articles
Crystal structure of a phosphorylation-coupled vitamin C transporter
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Ping Luo1,2,5, Xinzhe Yu2,3,5, Weiguang Wang1,2, Shilong Fan2, Xiaochun Li4 & Jiawei Wang1,2 Bacteria use vitamin C (l-ascorbic acid) as a carbon source under anaerobic conditions. The phosphoenolpyruvate-dependent phosphotransferase system (PTS), comprising a transporter (UlaA), a IIB-like enzyme (UlaB) and a IIA-like enzyme (UlaC), is required for the anaerobic uptake of vitamin C and its phosphorylation to l-ascorbate 6-phosphate. Here, we present the crystal structures of vitamin C–bound UlaA from Escherichia coli in two conformations at 1.65-Å and 2.35-Å resolution. UlaA forms a homodimer and exhibits a new fold. Each UlaA protomer consists of 11 transmembrane segments arranged into a ‘V-motif’ domain and a ‘core’ domain. The V motifs form the interface between the two protomers, and the core-domain residues coordinate vitamin C. The alternating access of the substrate from the opposite side of the cell membrane may be achieved through rigid-body rotation of the core relative to the V motif. Vitamin C (l-ascorbic acid or ascorbate) is an effective antioxidant and an essential cofactor in numerous enzymatic reactions1. Vitamin C deficiency leads to the debilitating disorder scurvy 2, and the species that do not synthesize ascorbate require it in the diet. Ascorbate enters the epithelial cells of the intestine in mammals mainly through a Na+-dependent cotransporter, SVCT1 (ref. 3), of which the Km to the substrate l-ascorbate has been reported to be 75–250 µM (ref. 4). E. coli and other enteric bacteria, normal inhabitants of the intestine, can also ferment vitamin C in an anaerobic environment5,6, with a transporter system that has a low-micromolar binding affinity of 9 µM (ref. 7). Therefore, humans and their intestinal bacteria may compete for the use of ascorbate in the intestine7. The catabolic pathway of vitamin C in E. coli was identified via the ‘utilization of l-ascorbate’ (ula) operon8. The PTS encoded by ula, comprising a transporter protein (UlaA), an enzyme IIB–like protein (UlaB) and an enzyme IIA–like protein (UlaC), is responsible for the anaerobic uptake of vitamin C from the intestine across the bacterial membrane and its phosphorylation to l-ascorbate 6-phosphate9–12. These three proteins as well as the energy-coupling PTS proteins including enzyme I and heat-stable phosphocarrier protein (HPr) are required for the anaerobic utilization and uptake of vitamin C13,14. PTSs are classified into two superfamilies with distinct evolutionary origins15: the AG (ascorbate and galactitol) superfamily16 and the GFL (glucose-glucoside, glucitol, fructose-mannitol and lactoseN,N′-diacetylchitobiose-β-glucoside) superfamily16–18. In contrast to the N,N′-diacetylchitobiose–specific ChbC19 from Bacillus cereus (a member of the Glc family within the GFL superfamily and also the only PTS membrane enzyme with a published structure), the ascorbatespecific PTS UlaABC belongs to the AG superfamily16,20. Homologs of
UlaA have been identified in a large number of evolutionarily divergent bacteria, including numerous Gram-negative and Gram-positive proteobacteria (Supplementary Fig. 1), but not in archaea or eukaryotes9,20–22. Except for species of Corynebacterium, Streptomyces and Bacillus, almost all organisms possessing UlaABC homologs are human or animal pathogens7. However, there has been no structure available for any member of this important PTS-AG superfamily so far. To gain more insights into UlaA, we set out to determine its structure. RESULTS Overview of the UlaA structure We overexpressed the UlaA from E. coli, purified the resulting protein to homogeneity and estimated the molecular weight of detergent-solubilized UlaA to be approximately 122.3 kDa, according to static light scattering. Because the calculated molecular weight of a monomer was ~51 kDa, detergent-solubilized UlaA therefore appeared to be a homodimer. We determined the vitamin C–bound structures of UlaA in two distinct crystallographic forms: C2 and P21. The C2-form crystal diffracted X-rays to 1.65 Å (Table 1 and Supplementary Fig. 2), with one protomer in each asymmetric unit (ASU). UlaA, and we designated UlaA and its homodimeric partner generated via the dyad symmetry as C2A and C2A′, respectively. The P21-symmetry crystal diffracted X-rays to 2.35 Å (Table 1), with a dimer in the ASU, designated P21A and P21B, respectively. We focused on the C2 form for structural description because of its high resolution. The structure in the P21 form will be discussed later. Each protomer contained 11 transmembrane segments (TMs), four reentrant hairpin–like structures (HP1, HP2, HP3 and HP4) and
1State
Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing, China. 2Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China. 3Ministry of Education Key Laboratory of Protein Science, School of Life Sciences, Tsinghua University, Beijing, China. 4Laboratory of Cell Biology, Rockefeller University, New York, New York, USA. 5These authors contributed equally to this work. Correspondence should be addressed to X.L. ([email protected]) or J.W. ([email protected]). Received 25 October 2014; accepted 14 January 2015; published online 16 February 2015; doi:10.1038/nsmb.2975
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articles Table 1 Data collection and refinement statistics UlaA C 2 form
K2Pt(NO2)4 derivative of C 2 form
KAu(CN)2 derivative of C2 form
UlaA P 21 form
C 2
C 2
C 2
P 21
114.18, 85.80, 83.44
83.78, 85.56, 88.95
Data collection Space group Cell dimensions a, b, c (Å) α, β, γ (°) Resolution (Å)
113.66, 85.81, 83.24 114.22, 86.28, 83.53 90, 127.97, 90
90, 128.31, 90
90, 128.14, 90
90, 96.73, 90
50–1.65 (1.71–1.65)
50–3.2 (3.31–3.2)
50–3.0 (3.11–3.0)
50–2.35 (2.43–2.35)
Rmerge
5.7 (60.3)
19.7 (78.1)
20.1 (81.5)
8.4 (54.1)
I / σI
15.26 (1.42)
12.97 (2.02)
6.7 (0.8)
13.03 (1.6)
94.7 (71.0)
99.6 (98.5)
98.9 (90.9)
96.1 (94.7)
2.0 (1.7)
14.3 (12.2)
4.6 (3.9)
2.1 (2.1)
Completeness (%) Redundancy Refinement Resolution (Å) No. reflections Rwork / Rfree
32.79–1.65 71,469 13.47 / 17.69
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No. atoms Protein
4,048
Ligand/ion
186
Water
371
B factors Protein
20.25
Ligand/ion
41.25
Water
46.11
r.m.s. deviations Bond lengths (Å)
0.011
Bond angles (°)
1.247
Values in parentheses are for highest-resolution shell.
three horizontal amphipathic α-helices (AH1, AH2 and AH3), representing a complex fold that, to our knowledge, is previously unreported (Fig. 1a–c and Supplementary Fig. 1). In the dimeric UlaA, the two protomers were oriented parallel to each other and were related by a two-fold axis perpendicular to the membrane (Fig. 1c). The dimension of the homodimer was 49 Å × 53 Å × 94 Å (Fig. 1c and Supplementary Fig. 3a). All TMs were helices, except TM4 and TM9, both of which concatenate an N-terminal β-strand and a C-terminal helix across the cell membrane (Supplementary Fig. 1). Each hairpin (HP) motif consisted of an N-terminal β-strand and a short C-terminal helix connected by an extended loop, which was central to the binding of substrates, as exemplified in the glutamate transporter Glt Ph23,24 and the diacetylchitobiose transporter ChbC19. The UlaA C terminus probably resided on the cytoplasmic side, as inferred from two aspects: first, the ‘positive-inside’ rule25 (Supplementary Fig. 3b); second, the fusion of cytosolic UlaB C terminal to UlaA within a single peptide7 in some species, for example, Vibrio cholerae, Pasteurella multocida and Mycoplasma pulmonis (alignment in Supplementary Fig. 1). Each protomer included an internal ‘inverted’ structural repeat, except TM11 and AH3 (Supplementary Fig. 1). The N-terminal half of the protomer, comprising AH1, TM1–5, HP1 and HP2, were structurally related to the C-terminal half, consisting of AH2, TM6–10, HP3 and HP4, with a two-fold pseudosymmetry axis parallel to the membrane (Supplementary Fig. 3c,d). The first two TMs of each repeat gave rise to V-shaped motifs (V motif 1 and V motif 2), both of which interlocked to form the V-motif domain (Fig. 1c). The rest of the TMs and HPs in each repeat formed the transporter cores (core 1 and core 2). These two transporter-core subdomains, together with TM11, formed the core domain of UlaA. It was noteworthy that the virulence-associated PTS permease VpeC26, in
the same family with UlaA, lacks the C-terminal TM11 according to the sequence alignment (Supplementary Fig. 1). The lengthy loop between HP2a and AH2 is responsible for the connection between the V motif and the core domains (Supplementary Fig. 3a). An extensive dimer interface was visible only between V motifs, with a buried surface area of 1,522 Å2 (Fig. 1c).
Vitamin C–binding site Vitamin C was caged in an ellipsoidshaped substrate-bound pocket of core domain that measured approximately 38.5–2.35 8.3Å × 10.6Å × 7.0Å (Supplementary 49,141 Fig. 4). The structure repeat–related 19.68 / 23.88 HP1 and HP3 framed the top and bottom sides of the cleft. The tips of HP2 6,650 and HP4 met in the middle of the mem45 brane bilayer and consolidated the base 194 of the binding pocket. The left and right side walls were constructed mainly by 43.88 residues from TM4a and TM9a, which 49.22 were also symmetry related. Therefore, 55.46 the substrate-bound pocket was located between two repeats, and all the non0.009 integral helical structural motifs (HP1 1.211 and HP3, HP2 and HP4, and TM4 and TM9) participated in the delimitation of the substrate. Vitamin C was recognized through a mixture of hydrogen bonds and van der Waals interactions (Fig. 1d). Most of the hydrogen bond–contributing residues came from the lower-left hemicube of the pocket, mainly TM4a, HP3 and HP2. However, van der Waals interactions from TM9a, HP1 and HP4 constituted the other opposite side of the cleft, except Tyr87 from HP1. Most of the residues that interact with Vitamin C were highly conserved (Supplementary Fig. 1). We generated several UlaA variants, each containing a missense mutation, and examined their ability to bind vitamin C by isothermal titration calorimetry (ITC)27,28. The binding affinity between vitamin C and wild-type UlaA was approximately 6.1 ± 0.9 µM (Fig. 1e and Supplementary Fig. 5). Replacement of His194, Gln195 or Asp314 by alanine or serine invariably abrogated vitamin C binding. By contrast, the other UlaA variants crippled the binding affinity for the substrate, as compared with that of wild-type UlaA (Fig. 1e).
UlaA outward-open and occluded conformations Molecule C2A was considerably more similar to P21A (r.m.s. deviation 0.24 Å over 442 Cα atoms) than to P21B (1.59 Å over 427 residues). According to the accessibility of the ligand-binding site from the periplasmic side of the membrane, C2A, C2A′ and P21A corres ponded to an outward-open conformational state, and in this conformational state, the ligand was accessible from only the periplasmic side (Fig. 2a). Interestingly, P21B exhibited an occluded state, with the ligand occluded from both sides of the membrane (Fig. 2b). The C2A–C2A′ homodimer superimposed onto the P21A–P21B dimer, and C2A and P21A are almost entirely identical (Supplementary Fig. 6a). C2A′ and P21B displayed a substantial conformational shift mainly due to the rigid-body rotation of the core domain with respect to the V motif (Supplementary Fig. 6a). The core domain rotated about
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articles b 1
2a
C2A′ V motif
c
AH
5
10
TM
Core 1 Vitamin C–binding site
TM11
53 Å
Core 2 V motif 2 94 Å
e 400
HP1 TM4a
Y87
I358
O2 O3
M410
I136 H135
Q195 F362
O5 O6
D314
HP3
350
T86
Q139
H194
HP2 A316
HP4 TM9a
300 250 80 60 40 20 0
No binding
W Y T H 87A 13 Q 5A 1 H 39A 1 Q 94A 19 D 5A 31 4 T8 S I3 6A 58 F3 A M 62A 41 0A
d
Kd (µM)
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9
V motif 1
Hydrogen bonds
van der Waals
the pocket and enters into the cytosol. In step 4, the core domain moves back to the default state7,21. With the progress of the movement of the core domain during the transport activity, a contracted lengthy loop connecting HP2a and AH2 (Supplementary Fig. 3a, blue loop) would probably stretch to keep the V-motif and core domains together. Our high-resolution UlaA structures revealed the architecture of the PTS-AG superfamily, the substrate-binding site and the motion of two conformations, shedding light on the probable mechanism of vitamin C translocation in bacteria. The structural fold of UlaA was completely different from that of ChbC19, a member of the PTS-GFL superP21B V motif family, thus suggesting distinctive evolutionary
Vitamin C in C2A′ Vitamin C in P21B
C2A′ and P21B cores before superposition
2
c
Occluded, substrate bound
Outward open, substrate bound
TM
TM11
8 TM
b
a
TM4
TM 7
HP 2b
AH3
TM6
DISCUSSION According to our structural analysis of UlaA in both C2 and P21 space groups, we propose that UlaA might use an ‘elevator’ mechanism29 (which is a special form of the general ‘alternatingaccess mechanism’30,31) to transport the vitamin C across the cell membrane, similar to the mechanism of sugar transport in ChbC19. The V motif remains largely unaltered while the core domain moves relative to the V motif, with the substrate-bound site accessible alternatively from both sides of the cell membrane (Fig. 2a,b). The speculative working model of the transport activity of UlaA might involve four sequential steps (Fig. 3). The default state is likely to be an open, outward-facing conformation, and the core 2 subdomain is closest to the V-motif 2 subdomain. In step 1, substrate binds to the pocket of the core domain (current C2-form structure). In step 2, UlaA switches to an occluded conformation, in which the core domain undergoes a rigid-body rotation relative to the V-motif domain (current P21-form structure). In step 3, the core domain rotates even further, with the core 1 subdomain close to the V-motif 1 subdomain in order to switch to the inward-facing state, and then the phosphorylated substrate leaves
TM
45°
HP
Vitamin C
4.33° relative to the V-motif domain. This conformational change was accompanied by a maximum atom translation of ~7 Å (Supplementary Fig. 6b). Nevertheless, the core domains of C2A′ and P21B could be superimposed perfectly, even for the bound substrate, vitamin C (Fig. 2c). Therefore, conformational transitions between outwardfacing-state C2A′ and occluded-state P21B were accomplished via the rigid-body rotation of the core domain with respect to the V-motif domain but did not affect the substrate-binding site (Fig. 2b).
TM
TM4a
1a
1
TM9 a
HP1b HP4 b
HP
AH
TM3
TM2
a
a HP4
Figure 1 Overall structure of UlaA and vitamin C coordination. (a,b) Ribbon representations of the UlaA protomer in rainbow colors, shown in two rotated views with the N terminus in blue and the C terminus in red; the bound vitamin C is shown in ball-and-stick representation. (c) View from the extracellular side of the UlaA dimer. One protomer is shown in cartoon, the other as electrostatic potential surface. UlaA is spatially organized into V motif 1 (cyan), core 1 (yellow), V motif 2 (magenta), core 2 (orange) and TM11 (gray) subdomains. (d) Polar and van der Waals contacts coordinating vitamin C. (e) Summary of ITC measurements of the binding affinities of UlaA variants to vitamin C. The data represent the mean and s.d. of three independent experiments; WT, wild type.
After r.m.s.d. superposition
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Figure 2 Accessibility of the ligand-binding site in the outward-open and occluded states. (a) The core domain of the outward-open C2A′ molecule, shown in surface electrostatic potential, with the V-motif domain in red and the vitamin C (green) indicated by the black ellipse. (b) The core domain of the occluded P21B molecule, shown in surface electrostatic potential, with the V motif in blue. The ligand has been occluded from both sides of the membrane, as indicated by the black ellipse. (c) Superposition of the core domains from C2A′ and P21B molecules. Short yellow arrows denote relative movement of the core domain in vitamin C transport. r.m.s.d., r.m.s. deviation.
articles VC
Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. Co
V motif 1
re
Co
V motif 1
1
re
VC
1 Co
V motif 2
re
Outward open, apo Rigidbody rotation of core domain
4
re
V motif 1
Co
1
r
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P
Co
2
2
V motif 1
e2
re
Outward open, substrate bound C2 crystal form
Core 1 VC
3 V motif 2
Co
V motif 2
2
1
V motif 2
VC
Inward open, apo
Core 2
Occluded, substrate bound P21 crystal form
Figure 3 Speculative model of the UlaA transport activity. Possible conformational changes to convert the outward-open, vitamin C–bound state observed in the C2 crystal structure, through the occluded, substrate-bound state in the P21 structure, to the inward-facing state are proposed. VC, vitamin C; P, phosphate.
paths between the PTS-AG and PTS-GFL superfamilies. On the basis of our structural observations, UlaA-mediated vitamin C transport might use the similar elevator mechanism to transport the substrate across the cell membrane29,32. Methods Methods and any associated references are available in the online version of the paper. Accession codes. Coordinates and structure factors for P21 and C2 forms have been deposited in the Protein Data Bank under accession codes 4RP8 and 4RP9, respectively. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments We thank Shanghai Synchrotron Radiation Source for access to beamline BL17U, SPring-8 for access to beamline BL41XU and the Brookhaven National Synchrotron Light Source for access to beamline X29A. We thank N. Yan and E. Coutavas for discussion and comments on the manuscript and D. King (University of California, Berkeley) for MS analysis. This work was supported by funds from the Ministry of Science and Technology of China (grant nos. 2011CB911102 and 2015CB910104), Tsinghua University 985 Phase II funds, National Natural Science Foundation of China (31321062) and Beijing Municipal Commissions of Education and Science and Technology to J.W. We acknowledge the China National Center for Protein Sciences, Beijing, for providing the facility support. X.L. is supported as the Gordon and Betty Moore Foundation Fellow of the Life Sciences Research Foundation. AUTHOR CONTRIBUTIONS P.L., X.Y., X.L. and J.W. designed all experiments. P.L., X.Y., W.W., S.F. and X.L. performed the experiments. All authors analyzed the data. P.L., X.Y., X.L. and J.W. contributed to manuscript preparation. J.W. wrote the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
1. Brown, L.A.S. & Jones, D.P. in Handbook of Antioxidants (eds. Cadenas, E. & Packer, L.) 117–156 (Marcel Dekker, New York, 1996). 2. Ellis, R.W. Vitamin-C deficiency and periostitis of both ulnae? Scurvy. Proc. R. Soc. Med. 32, 139–141 (1939). 3. Liang, W.-J., Johnson, D. & Jarvis, S.M. Vitamin C transport systems of mammalian cells. Mol. Membr. Biol. 18, 87–95 (2001). 4. Wang, H. et al. Human Na+-dependent vitamin C transporter 1 (hSVCT1): primary structure, functional characteristics and evidence for a non-functional splice variant. Biochim. Biophys. Acta 1461, 1–9 (1999). 5. Esselen, W.B. & Fuller, J.E. The oxidation of ascorbic acid as influenced by intestinal bacteria. J. Bacteriol. 37, 501–521 (1939). 6. Young, R.M. & James, L.H. Action of intestinal microorganisms on ascorbic acid. J. Bacteriol. 44, 75–84 (1942). 7. Zhang, Z., Aboulwafa, M., Smith, M.H. & Saier, M.H. Jr. The ascorbate transporter of Escherichia coli. J. Bacteriol. 185, 2243–2250 (2003). 8. Yew, W.S. & Gerlt, J.A. Utilization of l-ascorbate by Escherichia coli K-12: assignments of functions to products of the yjf-sga and yia-sgb operons. J. Bacteriol. 184, 302–306 (2002). 9. Postma, P.W., Lengeler, J.W. & Jacobson, G.R. Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. Microbiol. Rev. 57, 543–594 (1993). 10. Lengeler, J.W., Jahreis, K. & Wehmeier, U.F. Enzymes II of the phospho enol pyruvate-dependent phosphotransferase systems: their structure and function in carbohydrate transport. Biochim. Biophys. Acta 1188, 1–28 (1994). 11. Robillard, G.T. & Broos, J. Structure/function studies on the bacterial carbohydrate transporters, enzymes II, of the phosphoenolpyruvate-dependent phosphotransferase system. Biochim. Biophys. Acta 1422, 73–104 (1999). 12. Siebold, C., Flukiger, K., Beutler, R. & Erni, B. Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS). FEBS Lett. 504, 104–111 (2001). 13. Deutscher, J. et al. The bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system: regulation by protein phosphorylation and phosphorylation-dependent protein-protein interactions. Microbiol. Mol. Biol. Rev. 78, 231–256 (2014). 14. Deutscher, J., Francke, C. & Postma, P.W. How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria. Microbiol. Mol. Biol. Rev. 70, 939–1031 (2006). 15. Saier, M.H., Hvorup, R.N. & Barabote, R.D. Evolution of the bacterial phosphotransferase system: from carriers and enzymes to group translocators. Biochem. Soc. Trans. 33, 220–224 (2005). 16. Chang, A.B., Lin, R., Studley, W.K., Tran, C.V. & Saier, M.H. Phylogeny as a guide to structure and function of membrane transport proteins. Mol. Membr. Biol. 21, 171–181 (2004). 17. Chen, J.S. et al. Phylogenetic characterization of transport protein superfamilies: superiority of SuperfamilyTree programs over those based on multiple alignments. J. Mol. Microbiol. Biotechnol. 21, 83–96 (2011). 18. Nguyen, T.X., Yen, M.R., Barabote, R.D. & Saier, M.H. Jr. Topological predictions for integral membrane permeases of the phosphoenolpyruvate:sugar phosphotrans ferase system. J. Mol. Microbiol. Biotechnol. 11, 345–360 (2006). 19. Cao, Y. et al. Crystal structure of a phosphorylation-coupled saccharide transporter. Nature 473, 50–54 (2011). 20. Hvorup, R., Chang, A.B. & Saier, M.H. Bioinformatic analyses of the bacterial l-ascorbate phosphotransferase system permease family. J. Mol. Microbiol. Biotechnol. 6, 191–205 (2003). 21. Linster, C.L., Van Schaftingen, E. & Vitamin, C. Biosynthesis, recycling and degradation in mammals. FEBS J. 274, 1–22 (2007). 22. Saier, M.H. Jr. & Reizer, J. The bacterial phosphotransferase system: new frontiers 30 years later. Mol. Microbiol. 13, 755–764 (1994). 23. Yernool, D., Boudker, O., Jin, Y. & Gouaux, E. Structure of a glutamate transporter homologue from Pyrococcus horikoshii. 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ONLINE METHODS
Protein expression and purification. We selected several homologs of UlaA from different prokaryotic genomes. Their cDNAs were cloned into pET15b and pET21b (Novagen). After screening, UlaA with a C-terminal His8 tag from E. coli yielded higher than 1 mg/l cell culture and showed good solution behavior in gel filtration (Superdex-200, GE Healthcare). The transformed C43 (DE3) (Lucigen) cells were grown to an optical density of 1.2 at A600 nm and were induced with 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Cells were disrupted with a French Press with two passes at 15,000 p.s.i., in buffer A containing 25 mM Tris-Cl, pH 8.0, and 150 mM NaCl. After a low-speed centrifugation, the resulting supernatant was centrifuged at high speed to sediment a membrane fraction, which then was incubated in buffer A with 1% (w/v) n-nonyl-β-dmaltopyranoside (NM, Anatrace) for 1 h at 4 °C. The lysate was centrifuged again, and the supernatant was loaded onto a Ni2+-NTA affinity column (Qiagen). After two washes, the protein was eluted with 25 mM Tris-Cl, pH 8.0, 150 mM NaCl, 300 mM imidazole and 0.3% NM, and it was then concentrated by Centricon (Millipore) for subsequent gel filtration in buffer A with detergent and 2 mM ascorbic acid (Sigma-Aldrich). The peak fraction was collected for crystallization. Limited proteolysis was used to identify the structural core of UlaA. MS revealed that the C-terminal seven amino acid residues and His8 tag were removed by trypsin (Sigma-Aldrich) digestion. All mutations were generated with two-step PCR. Crystallization. Crystals were grown at 18 °C by the hanging-drop vapordiffusion method. Full-length UlaA protein purified in 0.4% (w/v) n-decyl-β-dmaltopyranoside (DM; Anatrace) gave rise to large crystals in multiple conditions, but the best data set collected at a synchrotron for these crystals, at a nominal resolution of 10 Å, was unsuitable for structural determination. The truncated UlaA purified in 1.0% (w/v) n-octyl-β-d-glucopyranoside (β-OG, Anatrace) gave rise to rod-shaped crystals. The crystals appeared after 5 d in the well buffer containing 0.1 M MES, pH 6.5, 0.1 M NaCl and 30% (v/v) PEG 400. The crystals in space group C2 diffracted to 4 Å at synchrotrons. To further improve the resolution, n-nonyl-β-d-glucopyranoside (β-NG, Anatrace) was added into crystallization buffer at 0.4% (v/v) final concentration to improve diffraction at 1.65 Å and 2.35 Å in C2 and P21 space groups. Platinum or gold derivatives were obtained by soaking native crystals for 2 h in mother liquor plus 10 mg/ml K2Pt(NO2)4 or 2 mg/ml KAu(CN)2. All crystals were directly flash frozen in a cold nitrogen stream at 100 K. Data collection and structure solution. All the data were collected at SSRF beamline BL17U, SPring-8 beamline BL41XU or NSLS beamline X29A
doi:10.1038/nsmb.2975
and processed with the HKL2000 package33. Further processing was carried out with programs from the CCP4 suite34,35. Data collection statistics are summarized in Table 1. The initial phases were obtained from the gold-derivative and platinumderivative crystals by multiple isomorphous replacement with anomalous scattering (MIRAS) with SOLVE36. The real-space constraints were applied to the electron density map in DM37. The model was built manually in COOT38,39, and the structure was refined with PHENIX40,41. Model validation was performed with PROCHECK42 and the WHATCHECK routine of WHAT IF43. All structure figures were prepared with PyMOL (http://www.pymol.org/). Isothermal titration calorimetry. The binding affinity between vitamin C and UlaA variants was measured by ITC. Full-length wild-type or mutant UlaA was extracted and purified through Ni-NTA resin in buffer containing 0.2% DM. The eluted UlaA was concentrated and underwent size-exclusion chromatography (Superdex 200, GE Healthcare). The peak fraction was pooled for ITC titration. The protein was adjusted to about 0.4 mM and was titrated with 6 mM vitamin C dissolved in the same buffer as used in the size-exclusion chromatography. All experiments were performed three times with a Microcal iTC200 (GE Healthcare) at 18 °C. The data were fitted with ORIGIN 7.0 (OriginLab). 33. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data. Methods Enzymol. 276, 307–326 (1997). 34. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011). 35. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994). 36. Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999). 37. Cowtan, K. dm: an automated procedure for phase improvement by density modification. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography 31, 34–38 (1994). 38. Emsley, P., Lohkamp, B., Scott, W. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010). 39. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004). 40. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010). 41. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002). 42. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993). 43. Vriend, G. WHAT IF: a molecular modeling and drug design program. J. Mol. Graph. 8, 52–56 (1990).
nature structural & molecular biology
Crystal structure of a phosphorylation-coupled vitamin C transporter
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Ping Luo1,2,5, Xinzhe Yu2,3,5, Weiguang Wang1,2, Shilong Fan2, Xiaochun Li4 & Jiawei Wang1,2 Bacteria use vitamin C (l-ascorbic acid) as a carbon source under anaerobic conditions. The phosphoenolpyruvate-dependent phosphotransferase system (PTS), comprising a transporter (UlaA), a IIB-like enzyme (UlaB) and a IIA-like enzyme (UlaC), is required for the anaerobic uptake of vitamin C and its phosphorylation to l-ascorbate 6-phosphate. Here, we present the crystal structures of vitamin C–bound UlaA from Escherichia coli in two conformations at 1.65-Å and 2.35-Å resolution. UlaA forms a homodimer and exhibits a new fold. Each UlaA protomer consists of 11 transmembrane segments arranged into a ‘V-motif’ domain and a ‘core’ domain. The V motifs form the interface between the two protomers, and the core-domain residues coordinate vitamin C. The alternating access of the substrate from the opposite side of the cell membrane may be achieved through rigid-body rotation of the core relative to the V motif. Vitamin C (l-ascorbic acid or ascorbate) is an effective antioxidant and an essential cofactor in numerous enzymatic reactions1. Vitamin C deficiency leads to the debilitating disorder scurvy 2, and the species that do not synthesize ascorbate require it in the diet. Ascorbate enters the epithelial cells of the intestine in mammals mainly through a Na+-dependent cotransporter, SVCT1 (ref. 3), of which the Km to the substrate l-ascorbate has been reported to be 75–250 µM (ref. 4). E. coli and other enteric bacteria, normal inhabitants of the intestine, can also ferment vitamin C in an anaerobic environment5,6, with a transporter system that has a low-micromolar binding affinity of 9 µM (ref. 7). Therefore, humans and their intestinal bacteria may compete for the use of ascorbate in the intestine7. The catabolic pathway of vitamin C in E. coli was identified via the ‘utilization of l-ascorbate’ (ula) operon8. The PTS encoded by ula, comprising a transporter protein (UlaA), an enzyme IIB–like protein (UlaB) and an enzyme IIA–like protein (UlaC), is responsible for the anaerobic uptake of vitamin C from the intestine across the bacterial membrane and its phosphorylation to l-ascorbate 6-phosphate9–12. These three proteins as well as the energy-coupling PTS proteins including enzyme I and heat-stable phosphocarrier protein (HPr) are required for the anaerobic utilization and uptake of vitamin C13,14. PTSs are classified into two superfamilies with distinct evolutionary origins15: the AG (ascorbate and galactitol) superfamily16 and the GFL (glucose-glucoside, glucitol, fructose-mannitol and lactoseN,N′-diacetylchitobiose-β-glucoside) superfamily16–18. In contrast to the N,N′-diacetylchitobiose–specific ChbC19 from Bacillus cereus (a member of the Glc family within the GFL superfamily and also the only PTS membrane enzyme with a published structure), the ascorbatespecific PTS UlaABC belongs to the AG superfamily16,20. Homologs of
UlaA have been identified in a large number of evolutionarily divergent bacteria, including numerous Gram-negative and Gram-positive proteobacteria (Supplementary Fig. 1), but not in archaea or eukaryotes9,20–22. Except for species of Corynebacterium, Streptomyces and Bacillus, almost all organisms possessing UlaABC homologs are human or animal pathogens7. However, there has been no structure available for any member of this important PTS-AG superfamily so far. To gain more insights into UlaA, we set out to determine its structure. RESULTS Overview of the UlaA structure We overexpressed the UlaA from E. coli, purified the resulting protein to homogeneity and estimated the molecular weight of detergent-solubilized UlaA to be approximately 122.3 kDa, according to static light scattering. Because the calculated molecular weight of a monomer was ~51 kDa, detergent-solubilized UlaA therefore appeared to be a homodimer. We determined the vitamin C–bound structures of UlaA in two distinct crystallographic forms: C2 and P21. The C2-form crystal diffracted X-rays to 1.65 Å (Table 1 and Supplementary Fig. 2), with one protomer in each asymmetric unit (ASU). UlaA, and we designated UlaA and its homodimeric partner generated via the dyad symmetry as C2A and C2A′, respectively. The P21-symmetry crystal diffracted X-rays to 2.35 Å (Table 1), with a dimer in the ASU, designated P21A and P21B, respectively. We focused on the C2 form for structural description because of its high resolution. The structure in the P21 form will be discussed later. Each protomer contained 11 transmembrane segments (TMs), four reentrant hairpin–like structures (HP1, HP2, HP3 and HP4) and
1State
Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing, China. 2Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China. 3Ministry of Education Key Laboratory of Protein Science, School of Life Sciences, Tsinghua University, Beijing, China. 4Laboratory of Cell Biology, Rockefeller University, New York, New York, USA. 5These authors contributed equally to this work. Correspondence should be addressed to X.L. ([email protected]) or J.W. ([email protected]). Received 25 October 2014; accepted 14 January 2015; published online 16 February 2015; doi:10.1038/nsmb.2975
nature structural & molecular biology advance online publication
articles Table 1 Data collection and refinement statistics UlaA C 2 form
K2Pt(NO2)4 derivative of C 2 form
KAu(CN)2 derivative of C2 form
UlaA P 21 form
C 2
C 2
C 2
P 21
114.18, 85.80, 83.44
83.78, 85.56, 88.95
Data collection Space group Cell dimensions a, b, c (Å) α, β, γ (°) Resolution (Å)
113.66, 85.81, 83.24 114.22, 86.28, 83.53 90, 127.97, 90
90, 128.31, 90
90, 128.14, 90
90, 96.73, 90
50–1.65 (1.71–1.65)
50–3.2 (3.31–3.2)
50–3.0 (3.11–3.0)
50–2.35 (2.43–2.35)
Rmerge
5.7 (60.3)
19.7 (78.1)
20.1 (81.5)
8.4 (54.1)
I / σI
15.26 (1.42)
12.97 (2.02)
6.7 (0.8)
13.03 (1.6)
94.7 (71.0)
99.6 (98.5)
98.9 (90.9)
96.1 (94.7)
2.0 (1.7)
14.3 (12.2)
4.6 (3.9)
2.1 (2.1)
Completeness (%) Redundancy Refinement Resolution (Å) No. reflections Rwork / Rfree
32.79–1.65 71,469 13.47 / 17.69
© 2015 Nature America, Inc. All rights reserved.
No. atoms Protein
4,048
Ligand/ion
186
Water
371
B factors Protein
20.25
Ligand/ion
41.25
Water
46.11
r.m.s. deviations Bond lengths (Å)
0.011
Bond angles (°)
1.247
Values in parentheses are for highest-resolution shell.
three horizontal amphipathic α-helices (AH1, AH2 and AH3), representing a complex fold that, to our knowledge, is previously unreported (Fig. 1a–c and Supplementary Fig. 1). In the dimeric UlaA, the two protomers were oriented parallel to each other and were related by a two-fold axis perpendicular to the membrane (Fig. 1c). The dimension of the homodimer was 49 Å × 53 Å × 94 Å (Fig. 1c and Supplementary Fig. 3a). All TMs were helices, except TM4 and TM9, both of which concatenate an N-terminal β-strand and a C-terminal helix across the cell membrane (Supplementary Fig. 1). Each hairpin (HP) motif consisted of an N-terminal β-strand and a short C-terminal helix connected by an extended loop, which was central to the binding of substrates, as exemplified in the glutamate transporter Glt Ph23,24 and the diacetylchitobiose transporter ChbC19. The UlaA C terminus probably resided on the cytoplasmic side, as inferred from two aspects: first, the ‘positive-inside’ rule25 (Supplementary Fig. 3b); second, the fusion of cytosolic UlaB C terminal to UlaA within a single peptide7 in some species, for example, Vibrio cholerae, Pasteurella multocida and Mycoplasma pulmonis (alignment in Supplementary Fig. 1). Each protomer included an internal ‘inverted’ structural repeat, except TM11 and AH3 (Supplementary Fig. 1). The N-terminal half of the protomer, comprising AH1, TM1–5, HP1 and HP2, were structurally related to the C-terminal half, consisting of AH2, TM6–10, HP3 and HP4, with a two-fold pseudosymmetry axis parallel to the membrane (Supplementary Fig. 3c,d). The first two TMs of each repeat gave rise to V-shaped motifs (V motif 1 and V motif 2), both of which interlocked to form the V-motif domain (Fig. 1c). The rest of the TMs and HPs in each repeat formed the transporter cores (core 1 and core 2). These two transporter-core subdomains, together with TM11, formed the core domain of UlaA. It was noteworthy that the virulence-associated PTS permease VpeC26, in
the same family with UlaA, lacks the C-terminal TM11 according to the sequence alignment (Supplementary Fig. 1). The lengthy loop between HP2a and AH2 is responsible for the connection between the V motif and the core domains (Supplementary Fig. 3a). An extensive dimer interface was visible only between V motifs, with a buried surface area of 1,522 Å2 (Fig. 1c).
Vitamin C–binding site Vitamin C was caged in an ellipsoidshaped substrate-bound pocket of core domain that measured approximately 38.5–2.35 8.3Å × 10.6Å × 7.0Å (Supplementary 49,141 Fig. 4). The structure repeat–related 19.68 / 23.88 HP1 and HP3 framed the top and bottom sides of the cleft. The tips of HP2 6,650 and HP4 met in the middle of the mem45 brane bilayer and consolidated the base 194 of the binding pocket. The left and right side walls were constructed mainly by 43.88 residues from TM4a and TM9a, which 49.22 were also symmetry related. Therefore, 55.46 the substrate-bound pocket was located between two repeats, and all the non0.009 integral helical structural motifs (HP1 1.211 and HP3, HP2 and HP4, and TM4 and TM9) participated in the delimitation of the substrate. Vitamin C was recognized through a mixture of hydrogen bonds and van der Waals interactions (Fig. 1d). Most of the hydrogen bond–contributing residues came from the lower-left hemicube of the pocket, mainly TM4a, HP3 and HP2. However, van der Waals interactions from TM9a, HP1 and HP4 constituted the other opposite side of the cleft, except Tyr87 from HP1. Most of the residues that interact with Vitamin C were highly conserved (Supplementary Fig. 1). We generated several UlaA variants, each containing a missense mutation, and examined their ability to bind vitamin C by isothermal titration calorimetry (ITC)27,28. The binding affinity between vitamin C and wild-type UlaA was approximately 6.1 ± 0.9 µM (Fig. 1e and Supplementary Fig. 5). Replacement of His194, Gln195 or Asp314 by alanine or serine invariably abrogated vitamin C binding. By contrast, the other UlaA variants crippled the binding affinity for the substrate, as compared with that of wild-type UlaA (Fig. 1e).
UlaA outward-open and occluded conformations Molecule C2A was considerably more similar to P21A (r.m.s. deviation 0.24 Å over 442 Cα atoms) than to P21B (1.59 Å over 427 residues). According to the accessibility of the ligand-binding site from the periplasmic side of the membrane, C2A, C2A′ and P21A corres ponded to an outward-open conformational state, and in this conformational state, the ligand was accessible from only the periplasmic side (Fig. 2a). Interestingly, P21B exhibited an occluded state, with the ligand occluded from both sides of the membrane (Fig. 2b). The C2A–C2A′ homodimer superimposed onto the P21A–P21B dimer, and C2A and P21A are almost entirely identical (Supplementary Fig. 6a). C2A′ and P21B displayed a substantial conformational shift mainly due to the rigid-body rotation of the core domain with respect to the V motif (Supplementary Fig. 6a). The core domain rotated about
advance online publication nature structural & molecular biology
articles b 1
2a
C2A′ V motif
c
AH
5
10
TM
Core 1 Vitamin C–binding site
TM11
53 Å
Core 2 V motif 2 94 Å
e 400
HP1 TM4a
Y87
I358
O2 O3
M410
I136 H135
Q195 F362
O5 O6
D314
HP3
350
T86
Q139
H194
HP2 A316
HP4 TM9a
300 250 80 60 40 20 0
No binding
W Y T H 87A 13 Q 5A 1 H 39A 1 Q 94A 19 D 5A 31 4 T8 S I3 6A 58 F3 A M 62A 41 0A
d
Kd (µM)
© 2015 Nature America, Inc. All rights reserved.
9
V motif 1
Hydrogen bonds
van der Waals
the pocket and enters into the cytosol. In step 4, the core domain moves back to the default state7,21. With the progress of the movement of the core domain during the transport activity, a contracted lengthy loop connecting HP2a and AH2 (Supplementary Fig. 3a, blue loop) would probably stretch to keep the V-motif and core domains together. Our high-resolution UlaA structures revealed the architecture of the PTS-AG superfamily, the substrate-binding site and the motion of two conformations, shedding light on the probable mechanism of vitamin C translocation in bacteria. The structural fold of UlaA was completely different from that of ChbC19, a member of the PTS-GFL superP21B V motif family, thus suggesting distinctive evolutionary
Vitamin C in C2A′ Vitamin C in P21B
C2A′ and P21B cores before superposition
2
c
Occluded, substrate bound
Outward open, substrate bound
TM
TM11
8 TM
b
a
TM4
TM 7
HP 2b
AH3
TM6
DISCUSSION According to our structural analysis of UlaA in both C2 and P21 space groups, we propose that UlaA might use an ‘elevator’ mechanism29 (which is a special form of the general ‘alternatingaccess mechanism’30,31) to transport the vitamin C across the cell membrane, similar to the mechanism of sugar transport in ChbC19. The V motif remains largely unaltered while the core domain moves relative to the V motif, with the substrate-bound site accessible alternatively from both sides of the cell membrane (Fig. 2a,b). The speculative working model of the transport activity of UlaA might involve four sequential steps (Fig. 3). The default state is likely to be an open, outward-facing conformation, and the core 2 subdomain is closest to the V-motif 2 subdomain. In step 1, substrate binds to the pocket of the core domain (current C2-form structure). In step 2, UlaA switches to an occluded conformation, in which the core domain undergoes a rigid-body rotation relative to the V-motif domain (current P21-form structure). In step 3, the core domain rotates even further, with the core 1 subdomain close to the V-motif 1 subdomain in order to switch to the inward-facing state, and then the phosphorylated substrate leaves
TM
45°
HP
Vitamin C
4.33° relative to the V-motif domain. This conformational change was accompanied by a maximum atom translation of ~7 Å (Supplementary Fig. 6b). Nevertheless, the core domains of C2A′ and P21B could be superimposed perfectly, even for the bound substrate, vitamin C (Fig. 2c). Therefore, conformational transitions between outwardfacing-state C2A′ and occluded-state P21B were accomplished via the rigid-body rotation of the core domain with respect to the V-motif domain but did not affect the substrate-binding site (Fig. 2b).
TM
TM4a
1a
1
TM9 a
HP1b HP4 b
HP
AH
TM3
TM2
a
a HP4
Figure 1 Overall structure of UlaA and vitamin C coordination. (a,b) Ribbon representations of the UlaA protomer in rainbow colors, shown in two rotated views with the N terminus in blue and the C terminus in red; the bound vitamin C is shown in ball-and-stick representation. (c) View from the extracellular side of the UlaA dimer. One protomer is shown in cartoon, the other as electrostatic potential surface. UlaA is spatially organized into V motif 1 (cyan), core 1 (yellow), V motif 2 (magenta), core 2 (orange) and TM11 (gray) subdomains. (d) Polar and van der Waals contacts coordinating vitamin C. (e) Summary of ITC measurements of the binding affinities of UlaA variants to vitamin C. The data represent the mean and s.d. of three independent experiments; WT, wild type.
After r.m.s.d. superposition
nature structural & molecular biology advance online publication
Figure 2 Accessibility of the ligand-binding site in the outward-open and occluded states. (a) The core domain of the outward-open C2A′ molecule, shown in surface electrostatic potential, with the V-motif domain in red and the vitamin C (green) indicated by the black ellipse. (b) The core domain of the occluded P21B molecule, shown in surface electrostatic potential, with the V motif in blue. The ligand has been occluded from both sides of the membrane, as indicated by the black ellipse. (c) Superposition of the core domains from C2A′ and P21B molecules. Short yellow arrows denote relative movement of the core domain in vitamin C transport. r.m.s.d., r.m.s. deviation.
articles VC
Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. Co
V motif 1
re
Co
V motif 1
1
re
VC
1 Co
V motif 2
re
Outward open, apo Rigidbody rotation of core domain
4
re
V motif 1
Co
1
r
© 2015 Nature America, Inc. All rights reserved.
P
Co
2
2
V motif 1
e2
re
Outward open, substrate bound C2 crystal form
Core 1 VC
3 V motif 2
Co
V motif 2
2
1
V motif 2
VC
Inward open, apo
Core 2
Occluded, substrate bound P21 crystal form
Figure 3 Speculative model of the UlaA transport activity. Possible conformational changes to convert the outward-open, vitamin C–bound state observed in the C2 crystal structure, through the occluded, substrate-bound state in the P21 structure, to the inward-facing state are proposed. VC, vitamin C; P, phosphate.
paths between the PTS-AG and PTS-GFL superfamilies. On the basis of our structural observations, UlaA-mediated vitamin C transport might use the similar elevator mechanism to transport the substrate across the cell membrane29,32. Methods Methods and any associated references are available in the online version of the paper. Accession codes. Coordinates and structure factors for P21 and C2 forms have been deposited in the Protein Data Bank under accession codes 4RP8 and 4RP9, respectively. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments We thank Shanghai Synchrotron Radiation Source for access to beamline BL17U, SPring-8 for access to beamline BL41XU and the Brookhaven National Synchrotron Light Source for access to beamline X29A. We thank N. Yan and E. Coutavas for discussion and comments on the manuscript and D. King (University of California, Berkeley) for MS analysis. This work was supported by funds from the Ministry of Science and Technology of China (grant nos. 2011CB911102 and 2015CB910104), Tsinghua University 985 Phase II funds, National Natural Science Foundation of China (31321062) and Beijing Municipal Commissions of Education and Science and Technology to J.W. We acknowledge the China National Center for Protein Sciences, Beijing, for providing the facility support. X.L. is supported as the Gordon and Betty Moore Foundation Fellow of the Life Sciences Research Foundation. AUTHOR CONTRIBUTIONS P.L., X.Y., X.L. and J.W. designed all experiments. P.L., X.Y., W.W., S.F. and X.L. performed the experiments. All authors analyzed the data. P.L., X.Y., X.L. and J.W. contributed to manuscript preparation. J.W. wrote the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
1. Brown, L.A.S. & Jones, D.P. in Handbook of Antioxidants (eds. Cadenas, E. & Packer, L.) 117–156 (Marcel Dekker, New York, 1996). 2. Ellis, R.W. Vitamin-C deficiency and periostitis of both ulnae? Scurvy. Proc. R. Soc. Med. 32, 139–141 (1939). 3. Liang, W.-J., Johnson, D. & Jarvis, S.M. Vitamin C transport systems of mammalian cells. Mol. Membr. Biol. 18, 87–95 (2001). 4. Wang, H. et al. Human Na+-dependent vitamin C transporter 1 (hSVCT1): primary structure, functional characteristics and evidence for a non-functional splice variant. Biochim. Biophys. Acta 1461, 1–9 (1999). 5. Esselen, W.B. & Fuller, J.E. The oxidation of ascorbic acid as influenced by intestinal bacteria. J. Bacteriol. 37, 501–521 (1939). 6. Young, R.M. & James, L.H. Action of intestinal microorganisms on ascorbic acid. J. Bacteriol. 44, 75–84 (1942). 7. Zhang, Z., Aboulwafa, M., Smith, M.H. & Saier, M.H. Jr. The ascorbate transporter of Escherichia coli. J. Bacteriol. 185, 2243–2250 (2003). 8. Yew, W.S. & Gerlt, J.A. Utilization of l-ascorbate by Escherichia coli K-12: assignments of functions to products of the yjf-sga and yia-sgb operons. J. Bacteriol. 184, 302–306 (2002). 9. Postma, P.W., Lengeler, J.W. & Jacobson, G.R. Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. Microbiol. Rev. 57, 543–594 (1993). 10. Lengeler, J.W., Jahreis, K. & Wehmeier, U.F. Enzymes II of the phospho enol pyruvate-dependent phosphotransferase systems: their structure and function in carbohydrate transport. Biochim. Biophys. Acta 1188, 1–28 (1994). 11. Robillard, G.T. & Broos, J. Structure/function studies on the bacterial carbohydrate transporters, enzymes II, of the phosphoenolpyruvate-dependent phosphotransferase system. Biochim. Biophys. Acta 1422, 73–104 (1999). 12. Siebold, C., Flukiger, K., Beutler, R. & Erni, B. Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS). FEBS Lett. 504, 104–111 (2001). 13. 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ONLINE METHODS
Protein expression and purification. We selected several homologs of UlaA from different prokaryotic genomes. Their cDNAs were cloned into pET15b and pET21b (Novagen). After screening, UlaA with a C-terminal His8 tag from E. coli yielded higher than 1 mg/l cell culture and showed good solution behavior in gel filtration (Superdex-200, GE Healthcare). The transformed C43 (DE3) (Lucigen) cells were grown to an optical density of 1.2 at A600 nm and were induced with 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Cells were disrupted with a French Press with two passes at 15,000 p.s.i., in buffer A containing 25 mM Tris-Cl, pH 8.0, and 150 mM NaCl. After a low-speed centrifugation, the resulting supernatant was centrifuged at high speed to sediment a membrane fraction, which then was incubated in buffer A with 1% (w/v) n-nonyl-β-dmaltopyranoside (NM, Anatrace) for 1 h at 4 °C. The lysate was centrifuged again, and the supernatant was loaded onto a Ni2+-NTA affinity column (Qiagen). After two washes, the protein was eluted with 25 mM Tris-Cl, pH 8.0, 150 mM NaCl, 300 mM imidazole and 0.3% NM, and it was then concentrated by Centricon (Millipore) for subsequent gel filtration in buffer A with detergent and 2 mM ascorbic acid (Sigma-Aldrich). The peak fraction was collected for crystallization. Limited proteolysis was used to identify the structural core of UlaA. MS revealed that the C-terminal seven amino acid residues and His8 tag were removed by trypsin (Sigma-Aldrich) digestion. All mutations were generated with two-step PCR. Crystallization. Crystals were grown at 18 °C by the hanging-drop vapordiffusion method. Full-length UlaA protein purified in 0.4% (w/v) n-decyl-β-dmaltopyranoside (DM; Anatrace) gave rise to large crystals in multiple conditions, but the best data set collected at a synchrotron for these crystals, at a nominal resolution of 10 Å, was unsuitable for structural determination. The truncated UlaA purified in 1.0% (w/v) n-octyl-β-d-glucopyranoside (β-OG, Anatrace) gave rise to rod-shaped crystals. The crystals appeared after 5 d in the well buffer containing 0.1 M MES, pH 6.5, 0.1 M NaCl and 30% (v/v) PEG 400. The crystals in space group C2 diffracted to 4 Å at synchrotrons. To further improve the resolution, n-nonyl-β-d-glucopyranoside (β-NG, Anatrace) was added into crystallization buffer at 0.4% (v/v) final concentration to improve diffraction at 1.65 Å and 2.35 Å in C2 and P21 space groups. Platinum or gold derivatives were obtained by soaking native crystals for 2 h in mother liquor plus 10 mg/ml K2Pt(NO2)4 or 2 mg/ml KAu(CN)2. All crystals were directly flash frozen in a cold nitrogen stream at 100 K. Data collection and structure solution. All the data were collected at SSRF beamline BL17U, SPring-8 beamline BL41XU or NSLS beamline X29A
doi:10.1038/nsmb.2975
and processed with the HKL2000 package33. Further processing was carried out with programs from the CCP4 suite34,35. Data collection statistics are summarized in Table 1. The initial phases were obtained from the gold-derivative and platinumderivative crystals by multiple isomorphous replacement with anomalous scattering (MIRAS) with SOLVE36. The real-space constraints were applied to the electron density map in DM37. The model was built manually in COOT38,39, and the structure was refined with PHENIX40,41. Model validation was performed with PROCHECK42 and the WHATCHECK routine of WHAT IF43. All structure figures were prepared with PyMOL (http://www.pymol.org/). Isothermal titration calorimetry. The binding affinity between vitamin C and UlaA variants was measured by ITC. Full-length wild-type or mutant UlaA was extracted and purified through Ni-NTA resin in buffer containing 0.2% DM. The eluted UlaA was concentrated and underwent size-exclusion chromatography (Superdex 200, GE Healthcare). The peak fraction was pooled for ITC titration. The protein was adjusted to about 0.4 mM and was titrated with 6 mM vitamin C dissolved in the same buffer as used in the size-exclusion chromatography. All experiments were performed three times with a Microcal iTC200 (GE Healthcare) at 18 °C. The data were fitted with ORIGIN 7.0 (OriginLab). 33. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data. Methods Enzymol. 276, 307–326 (1997). 34. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011). 35. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994). 36. Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999). 37. Cowtan, K. dm: an automated procedure for phase improvement by density modification. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography 31, 34–38 (1994). 38. Emsley, P., Lohkamp, B., Scott, W. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010). 39. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004). 40. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010). 41. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002). 42. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993). 43. Vriend, G. WHAT IF: a molecular modeling and drug design program. J. Mol. Graph. 8, 52–56 (1990).
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