E),
residue-wise mean difference in square fluctuations (red
curve), residue-wise mean Ca-Ca displacements (blue
curve) and residue-wise mean difference in Voronoi
packing densities (green curve) are plotted. Fluctuation
differences only weakly correlate with Ca-Ca displace-
ments (Spearman rank order correlation, mean
cc 5 0.37) and Voronoi packing density differences
(Spearman rank order correlation, mean cc 5 0.23).
Thus, the correlate of residue interactions specific to
AGC kinases, observed as differential fluctuations, is not
a trivial consequence of structure differences. Repeating
the analysis, considering only the Protein Kinase A (PKA
or c-AMP dependent protein kinase) family of AGC
group of kinases against non-AGC kinases (Fig. 5), also
yields similar results.
Case study: TK group (with special reference to nRTKs)
The second group of STY kinases enquired for group-
specific structural fluctuations is tyrosine kinases (TK).
TK group consists of kinases that transfer the phosphate
moiety from an ATP specifically to a tyrosine residue in
the substrate protein. It encompasses diverse families,
including those that form cell surface receptors (receptor
tyrosine kinases, or RTKs) and those that are localized in
the cytoplasm (non-receptor tyrosine kinases, or nRTKs).
Despite the diversity, a key regulatory feature commonly
found in many of the TKs is their binding to Src
Homology 2 (SH2) domains. In the case of RTKs, cyto-
plasmic target proteins72 and adapter proteins73 bind to
the activated kinase domains via their SH2 domains. In
the case of nRTKs, contiguous SH2 and SH3 domains
are involved in interaction with the kinase domain, thus
regulating the TK between active and inactive states.74
Thus, SH2 domain, a key regulator of TK function and
unknown to regulate any other group of STY kinase, was
investigated as a plausible regulation determinant of TKs
that could be reflected in the structural fluctuations.
Structural fluctuation patterns observed only in the
members of TK group were analyzed as described in the
previous section. The residue-wise interaction density of
the kinase domain (Pfam ID: PF07714) with SH2
domain (Pfam ID: PF00017), determined from 24 avail-
able crystal structures, is plotted onto the kinase fold in
Figure 6(A). We observe that aC-helix, loop connecting
b7-b8 strands and parts of activation loop, aF-helix,
aH-helix and aI-helix are the known regions of interac-
tion with SH2 domain. Figure 6(C) shows the residue-
wise mean normalized square fluctuations of 25 TKs
(green curve) and 48 non-TKs (purple curve) from our
dataset (Table I). Those residue positions that show dif-
ferential (unpaired t-test, P < 0.0005) structural fluctua-
tions between the TK and non-TK kinases are marked in
red below the zero ordinate. When mapped onto the
kinase fold [Fig. 6(B), red], these residues span aC-helix,
loop connecting b7-b8 strands and parts of activation
loop, aG-helix, aH-helix and aI-helix. We observe a
strong agreement between the residues which are found
to be involved in group-specific regulatory interactions
of TKs [Fig. 6(A)] and those that were derived from
structural fluctuation analysis [Fig. 6(B)]. As in the pre-
vious analyses, we see that the fluctuation differences
between TK and non-TK kinase pairs are only weakly
correlated with Ca-Ca displacements (Spearman rank
order correlation, mean cc 5 0.46) and Voronoi packing
density differences (Spearman rank order correlation,
mean cc 5 0.27). Analyses contrasting only the nRTKs
with non-TKs (Fig. 7) also yield similar results.
Flexibility features characteristic of a
family—A case study with CDKs
In the previous sections, we have successfully
delineated the structural fluctuations essential for all STY
kinases and those specific for AGC and TK groups. Fur-
ther, we investigated if the residues characteristic of a
kinase family reflect structural dynamics characteristic of
the family. For this analysis, cyclin-dependent kinase
(CDK) family of the CMGC group was considered since
PROTEINS 969
 R. Kalaivani et al.

Figure 5
Fluctuations specific to PKA are seen in residues involved in AGC-specific protein-protein interactions. (A) An example PKA STY kinase structure
(PDB ID: 1ATP_E) is depicted. The C-terminal tail is shown in sticks (green) and the catalytic domain is represented in cartoon. (B) The
C-terminal interaction density at every residue position, calculated from 1ATP_E, is plotted on the kinase fold in a red-gray scheme. The residues
in the kinase catalytic domain that interacted with the C-terminal (inter Ca distances of 6 Å or less) are colored red and the rest are colored gray.
(C) Upon analysis of 9 PKA and 56 non-AGC kinase structures in Table I, residues that showed PKA-specific structural fluctuations (unpaired
t-test, P < 0.005) are mapped in red color on the kinase fold. (D) Residue-wise mean of normalized square fluctuations of 9 PKA kinase structures
(green) and 56 non-AGC group kinase structures (purple) are plotted as a function of the aligned residue positions. Those alignment positions that
showed statistically different (unpaired t-test, P < 0.005) structural fluctuation distributions between the PKA and the non-AGC groups are marked
red below the ordinate. (E) Residue-wise mean of (i) normalized square fluctuation differences (red), (ii) Ca-Ca displacements (blue), and (iii)
Voronoi packing density differences (green) between all possible PKA-non-AGC kinase pairs are plotted as a function of the aligned residue posi-
tions. On an average, we observe only a weak correlation of fluctuation differences with Ca-Ca displacements (Spearman rank order correlation,
mean cc 5 0.33) and Voronoi packing density differences (Spearman rank order correlation, mean cc 5 0.22). [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]

they are stringently regulated with several protein-protein
interactions. Some of these interactions are observed
only in the kinases of CDK family, that is, a given pro-
tein interacts with most members of the CDK family,
and with none of any other family, in the CMGC group.
We identified four such CDK-family-specific interactions
from iPfam:60 Cyclin_N (Pfam ID: PF00134), Ank_2
(Pfam ID: PF12796), CDKN3 (Pfam ID: PF05706), and
CKS (Pfam ID: PF01111). Figure 8(A–D) illustrates the
cartoon representations of each of these interactions
along with the interacting residues (red) in the kinase
catalytic domain. Similar to the previous analyses, the
residues in seven structures of the CDK family [Fig.
8(E), green curve] in our dataset (Table I) that showed
differential (unpaired t-test, P < 0.05) structural fluctua-
tions when compared with the eight structures of the
non-CDK families [Fig. 8(E), purple curve] of CMGC
group were marked in red below the zero ordinate.

970 PROTEINS
 Structural Fluctuations in Protein Kinases

Figure 6
Fluctuations specific to TK group exist in residues involved in TK-specific protein-protein interactions. (A) The SH2 domain interaction density at
every residue position, calculated from 24 TK structures retrieved from iPfam, is plotted on the kinase fold in a red-gray scheme. The residues that
interacted with the SH2 domain the most number of times in all the 24 structures are colored red and those that interacted the least are repre-
sented in gray. (B) Twenty-five TK and 48 non-TK structures’ global mode normalized square fluctuations were analyzed. The residues that show
TK-specific structural fluctuations with significant difference from non-TK groups (unpaired t-test, P < 0.0005) are mapped (red) on the kinase
fold. (C) Residue-wise mean of normalized square fluctuations of 25 TK group kinase structures (green) and 48 non-TK group kinase structures
(purple) are plotted as a function of aligned positions. Those alignment positions that showed statistically different (unpaired t-test, P < 0.0005)
structural fluctuation distributions between the TK and the non-TK groups are marked red below the ordinate. (D) Residue-wise mean of (i) nor-
malized square fluctuation differences (red), (ii) Ca-Ca displacements (blue), and (iii) Voronoi packing density differences (green) between all pos-
sible TK-non-TK pairs are plotted as a function of the aligned residue positions. On an average, we observe only a weak correlation of fluctuation
differences with Ca-Ca displacements (Spearman rank order correlation, mean cc 5 0.46) and Voronoi packing density differences (Spearman rank
order correlation, mean cc 5 0.27). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

When mapped on to the kinase catalytic fold [Fig. 8(F),
red], we can see remarkable agreement between the resi-
dues involved in binding of only CDKs to other proteins
[Fig. 8(A–D)] and those derived from structural fluctua-
tions [Fig. 8(F)]. Taken together, our analyses strongly
advocate that the residues functionally specialized to
carry out protein-protein interactions show conserved
and differential fluctuations at the kinase, group and
family levels.
Prediction of group and family
We performed a classical linear discriminant analysis
to predict the group classification of 71 STY kinase

PROTEINS 971
 R. Kalaivani et al.

Figure 7
Fluctuations specific to nRTKs are seen in residues involved in TK-specific protein-protein interactions. (A) The SH2 domain interaction density at
every residue position, calculated from 22 nRTK structures retrieved from iPfam, is plotted on the kinase fold in a red-gray scheme. The residues
that interacted with the SH2 domain the most number of times in all the 22 structures are colored red and those that interacted the least are repre-
sented in gray. (B) The residues that show nRTK-specific structural fluctuations are mapped (red) on the kinase fold. The residues that showed
statistically different (unpaired t-test, P < 0.0005) structural fluctuations in the 18 nRTK structures, when compared with the 48 non-TK structures
in Table I is colored red. (C) Residue-wise mean of normalized square fluctuations of 18 nRTK structures (green) and 48 non-TK group kinase
structures (purple) are plotted as a function of the aligned residue positions. Those alignment positions that showed statistically different (unpaired
t-test, P < 0.0005) structural fluctuation distributions between the nRTK and the non-TK groups are marked red below the ordinate. (D) Residue-
wise mean of (i) normalized square fluctuation differences (red), (ii) Ca-Ca displacements (blue), and (iii) Voronoi packing density differences
(green) between all possible nRTK-non-TK pairs are plotted as a function of the aligned residue positions. On an average, we observe only a weak
correlation of fluctuation differences with Ca-Ca displacements (Spearman rank order correlation, mean cc 5 0.46) and Voronoi packing density
differences (Spearman rank order correlation, mean cc 5 0.32). [Color figure can be viewed in the online issue, which is available at wileyonlineli-
brary.com.]

structures (Supporting Information Fig. S6) using their
structural fluctuations at 326 alignment positions in a
leave-one-out method. The 71 STY kinases in the dataset
belonged to one of the five groups: AGC, CAMK, CMGC,
STE, and TK. Assuming equal distribution and sampling
of kinases in the five groups, a random group classification
would result in an accuracy of 20% based purely on
chance. If the distribution of the 71 kinases in the five
groups is taken into account, we expect an accuracy of
18.8% 6 9.7 by chance. However, the simplest classifier
could predict the groups of the STY kinases, based solely
on the normalized global mode structural fluctuations,

972 PROTEINS
 Structural Fluctuations in Protein Kinases

Figure 8
Fluctuations specific to CDK family are seen in residues involved in CDK-specific protein-protein interactions. CDK-specific protein-protein inter-
actions with (A) Cyclin_N (PDB ID: 1JST), (B) Ank_2 (PDB ID: 1BLX), (C) CDKN3 (PDB ID: 1FQ1), and (D) CKS (PDB ID: 1BUH) are
depicted. The CDK-specific interacting partners are represented in sticks (green). The kinase catalytic domain itself is represented in cartoon, with
the residues colored red if they interacted (inter Ca distances of 6 Å or less) with the CDK-specific partner and gray if they did not. (E) Residue-
wise mean of normalized square fluctuations of seven CDK family structures of CMGC group (green) and 8 non-CDK family structures of CMGC
group (purple) in Table I are plotted as a function of the aligned residue positions. Those alignment positions that showed statistically different
(unpaired t-test, P < 0.05) structural fluctuation distributions between the CDK and the non-CDK families are marked red below the ordinate. (F)
The residues that show CDK-specific structural fluctuations (unpaired t-test, P < 0.05) in the seven CDK structures, when compared with the eight
non-CDK structures, are mapped (red) on the kinase fold. (G) Residue-wise mean of (i) normalized square fluctuation differences (red), (ii) Ca-
Ca displacements (blue), and (iii) Voronoi packing density differences (green) between all possible CDK-non-CDK pairs are plotted as a function
of the aligned residue positions. On an average, we observe only a weak correlation of fluctuation differences with Ca-Ca displacements (Spearman
rank order correlation, mean cc 5 0.54) and Voronoi packing density differences (Spearman rank order correlation, mean cc 5 0.33).

with an accuracy of 90.14%, significantly better than
chance (v2 test, P 5 E-10). Similarly, prediction of family
was done on a dataset of 62 STY kinases (Supporting
Information Fig. S7). They belonged to 16 families: Akt,
DMPK, PKA, PKC, DAPK, PHK, CDK, GSK, MAPK,
SRPK, STE20, Abl, Ack, EGFR, InsR, and Src. A random
prediction of family would be of 6.25% accuracy. Consid-
eration of distribution of kinases in the families gives a
mean expected accuracy of 4.7% 6 4.2 by chance. Again, a
better than chance (v2 test, P 5 E-16) of 82.26% accuracy
was observed in the classifier predictions. This
discriminant analysis suggests that the information present
in the global mode fluctuations of the kinase residues is
sufficient to classify them into groups and families. Previ-
ously, we demonstrated that the group-specific and
family-specific residues, in terms of differential structural
fluctuations, often corresponded to regions of specific
modular interactions with other proteins/domains. Taken
together, we infer that those alignment positions that are
weighed maximally by the classifier would be likely sites of
interactions with other proteins/domains observed only in
the group or family concerned.

PROTEINS 973
 R. Kalaivani et al.
Application of structural fluctuations in
inhibition of kinases
Design of proper kinase inhibitors remains a difficult
task, especially in attaining selectivity of target. Although
many inhibitors have been designed75 for various STY
kinases, recent high throughput binding and functional
assays have revealed the extensive cross reactivity of the
drugs.76,77 One approach to obtain highly selective
drugs is to limit their binding to the target STY kinase
alone. This requires knowledge of regions in the STY
kinase that are specific to the target by way of differential
binding abilities. From our previous analyses, we derive
that these regions with differential binding abilities will
have differential structural fluctuations. We aim to pin-
point the regions of differential fluctuations using GNM
based NMA that could be potential drug targets with
selective binding modes. To this end, we analyzed the
milestone studies of Fabian et al.76 and Karaman et al.77
and noted that Src inhibitors frequently bind to off-
targets like Abl and EGFR kinases. We undertook the
case study of Src kinases to determine the regions that
are characterized by statistically distinct structural fluctu-
ations from that of Abl and EGFR kinases.
From our dataset (Table I), we compared the normal-
ized global mode square fluctuations of 10 Src kinases
[Fig. 9(A), green curve] against that of 4 Abl kinases
[Fig. 9(A), purple curve]. The residues with differential
fluctuations (unpaired t-test, P < 0.05) are marked in red
below the ordinate. When mapped to the Src kinase fold,
these residues [Fig. 9(B), red] correspond to the b-sheet
in the N-lobe and the aG-helix in the C-lobe. Likewise,
when compared with the three EGFR family kinases [Fig.
9(C), purple curve], the Src kinases [Fig. 9(C), green
curve] had differential (unpaired t-test, P < 0.01) struc-
tural fluctuations around the b-sheet in the N-lobe and
the aG-helix in the C-lobe [Fig. 9(D), red]. We propose
that these regions are potential targets for drugs with
increased selectivity imparted by specific binding.
Recently, the aG-helix has also been identified to be
essential for substrate recognition and binding in many
kinases.78,79 Thus, aG-helix is an attractive candidate
for drug target that will both inhibit the kinase activity
as well as impart selectivity. This prediction remains to
be validated experimentally.
DISCUSSION
Traditional methods that detect similarity in sequence,
structure and motifs in order to gauge the relatedness
among proteins have shortcomings. Dynamics is emerg-
ing as the key indicator of function, regulation and spec-
ificity of proteins. Indeed, proteins exist as fluid
molecules with constant motions inside the cell80 and
movements are crucial to function, regulation, stability
and evolution.81,82 For the first time, global mode
974 PROTEINS
fluctuations (derived from GNM-based NMA) of the
homologous STY kinase superfamily have been scruti-
nised. STY kinases have been experimentally and man-
ually curated over the years and categorized into groups,
subcategorized into families and further divided into
subfamilies. STY kinases within a subfamily are more
closely related to each other than those within a family
and across subfamilies, which are in turn more closely
related to each other than those within a group and
across families. The clear hierarchy is manually estab-
lished, taking into account the sequence similarities, sub-
strate specificities, concurring domain architectures,
cellular localization, participating biological pathways,
etc.21 This extensive understanding of the STY kinases
makes it possible to validate the effectiveness of GNM-
based NMA derived structural fluctuations in under-
standing protein relatedness.
STY kinases form a large family with diverse substrate
specificities, regulatory mechanisms and protein-protein
interactions. These properties are comparable for closely
related STY kinases (Within-Subfamily category) and
diverge progressively as we move through Within-Family,
Within-Group, and Across-Group categories. Consistent
with this, the global mode structural fluctuations are
most correlated in the Same-Protein and Within-
Subfamily categories, with hierarchical decrease through
Within-Family, Within-Group and Across-Group catego-
ries. This is the first pointer that suggests that the struc-
tural fluctuations derived from NMA reflect
diversification in regulatory features observed in homolo-
gous kinases. It is well known that the functional or reg-
ulation relatedness among proteins, in many cases,
corresponds to similar sequence and structure. Our study
points out that the similarity in structural fluctuations,
although derived solely from the Ca positions, shows
only weak correlations with structure similarity (such as
RMSD, Ca-Ca displacements and packing density differ-
ences) and sequence similarity. Taken together, the global
mode structural fluctuations correspond well with
domain/protein association attributes and weakly with
sequence and structure attributes. This trend has been
previously observed17,19 in globins and other homolo-
gous families. These evidences strongly suggest that the
fluctuations important for retention of attributes such as
specificity, regulation, and localization are selectively con-
served during evolution against those that are not.
If there is indeed conservation of structural fluctua-
tions, in order to preserve common regulatory attributes
of related kinases, we should be able to identify a con-
served flexibility signature in all the STY kinases that is
crucial for the common phosphotransfer function. Such
a conserved flexibility profile was identified around the
catalytic loop of the kinase fold. The residues three-
dimensionally in and around the catalytic loop enjoy
very little mobility when compared with the other resi-
dues. This result also corroborates with a previous
 Structural Fluctuations in Protein Kinases

Figure 9
Residues with fluctuations specific to Src family may be used for drug targeting. (A) Residue-wise mean of normalized square fluctuations of 10
Src family structures of TK group (green) and 4 Abl family structures of TK group (purple) in Table I are plotted as a function of the aligned resi-
due positions. Those alignment positions that showed statistically different (unpaired t-test, P < 0.05) structural fluctuation distributions between
the Src and Abl families are marked red below the ordinate. (B) The residues that showed differential fluctuations (unpaired t-test, P < 0.05) in the
10 Src structures, when compared with the four Abl structures, are mapped (red) on the kinase fold. (C) Residue-wise mean of (i) normalized
square fluctuation differences (red), (ii) Ca-Ca displacements (blue), and (iii) Voronoi packing density differences (green) between all possible Src-
Abl kinase pairs are plotted as a function of the aligned residue positions. On an average, we observe only a weak correlation of fluctuation differ-
ences with Ca-Ca displacements (Spearman rank order correlation, mean cc 5 0.38) and Voronoi packing density differences (Spearman rank order
correlation, mean cc 5 0.22). (D) Residue-wise mean of normalized square fluctuations of 10 Src family structures of TK group (green) and 3
EGFR family structures of TK group (purple) in Table I are plotted as a function of the aligned residue positions. Those alignment positions that
showed statistically different (unpaired t-test, P < 0.01) structural fluctuation distributions between the Src and the EGFR families are marked red
below the ordinate. (E) The residues that showed differential fluctuations (unpaired t-test, P < 0.01) in the 10 Src structures, when compared with
the 3 EGFR structures, are mapped (red) on the kinase fold. (F) Residue-wise mean of (i) normalized square fluctuation differences (red), (ii) Ca-
Ca displacements (blue), and (iii) Voronoi packing density differences (green) between all possible Src-EGFR kinase pairs are plotted as a function
of the aligned residue positions. On an average, we observe only a weak correlation of fluctuation differences with Ca-Ca displacements (Spearman
rank order correlation, mean cc 5 0.57) and Voronoi packing density differences (Spearman rank order correlation, mean cc 5 0.20). [Color figure
can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

study63 that reported relatively higher stability of cata- throughout the STY kinase superfamily. This conserved
lytic residues in other enzyme families. Further, the flexibility signature can be understood in the light of
restricted mobility of these residues is conserved function of STY kinases. The aspartate in the catalytic

PROTEINS 975
 R. Kalaivani et al.
loop is a completely conserved residue that accepts
hydrogen during the phosphotransfer. It would be essen-
tial for the residues around the phosphosite and nucleo-
side to be stable for efficient phosphate moiety transfer,
explaining the signature stability of the catalytic loop in
the kinases.
After describing the structural fluctuations common to
all the STY kinases, the present study identified struc-
tural fluctuations conserved in closely-related STY
kinases, such as AGC and TK groups. The structural
fluctuations which were differentially present in AGC
group, as compared with all other groups, were indeed
found in residues that were involved in AGC-group-
specific protein-protein interactions. Similarly, the fluctu-
ations that demarcated the TK group from all other
groups were observed in residues involved in TK-group-
specific protein-protein interactions. We report, for the
first time, that residues that accomplish group-specific
regulatory roles (like modular interaction with another
domain/protein) also possess fluctuations that are signifi-
cantly different from other groups. This indicates that
residues involved in group-specific regulatory mecha-
nisms can be identified by virtue of their differential
fluctuation patterns. This could find implications in
identifying regions of protein-protein interactions and
regulatory modulations from the structures alone. This
result is also true in a much narrower and more specific
category of family. CDK family of STY kinases, belonging
to the CMGC group, shows fluctuations remarkably dif-
ferent from other CMGC kinases in those residues that
are involved in CDK-family-specific protein-protein
interactions. Thus, we have identified structural fluctua-
tions essential for STY kinases, specific for AGC and TK
groups and unique to CDK family.
Present analyses suggest that global mode fluctuations
of STY kinases contain information specific to functional
attributes like protein binding, and thus are capable of
identifying the group and family classification of a kinase
structure. Upon testing the hypothesis using a linear dis-
crimination analysis, we find that it is possible to classify
a kinase structure to its group and family solely based on
its global mode square fluctuations. Finally, we present a
possible application of using the global mode square
fluctuations as a means to predict drug targets in STY
kinases. As a case study, we identified the aG-helix of
the Src kinases to be a potential drug target. We predict
that a drug designed to bind the Src kinase at the aG-
helix will not only inhibit the kinase, but also have mini-
mal cross reaction with similar kinases like Abl and
EGFR. Indeed, aG-helix has a sufficiently distinct fluctu-
ation pattern in Src, when compared with Abl and EGFR
kinases. As an extension, we could interpret that aG-
helix will result in distinct binding modes, and hence
curtail cross reaction. Laboratory experiments need to be
carried out in order to test this prediction.
976 PROTEINS
CONCLUSIONS
We have, for the first time, validated GNM based
NMA as a tool to understand the regulation attributes
and specificities in a protein superfamily. Taking the
well-studied STY kinases as the model system, we have
established that the global mode structural fluctuations
are conserved better in closely related proteins than in
distantly related proteins. Such a conservation of fluctua-
tion profiles is not a trivial consequence of sequence and
structure similarities, but has correspondence to func-
tional relatedness. The conservation of fluctuations is
reliably observed at different levels of hierarchy of STY
kinases: group, family, and subfamily. We identified fluc-
tuation profiles essential to all STY kinases and unique
to AGC group, TK group and CDK family of kinases.
Such group-characteristic and family-characteristic fluc-
tuation patterns strongly localized to group-specific and
family-specific protein-protein interaction sites. Finally,
we tested whether the global mode fluctuations con-
tained enough information to demarcate the groups and
families of STY kinases. After successful classification, we
identified, based on flexibility profile analysis, aG-helix
to be a potential drug target site in Src kinase that would
minimise cross reaction with Abl and EGFR kinases.
ACKNOWLEDGMENT
The authors thank the anonymous reviewers whose
comments helped improve the rigor and presentation of
the study.
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