proteins STRUCTURE O FUNCTION O BIOINFORMATICS
STRUCTURE NOTE
Crystal structure of NMA1982 ˚ from Neisseria meningitidis at 1.5 A resolution provides a structural scaffold for nonclassical, eukaryotic-like phosphatases S. Sri Krishna,1,2,3* Lutz Tautz,2* Qingping Xu,1,4 Daniel McMullan,1,5 Mitchell D. Miller,1,4 Polat Abdubek,1,5 Eileen Ambing,1,5 Tamara Astakhova,1,3 Herbert L. Axelrod,1,4 Dennis Carlton,1,6 Hsiu-Ju Chiu,1,4 Thomas Clayton,1,6 Michael DiDonato,1,5 Lian Duan,1,3 Marc-Andre´ Elsliger,1,6 Slawomir K. Grzechnik,1,3 Joanna Hale,1,5 Eric Hampton,1,5 Gye Won Han,1,6 Justin Haugen,1,5 Lukasz Jaroszewski,1,2,3 Kevin K. Jin,1,4 Heath E. Klock,1,5 Mark W. Knuth,1,5 Eric Koesema,1,5 Andrew T. Morse,1,3 Tomas Mustelin,2 Edward Nigoghossian,1,5 Silvya Oommachen,1,4 Ron Reyes,1,4 Christopher L. Rife,1,4 Henry van den Bedem,1,4 Dana Weekes,1,2 Aprilfawn White,1,5 Keith O. Hodgson,1,4 John Wooley,1,3 Ashley M. Deacon,1,4 Adam Godzik,1,2,3 Scott A. Lesley,1,5,6 and Ian A. Wilson1,6y 1 Joint Center for Structural Genomics (JCSG) 2 Burnham Institute for Medical Research, La Jolla, California 3 Center for Research in Biological Systems, University of California, San Diego, La Jolla, California 4 Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California 5 Genomics Institute of the Novartis Research Foundation, San Diego, California 6 The Scripps Research Institute, La Jolla, California
INTRODUCTION Protein phosphorylation–dephosphorylation is a universal mechanism used to regulate protein function. Historically, research efforts have focused on protein kinases, but recently the role of phosphatases that catalyze the equally important dephosphorylation step is becoming increasingly appreciated.1 The role of protein phosphorylation in eukaryotic signal transduction is well documented. Among bacteria, protein phosphatases such as Yersinia pestis YopH are virulence factors, which are secreted into human cells during pathogenic invasion. However, recent genomic studies have identified numerous protein phosphatase homologs in nonpathogenic C 2007 WILEY-LISS, INC. V
bacteria, suggesting intrinsic function within these organisms. Despite their ancient origin, which possibly predates the eukaryote/prokaryote split, and the resulting sequence and structural divergence, all known protein tyrosine phosphatases (PTPs) have a canonical functional motif consisting of Cys-(X)5-Arg. This motif is part of a
Grant sponsor: National Institutes of Health, Protein Structure Initiative; Grant numbers: P50 GM62411, U54 GM074898. *Sri Krishna and Lutz Tautz contributed equally to this work. y Correspondence to: Dr. Ian Wilson, JCSG, The Scripps Research Institute, BCC206, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail:
[email protected] Received 29 September 2006; Accepted 26 October 2006 Published online 16 July 2007 in Wiley InterScience (www.interscience.wiley. com). DOI: 10.1002/prot.21314
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critical loop that wraps around the tyrosine, serine, or threonine2 residues to be dephosphorylated. Neisseria meningitidis protein of unknown function NMA1982 has a molecular weight of 17,461 Da (residues 1-155) and a calculated isoelectric point of 6.91. It belongs to a large group of experimentally uncharacterized bacterial proteins that have been included in the Pfam database3 as DUF (domain of unknown function) No. 442. Close homologs of NMA1982 are found mostly in proteobacteria, including a number of human pathogens, such as Pseudomonas aeruginosa (cystic fibrosis), Bordetella pertussis (whooping cough), and Burkholderia pseudomallei (melioidosis). Other homologous sequences are from Mesorhizobium loti and Nitrosomonas europaea, which are soil bacteria responsible for nitrogen fixation. N. meningitidis itself is an important human pathogen responsible for causing meningitis and septicaemia, which are debilitating diseases that kill thousands of people every year. The DUF442 family is also well represented among environmental sequences, with over 200 homologs in the global ocean sampling data produced by the environmental sequencing project of the Craig Venter Institute.4 Furthermore, proteins from this family show very limited, but statistically significant, sequence similarity to eukaryotic PTPs. This similarity can be recognized only by very sensitive distant homology recognition algorithms, such as FFAS5 and ORFEUS.6 Interestingly, the hallmark Cys-(X)5-Arg motif of classical PTPs is replaced by a Cys-(X)4-Arg motif in NMA1982 and its homologs. This disparity cast doubts on the assignment of this protein family to the PTP superfamily and possibly led to its classification as a DUF in the Pfam database.3 Here, we report the crystal structure of NMA1982, which was determined using the semiautomated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG),7 part of the National Institute of General Medical Sciences (NIGMS) Protein Structure Initiative (PSI). Structural analysis confirms our predictions that it belongs to a family of eukaryotic-like PTPs. The overall fold shows a very strong structural similarity to type II PTPs, with conservation of all secondary structural elements. Furthermore, the critical active site residues responsible for phosphatase activity, along with several other residues that are conserved among classical PTPs, are similarly conserved in NMA1982. Our preliminary biochemical characterization of this protein also confirms a phosphatase activity. The primary goal of the PSI is to determine the threedimensional structure of proteins from protein families that lack structural coverage in order to rapidly expand the coverage of protein fold space. In addition, the JCSG also targets proteins from large protein families, such as the PTPs, which have homologs distributed across all kingdoms of life. Therefore, during target selection, we focus on proteins that are likely to represent major variants of the fold and function of important protein superfamilies. NMA1982 provides the first structure of a
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nonclassical Cys-(X)4-Arg active site motif in a functionally active protein phosphatase. MATERIALS AND METHODS Protein production and crystallization
The gene encoding NMA1982 was amplified by polymerase chain reaction (PCR) from Neisseria meningitides FAM18 genomic DNA using PfuTurbo (Stratagene) and primers corresponding to the predicted 50 - and 30 -ends. The serogroup C, FAM18 strain used here contains two amino acid substitutions (V45I and S102Y) as compared to the GenBank sequence (gi: 7380613; Swiss-Prot: Q9JT40) from the serogroup A, Z2491 strain. The PCR product was cloned into plasmid pSpeedET, which encodes an expression and purification tag followed by a tobacco etch virus (TEV) protease cleavage site (MGSDKIHHHHHHENLYFQG) at the amino terminus of the full-length protein. The cloning junctions were confirmed by DNA sequencing. Protein expression was performed in a selenomethionine-containing medium using the Escherichia coli strain GeneHogs (Invitrogen). At the end of fermentation, lysozyme was added to the culture to a final concentration of 250 lg/mL, and the cells were harvested. After one freeze/thaw cycle, the cells were sonicated in lysis buffer [50 mM HEPES pH 8.0, 50 mM NaCl, 10 mM imidazole, 1 mM Tris (2-carboxyethyl)phosphine hydrochloride (TCEP)], and the lysate was clarified by centrifugation at 32,500 3 g for 30 min. The soluble fraction was applied to nickel-chelating resin (GE Healthcare) preequilibrated with lysis buffer, the resin was washed with wash buffer [50 mM HEPES pH 8.0, 300 mM NaCl, 40 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP], and the target protein was eluted with elution buffer [20 mM HEPES pH 8.0, 300 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP]. The eluate was buffer exchanged with HEPES crystallization buffer [20 mM HEPES pH 8.0, 200 mM NaCl, 40 mM imidazole, 1 mM TCEP] using a PD-10 column (GE Healthcare) and treated with 1 mg of TEV protease per 10 mg of eluted protein. The digested eluate was applied to nickel-chelating resin (GE Healthcare) preequilibrated with HEPES crystallization buffer, and the resin was washed with the same buffer. The flow-through and wash fractions were combined and concentrated for crystallization assays to 15 mg/mL by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method8 with standard JCSG crystallization protocols.7 The crystallization reagent contained 0.2 M MgCl2, 30% (w/v) polyethylene glycol 4,000, and 0.1 M Tris pH 8.5. Initial screening for diffraction was carried out using the Stanford Automated Mounting system (SAM)9 at the Stanford Synchrotron Radiation Laboratory (SSRL, Stanford, CA). The crystals were indexed in monoclinic space group C2 (Table I). Molecular weight DOI 10.1002/prot
Crystal Structure of NMA1982
Table I Summary of Crystal Parameters, Data Collection, and Refinement Statistics for NMA1982 (PDB: 2f46)
Space group Unit cell parameters
Data collection Wavelength () Resolution range () Number of observations Number of reflections Completeness (%) Mean I/r(I) Rsym on I Highest resolution shell () Model and refinement statistics Resolution range () No. of reflections (total) No. of reflections (test) Completeness (% total) Stereochemical parameters Restraints (RMS observed) Bond length Bond angle Average isotropic B-value ESU based on Rfree value Protein residues/atoms Solvent molecules
C2 a ¼ 144.28 , b ¼ 33.47 , c ¼ 59.90 , a ¼ 90.008, b ¼ 96.188, g ¼ 90.008 k1MADSe 1.01995 30–1.41 145,442 46,247 83.4 (32.3)a 14.8 (2.1)a 0.041 (0.276)a 1.45–1.41
k2MADSe 0.97974 30–1.41 160,273 49,672 89.7 (48.9) 12.6 (1.9) 0.052 (0.335) 1.45–1.41
30–1.41 46,237b 2,352 83.4
Dataset used in refinement Cutoff criteria Rcryst Rfree
k1MADSe jF j > 0 0.200 0.228
0.016 1.458 16.8 2 0.089 286/2406 412
ESU, estimated overall coordinate error.10,11 Rsym ¼ SjIihIiij/SjIij where Ii is the scaled intensity of the ith measurement and hIii is the mean intensity for that reflection. Rcryst ¼ SjjFobsjjFcalcjj/SjFobsj where Fcalc and Fobs are the calculated and observed structure factor amplitudes, respectively. Rfree ¼ as for Rcryst, but for 5.0% of the total reflections chosen at random and omitted from refinement. a Highest resolution shell in parentheses. b Typically, the number of unique reflections used in refinement is slightly less than the total number that were integrated and scaled. Reflections are excluded due to systematic absences, negative intensities and rounding errors in the resolution limits and cell parameters.
and oligomeric state of NMA1982 were determined using a 1 cm 3 30 cm Superdex 200 column (GE Healthcare) in combination with static light scattering (Wyatt Technology). The mobile phase consisted of 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% (w/v) sodium azide. Phosphatase activity was confirmed using p-nitrophenyl phosphate (pNPP) as substrate. The reaction was carried out using different enzyme concentrations (21, 42, and 84 lM) in two buffers, namely, 150 mM Bis-Tris pH 6.0 and 100 mM Bis-Tris pH 7.5, each having an ionic strength of 150 mM adjusted with sodium chloride, and each containing 1 mM dithiothreitol. After a 1 h incubation of enzyme and pNPP at 308C, 50 lL of 1 M NaOH were added, and the absorbance of p-nitrophenol at 405 nm was measured, clearly showing concentrationdependent phosphatase activity. Background (no enzyme present) was subtracted. Data collection, structure solution, and refinement
Multi-wavelength anomalous diffraction (MAD) data were collected at the Advanced Light Source (ALS, Berkeley, USA) on beamline 8.3.1 at wavelengths corresponding to the inflection (k1) and low energy remote (k2) of DOI 10.1002/prot
a selenium MAD experiment using the BLU-ICE12 data collection environment. Datasets were collected at 100K using an ADSC CCD detector. The MAD data were integrated and reduced using Mosflm13 and then scaled with the program SCALA from the CCP4 suite.10 The structure was phased by SOLVE/RESOLVE,14 and the model was built using ARP/wARP15 and COOT16 and refined using REFMAC5.10 Since the data in the outer shell are incomplete, we define the nominal resolution as 1.5 A˚, which is the resolution of a dataset that is 100% complete and has the same number of reflections as observed in the current dataset.17 However, 3,603 reflections between 1.50 and 1.41 A˚ (39% complete for this shell) were included in the refinement. Data collection, phasing, and refinement statistics are summarized in Table I. Validation and deposition
Analysis of the stereochemical quality of the model was accomplished using AutoDepInputTool,18 MolProbity,19 SFcheck 4.0,20 and WHATIF 5.0.21 Protein quaternary structure analysis was performed using the PQS server.22 Figures were prepared with PyMOL (DeLano Scientific). Atomic coordinates and experimental structure factors for NMA1982 at 1.5 A˚ resolution have been deposited in PROTEINS
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Figure 1 Crystal structure of NMA1982: (A) Stereo ribbon diagram of NMA1982 monomer color-coded from N-terminus (blue) to C-terminus (red). Helices (H1-H6) and b-strands (b1-b5) are labeled. (B) Diagram showing the secondary structural elements superimposed on its primary sequence. The a-helices, b-strands of b-sheet A, and b-bulges are indicated. The b-hairpin is depicted as a red loop. The disordered region is displayed as a broken line.
the Protein Data Bank (PDB) and are accessible under the code 2f46. RESULTS AND DISCUSSION The crystal structure of NMA1982 [Fig. 1(A,B)] was determined to 1.5 A˚ resolution using the MAD method. The final model includes two monomers (chains A and B) in the asymmetric unit, each containing residues 13-155. The residual glycine residue from the TEV protease cleavage site and residues 1-12 of the protein are disordered in both monomers. In addition, density for two Cl ions, one unknown ligand (UNL), and 411 water molecules were observed in the asymmetric unit. The Matthews’ coefficient (Vm)23 is 2.01 A˚3/Da, and the estimated solvent content is 38.7%. The Ramachandran plot produced by MolProbity19 shows that 99.3% and 100% of the residues are in favored and allowed regions, respectively. NMA1982 is composed of five b-strands (b1-b5), six a-helices (H1-H6), and three 310-helices (H10 , H20 , H60 ) [Fig. 1(A,B)]. The total b-sheet, a-helical, and 310-helical
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content is 14.0%, 39.2%, and 4.2%, respectively. The overall structure of NMA1982 resembles a typical PTPase type II fold of the SCOP classification scheme.24 This PTPase type II fold is three-layered and is usually characterized by a parallel b-sheet with strand order 1423 flanked by a-helices. In NMA1982, the b-sheet contains five b-strands, where the additional b-strand is present at the N-terminus of the PTPase core. Furthermore, two additional a-helices are found at the C-terminal region. As suggested by profile-profile comparison methods, such as FFAS and ORFEUS, the NMA1982 protein has weak sequence similarity to eukaryotic PTPs. Subsequent structural comparison by DALI25 and FATCAT3 found similarities to several PTPs with statistically significant Zscores. DALI identified a human kinase-associated phosphatase (1fpz; Z ¼ 14.3) as the closest match in the PDB, while FATCAT found greater similarity to a putative plant phosphatase from Arabidopsis thaliana (1xri; Pvalue ¼ 5.6 E 11), whose structure was determined by the Center for Eukaryotic Structural Genomics (CESG). In fact, close structural similarity was found to all proDOI 10.1002/prot
Crystal Structure of NMA1982
Figure 2 Structural comparison of NMA1982 with other PTPs: (A) Multiple structural alignment of NMA1982 (grey), human kinase-associated phosphatase (1fpz; magenta), and putative phosphatase from Arabidopsis thaliana (1xri; lilac). Insertions specific to human and A. thaliana proteins are denoted by IHs and IAt, respectively. Cys and Arg residues from the active site loop are shown in ball-and-stick. (B) Structural alignment of NMA1982 (grey) and the putative phosphatase from Arabidopsis thaliana (1xri; lilac). Cys and Arg residues from the active site loop are shown in ball-and-stick representation, and the residue numbers correspond to NMA1982.
teins of the PTP family. While the RMSD in both comparisons is identical (2.8 A˚), the putative phosphatase from A. thaliana can be aligned to NMA1982 with less gaps, but with a slightly shorter alignment, than can the human phosphatase (16 gaps vs. 30 gaps, and 135 vs. 140 alignment length). Both A. thaliana phosphatase and the human phosphatase have a large loop insertion (at different locations) [Fig. 2(A)], as compared to NMA1982. In A. thaliana, this loop region is involved in inter-subunit interactions and is part of the hexameric interface. Furthermore, both A. thaliana phosphatase and human phosphatase contain a similar active site loop with a CysDOI 10.1002/prot
(X)5-Arg motif, which is shorter by one residue in NMA1982 [Fig. 2(B)]. The structural alignments also reveal that almost all residues in the phosphate-binding pocket are conserved and structurally superimposable among these three enzymes, although they share very low overall sequence similarity [Fig. 2(B)]. DALI also finds similarities to members of the dual specificity phosphatase family, which, prior to NMA1982, contained only structures of mammalian (human and mouse) proteins. However, these dual specificity phosphatases contain additional secondary structural elements, as compared to NMA1982, PROTEINS
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Figure 3 Comparison of the active site of classical PTPs with the nonclassical NMA1982: (A) Multiple sequence alignment of the active site loop of representative phosphatases with Cys-(X)4-Arg and Cys-(X)5-Arg motifs. Sequences are labeled according to the NCBI gene identification (gi) number, and an abbreviation of the species name is shown in brackets: Nm, Neisseria meningitidis; Bp33571275, Bordetella pertussis Tohama I; Ml, Mesorhizobium loti; Pa, Pseudomonas aeruginosa; Bp67672005, Burkholderia pseudomallei; Hs, Homo sapiens; Sp, Strongylocentrotus purpuratus; Ch, Cytophaga hutchinsonii; Gv, Gloeobacter violaceus. The start and end residue numbers are indicated before and after each sequence. The cysteine and arginine residues of the Cys-(X)4-Arg and Cys-(X)5-Arg motifs are boxed in black and glycines present between the Cys and Arg are colored red. (B) Structural alignment of NMA1982 (light green) and a classical protein tyrosine phosphatase, PTP1B (1pty; forest green). Side chains of functionally important residues in both enzymes are displayed as ball-and-stick and labeled (PTP1B residues are shown in brackets). The PTyr-loop of protein tyrosine phosphatase is colored red and indicated with an arrow.
and are classified into a separate family of higher-molecular-weight phosphotyrosine protein phosphatases in the SCOP database. A majority of PTPs exist as dimers in solution. However, both analytical size exclusion chromatography in combination with static light scattering and crystallographic packing using the PQS server22 indicate that a monomer is the biologically relevant form for NMA1982. The solvent-exposed loop of NMA1982, Cys108-Arg113 overlaps well with the highly conserved P-loop found in the active site of classical PTPs, such as PTP1B (PDB 1pty; Cys215-Arg221). In PTP1B, the backbone of the P-loop and the side chain of an invariant arginine form hydrogen bonds with the substrate’s phosphate group, while the sulfur of an invariant cysteine acts as the nucleophile during catalysis. Although the P-loop in NMA1982 differs in length by one amino acid, the corresponding residues, Cys108 and Arg113 in NMA1982, are similarly positioned to those in PTP1B and other PTP structures [Fig. 3(A,B)].
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The enzymatic mechanism of PTPs requires a general acid/base, which is usually an aspartate positioned in a loop that shifts towards the catalytic pocket upon substrate binding (Asp181 in PTP1B). Asp84 in NMA1982 is part of a similarly located loop and could potentially act as the general acid/base during catalysis. A highly conserved glutamic acid (Glu115 in PTP1B) forms hydrogen bonds with the invariant arginine in the P-loop. In NMA1982, Glu53 is in a corresponding position and also hydrogen bonds with Arg113. Further similarities include Gln28 as part of a highly conserved core structure surrounding the P-loop (Gln85 in PTP1B), as well as the hydrophobic core formed by Ile45/Ile46/Cys47, which corresponds to PTP1B’s Val107/Val108/Met109. However, other functional motifs, such as the phosphotyrosine recognition loop [43-46 in PTP1B; Fig. 3(B), shown in red] and an arginine residue (Arg257 in PTP1B) that hydrogen bonds with the P-loop and reduces the pKa of the catalytic cysteine, are absent from NMA1982 DOI 10.1002/prot
Crystal Structure of NMA1982
[Fig. 3(B)]. Nevertheless, given the conservation of several active site residues among these enzymes, it is likely that NMA1982 adopts a similar mechanism of dephosphorylation as PTPs and acts as a protein phosphatase, but it is unlikely to be tyrosine-specific. The only significant difference is the length of the P-loop, which in NMA1982 is shorter by one amino acid, so the sequence does not conform to the signature motif Cys-(X)5-Arg that is found in all PTPs known to date. The Cys-(X)4-Arg motif in NMA1982 defines a new class of PTPs for which no close structural template was previously available. The structure of NMA1982 can serve as a template for generating models of homologs of this novel hydrolase/ phosphatase subfamily. Models of these homologs and their sequences are accessible at http://www1.jcsg.org/ cgi-bin/models/get_mor.pl?key¼2f46. ACKNOWLEDGMENTS Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL) and the Advanced Light Source (ALS). The SSRL is a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences). The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory. The American Type Culture Collection (ATCC) provided genomic DNA used to clone NMA1982, ATCC Number: 700532D-5. REFERENCES 1. Alonso A, Sasin J, Bottini N, Friedberg I, Friedberg I, Osterman A, Godzik A, Hunter T, Dixon J, Mustelin T. Protein tyrosine phosphatases in the human genome. Cell 2004;117:699–711. 2. Dixon JE. Structure and catalytic properties of protein tyrosine phosphatases. Ann N Y Acad Sci 1995;766:18–22. 3. Bateman A, Coin L, Durbin R, Finn RD, Hollich V, Griffiths-Jones S, Khanna A, Marshall M, Moxon S, Sonnhammer EL, Studholme DJ, Yeats C, Eddy SR. The Pfam protein families database. Nucleic Acids Res 2004;32:D138–D141. 4. Yooseph S, Sutton G, Rusch DB, Halpern AL, Williamson SJ, Remington K, Eisen JA, Heidelberg KB, Manning G, Li W, Jaroszewski L, Cieplak P, Miller CS, Li H, Mashiyama ST, Joachimiak MP, van Belle C, Chandonia JM, Soergel DA, Zhai Y, Natarajan K, Lee S, Raphael BJ, Bafna V, Freidman R, Brenner SE, Godzik A, Eisenberg D, Dixon JE, Taylor SS, Strausberg RL, Frazier M, Venter JC. The Sorcerer II global ocean sampling expedition: expanding the universe of protein families. PLoS Biol 2007;5:e16. 5. Jaroszewski L, Rychlewski L, Li Z, Li W, Godzik A. FFAS03: a server for profile–profile sequence alignments. Nucleic Acids Res 2005;33: W284–W288.
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