Journal of Structural Biology 184 (2013) 182192

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Journal of Structural Biology

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Crystal structure of rat intestinal alkaline phosphatase Role of crown

domain in mammalian alkaline phosphatases
Kaushik Ghosh a,, Debarati Mazumder Tagore a, Rushith Anumula a, Basanth Lakshmaiah a,
P.P.B.S. Kumar a, Senthuran Singaram a, Thangavelu Matan a, Sanjith Kallipatti a, Sabariya Selvam a,
Prasad Krishnamurthy a, Manjunath Ramarao b,
a
Applied Biotechnology, Biocon Bristol-Myers Squibb Research and Development Center, Syngene International Ltd., Biocon Park, Bommasandra IV Phase, Jigani Link Road,
Bangalore 560099, India
b
Bristol-Myers Squibb India Ltd. and Biocon Bristol-Myers Squibb Research and Development Center, Bangalore 560099, India

a r t i c l e i n f o a b s t r a c t

Article history: Intestinal alkaline phosphatases (IAPs) are involved in the cleavage of phosphate prodrugs to liberate the
Received 2 July 2013 drug for absorption in the intestine. To facilitate in vitro characterization of phosphate prodrugs, we have
Received in revised form 20 September 2013 cloned, expressed, puried and characterized IAPs from rat and cynomolgus monkey (rIAP and cIAP
Accepted 21 September 2013
respectively) which are important pre-clinical species for drug metabolism studies. The recombinant
Available online 27 September 2013
rat and monkey enzymes expressed in Sf9 insect cells (IAP-Ic) were found to be glycosylated and active.
Expression of rat IAP in Escherichia coli (rIAP-Ec) led to 200-fold loss of activity that was partially recov-
Keywords:
ered by the addition of external Zn2+ and Mg2+ ions. Crystal structures of rIAP-Ec and rIAP-Ic were deter-
Rat intestinal alkaline phosphatase
structure
mined and they provide rationale for the discrepancy in enzyme activities. Rat IAP-Ic retains its activity in
Crown domain presence of both Zn2+ and Mg2+ whereas activity of most other alkaline phosphatases (APs) including the
Metal ion requirements cIAP was strongly inhibited by excess Zn2+. Based on our crystal structure, we hypothesized the residue
Glycosylation Q317 in rIAP, present within 7 of the Mg2+ at M3, to be important for this difference in activity. The
Q317H rIAP and H317Q cIAP mutants showed reversal in effect of Zn2+, corroborating the hypothesis. Fur-
ther analysis of the two structures indicated a close linkage between glycosylation and crown domain
stability. A triple mutant of rIAP, where all the three putative N-linked glycosylation sites were mutated
showed thermal instability and reduced activity.
2013 Elsevier Inc. All rights reserved.

1. Introduction
Alkaline phosphatases (APs), a class of hydrolase (EC 3.1.3.1)
common to all organisms, are involved in the hydrolysis of phos-
phate monoesters under alkaline conditions. They are metalloen-
zymes consisting of three metal ions, typically two Zn2+ and one
Mg2+ ions in their active site. Mammalian APs (mAPs) have low se-
quence identity with their bacterial counterparts (2530%) but the
active site residues and the residues coordinating the metal ions
Abbreviations: APs, alkaline phosphatases; EcAP, Escherichia coli alkaline phos-
phatase; PLAP, placental alkaline phosphatase; IAP, intestinal alkaline phosphatase;
GCAP, germ cell alkaline phosphatase; TNAP, tissue non-specic alkaline phospha-
tase; pNPP, p-nitrophenyl-phosphate; rIAP, rat intestinal alkaline phosphatase;
cIAP, cynomolgus monkey intestinal alkaline phosphatase; rIAP-Ic, rat intestinal
alkaline phosphatase expressed in Sf9 insect cell; rIAP-Ec, rat intestinal alkaline
phosphatase expressed in E. coli; TM, triple mutant; Tm, melting temperature; Nag,
N-acetyl glucosamine.
Corresponding authors.
E-mail addresses: Kaushik.Ghosh@syngeneintl.com (K. Ghosh), manjunath.
ramarao@bms.com (M. Ramarao).
are largely conserved. Escherichia coli AP (EcAP) is one of the most
well studied enzymes both biochemically and structurally (Kim
and Wyckoff, 1991). All APs have a basic aba fold and the biolog-
ically active species is dimeric. Among the eukaryotic AP homologs,
structural information is available for human placental AP (hPLAP)
(Le Du et al., 2001; Llinas et al., 2005) and shrimp AP (SAP) (De
Backer et al., 2002, 2004). The overall catalytic mechanism is well
conserved between species. One Zn2+ ion (M1) activates the cata-
lytic serine leading to the formation of phosphoserine intermedi-
ate. A water molecule activated by the second Zn2+ ion (M2)
hydrolyzes the phosphoserine intermediate. The phosphate then
gets released or transferred to an acceptor (Xu and Kantrowitz,
1991). The third metal site (M3) was originally thought to be
responsible for providing the catalytic base (hydroxide ion bound
to Mg2+) which deprotonates the catalytic Ser (Kim and Wyckoff,
1991). But recent studies implicate it in stabilizing the transferred
phosphate moiety in the transition state (Zalatan et al., 2008).
The differences between the mammalian and prokaryotic APs
have been well documented (Zhang et al., 2005). Mammalian APs
are more active and less thermostable in comparison to the EcAP.

1047-8477/$ - see front matter 2013 Elsevier Inc. All rights reserved.
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 K. Ghosh et al. / Journal of Structural Biology 184 (2013) 182192
Structurally, the major difference is the presence of a distinct
crown domain and an N-terminal helix swap in the mAPs (Le Du
et al., 2001; Bossi et al., 1993). Additionally, mAPs are allosteric
enzymes (Hoylaerts et al., 1997) and are susceptible to non-
competitive inhibition by L-amino acids like, L-Phe, L-Trp, L-Leu,
L-homoarginine and levamisole (Fishman and Sie, 1971; Van Belle,
1976). There are four major types of APs found in human germ
cell (GCAP), intestinal (IAP), placental (PLAP) and tissue non-spe-
cic (TNAP). The rst three of these are located on chromosome
2 and have 9098% homology in sequence. TNAP is located on
chromosome 1 and has 50% homology with the other three iso-
forms and is believed to be the ancestor of the tissue-specic
APs. Mutations in TNAP gene has been implicated in hypophospha-
tasia involving diseases like, rickets and osteomalacia (Weiss et al.,
1988; Henthorn et al., 1992; Mornet, 2000). PLAP, on the other
hand, is thought to be involved in the transfer of maternal IgG to
the fetus (Makiya and Stigbrand, 1992) and also is one of the rst
proteins to be ectopically expressed by cancer cells. IAPs have the
highest specic activity among the different isoforms and they
have been extensively used in diagnosis, immunoassays and
molecular biology (Jablonski et al., 1986; Sekiguchi et al., 2011).
In the drug discovery paradigm, in many instances, poor solubil-
ity of the lead molecule precludes its development into a drug.
Phosphate prodrugs have been used successfully in few such cases
to increase the solubility as well as bioavailability of the parent
drug (Fleisher et al., 1996). IAPs present in the intestinal brush bor-
der play an important role in drug metabolism by cleaving phos-
phate prodrugs and releasing the parent drug. This approach
works best for the so called class II compounds in biopharmaceuti-
cal classication, which have good permeability but poor solubility.
However, not all phosphate prodrugs undergo the desired rapid
bioconversion by the IAP and there is a need to better understand
this process in vitro to facilitate the design of phosphate prodrugs.
Rat and cynomolgus monkey are two of the most common pre-clin-
ical species in drug discovery and recently rat intestinal mucosal
scraps and Caco-2 monolayers have been used to characterize the
phosphate prodrugs in vitro (Haodan et al., 2009). A better and
more quantitative model will be to use pure IAP enzymes. Since
these are not commercially available, we have cloned, expressed
and puried IAPs from human, rat and cynomolgus monkey.
While most species studied have one isoform of IAP, rat is un-
ique in having two isoforms of IAP (I and II) (Xie and Alpers,
2000), whereas cow has four isoforms of IAP (Manes et al., 1998).
Previous work with the rat IAP suggested that the two isoforms
are different in terms of their metal ion requirement and showed
different inhibitory potential when titrated with high concentra-
tion of Zn2+ (Calhau et al., 2000). Jejunal mucosa expressing only
isoform I was not inhibited by 1 mM Zn2+ whereas the duodenal
mucosa expressing both the isoforms was inhibited by 41% in the
presence of 1 mM Zn2+ (Calhau et al., 2000; Harada et al., 2005).
Here, we focus on the biochemical and biophysical character-
ization of puried rat and cynomolgus monkey IAP and report
the rst crystal structure of rat IAP. Structural analysis provide a
rationale for the decreased activity of mammalian APs when ex-
pressed in bacteria and further identify a key residue involved in
making rIAP insensitive to Zn2+. We have also established an
in vitroin vivo correlation of phosphate prodrug cleavage in rat
discussed in a separate manuscript (Subramanian et al., 2013).
2. Results and discussion
2.1. Expression, purication and characterization of rat and
cynomolgus monkey IAP
The rat and cynomolgus monkey IAP genes were cloned as de-
tailed in the methods. For expression in insect cells, the original
183
N-terminal signal sequence was kept intact and the variable C-
terminus region (483-end) was replaced by a hexahistidine tag to
facilitate afnity purication of these proteins. The residue num-
bering throughout the manuscript is based on the mature protein
sequence. For bacterial expression, the construct was similar
to the one used for expression in insect cells except that the
N-terminal signal sequence was removed.
APs were puried using metal afnity and size exclusion chro-
matography as described in the methods. The puried protein was
a dimer based on the migration in SEC and as determined by the
dynamic light scattering (DLS). The puried insect cell expressed
rIAP protein was found to be heavily glycosylated and migrated
as two distinct bands on SDSPAGE (Fig. 1a). Both bands were po-
sitive in anti-His Western blots indicating them to be the target
protein (Fig. 1b). We have conrmed their identity by performing
an in-gel activity staining. The slower migrating band retained
activity in the presence of 1% SDS in the gel (Fig. 1c). Since mam-
malian APs are known to be glycosylated and dimeric, this suggests
that the slower migrating band corresponds to a dimer, which is
resistant to denaturation. When treated with deglycosylating en-
zyme PNGaseF under denaturing conditions, we obtained a single
faster migrating species on the gel indicating the deglycosylated
monomers (lane 2, Fig. 1d). The bacterially expressed rIAP protein
was positive in anti-His Western blots and behaved as dimer in SEC
(data not shown).
2.2. Activity of the proteins and effect of metal ions on the activity
Activity of puried APs were tested using p-nitrophenylphos-
phate (pNPP) as substrate as described in the methods. The pH
optimum for rIAP and cIAP activity was found to be pH 9.5 (Supple-
mentary Fig. S1). Specic activity of the rat, cyno, bovine IAPs and
human placental AP enzymes tested followed the order bIA-
P > rIAPcIAP > hPLAP (Table 1a). The effect of pH on the catalytic
activity of these enzymes is evident from a 10-fold reduction in
the kcat/Km with an increase in pH from 9.5 to 10.5 (Table 1b).
The difference in the rate constant comes from a 10-fold reduction
in the Km of the enzymatic reaction at pH 10.5.
The effect of exogenous metal ion on enzyme activity was stud-
ied using zinc acetate and magnesium acetate. The enzyme was di-
luted in 1 M DEA buffer, pH 9.5 and magnesium acetate was
titrated from 10 lM to 10 mM nal concentration. No effect of
exogenous magnesium (up to 10 mM) was observed on rIAP and
cIAP activity. Zinc acetate titration at a concentration range of
50 lM to 1 mM was performed in 1 M DEA buffer pH 9.5 using
pNPP (10 mM nal) as a substrate at 37 C. Zinc had no effect on
the activity of rIAP but had a pronounced inhibitory effect on cIAP
(Fig. 2). Activity of bIAP and hPLAP was also inhibited with increas-
ing concentration of Zn2+ (Supplementary Fig. S2). On the other
hand, excess Mg2+ (up to 10 mM) had no effect on the activity of
any of these enzymes under the reaction condition tested (data
not shown).
The inhibitory effect of Zn2+ on APs has been well documented
in the literature (Cathala and Brunel, 1975; Linden et al., 1977). It is
believed that at higher concentrations, Zn2+ displaces the Mg2+ ion
from M3 site causing a signicant reduction in the activity of the
enzyme (Hung and Chang, 2001). Similar to the Zn-bound SAP
structure (De Backer et al., 2002, 2004), rIAP isoform I is hypothe-
sized to contain Zn2+ in all 3 metal positions (Harada et al., 2005),
accounting for the lack of inhibitory effect when titrated with ex-
cess Zn2+. An alternative hypothesis is that rIAP can accommodate
either Zn2+ or Mg2+ in M3 position without much difference in
activity. To understand the metal binding in detail we determined
the crystal structures of rIAP isoform I expressed in both insect and
E. coli cells.
184 K. Ghosh et al. / Journal of Structural Biology 184 (2013) 182192

Fig.1. Purication and initial characterization of rIAP: (a) SDSPAGE analysis of the puried rIAP with coomassie blue staining; 3 and 6 lg of nal rIAP protein were loaded in
lanes 1 and 2 respectively; molecular weight marker was loaded in lane 3. (b) Anti-His Western blot to conrm the identity of the rIAP protein; both the bands were positive
indicating that may be they are from the same protein. (c) Gel on the left was treated with NBTBCIP reagent to check for AP activity. The blue band indicates active AP. The
protein was treated with PNGaseF with or without heat denaturation as noted in the picture. The protein (middle lane) lost activity when heat denatured and treated with
PNGaseF and (d) same gel post stained with coomassie blue reagent conrms presence of two different species. (For interpretation of the references to color in this gure
legend, the reader is referred to the web version of this article.)

Table 1a
Specic activity of the different AP enzymes in DEA buffer.

pH 9.5 (IU/mg) pH 10.5 (IU/mg)

Rat IAP-I 254.06 82.04
Rat IAP-II 186.83 30.06
Rat IAP-I Q319H 226.71 190.29
Cyno IAP H319Q 95.85 20.03
Cyno IAP 259.71 130.15
Human PLAP 0.39 0.43
Bovine IAP 652.87 330.47
Rat IAP-I E. coli 17.83

2.3. Structure of rIAP Fig.2. Effect of exogenous addition of Zn acetate on the activities of rIAP and cIAP:
the activity of rIAP and cIAP in the presence of increasing concentration of Zn-
acetate was measured by absorbance at 405 nm after 10 min of pNPP hydrolysis.
The rIAP-Ec and rIAP-Ic structures were determined to 2.21 and
2.38 resolution respectively (Table 2 and Fig. 3). To the best of
our knowledge these are the rst crystal structures of any intesti- site Ser (92) was covalently linked with phosphate. This could be a
nal AP.rIAP-Ec structure: In the rIAP-Ec structure, the overall aba AP crystallization artifact, potentially due to impurities from the re-
fold, N-terminal helix swap and the disulde linkages were similar agents, as phosphate was not present in the crystallization mixture
to that of hPLAP (PDB id 1EW2). However, the crown domain nor was rIAP-Ec crystallized in the presence of pNPP. Moreover, the
(F362-T431) was not visible in the electron density maps crystals were obtained at pH 6.5 which lowers the cleavage rate of
(Fig. 3a). The tight interaction between the two monomers keeps the phosphoserine bond. In addition, the absence of metal at M2
the C-terminal end of the protein together, which is clearly visible could also account for the trapping of the phosphoserine interme-
in the structure (colored purple and red in Fig. 3a). Two disulde diate.rIAP-Ic structure: In the rIAP-Ic structure, the overall aba AP
linkages were visible in each monomer between residues C121 fold, N-terminal helix swap and the disulde linkages were similar
C183 and between C467C474. Only one metal in the active site to that of rIAP-Ec and hPLAP (PDB code 1EW2). The rIAP-Ic protein
at M3 was visible and identied as Mg2+ together with another was co-crystallized with pNPP which resulted in a fully metalated
Mg2+ (M4) at the metal binding domain (Le Du et al., 2001) structure very similar to that of the hPLAP (Le Du et al., 2001)
(Fig. 3a). The nal model includes 2 protein chains, 4 magnesium (Fig. 3b and Supplementary Fig. S3). The nal model of rIAP-Ic in-
ions, 2 glycerols and 279 water molecules. Interestingly the active cludes 2 protein chains, 4 zinc ions, 6 magnesium ions, 2 pNP (p-ni-
tro phenol) molecules, 9 Nag (N-acetyl glucosamine) sugar
Table 1b
molecules and 390 water molecules. We could model all the resi-
Enzymatic characterization of different IAP enzymes. dues starting from V1 to E482 in the mature protein chains and
part of the C-terminal His-tag as well (4 His residues in monomer
Enzyme Km (mM) Vmax (mM/s) kcat (s1) kcat/Km (M1s1)
A and two His residues in monomer B). The crown domain (F362
pH 9.5 T431) was clearly observed in the electron density maps and was
Rat IAP 1.33 4.35E  04 1.67E + 03 1.27E + 06
modeled using the sequence information. Two disulde linkages
Cyno IAP 0.40 2.70E  04 1.77E + 03 4.46E + 06
similar to the rIAP-Ec structure (C121C183 and C467C474) were
pH 10.5
Rat IAP 11.896 4.15E  04 1.60E + 03 1.34E + 05
observed (Supplementary Fig. S3).
Cyno IAP 3.955 2.26E  04 1.48E + 03 3.75E + 05 Glycosylation for rIAP-Ic was observed in the electron density
Bovine IAP 7.87 7.55E  05 3.43E + 03 4.36E + 05 maps close to N281 and N408 in both the monomers. There are 4
Human PLAP 1.91 8.49E  05 1.98E + 01 1.04E + 04 Nag units attached to the N281 residue in monomer A and 3
 K. Ghosh et al. / Journal of Structural Biology 184 (2013) 182192 185

Table 2
Data collection and renement statistics for the two rIAP structures.

Structure renement
rIAP-Ec rIAP-Ic
Unit cell () a = 63.32, b = 75.73, c = 170.71 a = 90.31, b = 167.17, c = 71.84
Wave length () 1.22 0.9796
Resolution () 2.21 (2.282.20)a 2.38 (2.472.38)
Spacegroup P 21 21 21 P 21 21 21
Total No. of reections measured 251,307 247,490
No. of unique reections 42,064 44,083
No. of free reections 2109 2224
Completeness 99.9% 99.9%
I/Ir 17.2 (2.4) 10.1 (2.7)
R 16.74 16.43
Rfree 20.34 20.43
No. of protein atoms 6172 7343
No. of hetero atoms 313 546
No. of solvent atoms 277 390
R.m.s. deviation of bond lengths () 0.010 0.010
R.m.s. deviation of bond angles () 1.08 1.11
R.m.s. deviation of dihedral angles () 16.74 16.57
Wilson B factor (2) 42.31 30.20
a
Values in the parenthesis are for the last shell.

Fig.3. (a) Structure of rIAP-Ec at 2.21 resolution. Monomer A and B are colored as green and yellow respectively. The missing crown domain is circled and indicated by the
dotted lines and the rest of the C-terminus is indicated by purple (A) and red (B) respectively. The two disulde linkages in each monomer are marked by red circles. Mg2+ ions
are shown as green spheres and (b) structure of the rIAP-Ic determined at 2.38 resolution. Monomer A and B are colored as green and yellow respectively. Metal ions are
represented by spheres (Zn blue, Mg green), pNP molecule and sugar moieties by sticks.

attached to N281 in monomer B. One Nag unit was found attached
to N408 in the crown domain in each of the monomers. The glyco-
sylation at N408 is unique and observed for the rst time in this
rIAP-Ic structure and, as shown below, might have important
implication in stability of the dimeric protein. In the hPLAP struc-
ture, residues N122 and N249 were glycosylated (Le Du et al.,
2001). N122 is conserved between hPLAP and rIAP (Supplementary
Fig. S4) and is located on the surface of the protein. Though glyco-
sylation is not observed in the electron density maps at N122 for
rIAP-Ic, its position mimics that of hPLAP and is most likely glycos-
ylated in vivo. Furthermore, while the residue corresponding to
N249 of hPLAP is absent in rIAP (Supplementary Fig. S4), glycosyl-
ation of N281 could stabilize the additional metal binding site.
2.4. Active site architecture
The active site of each monomer of rIAP-Ic contained 2 Zn2+
atoms (occupancy of 1 and 0.75 for M1 and M2 respectively) and
1 Mg2+ ion (M3) along with pNP (Fig. 3b and Supplementary
Fig. S5). The protein was crystallized in presence of 50-fold molar
excess of pNPP. This might have facilitated the binding of pNP near
the active site after its generation from the cleavage reaction. The
O5 atom of the nitro group is at a distance of 2.20 from the Zn2+
ion at M1. The re-rened structure of hPLAP in presence of p-
nitrophenyl phosphonate (pNPPate), a non-hydrolysable substrate
analogue (PDB id 3MK1) also had a pNP molecule bound near
the active site (Llinas et al., 2005; Stec et al., 2010). Unlike the
re-rened hPLAP structure or the rIAP-Ec structure, no phosphate
densities were observed in the active site of rIAP-Ic structure.
The M3 site of rIAP-Ic structure was found to be occupied by
Mg2+ in contrast to a published structural model of rIAP where
M3 was modeled as a Zn2+ (Harada et al., 2005). There was no
exogenous Zn2+ in the crystallization trials (a common additive
for most AP crystallization conditions) or during expression and
purication but the purication buffers contained 1 mM MgCl2. It
is possible that continuous exposure to MgCl2 led to the replace-
ment of the Zn2+ at M3 with Mg2+ ion. There are structures of
SAP where M3 is either Zn2+ (PDB id 1SHN) or Mg2+ (PDB id
1SHQ) and in the SAP/Zn2+structure, H149 (H153 in rIAP) coordi-
nates Zn2+ at M3 (De Backer et al., 2002). In the Mg2+ bound SAP
structure (PDB id 1SHQ) De Backer et al. (2004) as well as
the rIAP-Ic structure, H149 in SAP and H153 in rIAP) are
186 K. Ghosh et al. / Journal of Structural Biology 184 (2013) 182192
superimposable (Fig. 4) and they are not in direct contact with M3
but coordinate the Mg2+ ion through water molecules. It was also
observed that the R162 side chain in the SAP/Zn2+ structure
(1SHN) is in a docked conformation by forming H-bonding
interactions with the bound phosphate (Fig. 4, red dotted lines)
(De Backer et al., 2004). On the other hand, in the SAP/Mg2+ struc-
ture (1SHQ), the R162 side chain is in a non-docked conformation.
The orientation of R166 side chain (in rIAP-Ic) is similar to the ori-
entation of the docked R162 side chain in the SAP/Zn2+ structure
(1SHN), even though there are no phosphate in the active site, pos-
sibly because of ionic interaction between NH1 of R166 and the O6
of the pNP (Fig. 4, blue dotted lines). But the H153 side chain in
rIAP is in a position similar to the SAP structure with Mg2+ at M3
(1SHQ) further indicating the presence of Mg2+ at M3 in our
rIAP-Ic structure (Fig. 4).
Two additional Mg2+ ions were observed in the rIAP-Ic struc-
ture. One of these Mg2+ (M4) ions (located 10 away from the ac-
tive site) was also observed in the rIAP-Ec structure and in the
hPLAP structure (PDB id 1EW2) in a site called the metal binding
domain (Le Du et al., 2001). Coordination of the Mg2+ at this site
by residues E216, E270 and D285 are similar between the rIAP
and hPLAP structures. As mentioned in the case of the hPLAP struc-
ture (Le Du et al., 2001), it was not possible to distinguish between
Mg2+ and Ca2+ at this position using the current data. However,
since excess Mg2+ was utilized during purication and crystalliza-
tion, it is most likely that the metal in that position is Mg2+. The 5th
Mg2+ ion (M5) is unique in the rIAP-Ic structure and was not
observed in the hPLAP structure. It is located near the base of the
protein, at the end of the a11 helix (Supplementary Fig. S6), with
an average B-factor of 35 2 and is coordinated by three water
molecules and backbone carbonyl atoms of S342 and T345. This
Mg2+ ion is at a distance of >30 from the active site and probably
plays a structural role.
2.5. Structural basis of the observed effect of Zn2+ on rIAP
As mentioned earlier, exogenous addition of Zn and Mg acetate
have a different effect on the rIAP in comparison to bIAP, cIAP and
hPLAP. Activities of these three IAPs are inhibited in the presence of
excess Zn2+ whereas there is no effect on rIAP (Fig. 2). The differen-
tial effect of excess Zn2+ can be explained by the structural analy-
sis. The residues contacting the 3 metal ions in the active sites of
the AP enzymes have been conserved from bacteria to human
(Supplementary Fig. S4). However, there is less conservation in
Fig.4. Comparison of SAP and rIAP active sites: the SAP structures 1SHN (orange
carbon) and 1SHQ (blue carbon) were superimposed onto the rIAP-Ic structure
(green carbon); His149 moves towards the Zn at M3 (1SHN) to have a favorable
interaction. R162 has two different orientations (docked and non-docked) and R166
in the rIAP structure matches that of the docked conformation (1SHQ). Metal ions
for 1SHQ are omitted from the picture for simplicity.
the second shell of residues surrounding the metal ions. The M1
(Zn2+) site has the highest afnity for the metal and it is mainly be-
cause of the coordination through two conserved His residues. The
M2 site has been shown to be less stringent at least in case of EcAP;
it has been shown that the M2 Zn2+ can be replaced by Mg2+ with-
out affecting the overall activity of the enzyme (kcat/Km) (Tibbitts
et al., 1996). There is a decrease in afnity for substrate but con-
comitantly there is an increase in kcat due to ease of phosphate re-
lease in the presence of Zn2+ in M2 (Tibbitts et al., 1996). The M3
metal ion, in comparison, is a bit more accessible and, presumably,
is the rst metal ion to be displaced during titration (Coleman,
1992).
Comparison of the structure of hPLAP with rIAP revealed that all
the residues that are in direct contact of the metal ions are con-
served and the only difference is in the second sphere of residues
(Fig. 5a), where residue 317 was different in rat compared to other
IAPs and hPLAP (Supplementary Fig. S4, position indicated by a
star). This was also observed by Harada et al. while discussing a
structural model of rIAP (Harada et al., 2005). The side chain of
Q317 is at a distance of 6.64 from M3 Mg2+ ion and occupies sim-
ilar position as that of H317 in hPLAP (Fig. 5a). In order to conrm
the role of residue 317, we generated the Q317H mutation in rIAP
and H317Q mutation in cIAP. They showed a reversal of inhibitory
effect of Zn2+, i.e. the Q317H mutant rIAP was strongly inhibited by
excess Zn2+ and the H317Q cIAP was resistant to inhibition by
Zn2+ (Fig. 5b). This indicates that the Q317 residue has a direct
role in determining the effect of exogenous Zn2+ on the activity
of rIAP.
2.6. Comparison of the two rIAP structures
Based on the superposition of the two rIAP structures (rIAP-Ec
and rIAP-Ic) (Supplementary Fig. S6), the TLR tripeptide fragment
(residues 368370) was modeled into the additional 2FoFc density
in the rIAP-Ec structure near the missing crown domain (Supple-
mentary Fig. S7). This tripeptide is visible in both the subunits of
the rIAP-Ec structure and is not connected to the rest of the protein
in the structure. The tripeptide is held in its place by a network of
ionic interactions and the tripeptide from monomer B is present
close to monomer A and vice versa (Fig. 6). The NH1 and NH2 of
the R370 side chain of monomer B is stabilized by interactions with
the OE1 of E6 of monomer B and the backbone of V85 from mono-
mer A. The NE of R370 interacts with E66 OE1 of monomer B. The
backbone of R370 was stabilized by interaction with the backbone
of D86 from monomer A. Calculation of omit map showed clear
densities for the tripeptide (Fig. 6b and c). Overall, the structures
of rIAP-Ec and rIAP-Ic are very similar. Excluding the crown do-
main, the r.m.s.d. in Ca main chain atoms between the two struc-
tures is 0.58 . The number of residues in the dimer interface for
the rIAP-Ic structure is 129 and 80 for the rIAP-Ec structure. The to-
tal buried surface areas in the rIAP-Ec and rIAP-Ic structures are
2820 (16.7% of total accessible surface area) and 4475 2 (23.1%
of total accessible surface area) respectively. The difference in the
number of residues at the interface and consequently the amount
of buried surface area stems from the lack of the crown domain in
the rIAP-Ec structure.
In the rIAP-Ic structure, H320, H432 and D316 coordinate the
M1 Zn2+ ion while the M2 Zn2+ is stabilized by interactions with
D42 and D316. The crown domain is further stabilized by the pres-
ence of the Y367 side chain from the other monomer. Additionally,
there are two ionic interactions between the backbone of the
residues present in the loop between helices a11 and a12
(A321A324) and residues from the crown domain which stabilize
the region. The side chain NZ of K420 interacts with carbonyl of
A321 and the NE2 of Q421 makes polar interaction with the
backbone carbonyl of G322 (Fig. 7). The Mg2+ at M3 is stabilized
 K. Ghosh et al. / Journal of Structural Biology 184 (2013) 182192 187

Fig.5. Residue 317: (a) superposition of rIAP (in green) and the hPLAP (in yellow) structures showing the active site. All the residues near the metal ions are conserved except
for the change in residue 317 which is about 6.6 away from M3 and (b) effect of addition of Zn acetate on the activity of rat Q317H and cynomolgus monkey H317Q IAP
mutants.

Fig.6. TLR tripeptide: (a) stabilizing interaction of R370 (magenta) of monomer B (yellow) with neighbouring residues of both monomers A (green) and B are shown by red
dotted lines. The distances between the interacting atoms and the residues are labelled. The omit map densities for the TLR tripeptide of monomers A and B at Ir is shown in
(b) and (c) respectively.

by interaction with D42, S155 and E311 side chains together with
water molecules. On the contrary, in the rIAP-Ec structure, H320
and H432 are disordered (Fig. 7) and D316 is moved away
(2.84 distance between CG atoms of D316 between the two
structures). Further, owing to the absence of crown domain, the
anchoring interaction mediated through residues in the a11 and
a12 helices are lost and the loop is disordered allowing the a11 he-
lix to move (10). This resulted in the displacement of the side
chains of D316 and H320 leading to loss of metal ion at the M2 site.
The M3 metal (Mg2+) binding site is intact as the metal coordinat-
ing residues are not affected by the loss of the crown domain.
2.7. Why is the crown domain missing from the rIAP-Ec structure?
The puried rIAP-Ec protein was intact and its mass was con-
rmed by SDSPAGE and mass spectrometry (MS) analysis. The
initial hypothesis for the missing crown domain was that the do-
main is oppy in nature and hence it was not visible in the electron
density maps. Another hypothesis to explain the missing crown
domain was that protein was being cleaved during storage and
or crystallization. To explore protein cleavage, crystals were run
on SDSPAGE gels after extensive washing in stabilizing buffer.
We found that the protein migrated faster (at 42kDa) indicating
cleavage during crystallization trials at 20 C (Fig. 8a). The cleavage
was conrmed by incubating puried protein at room temperature
for 60 h (Fig. 8b, lane 1) and analyzing samples by mass spec. The
mass of the major species was determined to be 42062.7 Da. We
have also demonstrated that addition of protease inhibitors (Sig-
ma) prevents the cleavage. Crystallization attempts in the presence
of the protease inhibitors were unsuccessful indicating cleavage of
rIAP-Ec protein is important in crystallization. This led us to
hypothesize that the absence of glycosylation in bacterial expres-
sion systems for rIAP may lead to improperly folded crown domain
and that this domain, consequently, is more prone to protease
cleavage. As mentioned earlier, there is a T-L-R tripeptide (368
370) fragment from the crown domain visible in the rIAP-Ec struc-
ture (Fig. 6). Analysis of the sequence revealed that, cleavage at
K388 will produce a fragment with a molecular weight of
42,031 Da, which is very close to that observed after MS analysis
of the cleaved protein. Under non-denaturing conditions, this rst
cleavage is followed by a second cleavage after the R370 (part of
TLR) which becomes exposed leading to the removal of the crown
domain. Chakraborty et al. (2011) recently used CLASP (CataLytic
Active Site Prediction) software to show that APs, at least, SAP
188 K. Ghosh et al. / Journal of Structural Biology 184 (2013) 182192

On the other hand, the K388 residue in the rIAP-Ic structure is

Fig.7. Active site comparison between rIAP-Ic and rIAP-Ec structures: superposition
of the rIAP-Ec (blue carbon) and rIAP-Ic (green carbon) structures showing the
movement of the a11 helix near the active site of monomer A. The crown domains
are colored in green (A) and yellow (B) respectively for the two monomers of rIAP-
Ic. The loop connecting a11 and a12 helices is disordered in rIAP-Ec structure. In
the rIAP-Ic this loop is stabilized by hydrogen bonding interactions between the
crown domain residues K420 and Q421 and the backbone carbonyl of A321 and
G322 respectively. Residues directly contacting M1 (Zn), H320, D316 and H432 side
chains are displaced for the rIAP-Ec structure (shown in blue carbon) as well as D42
which contacts both M2 and M3. The Y367 present close to the TLR peptide from
monomer B (yellow) is also shown in the rIAP-Ic structure.
has partial proteolytic activity where it can cleave self as well as
other proteins. Moreover, its autocatalytic activity increases when
the metal ions are removed using EDTA. This indicates that the
rIAP-Ec in absence of the fully folded crown domain can potentially
auto-cleave to release the crown domain.
shielded by the glycosylation at the N408 residue in the other sub-
unit. It could be the reason why the crown domain is protected
when expressed in Sf9 cells. Further analysis of the rIAP protein se-
quence revealed that there are three possible N-linked glycosyla-
tion motifs (N-X-S/T) at residues N122, N281 and N408
respectively (Supplementary Fig. S4). To test this hypothesis, these
asparagine residues were mutated individually to aspartic acid and
a triple mutant was also generated (N122D/N281D/N408D). They
were expressed in Sf9 cells and puried from media. Stability of
the puried proteins was monitored using a uorescence based
thermal shift assay (TSA) (Pantoliano et al., 2001; Lo et al., 2004).
The results are summarized in Table 3. The Tm of IAPs puried from
insect cells were dependent on the buffer conditions and, in most
cases, the proteins were less stable in DEA buffer (di-ethyl amine)
pH 9.5 and were stabilized with the addition of Zn2+. The proteins
were most stable in the storage buffer (100 mM Tris (pH 7.4),
300 mM NaCl, 1 mM MgCl2 and 10% Glycerol (v/v)). Interestingly,
the rIAP-Ec was found to be signicantly less stable specically
in the DEA buffer pH 9.5 (Tm = 37 C). The Tm of the N to D mutants
demonstrated reduced thermal stability in comparison to the wild
type rIAP (Table 3) and thermal stability prole matches exactly to
the rIAP expressed in E. coli. The reduction in thermal stability
could possibly be due to the lack of N-linked glycosylation. The
activity of the triple mutant is reduced to half of that observed
for the wild type (data not shown). The activity measurements
were performed at 37 C and the Tm of the protein is also 37 C.
This could possibly be explained by two hypotheses one where
the insect cell is better equipped to fold the crown domain even
in the absence of glycosylation or the glycosylation is not essential
for proper folding of the crown domain. Based on our results we
believe that glycosylation plays an important role in the proper
folding of the crown domain.
We have isolated the rIAP-Ec protein without the crown domain
(Fig. 8b) and compared its activity with that of the intact rIAP-Ec
(either at 4 C or incubated with protease inhibitor cocktail at room
temperature). The specic activity of the intact rIAP-Ec protein is
similar to that of the cleaved product in presence of 1 mM Zn ace-
tate (16 IU/mg). The protease inhibitor did not alter the activity
of the rIAP-Ic protein when tested under similar conditions indicat-
ing that the phosphatase activity is not affected by the protease
inhibitor. This result indicates that the activity of rIAP-Ec protein
is unaffected by the presence or absence of the crown domain,
leading us to believe that the crown domain is not folded properly
in the rIAP-Ec protein. Consequently, the active site was not
formed properly and the afnity towards the metal ions was de-
creased leading to lowering of activity. In presence of excess Zn2+

Table 3
Thermal stability of different APs.

Name of Protein Tm DEA pH Tm DEA pH Tm SBa pH

9.5 (C) 9.5 + 1 mM Zn2+ (C) 7.4 (C)
hPLAP 71 63 NDb
bIAP 60 59 65.5
rIAP-I 60 66.5 65.5
rIAP-II 39 62.5 ND
rIAP-Ec 37 64 57
cIAP 38.5/60.5 62.5 41/65
rIAPN281D 42/54.5 66 64.5
Fig.8. Why crown domain is missing from rIAP-Ec structure? (a) one of the rIAP-Ec rIAPN408D 41/54.5 65.5 55.5/63
crystals is loaded onto a SDSPAGE (lane 1) after washing it thoroughly in reservoir rIAPN281D N408D 39.5/54 65.5 50/62.5
buffer. It migrated corresponding to molecular weight of 40 kDa protein distinctly rIAP TM (N122D 37/47 65.5 51/61.5
faster than the original puried protein (lane 2) which migrates as 55 kDa and (b) N281D N408D)
result of incubating the rIAP-Ec protein with and without protease inhibitors for
Two values in a single column for some of the constructs indicate dual transitions.
66 h. Lane 1 rIAP incubated at 20 C without PI; lane 2 rIAP with PI incubated at a
SB storage buffer.
20 C; lane 3 rIAP without PI incubated at 4 C. The faster migrating band in lane 1 b
ND not determined.
was later conrmed by ESI-MS analysis as 42062.7 Da.
 K. Ghosh et al. / Journal of Structural Biology 184 (2013) 182192
Fig.9. Protease cleavage sites: superposition of the two rIAP structures showing location of the K386, N408 and R370 residues. The K386 in ball and stick is shielded by the
glycosylation (sticks) at N408. Cleavage after K386 will lead to a fragment with theoretical molecular weight of 42,031 Da.
the active site could be partially occupied by Zn2+ leading to partial
recovery of activity (4-fold for intact rIAP-Ec and 30-fold for rIAP-
Ec without the crown domain, data not shown).
3. Conclusion
Rat and cynomolgus monkey IAPs were cloned, expressed, puri-
ed and characterized. These enzymes are being used to test the
cleavage efciency of phosphate prodrugs. The generated data will
help us understand the difference in cleavage efciency between
species. The crystal structure of rIAP was determined and, to the
best of our knowledge, this is the rst report of expression and
crystallization of recombinant rIAP. The crown domain in the
E. coli expressed rIAP protein is not properly folded and is prone
to protease cleavage. Based on recent literature reports, the loss
of crown domain due to protease cleavage could result from self-
cleavage of the protein, specically when metal sites are not fully
occupied (Chakraborty et al., 2011). Lack of properly folded crown
domain leads to the movement of key residues involved in interac-
tion with the active site metal ions which in turn is responsible for
the decrease in activity. The susceptibility to proteases is decreased
in case of rIAP expressed in Sf9 cells because of N408 glycosylation
which protects K386 from cleavage (Fig. 9).
Activity of rIAP was not inhibited by excess Zn2+ leading to the
hypothesis that rIAP has Zn2+ in all three metal positions. Crystal
structure of rIAP protein contained Mg2+ at the M3 position, but
it does not rule out the possibility that Zn2+ can also occupy this
position without changing the activity of the enzyme. The lack of
inhibition by excess Zn2+ in rIAP was reversed in the Q317H mu-
tant. On the other hand, the corresponding H317Q mutant of cIAP
was not inhibited by excess Zn2+ supporting the hypothesis that
the glutamine residue at position 317 renders the rIAP insensitive
to Zn2+ probably by indirect interaction mediated by water mole-
cules. The structural and biochemical data together suggests two
possibilities one indicating rIAP might be able to accommodate
either Zn2+ or Mg2+ in its active site without losing activity and
the other indicating rIAP has reduced afnity for Zn2+ specically
at the M3 site, hence its activity is not affected by excess Zn2+.
189
Residue Q317 in rIAP which is >6 away from the M3 site and
4 from H153, plays a role in this process (indicated by our
mutagenesis data) plausibly through one or more water molecules.
4. Materials and methods
Sequence of all the primers used in this study can be found in
the Supplementary document 1.
4.1. Denovo cloning
Rat and cynomolgus monkey total intestinal RNA was pur-
chased from Biochain. Rat IAP sequence from gene bank
(NM_022665) was used to design primers for cloning. For cyno-
molgus monkey, primers were designed in conserved regions using
multiple sequence alignment of gorilla, rhesus, human and chimp
sequences (all primers used in the study are listed in Supplemen-
tary document 1). First strand cDNA was synthesised using Super-
script III cDNA synthesis kit (Invitrogen). Independent amplicons of
expected size (1.7 kb) obtained from PCR amplication was
cloned into pTZ57R/T vector (InsTAcloning kit, Thermo Scientic)
and sequence conrmed (pTZ57RT/AP). For expression in insect
cells, the wild type IAP construct (up to residue 482 of mature se-
quence, Supplementary Fig. S4) was subcloned into pFastBac (pFB)
vector (Invitrogen). An NdeI site was created just upstream of the
ATG start codon for cloning purposes in the 50 -primer and the
30 -primer had an XhoI site and the His6 tag and the IAP gene was
PCR amplied using pTZ57RT/AP as template. The resultant NdeI
and XhoI fragment was inserted into the NdeI and XhoI sites of
the pFastBac expression vector. The recombinant bacmid DNA
were transfected into Sf9 cells. After two rounds of amplication,
the baculovirus stock was transfected into Sf9 cells for protein
expression.
4.2. Subcloning for expression in E. coli
Region comprising residues 1482 of rat IAP without the
N-terminal signal sequence was subcloned into pET28 vector for
190 K. Ghosh et al. / Journal of Structural Biology 184 (2013) 182192
expressing the IAP protein in E. coli Rosetta-gami cell. Six His res-
idues were included from the vector in the C-terminus for afnity
purication.
4.3. IAP mutagenesis
The pFastBac clones of rIAP and cIAP were used for mutagenesis
using QuikChange Site Directed Mutagenesis Kit (Agilent) as per
manufacturers recommended protocol (primers used are listed
in Supplementary document 1). Mutations (Rat: Q317H, N301D,
N428D, N142D, Cyno: H317Q) were conrmed by DNA sequencing.
Double and triple mutants were generated subsequently as re-
quired using the single mutants as templates. Bacmids were gener-
ated following similar protocol.
4.4. Expression and purication in insect cell
Sf9 cells at 2  106 cells/ml was infected at 27 C for 66 h with
P2 virus stock (30 ml/L) of respective IAP constructs. Cells were
harvested by centrifugation at 2500g for 25 min. Secretion of the
IAP protein into the media was conrmed by measuring activity
using NBT/BCIP reagents (see below). Supernatant was ltered
through 0.22 lm bottle top lter(s) and pass onto Q-Sepharose
chromatography. The ow through containing the AP activity
was subjected to Ninitrilotriacetic acid (NiNTA) chromatogra-
phy. The NiNTA eluted fractions containing AP activity were
pooled and subjected to size exclusion chromatography (SEC)
using Superdex 200 HR 26/60 column (GE Healthcare) in storage
buffer (100 mM Tris (pH 7.4), 300 mM NaCl, 1 mM MgCl2 and
10% Glycerol (v/v)). All the buffers used in purication had 1 mM
MgCl2.
4.5. In-gel activity assay
Reaction of AP with the combination of BCIP (5-Bromo-4-
Chloro-30 -Indolylphosphate p-Toluidine Salt) and NBT (Nitro-Blue
Tetrazolium Chloride) to yield an intense, insoluble black-purple
precipitate was used to measure AP activity in-gel. The protein
samples were ran on Novex 420% pre-cast TrisGlycine gradient
gels (Invitrogen). The samples were not heat denatured but had
0.5% SDS in the sample loading dye along with 5 mM 2-mercap-
toethanol. Gels were run on the recommended TrisGlycine run-
ning buffer containing 1% SDS. Post completion of the run, the
gel was incubated in 10 ml 1 PBS containing 100 ll Reagent-A
and 100 ll Reagent B (AP Conjugate Substrate Kit #170-6432, Col-
orimetric AP substrate reagent kit, includes premixed BCIP and
NBT solutions, Bio-Rad Laboratories) as per manufacturers guide-
lines. Active proteins were indicated by the appearance of blue col-
or bands on the gel after some time. The gel was then washed with
water, scanned and subsequently stained with coomassie brilliant
blue staining (Invitrogen).
4.6. Deglycosylation with PNGaseF
Deglycosylation reaction was performed with PNGaseF (P7367
Sigma) under native and denatured conditions. 10 lg of respective
IAP samples were used for each reaction. For denaturation, samples
were boiled at 100 C for 5 min in presence of 5% SDS and 0.4 M
DTT. 2 ll of 50 mM sodium phosphate buffer pH 7.5 and 2.0 ll of
10% NP-40 was added to the sample and mixed followed by addi-
tion of 2 ll PNGaseF (1 Unit). Total volume of the reaction was
made up to 20 ll with water and the mixture was incubated at
room temperature for 3 h. For native conditions, 10 lg of IAP sam-
ples was mixed with 50 mM sodium phosphate buffer, pH 7.5 and
incubated with 2 Units of PNGaseF at room temperature for 3 h
prior to run on a SDSPAGE followed by in-gel activity test as men-
tioned above.
4.7. Thermal stability analysis
Thermal stability of the different variants of IAPs was per-
formed using Sypro Orange dye (Molecular Devices) and Bio-Rad
i5 thermal cycler. The protein concentration was constant at
0.4 mg/ml in all the measurements and each data point was repli-
cated at least twice.
Dynamic light scattering measurements were performed using
Dynapro 96-well plate reader from Wyatt Technology Corpora-
tions. All measurements were made at protein concentration of
1 mg/ml.
Mass spectrometric analysis: Total mass estimation of protein
samples were performed on a Bruker HCT ultra ESI-MS coupled
with Agilent 1200 series HPLC system.
4.8. Activity measurements
Bovine intestinal alkaline phosphatase (Bovine IAP) and human
placental alkaline phosphatase (hPLAP) were purchased from
Sigma (P6774, P1391 respectively). The specic activity of the
enzymes was determined in 1 M DEA (diethanolamine), 5 mM
Mg-acetate buffer using 10 mM nal concentration of p-nitrophe-
nylphosphate (pNPP) as a substrate and at respective pH condi-
tions at 37 C. The reaction was monitored at 405 nm using a
Spectramax plate reader for the p-nitrophenol product. One unit
of alkaline phosphatase activity is dened as the amount of
enzyme that hydrolyzes 1 lmol of pNPP per min at 37 C at a given
pH. The kinetics of phosphate hydrolysis was measured for rat IAP,
cyno IAP, human placental AP and bovine IAP in 1 M DEA, 5 mM
Mg acetate buffer using pNPP. The effect of pH on enzyme activity
was monitored using 10 mM pNPP (nal) in 100 mM citrate (pH
6.0), 100 mM TrisHCl (pH 7.0, 7.4), 100 mM Tris- (pH 8.0, 8.5,
9.0), 1 M DEA (pH 9.5, 10, 10.5, 11.0) respectively.
The effect of exogenous metal ion on enzyme activity was stud-
ied using zinc acetate and magnesium acetate. 0.0012 IU of each
enzyme in 1 M DEA, pH 9.5 buffer was used to assess the effect
of exogenous metal ions (Zn2+ and Mg2+) on activity. Magnesium
acetate (Sigma) was titrated from 10 lM to 10 mM nal concentra-
tion. Zinc acetate (Sigma) was titrated from 50 lM to 1 mM in 1 M
DEA buffer pH 9.5 using pNPP (10 mM nal) as a substrate at 37 C.
4.9. Crystallization of rIAP
Initial crystallization trials were performed with 10 mg/ml pro-
tein at 20 C by sitting-drop vapor diffusion method in 96-well
crystallization plates with commercially available crystallization
screens (Hampton Research). Both rIAP-Ec as well as rIAP-Ic
showed signs of crystallization in several conditions. The best
rIAP-Ec crystals were obtained with 0.1 M NH4Cl, 0.1 M MES,
pH 6.5 and 20% PEG MME 2000 as the reservoir solution. The
rIAP-Ic was co-crystallized with 10 mM pNPP using 1.4 M sodium
citrate, 0.1 M Hepes, pH 7.5 as the reservoir solution. Rod shaped
crystals appeared for rIAP-Ec and needle shaped crystals were
obtained for rIAP-Ic after 4 days. The crystals were soaked in the
reservoir solution containing 20% glycerol and ash frozen in liquid
nitrogen for data collection.
4.10. Data collection
X-ray diffraction images for rIAP-Ec were obtained at 2.20 res-
olution using SER-CAT 22ID beam line at Argonne National Labora-
tory, Argonne IL, USA. Data for rIAP-Ic crystal was collected using
CLS_CMCF1_08ID beamline of Canadian light source at 2.38
 K. Ghosh et al. / Journal of Structural Biology 184 (2013) 182192
resolution. The images were processed using Denzo and Scalepack
in the HKL-2000 program package (Otwinowski and Minor, 1997).
4.11. Structure solution and renement
Conversion of the integrated intensities (I) to the structure fac-
tors (F) was done using TRUNCATE and molecular replacement was
done with PHASER, both from the CCP4 program suite (Collabora-
tive Computational Project N, 1994).
4.12. rIAP-Ec
Initial trials of Molecular replacement with hPLAP (1EW2) were
unsuccessful yielding no solution. A new model of hPLAP (1EW2)
was made where residues at N-terminal and C-terminal ends were
trimmed down and where the crown domain was removed. This
yielded a solution with a dimer in the asymmetric unit and solvent
content of 47%. All the model building was done in COOT (Emsley
and Cowtan, 2004). Program Auto Buster (version 1.11.1) (Blanc
et al., 2004) was used for the positional renement. Presence of
unmodeled density near the M3 site in the 2FoFc and FoFc map
indicated presence of metal ion. Initially, Zn2+ was modeled into
the density and rened. Later Zn2+ was replaced with Mg2+ ion
based on the B-factors (Mg2+ B-fac 70 2, Zn2+ B-fac 130 2),
electron density and co-ordination. Crown domain (Phe 362Thr
461) was completely absent in the electron density maps, hence
left unmodeled. After several round of the model building/correc-
tions and renement the nal Rwork and Rfree of the structure were
16.74 and 20.34 respectively.
4.13. rIAP-Ic
Initial trials of molecular replacement with hPLAP (1EW2) using
MOLREP (Vagin and Teplyakov, 1997) yielded a solution with a di-
mer in asymmetric unit and solvent content of 50%. Rigid body
renement resulted in Rwork and Rfree of 36 and 36 respectively.
The electron density maps were evaluated with the program COOT
(Emsley and Cowtan, 2004) and the model was rened with Auto
Buster (version 1.11.1) (Blanc et al., 2004). After several round of
the model building/corrections and renement the nal Rwork
and Rfree of the structure were 16.60 and 20.32 respectively. The -
nal model includes 2 protein chains, 4 zinc ions, 6 magnesium ions,
2 pNP molecules, 9 Nag sugar molecules and 390 water molecules.
The data collection and renement statistics are summarized in
Table 2.
4.14. Quality of the models
Program PROCHECK (Laskowski et al., 1993) in the CCP4 pack-
age was used to check the geometry of the rened model. The
Ramachandran statistics shows 93.3% of residues in core region
and 0.0% of residues in disallowed regions for rIAP-Ec and 90.9%
of residues in core region and 0.0% of residues in disallowed re-
gions for rIAP-Ic. No D-amino acids or incorrect chiral volumes
were observed in the nal models. Models were validated using
Molprobity (Davis et al., 2007). Pictures of the structural models
were generated using Pymol (DeLano, 2002).
4.15. Protein data bank entry codes
The coordinates have been deposited in the PDB at Brookhaven
with entry codes: rIAP-Ec, 4KJD; rIAP-Ic, 4KJG.
191
Acknowledgments
The authors would like to thank Shamrock Structures LLC for
their high quality crystallographic data collection services. We
are thankful to Yoganand Vadari for his help in the initial stages
of the project and Drs. Mike Sinz, Jodi Muckelbauer and Sucharita
Bose for critical reading of the manuscript. We would also like to
acknowledge inputs and advices from Drs. James Bryson, Mian
Gao, Sandhya Mandlekar, Kimberley Lentz and David Rodrigues
at different stages of the project.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jsb.2013.09.017.
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