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• W2008232775 abstract "Penicillin-binding proteins (PBPs) are membrane-associated enzymes which perform critical functions in the bacterial cell division process. The single d-Ala,d-Ala (d,d)-carboxypeptidase in Streptococcus pneumoniae, PBP3, has been shown to play a key role in control of availability of the peptidoglycal substrate during cell growth. Here, we have biochemically characterized and solved the crystal structure of a soluble form of PBP3 to 2.8 Å resolution. PBP3 folds into an NH2-terminal, d,d-carboxypeptidase-like domain, and a COOH-terminal, elongated β-rich region. The carboxypeptidase domain harbors the classic signature of the penicilloyl serine transferase superfamily, in that it contains a central, five-stranded antiparallel β-sheet surrounded by α-helices. As in other carboxypeptidases, which are present in species whose peptidoglycan stem peptide has a lysine residue at the third position, PBP3 has a 14-residue insertion at the level of its omega loop, a feature that distinguishes it from carboxypeptidases from bacteria whose peptidoglycan harbors a diaminopimelate moiety at this position. PBP3 performs substrate acylation in a highly efficient manner (kcat/Km = 50,500 m–1·s–1), an event that may be linked to role in control of pneumococcal peptidoglycan reticulation. A model that places PBP3 poised vertically on the bacterial membrane suggests that its COOH-terminal region could act as a pedestal, placing the active site in proximity to the peptidoglycan and allowing the protein to “skid” on the surface of the membrane, trimming pentapeptides during the cell growth and division processes. Penicillin-binding proteins (PBPs) are membrane-associated enzymes which perform critical functions in the bacterial cell division process. The single d-Ala,d-Ala (d,d)-carboxypeptidase in Streptococcus pneumoniae, PBP3, has been shown to play a key role in control of availability of the peptidoglycal substrate during cell growth. Here, we have biochemically characterized and solved the crystal structure of a soluble form of PBP3 to 2.8 Å resolution. PBP3 folds into an NH2-terminal, d,d-carboxypeptidase-like domain, and a COOH-terminal, elongated β-rich region. The carboxypeptidase domain harbors the classic signature of the penicilloyl serine transferase superfamily, in that it contains a central, five-stranded antiparallel β-sheet surrounded by α-helices. As in other carboxypeptidases, which are present in species whose peptidoglycan stem peptide has a lysine residue at the third position, PBP3 has a 14-residue insertion at the level of its omega loop, a feature that distinguishes it from carboxypeptidases from bacteria whose peptidoglycan harbors a diaminopimelate moiety at this position. PBP3 performs substrate acylation in a highly efficient manner (kcat/Km = 50,500 m–1·s–1), an event that may be linked to role in control of pneumococcal peptidoglycan reticulation. A model that places PBP3 poised vertically on the bacterial membrane suggests that its COOH-terminal region could act as a pedestal, placing the active site in proximity to the peptidoglycan and allowing the protein to “skid” on the surface of the membrane, trimming pentapeptides during the cell growth and division processes. Bacterial division is a complex phenomenon that requires the coordination of diverse processes including chromosomal segregation, FtsZ ring-dependent membrane constriction, and cell wall synthesis at the site of septation. The latter process involves the polymerization of glycan chains and transpeptidation of pentapeptidic moieties within the structure of the peptidoglycan, a highly cross-linked mesh that is crucial for maintaining bacterial shape and providing protection from osmotic shock and lysis (1Nanninga N. Microbiol. Mol. Biol. Rev. 1998; 62: 110-129Crossref PubMed Google Scholar). Both reactions are catalyzed by penicillin-binding proteins (PBPs), 1The abbreviations used are: PBP, penicillin-binding protein; hmm, high molecular mass; lmm, low molecular mass; d,d, d-Ala,d-Ala; Ac2-KAA, N,N-diacetyl-l-Lys-d-Ala-d-Ala; SeMet, selenomethionine; r.m.s., root mean square. membrane-associated molecules, which can be classified as high molecular mass (hmm; often bifunctional) and low molecular mass (lmm; monofunctional) and play key roles in the bacterial life cycle. The pathogenic bacterium Streptococcus pneumoniae offers a unique opportunity for the study of the relationship between cell division and cell wall synthesis, since it carries a relatively simple set of six PBPs, compared with other well studied organisms which present much higher complexity (2Williamson R. Hakenbeck R. Tomasz A. Antimicrob. Agents Chemother. 1980; 18: 629-637Crossref PubMed Scopus (72) Google Scholar). In this organism, PBP1a, -1b, and -2a catalyze both glycosyltransfer and transpeptidation; PBP2b and -2x only catalyze the latter reaction, and PBP3, the single lmm PBP in S. pneumoniae, has been shown to act as a d-Ala,d-Ala (d,d) carboxypeptidase (3Hakenbeck R. Kohiyama M. Eur. J. Biochem. 1982; 127: 231-236Crossref PubMed Scopus (35) Google Scholar). The central role of hmm PBPs in the cell growth and division processes has been recently confirmed through the study of their localization within the cell cycle through the employment of immunofluorescence techniques (4Morlot C. Zapun A. Dideberg O. Vernet T. Mol. Microbiol. 2003; 50: 845-855Crossref PubMed Scopus (104) Google Scholar). In S. pneumoniae, the constriction of the FtsZ-ring is spatially coupled to PBP2x- and PBP1a-mediated septal peptidoglycan synthesis, with the former process preceding the latter by approximately 5 min (4Morlot C. Zapun A. Dideberg O. Vernet T. Mol. Microbiol. 2003; 50: 845-855Crossref PubMed Scopus (104) Google Scholar). At the beginning of the cell cycle, PBP3 localizes throughout the whole bacterial surface but seems to be absent from the future division site (5Morlot C. Noirclerc-Savoye M. Zapun A. Dideberg O. Vernet T. Mol. Microbiol. 2004; 51: 1641-1648Crossref PubMed Scopus (88) Google Scholar). Since the d,d-carboxypeptidase activity of PBP3 removes the COOH-terminal d-alanine of the peptidoglycan pentapeptide side chains, its hemispheric localization implies that the cellular region neighboring the future division site will be the only one where full-length pentapeptides will be available as substrates for other PBPs. Interestingly, a mutant pneumococcal strain which lacks PBP3 displays abnormal morphology and exhibits multiple septa initiated at aberrant locations (6Schuster C. Dobrinski B. Hakenbeck R. J. Bacteriol. 1990; 172: 6499-6505Crossref PubMed Google Scholar). Thus, it is likely that the availability of intact pentapeptidic substrates dictates the localization of the hmm PBPs. Therefore, by guaranteeing that pentapeptides are available uniquely at the future division site, PBP3 may ensure the spatial coordination of the FtsZ-ring with the septum synthesis machinery. PBP3 is associated to the bacterial membrane through a COOH-terminal amphiphilic helix. In the d,d-carboxypeptidase reaction catalyzed by PBP3, an active serine residue reacts with the d-Ala-d-Ala COOH terminus of a peptide chain of the peptidoglycan to form a transient acyl-enzyme complex that is subsequently hydrolyzed. The reaction results in the formation of a tetrapeptide that can only serve as an acceptor for a subsequent transpeptidation reaction by other PBPs (3Hakenbeck R. Kohiyama M. Eur. J. Biochem. 1982; 127: 231-236Crossref PubMed Scopus (35) Google Scholar). As in the case of the d,d-transpeptidase activity, d,d-carboxypeptidation is inhibited by penicillin and other β-lactam antibiotics that mimic the structure of the d-Ala-d-Ala carboxyl terminus of the pentapeptide chain (7Tipper D.J. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1133-1141Crossref PubMed Scopus (646) Google Scholar). These antibiotics react with PBPs to form a stable acyl-enzyme complex, resulting in prolonged inhibition of the enzymes. Recently, the structure of PBP5, the soluble, signal peptide-lacking form of the d,d-carboxypeptidase from Escherichia coli, was reported in wild type and mutant forms to high resolution (1.85 and 1.9 Å, respectively; Refs. 8Davies C. White S.W. Nicholas R.A. J. Biol. Chem. 2001; 276: 616-623Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar and 9Nicholas R.A. Krings S. Tomberg J. Nicola G. Davis C. J. Biol. Chem. 2003; 278: 52826-52833Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Wild type PBP5 deacylates its acyl-enzyme complex at a very high rate, which is reminiscent of that of a class A β-lactamase (the latter with a poor substrate). Although these reports shed light on the enzymology of d,d-carboxypeptidation and the two-domain fold of the enzyme, several points still remain unclear, including the nature of the d,d-carboxypeptidase substrates and the enzymatic role of lmm PBPs in the cell division process. In light of our previous reports on the localization of PBP3 in the cell cycle (5Morlot C. Noirclerc-Savoye M. Zapun A. Dideberg O. Vernet T. Mol. Microbiol. 2004; 51: 1641-1648Crossref PubMed Scopus (88) Google Scholar) and in an effort to answer some of the questions above, we performed the enzymatic characterization of pneumococcal PBP3 and solved the structure of a soluble form of the enzyme at 2.8 Å resolution. Although the general folds of the pneumococcal and E. coli enzymes are similar, PBP3 harbors a significantly longer omega-like loop, a feature subsequently identified as a telltale motif in enzymes present in bacteria whose peptidoglycan structures contain an l-lysine group in the third stem peptide position. Interestingly, its carboxypeptidase domain is highly reminiscent of that of transpeptidase K15 from Streptomyces spp. but shares structural resemblance with other peptidoglycan biosynthetic enzymes only in the immediate vicinity of the active site. PBP3 is a highly efficient d,d-carboxypeptidase, hydrolyzing a synthetic peptide substrate 180 times more efficiently than E. coli PBP5; however, acyl-enzyme deacylation is 20-fold slower than for its E. coli counterpart, suggesting that PBP3 plays a particular role in control of peptidoglycan reticulation in the Gram-positive cell wall. Last, the positioning of the active site on the opposite face of the molecule from the COOH-terminal, membrane-interacting region may place it in optimal position to contact the peptidoglycan layer throughout the two cellular hemispheres. Measurement of Kinetic and Antibiotic Recognition Parameters—The construction of plasmid pGEX-sPBP3* encoding the soluble form of wild type PBP3, which lacks both the COOH-terminal helix and the signal peptide (sPBP3*), was described previously (5Morlot C. Noirclerc-Savoye M. Zapun A. Dideberg O. Vernet T. Mol. Microbiol. 2004; 51: 1641-1648Crossref PubMed Scopus (88) Google Scholar). d,d-Carboxypeptidase activity was assayed with N,N-diacetyl-l-Lys-d-Ala-d-Ala (Ac2-KAA). sPBP3* (10 nm) was incubated at 37 °C in 50 mm Tris-HCl (pH 8.5), 50 mm NaCl, 1 mm EDTA, 0.5 mg/ml bovine serum albumin, and Ac2-KAA at concentrations ranging from 37.5 to 24,000 μm. After various time intervals, the reaction was stopped by addition of penicillin G to 0.1 mm, and released d-Ala was measured by the method described by Johnson et al. (10Johnson K. Duez C. Frere J.M. Ghuysen J.M. Methods Enzymol. 1975; 43: 687-698Crossref PubMed Scopus (19) Google Scholar). The functional homogeneity of the protein sample was determined by titrating the active sites present in the preparation using [3H]benzylpenicillin (20 Ci/mmol, 1 mCi/ml; Amersham Biosciences) as a reporter. sPBP3* solutions at 2 and 5 μm were incubated for 15 min at 37 °C in 50 mm Tris-HCl (pH 8.0), 200 mm NaCl containing 0.01–20 μm [3H]benzylpenicillin. The samples were subsequently submitted to SDS-12% PAGE electrophoresis, and estimation of [3H]benzylpenicillin bound to proteins was monitored by two different procedures. The gel was stained with Coomassie Blue, destained, incubated with Amplify (Amersham Biosciences), dried, and either exposed to film for 16 h or cut around the protein bands. In the latter case, the gel slices were mixed with 5 ml of LSC mixture (Picofluor 15, Packard), and their radioactivity was measured using a liquid scintillation analyzer (Packard model 2100TR). To analyze the kinetics of the deacylation reaction, 2 μm purified sPBP3* was labeled with 1 μm [3H]benzylpenicillin at 37 °C during 15 min in 50 mm Tris-HCl (pH 8.0), 200 mm NaCl. Excess of cold benzylpenicillin (15 mm) was then added, and the reaction was continued at 37 °C. Aliquots were regularly removed, submitted to SDS-PAGE electrophoresis, and the amount of radioactivity was measured in the protein bands as mentioned above. The ability of sPBP3* to hydrolyze the pseudo substrate N-benzoyl-d-alanylmercaptoacetic acid (S2d), which is a thioester analog of the stem wall peptide, was explored to generate a comparison profile of hydrolysis rates for other, previously characterized pneumococcal PBPs. Hydrolysis of S2d was followed by monitoring the amount of thiol group released using the method described by Zhao et al. (11Zhao G. Yeh W.K. Carnahan R.H. Flokowitsch J. Meier T.I. Alborn Jr., W.E. Becker G.W. Jaskunas S.R. J. Bacteriol. 1997; 179: 4901-4908Crossref PubMed Google Scholar). Crystallization and Structure Solution—Selenomethionine (SeMet)-substituted sPBP3* was expressed in E. coli B834. Cells were grown in LeMaster medium (12LeMaster D.M. Richards F.M. Biochemistry. 1985; 24: 7263-7268Crossref PubMed Scopus (212) Google Scholar) containing 40 mg·l–1 methionine that was progressively replaced by SeMet. Expression was induced at A600 ∼0.3 and the purification of the protein was carried out as described previously (5Morlot C. Noirclerc-Savoye M. Zapun A. Dideberg O. Vernet T. Mol. Microbiol. 2004; 51: 1641-1648Crossref PubMed Scopus (88) Google Scholar), except that all of the buffers were supplemented with 10 mm dithiothreitol. Complete replacement of methionine residues by selenomethionine was confirmed by electrospray mass spectrometry. SeMet-labeled sPBP3* crystals were grown by hanging drop vapor diffusion using 1.5 μl of protein solution (4 mg·ml–1), 1.5 μl of well solution (0.2 m K,Na-tartrate, 0.1 m trisodium citrate (pH 5.6), 1.7 m (NH4)2SO4, and 3% (v/v) polyethylene glycol 400) and 0.5 μl of 142 mm NaI per drop. Orthorhombic crystals grew at 20 or 15 °C within 1 week. The crystals belong to space group P212121 with cell dimensions a = 87.57 Å, b = 120.69 Å, and c = 176.92 Å and have four molecules in the asymmetric unit. Prior to data collection, crystals were cryoprotected by transfer into 20% (v/v) glycerol, 2% ethylene glycol, 0.2 m K,Na-tartrate, 0.1 m trisodium citrate (pH 5.6), and 1.9 m (NH4)2SO4. Multiwavelength anomalous diffraction data were collected at 100 K from a single SeMet crystal at peak, inflection, and remote wavelengths, on an ADSC Quantum 4R CCD detector at beamline ID14EH4 at the European Synchrotron Radiation Facility, Grenoble, France. 140 degrees of data collected for each wavelength were processed using MOSFLM (13Leslie A.G.M. MOSFLM User Guide. MRC Laboratory of Molecular Biology, Cambridge, UK2002Google Scholar) and CCP4 (14Project Collaborative Computing Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-766Crossref PubMed Scopus (19797) Google Scholar). Using the peak anomalous data, the selenium sites were located with ShelX-d (15Sheldrick G.M. SHELXL97: Program for the Refinment of Crystal Structure. University of Göttingen, Göttingen, Germany1997Google Scholar) and refined with SHARP (16de La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar). The resulting 44 sites were used for phasing with SHARP. Phase improvement with DM and SOLOMON to 2.8 Å produced a clearly interpretable electron density map, from which an initial model was built using ARPwARP (17Morris R.J. Perrakis A. Lamzin V.S. Methods Enzymol. 2003; 374: 229-244Crossref PubMed Scopus (475) Google Scholar). The four molecules in the orthorhombic asymmetric unit were sufficiently different so that non-crystallographic symmetry averaging was not helpful. The model was improved by iterative rounds of manual fitting using O and QUANTA and refinement in CNS (18Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Structural superpositions were performed using LSQKAB (14Project Collaborative Computing Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-766Crossref PubMed Scopus (19797) Google Scholar). Figs. 1, 2, 3 were prepared with Molscript (19Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and Raster3D (20Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2859) Google Scholar).Fig. 2Active site architecture of sPBP3* from S. pneumoniae (A) (stereo view), PBP5 from E. coli (B), and the Streptomyces K15d,d-transpeptidase (C). The main catalytic residues are represented in ball and stick and are colored by atom type. Several potential hydrogen bonds are denoted by orange dashed lines. The secondary structures are shown with ribbon diagram; the helices are colored red, and the β-sheets are green. Proteins were crystallized at pH values of 5.6, 7.0, and 7.2, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Comparison of the structure of the NH2-terminal carboxypeptidase domain of sPBP3* from S. pneumoniae with: the same domain from PBP5 from E. coli (A), Streptomyces K15 transpeptidase (B), TEM-1 β-lactamase from E. coli (C), the transpeptidase domain of PBP2a from S. aureus (D), and the transpeptidase domain of PBP2x from S. pneumoniae R6 (E). The overall fold of sPBP3* is in green, with the omega-like loop colored in yellow. The other structures are in violet, with the omega-like loop colored in blue (except for PBP2a and PBP2x, which do not harbor classical omega loops).View Large Image Figure ViewerDownload Hi-res image Download (PPT) sPBP3* Is a Highly Efficient d,d-Carboxypeptidase—The interaction between PBPs and peptidoglycan substrates or β-lactam antibiotics obeys a three-step reaction that is represented as follows, E+I⇄k−1k1EI→k2EI*→k3E+Peq.1 where E is the active PBP enzyme, I is the substrate, EI is the non-covalent Michaelis-Menten complex, EI* is the acyl-enzyme covalent complex, and P is the product of the reaction (21Frère J.M. Ghuysen J.M. Iwatsubo M. Eur. J. Biochem. 1975; 57: 343-351Crossref PubMed Scopus (52) Google Scholar). Enzymatic parameters for carboxypeptidation were estimated by measuring the initial velocities (ν) at various concentrations of Ac2-KAA. Inhibition of sPBP3* by its own substrate was observed above a ligand concentration of 15 mm, a phenomenon that has been previously reported for a the d,d-carboxypeptidase from Neisseria gonorrhoeae (22Stefanova M.E. Tomberg J. Olesky M. Holtje J.V. Gutheil W.G. Nicholas R.A. Biochemistry. 2003; 42: 14614-14625Crossref PubMed Scopus (48) Google Scholar). The values and standard error of kcat = 110 ± 10 s–1 and Km = 19 ± 3 mm 1·s–1 (thus, kcat/Km = 5689 m–1) were obtained by fitting the data points, from zero to peak activity, against the equation ν/[sPBP3*]T = kcat [Ac2-KAA]/(Km + [Ac2-KAA]); these estimates may be lower than the true kcat and Km due to the inhibition of the enzyme at high ligand concentrations. By comparison, the kcat/Km value measured with this substrate for E. coli PBP5 is 32 m–1·s–1 (9Nicholas R.A. Krings S. Tomberg J. Nicola G. Davis C. J. Biol. Chem. 2003; 278: 52826-52833Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The k3 deacylation rate calculated for sPBP3* is comparable with those reported for other S. pneumoniae PBPs (Table I) but is 20–30-fold lower than that of E. coli PBP5 (9Nicholas R.A. Krings S. Tomberg J. Nicola G. Davis C. J. Biol. Chem. 2003; 278: 52826-52833Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In addition, sPBP3* possesses an efficiency of hydrolysis of 50 500 ± 2500 m–1·s–1 for the pseudo substrate S2d. This value is 200–1000-fold higher than for PBP1a, PBP2a, and PBP2b and 20-fold higher than for PBP2x (Table II) (23Di Guilmi A.M. Mouz N. Andrieu J.P. Hoskins J. Jaskunas S.R. Gagnon J. Dideberg O. Vernet T. J. Bacteriol. 1998; 180: 5652-5659Crossref PubMed Google Scholar, 24Di Guilmi A.M. Mouz N. Martin L. Hoskins J. Jaskunas S.R. Dideberg O. Vernet T. J. Bacteriol. 1999; 181: 2773-2781Crossref PubMed Google Scholar, 25Pagliero E. Chesnel L. Hopkins J. Croize J. Dideberg O. Vernet T. Di Guilmi A.M. Antimicrob. Agents Chemother. 2004; 48: 1848-1855Crossref PubMed Scopus (42) Google Scholar, 26Mouz N. Di Guilmi A.M. Gordon E. Hakenbeck R. Dideberg O. Vernet T. J. Biol. Chem. 1999; 274: 19175-19180Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 27Lobkovsky E. Moews P.C. Liu H. Zhao H. Frère J.M. Knox J.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11257-11261Crossref PubMed Scopus (222) Google Scholar). These high hydrolytic efficiency values suggest that PBP3 may play an important hydrolytic role during the peptidoglycan biosynthetic process.Table IDeacylation rates of different [3H]benzylpenicillin-PBP complexesProteink3t½ of the acyl-enzyme complexRef.× 10-5 s-1hsPBP3*5.73.38This workPBP2x*3.55.8026Mouz N. Di Guilmi A.M. Gordon E. Hakenbeck R. Dideberg O. Vernet T. J. Biol. Chem. 1999; 274: 19175-19180Abstract Full Text Full Text PDF PubMed Scopus (77) Google ScholarPBP1a*1.019.2023Di Guilmi A.M. Mouz N. Andrieu J.P. Hoskins J. Jaskunas S.R. Gagnon J. Dideberg O. Vernet T. J. Bacteriol. 1998; 180: 5652-5659Crossref PubMed Google ScholarPBP2a*3.26.0024Di Guilmi A.M. Mouz N. Martin L. Hoskins J. Jaskunas S.R. Dideberg O. Vernet T. J. Bacteriol. 1999; 181: 2773-2781Crossref PubMed Google ScholarPBP1b*5.63.4442Di Guilmi A.M. Dessen A. Dideberg O. Vernet T. J. Bacteriol. 2003; 185: 1650-1658Crossref PubMed Scopus (39) Google ScholarSPBP5 (E. coli)78.00.259Nicholas R.A. Krings S. Tomberg J. Nicola G. Davis C. J. Biol. Chem. 2003; 278: 52826-52833Abstract Full Text Full Text PDF PubMed Scopus (77) Google ScholarSPBP5′ (E. coli)3.06.40K15 (Streptomyces)101.9243Leyh-Bouille M. Nguyen-Disteche M. Pirlot S. Veithen A. Bourguignon C. Ghuysen J.M. Biochem. J. 1986; 235: 177-182Crossref PubMed Scopus (23) Google Scholar Open table in a new tab Table IIComparison of the hydrolysis efficiency of the pseudo substrate S2d by five different PBPs from S. pneumoniaeProteinkcat/KmRef.m-1 s-1SPBP3*50,500 ± 2500This workPBP2x*250044Mouz N. Gordon E. Di Guilmi A.M. Petit I. Petillot Y. Dupont Y. Hakenbeck R. Vernet T. Dideberg O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13403-13406Crossref PubMed Scopus (76) Google ScholarPBP2b*8025Pagliero E. Chesnel L. Hopkins J. Croize J. Dideberg O. Vernet T. Di Guilmi A.M. Antimicrob. Agents Chemother. 2004; 48: 1848-1855Crossref PubMed Scopus (42) Google ScholarPBP1a*25623Di Guilmi A.M. Mouz N. Andrieu J.P. Hoskins J. Jaskunas S.R. Gagnon J. Dideberg O. Vernet T. J. Bacteriol. 1998; 180: 5652-5659Crossref PubMed Google ScholarPBP2a*220 ± 2024Di Guilmi A.M. Mouz N. Martin L. Hoskins J. Jaskunas S.R. Dideberg O. Vernet T. J. Bacteriol. 1999; 181: 2773-2781Crossref PubMed Google Scholar Open table in a new tab Overview of the sPBP3* Structure—The structure of sPBP3* was determined by multiwavelength anomalous diffraction using seleno-methionyl-substituted protein; data collection, phasing, and refinement statistics are shown in Table III. The final structure, which includes four 360-amino acid monomers in the asymmetric unit, has an R factor of 21.2% (Rfree = 26.2%) at 2.8 Å; 83.1% of the residues lie within the most favored region of the Ramachandran plot and 477 water molecules are included in the model.Table IIIData collection, phasing, and refinement statisticsData collectionCell dimensions (Å)a = 87.57, b = 120.69, c = 176.92Space groupP212121PeakInflectionRemoteWavelength (Å)0.97920.97940.9393Resolution range (Å)2.83.02.8No. of unique/total reflections47,073/255,27838,767/200,35447,278/249,511Completeness (%)99.9 (100)99.8 (99.7)99.8 (100)Average multiplicity5.4 (5.0)5.2 (4.8)5.3 (5.0)Rsym (%)10.2 (27.9)16.1 (50)13.9 (46.6)I/σI (last shell)12.7 (4.7)9.65 (2.93)9.94 (2.99)Rano (%)7.9 (16.0)9.0 (25.5)7.8 (23.6)RefinementResolution (Å)2.80Rwork/Rfree (%)21.2 / 26.2Number of residuesChain A/B/C/D369/350/368/340Number of water molecules477Number of iodines16Number of sulfates4Average B factor (Å2)Protein29.51Solvent33.30r.m.s. bond deviation (Å)0.01r.m.s. angle deviation (°)2.00 Open table in a new tab The sPBP3* monomer consists of a single polypeptide chain organized into two domains that are orientated approximately at right angles to each other. The spatial relationship between the domains is reminiscent of that observed for its E. coli homolog, the PBP5 d,d-carboxypeptidase, with which PBP3 shares 27% sequence identity (Fig. 1). Domain I (cyan in Fig. 1, A and B) comprises residues 25–292 and bears the signature fold topology of the penicilloyl-serine transferase superfamily, thus harboring the active site of PBP3. Using the standard secondary structure classification of the class A and class C β-lactamases as well as PBP2x (27Lobkovsky E. Moews P.C. Liu H. Zhao H. Frère J.M. Knox J.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11257-11261Crossref PubMed Scopus (222) Google Scholar, 28Parès S. Mouz N. Petillot Y. Hakenbeck R. Dideberg O. Nat. Struct. Biol. 1996; 3: 284-289Crossref PubMed Scopus (165) Google Scholar), the first domain is principally constituted by a central five-stranded antiparallel β-sheet (β3/β4/β5/β1/β2) and two main helices (α8/α11). In addition, this domain contains three two-stranded antiparallel β-sheets (β2a/β2d, β2b/β2c, and β2e/β2f) and three helices (α2a/α4/α5). The COOH-terminal domain II bears an elongated structure which comprises residues 293–393 and is formed by a sandwich between two anti-parallel β-sheets. Comparison of the four molecules, which exist in the asymmetric unit, reveals that the greatest differences at the Cα level map to loop regions within the COOH-terminal domain, where the electron density is of poor quality in certain regions. Notably, the large area of interaction between the surfaces of domains I and II (∼800 Å2, including six potential hydrogen bonds) guarantees stability for the full-length molecule, which is reflected by the slight variation (<1°) of the angle between domains I and II in all four molecules of the asymmetric unit. The sPBP3* Active Site—The sPBP3* active site is at the distal end from the COOH terminus of the molecule. As observed with the other carboxypeptidases and penicillin-metabolyzing enzymes, the active site is mainly defined by three conserved structural motifs: SXXK (Ser56-Ile57-Thr58-Lys59), which includes the nucleophilic Ser56 residue, positioned at the NH2-terminal end of helix α2; SXN (Ser119-Ala120-Asn121), which forms the turn between helices α4 and α5onthe left side of the cavity, and K(T/S)G (Lys239-Thr240-Gly241), which lines strand β3 (Fig. 2A). In addition, the backbone NH groups of the essential Ser56 and Thr242 residues occupy positions that are compatible with the oxyanion hole function required for catalysis. The NH2 terminus of helix α11 and the loop between α6 and β2d also contribute residues to the active site. These include Arg278, located at the right top angle of the cavity, Thr160, and the structural Gly161, present on the extended loop at the bottom of the cavity. The hydrogen bonding network within the active site is extensive (Fig. 2A) and is identical in all four molecules in the asymmetric unit. The ϵ-NH2 group of Lys59 plays a central role in this network, forming hydrogen bonds with the hydroxyl group of Ser56 and Ser119, the side chain carbonyl group of Asn121 and the backbone carbonyl group from Thr160. Two water molecules are observed within the hydrogen bonding network, one of which (O-26) is conserved in the K15 transpeptidase (29Fonzé E. Vermeire M. Nguyen-Disteche M. Brasseur R. Charlier P. J. Biol. Chem. 1999; 274: 21853-21860Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), as well as in E. coli PBP5 (8Davies C. White S.W. Nicholas R.A. J. Biol. Chem. 2001; 276: 616-623Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 9Nicholas R.A. Krings S. Tomberg J. Nicola G. Davis C. J. Biol. Chem. 2003; 278: 52826-52833Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Although the architecture of the active site of the three enzymes is similar (compare Fig. 2, A–C), some significant differences can be observed in the orientation of three important catalytic residues: the side chains of Ser110, Lys213, and Thr214 of PBP5 are oriented differently from the equivalent residues in sPBP3*, Ser119, Lys239, and Thr240, respectively. In particular, in PBP5, Ser110 and Lys213 point away from the active site, and consequently the classical hydrogen bonding network within the active site is not formed in this molecule. It is of interest that all three molecules, w" @default.
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• W2008232775 title "Crystal Structure of a Peptidoglycan Synthesis Regulatory Factor (PBP3) from Streptococcus pneumoniae" @default.
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