i An update to this article is included at the end
Molecular Cell, Vol. 11, 571–575, March, 2003, Copyright 2003 by Cell Press
Kleisins: A Superfamily
of Bacterial and Eukaryotic
SMC Protein Partners
We describe a superfamily of eukaryotic and prokary-
otic proteins (kleisins) that includes ScpA, Scc1, Rec8,
and Barren. Scc1 interacts with SMC proteins through
N- and C-terminal domains to form a ring-like struc-
ture. Since these are the only domains conserved
among kleisins, we suggest that ring formation with
SMC proteins may define this family.
SMC (structural maintenance of chromosomes) proteins
have key functions in organizing chromosomal DNA in
most living organisms including bacteria (Hirano, 2002).
They interact with several other proteins (called non-SMC
components) to form different complexes (Jessberger,
2002). Their functions range from mitotic chromatin con-
densation (condensin complex) to sister chromatid co-
hesion (cohesin complex), DNA repair, and gene regula-
tion (Hirano, 2002; Nasmyth, 2002). Altogether, six
paralogous groups of eukaryote SMC proteins (Smc1-6)
are known. They have the same molecular architecture
(1000–1500 residues) involving an N- and a C-terminal
globular domain (corresponding to the Walker A and B
box-related segments of an ATPase), a central globular
dimerization domain and two interconnecting regions
with preference for a helical secondary structure.
Whereas the N- and C-terminal arms form an intramolec-
ular coiled coil that brings the two terminal ATPase seg-
ments spatially together, the central domain is responsi-
ble for heterodimerization with the equivalent domain
of another SMC protein (Haering et al., 2002). Cohesin
contains an Smc1/Smc3 heterodimer, condensin, an
Smc4/Smc2 heterodimer, and the recently discovered
DNA repair complex, an Smc5/Smc6 heterodimer (Hir-
ano, 2002).
Most eubacteria and archaea have a single SMC ho-
molog (Soppa, 2001) that forms homodimers (Melby et
al., 1998). Such proteins have been implicated in the
segregation and compaction of chromosomes both in
Bacillus subtilis (Britton et al., 1998) and Caulobacter
crescentus (Jensen and Shapiro, 1999). The B. subtilis
SMC protein forms a complex with two additional pro-
teins, ScpA and ScpB. Both are presumed to be essen-
tial for SMC function, as deletion of either of them pro-
duces a phenotype similar to that of smc (Mascarenhas
et al., 2002; Soppa et al., 2002). A functional connection
between SMC/ScpA/ScpB is also supported by their
clustering in the genome and, possibly, joint operon
regulation in a variety of bacteria (see http://
www.ncbi.nlm.nih.gov/COG for COG1196, COG1354,
and COG1386 [Tatusov et al., 2001]).
Our initial aim was to search for homologs of B. subtilis
ScpA (ypuG, NP_390203, 251 AA). The architecture of
the protein is tripartite. Its central region is probably
Letter to the Editor
unstructured in solution because it has lowered se-
quence complexity (residues 74–123, detected by SEG
with relaxed parametrization “25-3.0-3.3” [Wootton and
Federhen, 1996]) and contains polar segments (26—
often negatively—charged residues in the sequence
segment 76–123). In contrast, domains at the N and C
termini probably form a globular structure.
Independent PSI-BLAST (Altschul et al., 1997)
searches (inclusion E value ⫽ 0.001, compositionally
corrected statistics) in the nonredundant protein data-
base were started separately with the N and the C termini
of B. subtilis ScpA (see also protocols at http://mendel.
imp.univie.ac.at/SEQUENCES/kleisins/). Both searches
detected numerous prokaryote orthologs. Surprisingly,
the search with the C-terminal domain also hits a hypo-
thetical protein from A. thaliana (NP 188295) with signifi-
cance (E value ⫽ 0.0002 in round 4). Querying the protein
database with the plant sequence detected orthologs
not only in other plants but also in human, mouse, fly,
and protozoa.
We continued with a fan-like family collection strat-
egy, using every homologous sequence segment of a
significantly established new prokaryote family member
as the starting point for a new PSI-BLAST search.
Searches with N-terminal ScpA segments remained
within the prokaryotic clade, whereas the C-terminal
queries linked to eukaryotic proteins. Surprisingly, a PSI-
BLAST search with the C-terminal region of Staphylo-
coccus aureus ScpA (amino acids 147–249) detected at
a significant level Scc1/Rad21 of C. elegans (in round
5, E value ⫽ 0.0006). Additional iterations found another
six members of the Scc1/Rec8 family (convergence at
round 11). The Pseudomonas aeruginosa ScpA
(NP_251888) also significantly linked to the Scc1/Rec8
family (hit of Rec8 from mouse [AAF69524] with E value ⫽
0.001 in round 4).
The superfamily consisting of (1) ScpAs, (2) Scc1s/
Rec8s, and (3) the new sequence family, including the
hypothetical A. thaliana protein NP_188295, was called
kleisin (from the Greek word for closure: ␬␭⑀ı́␴ı␮␱ or
kleisimo). We named the Scc1/Rec8 subfamily kleisin-␣
and the newly identified eukaryotic family kleisin-␤.
We started searches for additional kleisin superfamily
members from a multiple alignment of the C termini
of ScpA/Scc1/Rec8 proteins and constructed a hidden
Markov model (HMM). No significant hits were identified
in the nonredundant database that had not already been
included in the ScpA/Scc1/Rec8 family. Nevertheless,
several Barren proteins appeared below the level of sig-
nificance immediately following the last verified hit, the
Rec8 from S. pombe. RPS-BLAST searches with mem-
bers of the Barren family against the CD database
(Marchler-Bauer et al., 2002) resulted in nonsignificant
(0.35 for the Drosophila Barren and much higher E values
for other homologs) hits to the PF2616/DUF173 domain.
However, we built a position-specific scoring matrix
from a nonredundant set of C-terminal segments of the
collected ScpA/Scc1/Rec8 sequences. RPS-BLAST
searches with this matrix resulted in significant hits
within the C termini of eight Barren proteins (Encephali-
Molecular Cell
572

Figure 1. N- and C-Terminal Domains of the Kleisin Superfamily

(A) C-terminal alignment of the kleisin protein family obtained after superposition of family alignments with selected member sequences from
the prokaryote kleisin (ScpA), the kleisin-␣ (Scc1/Rec8), kleisin-␤, and kleisin-␥ (Barren) groups. The numbers in parentheses are the correspond-
ing NCBI gi entries. (#) Conceptual translation from UniGene cluster Hs.180903 and gi 23097285. ($) Conceptual translation from
gnl|CVMUMN_5807 (preliminary sequence data was obtained from the University of Minnesota Cryptosporidium parvum Genome [MCPG]
Project website at http://www.cbc.umn.edu/researchprojects/agac/cp/). Positions with a conservational trend for functional residues are
marked with an asterisk. The secondary structure assignments at the bottom are for B. subtilis ScpA and correspond to predictions with PHD
(Rost, 1996). Helices are in red and ␤ strands are in green.
(B) N-terminal alignment of the kleisin protein family.
Letter to the Editor
573

Table 1. Nomenclature and Major Homology Relationships of Kleisins and Related SMC Proteins in Model Organisms

SMC Proteins

Kleisin Family Species Kleisin 1st SMC 2nd SMC

Prokaryote kleisin B. subtilis ScpA SMC SMC

E. coli ? (MukE or MukF) (MukB) (MukB)

Kleisin-␣ S. cerevisiae Scc1 Smc1 Smc3

Rec8a
S. pombe Rad21 Psm1 Psm3
Rec8a
A. thaliana SYN2 CAB77587 AAD26882
SYN3
SYN1a
C. elegans COH-1 F28B3.7 Y47D3A
COH-2
COH-3
REC-8a
D. melanogaster DmRad21 DmSMC1 DmSMC3
Mei-910ac
X. laevis XRAD21 XCMC1 XSMC3
H. sapiens Hrad21 hSMC1␣ hSMC3
Rec8a hSMC1␤a
Kleisin-␥ S. cerevisiae Brn1 Smc4 Smc2
S. pombe Cnd2 Cut3 Cut14
A. thaliana AAC25941 BAB10693 BAB11491
C. elegans Kle-2dc SMC-4 MIX-1
(DPY-26)b DPY-27b MIX-1
D. melanogaster Barren Gluon DmSMC2
X. laevis XCAP-H XCAP-C XCAP-E
H. sapiens HCAP-H hCAP-C hCAP-E

Postulated kleisins S. cerevisiae ? Smc5 Rhc18

S. pombe ? Spr18 Rad18
A. thaliana ? CAC01791 MIM
C. elegans ? F54D5.14 C23H4.6
D. melanogaster ? CG32438 CG5524
H. sapiens ? hSMC5 hSMC6

Names in SMC protein complexes follow published nomenclature (Dong et al., 2001; Hirano and Hirano, 2002; Jessberger, 2002) but are
rearranged to emphasize homology relationships. More closely related proteins (all kleisins, Smc1/Smc4 homologs, Smc2/Smc3 homologs)
are collated in a common column. Smc5/Smc6 are about equally distant to the remaining SMCs (Jones and Sgouros, 2001). Functional
replacements by proteins that are not shown to be homologous to kleisins or SMC proteins are enclosed in parentheses. According to the
results of Gruber et al. (2003), it can be assumed that Smc1 and Smc4 homologs interact with the C terminus and Smc3 and Smc2 homologs
with the N terminus of the corresponding kleisin.
a
Kleisins with a known meiotic chromosome segregation phenotype.
b
Protein with a role in gene dosage compensation.
c
Added in this study.
d
Functional replacement by a kleisin-␤ family member.

tozoon cuniculi, O. sativa, D. melanogaster, A. gambiae,
H. sapiens, P. falciparum, S. cerevisiae, and S. pombe,
E values between 0.01 and 3e-05). Therefore, the Barren
protein family can be safely included into the kleisin
superfamily as kleisin-␥.
An alignment of the N- and C-terminal domains of the
kleisin superfamily shows almost complete identity of
the hydrophobic pattern and some conservation of func-
tional residues (Figure 1). ScpA orthologs (prokaryote
kleisins) were detected throughout the bacterial king-
dom except for a few subdivisions: Crenarchaeota,
Methanothermobacteria, ␥-proteobacteria (including En-
terobacteriaceae, Pasteurellaceae, and Vibrionaceae),
⑀-proteobacteria, Chlamydia, and Rickettsia (see Sup-
plemental Table S1 at http://www.molecule.org/cgi/
content/full/11/3/571/DC1). The loss of the SMC/kleisin
pair might be explained by nonorthologous displace-
ment (Koonin et al., 1996) by the MukB/E/F system in
some proteobacteria. As far as currently available se-
quence data suggest, ScpAs and SMCs exist concomi-
tantly in many Prokarya but never together with MukB/E/F.
All eukaryotic families (kleisin ␣, ␤, and ␥) exist in
parallel, but some kleisins are missing in certain branches
of the phylogenetic tree (kleisin-␤ in fungi, kleisin-␥ in
nematodes) (Wildpaner et al., 2001). The absence of
kleisin-␥ in C. elegans raised the possibility that its newly
identified kleisin-␤ member KLE-2 (C29E4.2) might func-
tion during mitosis in a condensin-like SMC complex.
Like depletion of MIX-1 (SMC2-like) or SMC-4 (Kaitna
et al., 2002), kle-2(RNAi) prevents individualization of
chromosomes during prophase and induces extensive
chromosome bridges during anaphase (see supplemen-
tal data at http://www.molecule.org/cgi/content/full/11/
3/571/DC1).
Table 1 lists kleisins and their putative interacting
SMCs from several model organisms. There is a building
Molecular Cell
574
block common to all kleisin members, which involves
conserved N- and C-terminal globular domains sepa-
rated by a variable linker region with many polar resi-
dues. However, each family has distinctive features. The
prokaryote kleisins have typical sequence length of
ⵑ250 residues. The kleisin-␣ and kleisin-␤ family se-
quences are typically between 550 and 700 amino acids
(except in the parasitic organisms E. histolytica and E.
cuniculi). The variable-length central linker region con-
tains a conserved, partly hydrophobic segment of 20–30
residues. The kleisin-␥ family sequences are the most
divergent group (700–1000 residues). They have an ex-
tended central linker with alternating regions of lowered
complexity and many polar residues followed by inter-
spersed conserved motifs with hydrophobic residues.
It is quite likely that we have not yet found all super-
family members. Previous studies have established that
DPY-26 contains short N- and C-terminal motifs that
manually align with the Barren termini (Hirano et al.,
1997; Lieb et al., 1996). DPY-26 functions exclusively in
dosage compensation together with the SMC proteins
MIX-1 (an Smc2 ortholog) and DPY-27 (Smc4-like para-
log) and it could conceivably be a kleisin descendant.
Our searches have also not yet revealed any obvious
homology between kleisins and the MukE or MukF pro-
teins, which associate with the SMC-like MukB protein
of E. coli. Given a similar 3D structure and molecular
function of SMCs and MukB, it is possible that MukF
(which is more tightly associated with MukB than is
MukE [Yamazoe et al., 1999]) might perform the same
function for MukB as kleisins do for SMCs.
Where investigated, all kleisins appear to function to-
gether with SMC proteins. The B. subtilis ScpA protein
associates with an SMC protein, and deletion of either
of them produces very similar phenotypes (Mascaren-
has et al., 2002; Soppa et al., 2002). Kleisin-␣ proteins
are part of the cohesin complex and mediate sister chro-
matid cohesion together with Smc1/Smc3 heterodimers
(Nasmyth, 2002). Kleisin-␥ proteins are found associated
with Smc2/Smc4 heterodimers in the condensin com-
plex, and their inactivation in flies, frogs, and yeast have
similar consequences to that of smc2 or smc4 (Hirano,
2002). Finally, the depletion phenotype of the KLE-2
member of the kleisin-␤ family in C. elegans (this work)
suggests that in worms, which lack any obvious member
of the kleisin-␥ family, an Smc2/Smc4/kleisin-␤ complex
may fulfill a function similar to that of the Smc2/Smc4/
kleisin-␥ complex (condensin). Mammals and flies pos-
sess both types of complexes, which presumably have
different functions. Furthermore, kleisin-like proteins
(possibly kleisin-␤) may be part of an Smc5/Smc6
complex.
What then are the functions of SMC/kleisin devices
and what are the specific roles of kleisins? Yeast Scc1’s
N- and C-terminal domains have been shown to bridge
the heads of Smc3 and Smc1, respectively (Gruber et
al., 2003; Haering et al., 2002). These two domains are
the very same sequences that are conserved between
all members of the kleisin family. The Smc1 and Smc3
molecules to which Scc1 binds are also associated via
their dimerization domains situated at the other end of
the extended antiparallel coiled coil (Haering et al.,
2002). As a consequence, the connection of Smc1 and
Smc3 heads by Scc1 creates a large ring. The eukaryotic
SMC/kleisin rings so created are asymmetric structures.
In contrast, bacterial SMC proteins form homodimers,
which potentially can bind to two ScpA molecules.
We suggest that SMC and kleisin proteins cooperate
as part of the same molecular ring device. The conserva-
tion of SMC arm length suggests that a conserved mac-
romolecular complex is enclosed by the SMC/kleisin
rings. Cohesin is so tightly associated with chromatin
that it cannot be removed by 1.6 M KCl (Ciosk et al.,
2000). Spatial closeness of DNA and SMC/kleisin ring
is also demonstrated by chemical crosslinking (Tanaka
et al., 1999) and binding assays between B. subtilis SMC
and DNA (Hirano and Hirano, 2002). Yet, proteolytic
cleavage of its Scc1 (kleisin-␣) subunit by separase at
the metaphase-to-anaphase transition causes the SMC/
kleisin ring to dissociate from chromosomes (Nasmyth,
2002). Mutations in the hinge domain of B. subtilis SMC
inhibit SMC dimerization and its ability to interact with
DNA (Hirano and Hirano, 2002). Because cleavage of
Smc3⬘s coiled coil also causes cohesin to fall off chro-
mosomes and destroys sister chromatid cohesion
(Gruber et al., 2003), it has been proposed that cohesin’s
association with DNA arises from the passage of nucleo-
some strands through an Smc1/Smc3 ring closed by
Scc1 (Haering et al., 2002). If so, the association of SMC/
kleisin devices with chromatin is topological and not
chemical.
SMC/kleisin complexes may be primarily topological
devices that organize chromosomal DNA by bundling
strands. While cohesin’s function may be to hold to-
gether sister DNA molecules (interchromosomal cross-
linking), the function of condensin may be to “secure”
coils or link distant sequences on the same molecule
(intrachromosomal crosslinking). Bacterial SMC/kleisin
devices could in principle perform either of these func-
tions.
Acknowledgments
We thank Eugene Koonin for comments and the University of Minne-
sota Cryptosporidium parvum sequencing project. This work was
supported by Boehringer Ingelheim, FWF Österreich (P15037), and
Österreichische Nationalbank.
Alexander Schleiffer,* Susanne Kaitna,
Sebastian Maurer-Stroh, Michael Glotzer,
Kim Nasmyth, and Frank Eisenhaber*
Research Institute of Molecular Pathology
Dr. Bohr-Gasse 7
A-1030 Vienna
Austria
*Correspondence: schleiffer@imp.univie.ac.at (A.S.), frank.eisenhaber@
imp.univie.ac.at (F.E.)
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Molecular Cell
Volume 5, Issue 4, April 2000, Page 767

DOI: https://doi.org/10.1016/S1097-2765(04)70058-1