Crystal structure of lipid phosphatase Escherichia coli

phosphatidylglycerophosphate phosphatase B
Junping Fana,b,1, Daohua Jianga,c,1, Yan Zhaoa,d, Jianfeng Liuc, and Xuejun Cai Zhanga,2
a
National Laboratory of Macromolecules, National Center of Protein Science-Beijing, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101,
China; bGraduate School of the University of Chinese Academy of Sciences, Beijing 100049, China; cSchool of Life Science and Technology, Huazhong
University of Science and Technology, Wuhan, Hubei 430074, China; and dSchool of Life Sciences, University of Science and Technology of China, Hefei, Anhui
230027, China

Edited by Brian W. Matthews, University of Oregon, Eugene, OR, and approved April 1, 2014 (received for review February 19, 2014)

Membrane-integrated type II phosphatidic acid phosphatases the periplasmic side by a phosphatase, for example, phosphati-
(PAP2s) are important for numerous bacterial to human biological dyl-glycero-phosphatase B (PgpB, EC 3.1.3.27) (12), which may
processes, including glucose transport, lipid metabolism, and also be involved in the biogenesis of phosphatidylglycerol from
signaling. Escherichia coli phosphatidylglycerol-phosphate phos- phosphatidylglycerol phosphate (13). Similar dephosphorylation
phatase B (ecPgpB) catalyzes removing the terminal phosphate mechanisms of carrier lipids (e.g., dolichyl pyrophosphates) for
group from a lipid carrier, undecaprenyl pyrophosphate, and is glycan transport have been observed in yeast (14) and mamma-
essential for transport of many hydrophilic small molecules across lian cells (4), and the corresponding PAP2 phosphatases (Cwh8
the membrane. We determined the crystal structure of ecPgpB at and DOLPP1, respectively) have been found to be located in the
a resolution of 3.2 Å. This structure shares a similar folding topol- endoplasmic reticulum and are essential for luminal N-glycosylation
ogy and a nearly identical active site with soluble PAP2 enzymes. of newly synthesized proteins in eukaryotic cells. Therefore, PAP2-
However, the substrate binding mechanism appears to be funda- facilitated recycling of carrier lipids is of fundamental interest to
mentally different from that in soluble PAP2 enzymes. In ecPgpB, cell biology.
the potential substrate entrance to the active site is located in Based on amino acid sequence analysis, members of the PAP2
a cleft formed by a V-shaped transmembrane helix pair, allowing family share a signature sequence “KX6RPX12–54PSGHX31–54
lateral movement of the lipid substrate entering the active site SRX5HX3D,” which is often divided into three motifs: C1,
from the membrane lipid bilayer. Activity assays of point muta- “KX6RPF”; C2, “PSGH”; and C3, “SRX5HX3D” (1, 2) (Fig. S1).
tions confirmed the importance of the catalytic residues and po- The PAP2 family also includes one group of haloperoxidases
tential residues involved in phosphate binding. The structure also [e.g., vanadium-dependent chloroperoxidase (CPO)] (1). The CPO
suggests an induced-fit mechanism for the substrate binding. The of the fungus Curvularia inaequalis (ciCPO) has also been shown
3D structure of ecPgpB serves as a prototype to study eukaryotic to possess phosphatase activity (15). Crystal structures of soluble
PAP2 enzymes, including human glucose-6-phosphatase, a key en- ciCPO [Protein Data Bank (PDB) ID code 1VNC] (16) and
zyme in the homeostatic regulation of blood glucose concentrations. NSAP from Escherichia blattae (ebNSAP; PDB ID code 1D2T)
(17) from the PAP2 family have been reported. These 3D

T ype II phosphatidic acid phosphatases (PAP2s) are a large

family of phosphatases important for lipid metabolism and
signaling (1, 2). PAP2 proteins have been found in all life king-
structures share a similar folding topology for a core helix bun-
dle, with residues from the signature sequence residing on the
same end of the helix bundle. Structural variations may occur as
doms from bacteria to mammals. They catalyze dephosphory- insertions in the connecting loops and even within an essential
lation of broad substrates by specifically hydrolyzing phosphoric
monoester bonds. Their substrates include variety of phosphor- Significance
ylated carbohydrates, peptides, and lipids. PAP2s are involved in
vesicular trafficking, secretion, and endocytosis (e.g., the enzyme Phosphatases regulate many aspects of cellular function-
phosphatidate phosphatase APP1 in yeast) (3); protein glyco- homeostasis and signal transduction. Using X-ray crystallography
sylation [e.g., dolichyl pyrophosphate phosphatase 1 (DOLPP1) methods, we determined the structure of phosphatidylgly-
in the mouse] (4); energy storage (e.g., triacylglycerol bio- cerol-phosphate phosphatase B (PgpB) from Escherichia coli,
synthesis) (5); and stress response (6). In contrast to type I PAP a member of the type II phosphatidic acid phosphatase (PAP2)
enzymes, which are Mg2+-dependent and usually soluble, PAP2 family and a homologue of human glucose-6-phosphatase,
proteins are Mg2+-independent, and many of PAP2 enzymes are which plays a variety of physiopathological roles. Our structure
integral transmembrane (TM) proteins (7). Whereas the sol- of PgpB showed that the membrane-integrated and soluble
uble branch of PAP2s is called class A nonspecific acid phos- members of the PAP2 family share the same catalytic mecha-
phatases (NSAPs) (8), the TM branch of the PAP2 family is also nism. The mechanism of recognition of lipid substrates was
called the lipid phosphatase/phosphotransferase family (2) or postulated based on analyses of enzymatic activities and ther-
lipid phosphate phosphatase family (9). Human glucose-6-phos- mal stabilities of PgpB variants. This work presents an impor-
phatase (G6Pase), the key enzyme in the homeostatic regulation tant structural model for studying eukaryotic PAP2s.
of blood glucose concentrations, belongs to the TM PAP2 sub-
Author contributions: X.C.Z. designed research; J.F. and D.J. performed research; J.F., D.J.,
family (10). Thus, the TM property is unique to the PAP2 family. Y.Z., J.L., and X.C.Z. analyzed data; and J.F., D.J., and X.C.Z. wrote the paper.
In Escherichia coli, undecaprenyl phosphate (C55-P), a 55- The authors declare no conflict of interest.
carbon single-lipid chain phospholipid, serves as a carrier lipid to This article is a PNAS Direct Submission.
transfer a variety of phosphate-linked polymers across the peri-
Data deposition: Coordinates of the crystal structure of Escherichia coli phosphatidylglycerol-
plasmic membrane from the cytosol to the periplasmic space. phosphate phosphatase B have been deposited in the Protein Data Bank, www.pdb.org
Recently, the crystal structure of phospho–N-acetylmuramic (PDB ID code 4PX7).
acid–pentapeptide translocase (MraY) from Aquifex aeolicus, 1
J.F. and D.J. contributed equally to this work.
which catalyzes the transfer of substrates to C55-P, was reported 2
To whom correspondence should be addressed. E-mail: [email protected].
(11). For this process to be sustainable, the transport intermediate, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
undecaprenyl pyrophosphate, needs to be dephosphorylated on 1073/pnas.1403097111/-/DCSupplemental.

7636–7640 | PNAS | May 27, 2014 | vol. 111 | no. 21 www.pnas.org/cgi/doi/10.1073/pnas.1403097111

α-helix (e.g., in 1VNC), conferring variation of substrate speci- P212121 space group and contains one protein molecule per asym-
ficity. Although no 3D structures of TM PAP2 have been de- metrical unit with 60% solvent content (Matthews coefficient of
termined experimentally until now, they were predicted to share 3.1 Å3/Da); thus, the ecPgpB molecules are packed in the crystal as
a similar folding topology as well as a conserved active site with monomers. The crystal form is a type I membrane protein crystal
known structures of soluble PAP2 members (18). Consistent with with the TM helices aligned along the crystallographic c axis. Pep-
other TM PAP2 enzymes, E. coli PgpB (ecPgpB) was predicted tide segments of the residue ranges 2–32, 35–139, 144–239, and
to contain six TM helices, and this folding topology has been 242–254 were built in the final refined model, with a few short
confirmed experimentally. In particular, the catalytic center regions as well as a C-terminal His tag omitted because of weak
of ecPgpB has been shown to be located near the solvent– electron density. The structure model was refined to Rwork of
membrane interface on the periplasmic side (12), and both N 27.0% (Rfree of 30.2%). No disulfide bonds were found in the
and C termini are located on the cytosol side of the periplasmic crystal structure. One of the two native Cys residues, Cys67, was
membrane. The precise arrangement of the six TM helices; the partially buried, and the other Cys residue, Cys234, was exposed
3D structure of a predicted 70-residue periplasmic domain, in- to the solvent. The statistics of data collection and refinement
cluding part of the conserved sequence motifs; and the mecha- are summarized in Table S1.
nism of substrate recognition of ecPgpB remain to be identified. The 3D structure of PgpB is composed of six TM helices (TMs
To prove that TM PAP2 proteins share similar 3D folding with 1–6) and a small periplasmic domain consisting of 70 amino acid
soluble PAP2 proteins in general and to illustrate the structural residues (i.e., 92–161) (Fig. 1). The N and C termini are shown to
basis of the lipid substrate binding of PgpB in particular, we be located on the cytosol side, and the putative active site is on
determined the crystal structure of ecPgpB. Our results show the periplasmic side as predicted previously (12). Overall, the
that PgpB does indeed possess a folding topology similar to that positive-inside rule of charge distribution (19) is followed, with
of the two soluble PAP2 enzymes, namely, ebNSAP and ciCPO. 10 Arg or Lys residues but no acidic residue being located at the
The arrangements of the catalytic residues in the three available cytosol ends of the TM helices and in the connecting loops (Fig.
PAP2 crystal structures are nearly identical, whereas their substrate S1B). TMs 4–6 form the core of the TM region, with TMs 1–3
binding mechanisms are fundamentally different. In ecPgpB, the surrounding the core. TM3 is loosely packed with the rest of the
potential substrate entrance to the active site is in the cleft of TM domain. Its cytosolic N-terminal end is connected with TM2
a V-shaped TM helix pair. In addition, activity assays of point through a short five-residue loop, whereas its periplasmic C-terminal
mutations confirmed the importance of a number of residues po- end is connected to the periplasmic domain through a rather flex-
tentially involved in catalysis as well as substrate binding. ible 10-residue loop. The periplasmic domain is inserted between
TMs 3 and 4, and it contains four α-helices (i.e., α2–α5). The
Results and Discussion putative active site is formed by PAP2 signature motifs, which is
Overall Structure of ecPgpB. EcPgpB contains 254 amino acid resi- located in the primary sequence from the C terminus of TM3 to
dues (29-kDa molecular mass). The crystal structures of recombi- the N-terminal end of TM6. In the 3D structure, this active site is
nant WT ecPgpB and its variant containing a double point muta- located in the membrane–periplasm interface region and is
tion, I116M/E120K, were solved in the same crystal form, with a highly positively charged (Fig. 1B). Moreover, TMs 2 and 3 form
better resolution for the latter, however. Both mutation sites of this a V-shaped cleft, with the periplasmic opening side being over 10 Å
crystallized ecPgpB variant are located outside of both TM regions wide. This periplasmic opening is located close to the putative
and the signature motifs, and the protein sample showed identical catalytic site, and it is likely to be the binding site of the polar
properties as the WT otherwise (Fig. S2). Thus, the following dis- head of the substrate.
cussion is based on the refined structure of the mutant. The phases This structure study demonstrates experimentally that the in-
of the crystal structure were determined using a Se-Met–based tegral membrane PAP2 protein ecPgpB shares a similar folding
single-wavelength anomalous dispersion (SAD) method and were topology with soluble PAP2 proteins in the core helix bundle,
refined at a resolution of 3.2 Å. The crystal form belongs to the although their overall sequence homology is low (∼15% sequence

BIOCHEMISTRY

Fig. 1. Overall structure of ecPgpB. (A) Cartoon representation of the ecPgpB crystal structure. The structure is rainbow-colored, from the N terminus in blue
to C terminus in red. The conserved motifs C1, C2, and C3 are marked as ovals. Selected helices and the C terminus are labeled. Positions 116 and 120
(mutation sites) are marked as red spheres, and positions 67 and 234 (Cys residues) are marked as brown spheres. Estimated membrane boundaries are
indicated by dashed lines. In the periplasm view, helices α2, α3, and α4 are removed for clarity of TM helices. (B) Mapping of the electrostatic potential onto
the molecular surface. Positively charged regions are colored in blue, and negatively charged regions are colored in red. (C) Topology diagram of ecPgpB.

Fan et al. PNAS | May 27, 2014 | vol. 111 | no. 21 | 7637
identity). For instance, the α1-helice and TMs 4, 5, and 6 of two ends of the periplasmic insertion connecting TMs 3 and 4 (Fig.
ecPgpB showed a 1.4-Å rmsd with ebNSAP (PDB ID code 1EOI) 2B). The C3 motif forms the region from the C-terminal end of
(17) for ∼60 Cα atom pairs, including most of the signature motifs TM5 to the N-terminal end of TM6. In the following, we refer to
except the N-terminal region of the C1 motif (see below). On the a residue from a given motif by its motif number (1, 2, or 3),
other hand, TMs 1, 2, and 3 and most of the periplasmic domain combined with the position number in that motif, for the conve-
significantly deviate from the corresponding structural elements nience of structural comparison between different PAP2 enzymes.
of the soluble counterparts in ebNSAP/1EOI (Fig. 2). These For example, the catalytic His163 of ecPgpB is located at the fourth
differences are partially due to the structure requirement of position in the C2 motif and is referred to as His163(2.04). Catalytic
membrane proteins [e.g., the length of TM helices and the pos- residues His163(2.04) and His207(3.08), and most other residues es-
itive-inside rule (19)], and may also be required by the lipid sential for the activity, are located near the putative solvent–mem-
substrates. Similarly, the crystal structure of soluble ciCPO (PDB brane interface, and both their main-chain and side-chain atoms are
ID code 1VNC) shares a homologous core structure around the able to be superimposed with those in the crystal structure of
active site with ecPgpB as well as ebNSAP (Fig. 2). Nevertheless, ebNSAP/1EOI (Fig. 2B). An exception is the conserved Lys97(1.01),
ciCPO has an extra N-terminal region of over 230 amino acid which may be involved in an induced-fit mechanism. Therefore,
residues compared with ecPgpB, and this extra structural region ecPgpB is likely to share a similar phosphate hydrolysis mechanism
essentially blocks the surface corresponding to the lipid substrate with both ebNSAP and ciCPO.
entrance in ecPgpB (i.e., the front side in Fig. 1A). In addition, According to the established two-step catalytic mechanism
ciCPO contains a 48-residue insertion inside the helix corre- of soluble PAP2 enzymes, of the residues critical to catalysis,
sponding to TM6 of ecPgpB and C-terminal to the conserved His207(3.08) and Asp211(3.12) of ecPgpB form a charge–relay pair
motif C3 (i.e., the back side in Fig. 1A). Between TMs 4 and 5, and are responsible for nucleophilic attacking and formation of
ciCPO contains another insertion of ca. 50 amino acid residues a phosphate/enzyme intermediate in the first step of the reaction
(i.e., the bottom). Thus, the conserved core of TMs 4–6 is essential (20). His163(2.04) stabilizes the transition state intermediate and
for the active site formation of PAP2 enzymes. catalyzes the cleavage of the terminal PO4 group from the sub-
The overall sequence homology for TM PAP2 proteins is strate (18). Mutations at the equivalent position of His119(2.04) in
usually low (ca. 10−15% identity). However, based on sequence human G6Pase to any other amino acid residue have been shown
analyses and prediction of locations of TM helices, all known to abolish its enzyme activity (21). In ecPgpB, conserved Arg104(1.08)
TM PAP2 proteins are likely to share the same folding topology interacts with the catalytic His207(3.08) and potentially with the
with ecPgpB, assuming that the three conserved motifs, C1–C3, substrate. Mutations at the equivalent position of Arg83(1.08) in
have similar locations in their 3D structures (Fig. S1A). Human human G6Pase (alignment is shown in Fig. S1A) to any other
glucose-6 phosphatase possesses an additional four TM helices amino acid residue, including Lys, also abolished enzyme activity
C-terminal to the “canonical” six-TM helix topology. Therefore, our (21). In particular, mutation of R83C has been directly related to
crystal structure of ecPgpB strongly supports the notion that PAP2 glycogen storage disease type 1a (10, 22). Similarly, in ecPgpB,
proteins, both soluble and TM forms, are evolutionarily related. Arg201(3.02) interacts with His163(2.04). The N-terminal end of
TM4 also potentially contributes to binding of the PO4 group in
Conserved Active Site in ecPgpB. To verify that our recombinant a charge–dipole interaction. In particular, lack of the side chain
protein of ecPgpB was functional, we performed an in vitro ki- of Gly162(2.03) at the N-terminal end of TM4 favors binding of
netic analysis of the dephosphorylation catalysis of WT ecPgpB the substrate PO4 group. In agreement, active site mutations
toward 1,2-dioleoyl-sn-glycero-3-phosphate [PA (18:1)] using H163(2.04)A, H207(3.08)A, D211(3.12)E, R104(1.08)A, and R201(3.02)A
detergent-solubilized protein (Fig. 3A). The results showed that of ecPgpB showed only background level activity toward the sub-
the kcat was 24 (±2) s−1 and the Km was 0.30 (±0.09) mM), which strates (Fig. S3B) dioleoylglycerol pyrophosphate [DGPP (18:1)],
are roughly comparable with previously reported data (kcat = 61 s−1 dioctanoylglycerol pyrophosphate [DGPP (8:0)], 1-oleoyl-2-
and Km = 1.7 mM) (12). This catalytic activity is believed to be hydroxy-sn-glycero-3-phosphate [LPA (18:1)], and PA (18:1), sim-
associated with the signature motifs of the PAP2 family (1). In the ilar to negative controls (Fig. 3B). These results confirmed the
crystal structure of ecPgpB, the C1 and C2 motifs are located at the catalytic roles of the corresponding residues in the WT ecPgpB.

Fig. 2. Structural comparison of ecPgpB with soluble PAP2 enzymes. (A) Superposition of the core helices of ecPgpB (blue) with ebNSAP/1EOI (wheat) and
ciCPO/1VNC (cyan, with large insertions omitted). Backbones of the core helices and signature motifs are shown in ribbons, and the remaining parts are shown
in thin lines. A molybdate group in the active site from the ebNSAP crystal structure is shown in a sphere model. (B) Superposition of the active site of ecPgpB
with ebNSAP/1EOI. Conserved residues in signature motifs are labeled. Residue numbers of ebNSAP are shown in parentheses. The figures were generated
with the program PyMol.

7638 | www.pnas.org/cgi/doi/10.1073/pnas.1403097111 Fan et al.

Fig. 3. Activity measurements. (A) Enzyme activity of recombination WT ecPgpB. The protein concentration was 0.1 μM, and the reaction time was set to
1 min. (B) Phosphatase activity of variants of ecPgpB with different substrates. The substrates used were DGPP (18:1) (blue), DGPP (8:0) (red), LPA (18:1)
(green), and PA (18:1) (purple). Because most mutations showed only background activity, the reaction time was set to 10 min. Absorbance at 795 nm (A795 nm)
was reported as a measurement of the relative activity. Multiple experiments were performed (Methods), and the average results and SDs are shown.

In thermofluor assays, most purified variants showed compa- PAP2 enzymes and ecPgpB include TMs 1–3, which surround the
rable melting temperatures (Tms) with that of the WT Tm in the core formed by TMs 4–6 and the periplasmic insertion between
absence of substrate, ranging between 39 °C and 48 °C (Table TMs 3 and 4. In particular, we observed a V-shaped TM helix pair
S2). Interestingly, only the WT protein, but not its active site (TMs 2 and 3) with its periplasmic opening close to the active site
mutant forms H163(2.04)A, H207(3.08)A, and D211(3.12)E variants, (Fig. 4A). This TM helix pair forms a surface cleft, presumably
was further stabilized by a transition state analog, molybdate allowing membrane-associated lipid substrates to enter the active
(Na2MoO4) (Table S2), whereas Na2SO4 showed no effect and site. With such an accessing mode, a lipid substrate would insert
NaH2PO4 destabilized all variants slightly. In particular, in the its phosphate head into the bottom of the active pocket and to-
presence of 10 mM Na2MoO4, the Tm of WT ecPgpB increased ward the nucleophilic residue His207(3.08) to form the phosphate–
from 43 °C to 55 °C. This observation is in agreement with the enzyme complex and would allow His163(2.04) to complete the
previously reported crystal structure of the ebNSAP–Mo com- second step of dephosphorylation by recruiting a water molecule
plex (PDB ID code 1EOI), showing covalent bonding of the from the solvent-accessible side of the active site. To test this
MoO4−2 group with His189(3.08) (17). Moreover, the H207(3.08)A hypothesis, we constructed a few more point mutations at the
variant of ecPgpB, which is presumably not able to form the putative entrance cleft of this substrate binding pocket (Fig. 4A).
phosphate/enzyme intermediate, could be stabilized by addi- Most of the variants in this group (e.g., V54F, F61W, V88F,
tional substrate [1 mM PA (18:1)] with a melting temperature Q90A, K93E) showed reduced activity to some extent (Fig. 4B).
change (ΔTm) of approximately +12 °C (Fig. S3A), whereas As a control, Leu58 is located outside of the V-shaped opening,
other variants, as well as the WT, displayed a smaller change of and the H57F and L58F variants behave essentially like WT. In
thermal stability (ΔTm of ∼5 °C) in response to substrate bind- contrast, the A83F variant was predicted to block the opening
ing. These observations suggest that although most of the resi- from the TM3 side, and it did indeed exhibit significantly reduced
dues within the conserved active site contribute to the substrate activity. However, this variant remained capable of binding mo-
loading, His207(3.08) is more important for catalysis in ecPgpB. lybdate (Table S2), suggesting that the reduction in activity is the
result of weaker binding of the lipid chain(s) of the substrate.
Cleft of Substrate Entrance Is Located in the Membrane–Solvent Similarly, the G89L variant lost nearly all activity toward all four
Interface of PgpB. One of the fundamental questions about TM substrates tested. Therefore, we conclude that the opening of the
PAP2 enzymes is about the entrance for their lipid substrates. As V-shaped cleft that faces the outer leaflet of the lipid bilayer is
mentioned above, the main structural differences between soluble the substrate entrance of ecPgpB. The wide cleft formed by the

BIOCHEMISTRY

Fig. 4. Putative substrate entrance. (A) V-shaped substrate entrance. The main body of the enzyme is shown as a molecular surface model. The V-shaped TM
helix pair, TMs 2 and 3, are shown as cylinders. Putative movements of the substrate and TM3 are indicated as open arrows. Selected residues that were
mutated to define the substrate entrance are shown as sphere models. The positions of those residues that showed an effect on enzyme activity are
highlighted in yellow; otherwise, they are wheat-colored. (B) Relative activities of the variants in the putative substrate entrance. The reaction time was set to
1 min to compare initial reaction speeds of the variants. All substrate concentrations were 1 mM, and all protein concentrations were 0.1 μM.

Fan et al. PNAS | May 27, 2014 | vol. 111 | no. 21 | 7639
V-shaped TM helix pair may explain the promiscuous substrate without disturbing the conformation observed in the crystal
preference of ecPgpB. Similar loading mechanisms of lipid sub- structure, behaved in the same way as the Lys97(1.01)A variant in
strates have been proposed for several membrane-integrated terms of changes in thermal stability and activity (Fig. 4 and Table
enzymes (23, 24). S2). A potential advantage of such an induced-fit mechanism is to
Compared with known structures of soluble PAP2 proteins, restrict the enzyme activity to properly bound lipid substrates only.
our crystal structure appears to represent an apo-form of ecPgpB.
Specifically, TM3 packed loosely with the rest of the protein (Fig. Methods
1A). As a consequence, the conserved Lys97(1.01) residue is lo- The gene of full-length PgpB was cloned from the E. coli BL21 genome.
cated ca. 9 Å away from its putative active position near the C2 Recombinant PgpB protein was expressed in E. coli and purified in a de-
motif at the N-terminal end of TM4. Although we cannot rule out tergent combination of nonyl-β-D-glucopyranoside (Jiejing Tech, Inc.) and
the possibility that the observed Lys97(1.01) conformation is n-dodecyl-N,N-dimethylamine-N-oxide (Anatrace). The protein sample was
a crystallization artifact, the region surrounding this residue is not crystallized using the vapor diffusion method. Initial phases of the structure
involved in crystal packing. Thus, we attempt to postulate that factors were determined using the selenium-based SAD method, and the
Lys97(1.01) of ecPgpB must move from its current position in the structure model was refined at 3.2 Å (Fig. S4). Thermal stability of PgpB
crystal structure to complete the formation of the active site and variants was analyzed using a count per second-based thermofluor method,
to interact directly with the substrate PO4 group. Such a move- and activities of the PgpB variants were analyzed using a phosphor-molybdic
ment of Lys97(1.01) is likely to be driven by both substrate binding acid colorimetric method. More information on materials and methods can
to the V-shaped cleft and a subsequent movement of TM3 toward be found in SI Methods.
TM2. This hypothesis of conformational change upon induction
by a substrate is supported by the following observations: (i) ACKNOWLEDGMENTS. We thank the staff of the Protein Research Core
Facility at the Institute of Biophysics, Chinese Academy of Sciences, for their
Mutation K97(1.01)A lost its ability to bind the transition-state excellent technical assistance. We also thank staff members of the High
analog MoO4−2 (Table S2), which suggests that the side-chain tip Energy Accelerator Research Organization (Japan), Super Photon Ring-8
of Lys97(1.01) in ecPgpB directly interacts with MoO4−2; (ii) (Japan), and Shanghai Synchrotron Radiation Facility (China) synchrotron
K97(1.01)A lost the enzyme activity (Fig. 3B), suggesting that the facilities for their assistance in collecting diffraction data. This work was
supported by Ministry of Science and Technology (China) “973” Project Grant
conformation of the canonical active site observed in soluble 2011CB910301 (to X.C.Z.), Chinese Academy of Sciences Grant XDB080203
enzymes also functions in ecPgpB; and (iii) the variant G89L, (to X.C.Z.), and National Natural Science Foundation of China Grants
which was designed to block the movement of TM3 toward TM2 31130028 and 31225011 (to J.L.).

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