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• W2171970664 abstract "Widespread use of β-lactam antibiotics has promoted the evolution of β-lactamase mutant enzymes that can hydrolyze ever newer classes of these drugs. Among the most pernicious mutants are the inhibitor-resistant TEM β-lactamases (IRTs), which elude mechanism-based inhibitors, such as clavulanate. Despite much research on these IRTs, little is known about the structural bases of their action. This has made it difficult to understand how many of the resistance substitutions act as they often occur far from Ser-130. Here, three IRT structures, TEM-30 (R244S), TEM-32 (M69I/M182T), and TEM-34 (M69V), are determined by x-ray crystallography at 2.00, 1.61, and 1.52 Å, respectively. In TEM-30, the Arg-244 → Ser substitution (7.8 Å from Ser-130) displaces a conserved water molecule that usually interacts with the β-lactam C3 carboxylate. In TEM-32, the substitution Met-69 → Ile (10 Å from Ser-130) appears to distort Ser-70, which in turn causes Ser-130 to adopt a new conformation, moving its Oγ further away, 2.3 Å from where the inhibitor would bind. This substitution also destabilizes the enzyme by 1.3 kcal/mol. The Met-182 → Thr substitution (20 Å from Ser-130) has no effect on enzyme activity but rather restabilizes the enzyme by 2.9 kcal/mol. In TEM-34, the Met-69 → Val substitution similarly leads to a conformational change in Ser-130, this time causing it to hydrogen bond with Lys-73 and Lys-234. This masks the lone pair electrons of Ser-130 Oγ, reducing its nucleophilicity for cross-linking. In these three structures, distant substitutions result in accommodations that converge on the same point of action, the local environment of Ser-130. Widespread use of β-lactam antibiotics has promoted the evolution of β-lactamase mutant enzymes that can hydrolyze ever newer classes of these drugs. Among the most pernicious mutants are the inhibitor-resistant TEM β-lactamases (IRTs), which elude mechanism-based inhibitors, such as clavulanate. Despite much research on these IRTs, little is known about the structural bases of their action. This has made it difficult to understand how many of the resistance substitutions act as they often occur far from Ser-130. Here, three IRT structures, TEM-30 (R244S), TEM-32 (M69I/M182T), and TEM-34 (M69V), are determined by x-ray crystallography at 2.00, 1.61, and 1.52 Å, respectively. In TEM-30, the Arg-244 → Ser substitution (7.8 Å from Ser-130) displaces a conserved water molecule that usually interacts with the β-lactam C3 carboxylate. In TEM-32, the substitution Met-69 → Ile (10 Å from Ser-130) appears to distort Ser-70, which in turn causes Ser-130 to adopt a new conformation, moving its Oγ further away, 2.3 Å from where the inhibitor would bind. This substitution also destabilizes the enzyme by 1.3 kcal/mol. The Met-182 → Thr substitution (20 Å from Ser-130) has no effect on enzyme activity but rather restabilizes the enzyme by 2.9 kcal/mol. In TEM-34, the Met-69 → Val substitution similarly leads to a conformational change in Ser-130, this time causing it to hydrogen bond with Lys-73 and Lys-234. This masks the lone pair electrons of Ser-130 Oγ, reducing its nucleophilicity for cross-linking. In these three structures, distant substitutions result in accommodations that converge on the same point of action, the local environment of Ser-130. inhibitor-resistant TEM β-lactamase wild type TEM M182T, a thermostable iso-functional mutant TEM-1 β-lactamase is the predominant source of resistance to β-lactams, such as the penicillins. TEM-1 and related class A β-lactamases confer resistance by hydrolyzing the β-lactam ring of these antibiotics; bacteria expressing these enzymes have become widespread in hospitals and in the community. Beginning in 1980s, three mechanism-based class A β-lactamase inhibitors, clavulanate, tazobactam, and sulbactam, have been used in combination with conventional penicillins to reverse this resistance (Fig.1, A–C). However, since 1992, more than 26 so-called inhibitor-resistant TEM (IRT)1 mutants have been selected, reversing susceptibility to these three mechanism-based inhibitors in the clinic (www.lahey.org/studies/temtable.stm) (1Blazquez J. Baquero M.R. Canton R. Alos I. Baquero F. Antimicrob. Agents Chemother. 1993; 37: 2059-2063Crossref PubMed Scopus (138) Google Scholar,2Belaaouaj A. Lapoumeroulie C. Canica M.M. Vedel G. Nevot P. Krishnamoorthy R. Paul G. FEMS Microbiol. Lett. 1994; 120: 75-80PubMed Google Scholar). The inhibition mechanisms of these three inhibitors are similar, involving a secondary cross-linking reaction after the initial primary nucleophilic attack by the enzyme on the β-lactam ring (Fig.2) (3Knowles J.R. Acc. Chem. Res. 1985; 18: 97-104Crossref Scopus (221) Google Scholar, 4Kuzin A.P. Nukaga M. Nukaga Y. Hujer A. Bonomo R.A. Knox J.R. Biochemistry. 2001; 40: 1861-1866Crossref PubMed Scopus (82) Google Scholar, 5Yang Y. Janota K. Tabei K. Huang N. Siegel M.M. Lin Y.I. Rasmussen B.A. Shlaes D.M. J. Biol. Chem. 2000; 275: 26674-26682Abstract Full Text Full Text PDF PubMed Google Scholar, 6Imtiaz U. Billings E.M. Knox J.R. Manavathu E.K. Lerner S.A. Mobashery S. J. Am. Chem. Soc. 1993; 115: 4435-4442Crossref Scopus (121) Google Scholar, 7Brown R.P. Aplin R.T. Schofield C.J. Biochemistry. 1996; 35: 12421-12432Crossref PubMed Scopus (101) Google Scholar). The mechanism of inhibition has been studied kinetically (6Imtiaz U. Billings E.M. Knox J.R. Manavathu E.K. Lerner S.A. Mobashery S. J. Am. Chem. Soc. 1993; 115: 4435-4442Crossref Scopus (121) Google Scholar, 8Fisher J. Charnas R.L. Knowles J.R. Biochemistry. 1978; 17: 2180-2184Crossref PubMed Scopus (155) Google Scholar, 9Charnas R.L. Knowles J.R. Biochemistry. 1981; 20: 3214-3219Crossref PubMed Scopus (53) Google Scholar, 10Chaibi E.B. Peduzzi J. Farzaneh S. Barthelemy M. Sirot D. Labia R. Biochim. Biophys. Acta. 1998; 1382: 38-46Crossref PubMed Scopus (34) Google Scholar, 11Delaire M. Labia R. Samama J.P. Masson J.M. J. Biol. Chem. 1992; 267: 20600-20606Abstract Full Text PDF PubMed Google Scholar, 12Bret L. Chaibi E.B. Chanal-Claris C. Sirot D. Labia R. Sirot J. Antimicrob. Agents Chemother. 1997; 41: 2547-2549Crossref PubMed Google Scholar, 13Bush K. Macalintal C. Rasmussen B.A. Lee V.J. Yang Y. Antimicrob. Agents Chemother. 1993; 37: 851-858Crossref PubMed Scopus (237) Google Scholar, 14Zafaralla G. Manavathu E.K. Lerner S.A. Mobashery S. Biochemistry. 1992; 31: 3847-3852Crossref PubMed Scopus (110) Google Scholar), by crystallography (4Kuzin A.P. Nukaga M. Nukaga Y. Hujer A. Bonomo R.A. Knox J.R. Biochemistry. 2001; 40: 1861-1866Crossref PubMed Scopus (82) Google Scholar, 15Chen C.C. Herzberg O. J. Mol. Biol. 1992; 224: 1103-1113Crossref PubMed Scopus (102) Google Scholar), by simulation (10Chaibi E.B. Peduzzi J. Farzaneh S. Barthelemy M. Sirot D. Labia R. Biochim. Biophys. Acta. 1998; 1382: 38-46Crossref PubMed Scopus (34) Google Scholar, 11Delaire M. Labia R. Samama J.P. Masson J.M. J. Biol. Chem. 1992; 267: 20600-20606Abstract Full Text PDF PubMed Google Scholar, 16Imtiaz U. Manavathu E.K. Mobashery S. Lerner S.A. Antimicrob. Agents Chemother. 1994; 38: 1134-1139Crossref PubMed Scopus (40) Google Scholar), and by mass spectrum analysis (5Yang Y. Janota K. Tabei K. Huang N. Siegel M.M. Lin Y.I. Rasmussen B.A. Shlaes D.M. J. Biol. Chem. 2000; 275: 26674-26682Abstract Full Text Full Text PDF PubMed Google Scholar, 7Brown R.P. Aplin R.T. Schofield C.J. Biochemistry. 1996; 35: 12421-12432Crossref PubMed Scopus (101) Google Scholar) and is well understood (Fig. 2). The mechanism is thought to involve 8–10 different intermediates with the inactivation pathway (intermediate2 → 4) branching from conventional acyl intermediate hydrolysis (3Knowles J.R. Acc. Chem. Res. 1985; 18: 97-104Crossref Scopus (221) Google Scholar, 4Kuzin A.P. Nukaga M. Nukaga Y. Hujer A. Bonomo R.A. Knox J.R. Biochemistry. 2001; 40: 1861-1866Crossref PubMed Scopus (82) Google Scholar, 5Yang Y. Janota K. Tabei K. Huang N. Siegel M.M. Lin Y.I. Rasmussen B.A. Shlaes D.M. J. Biol. Chem. 2000; 275: 26674-26682Abstract Full Text Full Text PDF PubMed Google Scholar, 6Imtiaz U. Billings E.M. Knox J.R. Manavathu E.K. Lerner S.A. Mobashery S. J. Am. Chem. Soc. 1993; 115: 4435-4442Crossref Scopus (121) Google Scholar, 7Brown R.P. Aplin R.T. Schofield C.J. Biochemistry. 1996; 35: 12421-12432Crossref PubMed Scopus (101) Google Scholar). All three inhibitors ultimately form a covalent cross-link between Ser-70 and Ser-130. This latter residue is attacked by the reactive intermediate 2, which can, alternatively, be hydrolyzed to give intermediate 3, using the hydrolytic pathway (Fig. 2) (3Knowles J.R. Acc. Chem. Res. 1985; 18: 97-104Crossref Scopus (221) Google Scholar, 4Kuzin A.P. Nukaga M. Nukaga Y. Hujer A. Bonomo R.A. Knox J.R. Biochemistry. 2001; 40: 1861-1866Crossref PubMed Scopus (82) Google Scholar, 5Yang Y. Janota K. Tabei K. Huang N. Siegel M.M. Lin Y.I. Rasmussen B.A. Shlaes D.M. J. Biol. Chem. 2000; 275: 26674-26682Abstract Full Text Full Text PDF PubMed Google Scholar, 6Imtiaz U. Billings E.M. Knox J.R. Manavathu E.K. Lerner S.A. Mobashery S. J. Am. Chem. Soc. 1993; 115: 4435-4442Crossref Scopus (121) Google Scholar, 7Brown R.P. Aplin R.T. Schofield C.J. Biochemistry. 1996; 35: 12421-12432Crossref PubMed Scopus (101) Google Scholar). The inactivation and hydrolytic pathways compete during each reaction cycle. Although the hydrolytic pathway is over 100-fold faster than the inactivation pathway (3Knowles J.R. Acc. Chem. Res. 1985; 18: 97-104Crossref Scopus (221) Google Scholar, 8Fisher J. Charnas R.L. Knowles J.R. Biochemistry. 1978; 17: 2180-2184Crossref PubMed Scopus (155) Google Scholar,9Charnas R.L. Knowles J.R. Biochemistry. 1981; 20: 3214-3219Crossref PubMed Scopus (53) Google Scholar), inactivation is irreversible, and the enzyme is eventually completely inhibited. The IRT enzymes that have been selected involve substitutions to several different residues. Some, such as S130G (TEM-76), are simple to understand: the cross-linking residue is simply replaced. Others, such as substitutions to Met-69, Trp-165, Met-182, Arg-244, Arg-275, and Asn-276 (17Chaibi E.B. Sirot D. Paul G. Labia R. J. Antimicrob. Chemother. 1999; 43: 447-458Crossref PubMed Scopus (194) Google Scholar), are often distant from Ser-130, as far as 20 Å, making them harder to comprehend based on the wild type (WT) structure alone. Extensive site-directed mutagenesis and kinetic studies have been carried out on these mutant enzymes (see reviews by Chaibi et al. (17Chaibi E.B. Sirot D. Paul G. Labia R. J. Antimicrob. Chemother. 1999; 43: 447-458Crossref PubMed Scopus (194) Google Scholar), Knox (18Knox J.R. Antimicrob. Agents Chemother. 1995; 39: 2593-2601Crossref PubMed Scopus (301) Google Scholar), and Yang et al. (19Yang Y. Rasmussen B.A. Shlaes D.M. Pharmacol. Ther. 1999; 83: 141-151Crossref PubMed Scopus (91) Google Scholar)). Although several molecular modeling studies of IRTs have been undertaken (6Imtiaz U. Billings E.M. Knox J.R. Manavathu E.K. Lerner S.A. Mobashery S. J. Am. Chem. Soc. 1993; 115: 4435-4442Crossref Scopus (121) Google Scholar,10Chaibi E.B. Peduzzi J. Farzaneh S. Barthelemy M. Sirot D. Labia R. Biochim. Biophys. Acta. 1998; 1382: 38-46Crossref PubMed Scopus (34) Google Scholar), an atomic resolution structure is only available for one of them, TEM-84 (N276D) (20Swaren P. Golemi D. Cabantous S. Bulychev A. Maveyraud L. Mobashery S. Samama J.P. Biochemistry. 1999; 38: 9570-9576Crossref PubMed Scopus (45) Google Scholar). To further explore the structural bases of resistance to inhibition, the structures of TEM-30 (R244S), TEM-32 (M69I/M182T), and TEM-34 (M69V) were determined by x-ray crystallography. These structures reveal a subtle set of accommodations that, in different ways, all end up disrupting the local environment of the cross-linking residue, Ser-130, while leaving the hydrolytic mechanism less affected. Site-directed mutagenesis was carried out using a modified two-step PCR protocol (21Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar, 22Wang X. Minasov G. Shoichet B.K. Proteins. 2002; 47: 86-96Crossref PubMed Scopus (45) Google Scholar). TEM-30, TEM-32, and TEM-34 were expressed and purified in a procedure modified from Dubus et al. (23Dubus A. Wilkin J.M. Raquet X. Normark S. Frere J.M. Biochem. J. 1994; 301: 485-494Crossref PubMed Scopus (58) Google Scholar). The protein was produced at room temperature in 2× YT medium. Cells were collected by centrifugation and resuspended in 5 mm Tris/HCl, pH 8.0, containing 1 mm EDTA and 20% (w/v) sucrose in room temperature for 10 min. Cells were then collected and resuspended in ice-cold 5 mm MgCl2 for 10 min. The supernatant was saved as the periplasmic contents, concentrated to about 100 ml, and dialyzed against 5 mm Tris/HCl, pH 8.0. The crude extract was applied to a Q-Sepharose Fast Flow column (Amersham Biosciences) equilibrated with 5 mm Tris/HCl, pH 8.0. The column was then washed extensively with 5 mm Tris/HCl, pH 8.0. The enzyme was eluted by 5 mm Tris/HCl, pH 8.0, containing 100 mm NaCl. The active fractions were pooled and dialyzed against 100 mm sodium Pi, pH 8.0. The protein was then applied to a Ni2+-nitrilotriacetate-agarose column (Qiagen, Valencia, CA) equilibrated in the same buffer. The enzyme was eluted by 40 mm imidazole. The enzyme solution was concentrated to 6 mg/ml and stored in 200 mm potassium Pi, pH 7.0, 50% glycerol at −20 °C. The enzyme was denatured by raising the temperature in 0.1 °C increments at a ramp rate of 2 °C/min in 200 mm potassium Pi, pH 7.0, using a Jasco 715 spectropolarimeter with a Peltier effect temperature controller and an in-cell temperature monitor (22Wang X. Minasov G. Shoichet B.K. Proteins. 2002; 47: 86-96Crossref PubMed Scopus (45) Google Scholar). Denaturation was marked by an obvious transition in both the far-UV CD (223 nm) and fluorescence signals (maximum at 340 nm measured using a 300-nm cut-on filter). Both fluorescence and CD signals were monitored simultaneously. All melts were reversible and apparently two-state (22Wang X. Minasov G. Shoichet B.K. Proteins. 2002; 47: 86-96Crossref PubMed Scopus (45) Google Scholar,24Raquet X. Vanhove M. Lamotte-Brasseur J. Goussard S. Courvalin P. Frere J.M. Proteins. 1995; 23: 63-72Crossref PubMed Scopus (44) Google Scholar). Temperature of melting (Tm) and van't Hoff enthalpy of unfolding (ΔHVH) values were calculated using EXAM (25Kirchoff W. EXAM: A Two-state Thermodynamic Analysis Program. Gaithersburg, MD1993Crossref Google Scholar). The free energy of unfolding relative to WT was calculated using the method proposed by Becktel and Schellman (26Becktel W.J. Schellman J.A. Biopolymers. 1987; 26: 1859-1877Crossref PubMed Scopus (949) Google Scholar): ΔΔGu = ΔTm·ΔSuWT (26Becktel W.J. Schellman J.A. Biopolymers. 1987; 26: 1859-1877Crossref PubMed Scopus (949) Google Scholar). A positive value of ΔΔGu indicates a stability gain, and a negative value indicates stability loss. The ΔSuWT was 0.43 ± 0.02 kcal/mol·K. TEM-30, TEM-32, and TEM-34 were crystallized using the hanging drop vapor diffusion technique, equilibrating an 8-μl droplet containing 5 mg/ml protein and 0.65 m sodium potassium Pi buffer, pH 8.0, against a reservoir solution of 0.75 ml of 1.4 msodium potassium Pi buffer, pH 8.0 (22Wang X. Minasov G. Shoichet B.K. Proteins. 2002; 47: 86-96Crossref PubMed Scopus (45) Google Scholar). Droplets were initially seeded with microcrystals of the TEM mutant M182T (27Wang X. Minasov G. Shoichet B.K. J. Mol. Biol. 2002; 320: 85-95Crossref PubMed Scopus (368) Google Scholar). Crystals were cryoprotected by 25% sucrose in 1.6 mpotassium Pi buffer, pH 8.0. Crystals were mounted in a nylon loop and flash-frozen in liquid nitrogen (100 K). X-ray diffraction data were collected on the 5-ID beamline (λ = 1.0000 Å) of the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) at the Advanced Photon Source (Argonne, IL) using a MARCCD detector. Data were processed and merged using the DENZO/SCALEPCK suite (28Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38570) Google Scholar). The M182T structure (Protein Data Bank accession number 1JWP (22Wang X. Minasov G. Shoichet B.K. Proteins. 2002; 47: 86-96Crossref PubMed Scopus (45) Google Scholar)) was used as an initial model for molecular replacement. After rigid body refinement and torsion angle annealing using CNS (29Brünger 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 (16966) Google Scholar), mutated residues were fit intoFo − Fc difference density maps. The models were refined by cycles of Cartesian and B-factor refinement (30Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2545) Google Scholar) followed by manual corrections using the program Turbo (31Cambillau C. Roussel A. Turbo Frodo. OpenGL Ed. Universite Aix-Marseille II, Marseille, France1997Google Scholar). In the following discussion, we compare the IRT structures with that of a laboratory variant of TEM-1, TEM M182T, which is kinetically and structurally nearly identical to TEM-1 (22Wang X. Minasov G. Shoichet B.K. Proteins. 2002; 47: 86-96Crossref PubMed Scopus (45) Google Scholar). We will refer to this enzyme, M182T, as WT*. We use WT* as a point of comparison for the IRT enzymes for several reasons. First, we have ourselves determined the structure of WT* in the same buffer, space group, and cryo-conditions as for the IRT mutants. We also have refined WT* and the IRTs using the same protocols. This reduces small crystallographic differences that could otherwise occur when comparing the IRT structures with “true” WT, determined previously in other laboratories (32Short J.M. Fernandez J.M. Sorge J.A. Huse W.D. Nucleic Acids Res. 1988; 16: 7583-7600Crossref PubMed Scopus (1080) Google Scholar, 33Chaibi E.B. Peduzzi J. Barthelemy M. Labia R. J. Antimicrob. Chemother. 1997; 39: 668-669Crossref PubMed Scopus (8) Google Scholar, 34Strynadka N.C. Adachi H. Jensen S.E. Johns K. Sielecki A. Betzel C. Sutoh K. James M.N. Nature. 1992; 359: 700-705Crossref PubMed Scopus (529) Google Scholar, 35Jelsch C. Mourey L. Masson J.M. Samama J.P. Proteins. 1993; 16: 364-383Crossref PubMed Scopus (361) Google Scholar). Second, WT* is 2.5 kcal/mol more stable than TEM-1 (22Wang X. Minasov G. Shoichet B.K. Proteins. 2002; 47: 86-96Crossref PubMed Scopus (45) Google Scholar). This higher stability makes it express (36Huang W. Palzkill T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8801-8806Crossref PubMed Scopus (123) Google Scholar, 37Farzaneh S. Chaibi E.B. Peduzzi J. Barthelemy M. Labia R. Blazquez J. Baquero F. Antimicrob. Agents Chemother. 1996; 40: 2434-2436Crossref PubMed Google Scholar), crystallize, and diffract better than WT TEM-1 such that we have determined its structure to a 0.85-Å resolution (38Minasov G. Wang X. Shoichet B.K. J. Am. Chem. Soc. 2002; 124: 5333-5340Crossref PubMed Scopus (185) Google Scholar). We note that such stable iso-functional mutants are often used as pseudo-wild type in other enzymes, such as lysozyme (39Matsumura M. Matthews B.W. Science. 1989; 243: 792-794Crossref PubMed Scopus (186) Google Scholar). Moreover, many reported TEM-1 wild type structures derive from the Bluescript plasmid (32Short J.M. Fernandez J.M. Sorge J.A. Huse W.D. Nucleic Acids Res. 1988; 16: 7583-7600Crossref PubMed Scopus (1080) Google Scholar, 33Chaibi E.B. Peduzzi J. Barthelemy M. Labia R. J. Antimicrob. Chemother. 1997; 39: 668-669Crossref PubMed Scopus (8) Google Scholar, 34Strynadka N.C. Adachi H. Jensen S.E. Johns K. Sielecki A. Betzel C. Sutoh K. James M.N. Nature. 1992; 359: 700-705Crossref PubMed Scopus (529) Google Scholar, 35Jelsch C. Mourey L. Masson J.M. Samama J.P. Proteins. 1993; 16: 364-383Crossref PubMed Scopus (361) Google Scholar) and contain two substitutions, Ile-84 → Val and Val-182 → Ala, relative to WT TEM-1 found in clinical isolates. The substitution Met-182 → Thr, which occurs in TEM-32, takes place over 15 Å from the active site. This substitution has been shown to be a “global stabilizer” in extended spectrum β-lactamase mutants (27Wang X. Minasov G. Shoichet B.K. J. Mol. Biol. 2002; 320: 85-95Crossref PubMed Scopus (368) Google Scholar, 36Huang W. Palzkill T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8801-8806Crossref PubMed Scopus (123) Google Scholar). To investigate the effects on enzyme stability of this substitution and its coupling to the IRT substitution Met-69 → Ile and Met-69 → Leu, the relative thermostabilities of M69I/M182T (TEM-32), M69L (TEM-33), M69V (TEM-34), and M69I (TEM-40) were determined by reversible, two-state denaturation (22Wang X. Minasov G. Shoichet B.K. Proteins. 2002; 47: 86-96Crossref PubMed Scopus (45) Google Scholar) and analyzed for the change in free energy of folding using the method of Becktel and Schellman (26Becktel W.J. Schellman J.A. Biopolymers. 1987; 26: 1859-1877Crossref PubMed Scopus (949) Google Scholar). When compared with WT, M69I and M69V are 2.9 and 0.3 °C (1.3 and 0.1 kcal/mol) less stable than WT, respectively (Fig. 3). On the other hand, M69L is 2.3 °C (1.0 kcal/mol) more stable than WT. The perturbation of stability by the different Met-69 mutants is qualitatively consistent with the modeling studies by Labia and colleagues (10Chaibi E.B. Peduzzi J. Farzaneh S. Barthelemy M. Sirot D. Labia R. Biochim. Biophys. Acta. 1998; 1382: 38-46Crossref PubMed Scopus (34) Google Scholar). TEM-32 (M69I/M182T) is 3.7 °C (1.6 kcal/mol) more stable than WT but 2.4 °C (1.3 kcal/mol) less stable than WT* (Fig. 3). Crystallographic structures of TEM-30 (R244S), TEM-32 (M69I/M182T), and TEM-34 (M69V) were determined at 2.00-, 1.61-, and 1.52-Å resolution, respectively (Table I). The WT* structure (Protein Data Bank accession number 1JWP (22Wang X. Minasov G. Shoichet B.K. Proteins. 2002; 47: 86-96Crossref PubMed Scopus (45) Google Scholar)) was used as an initial model. For all three structures, the unit cell parameters are similar to WT* (Table I). After rigid body refinement and torsion angle annealing in CNS (29Brünger 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 (16966) Google Scholar), substituted residues were fit into the Fo −Fc difference density maps. Several rounds of Cartesian and B-factor refinement resulted in models with finalR-factors and Rfree values of 17.6 and 21.2%, 19.7 and 21.7%, and 17.7 and 18.9% for TEM-30, TEM-32, and TEM-34, respectively. All three structures closely resemble the WT* structure with root mean square deviation of all Cα atoms of 0.28, 0.41, and 0.30 Å for TEM-30, TEM-32, and TEM-34, respectively. The stereochemistry of the models was evaluated by the program Procheck (40Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). All residues except Leu-220, Ile-69 in TEM-32, and Val-69 in TEM-34 were in the most favored region of the Ramachandran plot, and Leu-220, Ile-69 in TEM-32, and Val-69 in TEM-34 were in the additionally allowed region.Table IData processing and refinement statisticsTEM-30TEM-32TEM-34Unit cell parameters (a, b, c, (Å))41.53, 59.67, 88.1041.22, 60.50, 88.7141.37, 59.60, 88.29Resolution range for refinement (Å)17.0–2.00 (2.07–2.00)1-aValues in parentheses are for the highest resolution shell.17.0–1.61 (1.67–1.61)17.0–1.52 (1.57–1.52)Unique reflections14,82829,38534,443Total observations75,684181,694208,926Rmerge(%)8.8 (34.2)6.1 (27.3)5.4 (34.5)Completeness (%)96.4 (98.4)99.2 (99.8)99.7 (100)〈I〉/〈ς(I)〉16.6 (3.6)24.6 (6.1)29.5 (4.7)Number of protein residues263263263Number of ions1 HPO42−1 HCO3−, 2 K+2 HPO42−, 1 K+Number of water molecules224328398r.m.s.d.1-cr.m.s.d., root mean square deviation. bond lengths (Å)0.0090.0090.009r.m.s.d. bond angles (°)1.51.61.5R-factor (%)17.619.717.7Rfree(%)1-bRfree was calculated with 10% of reflections set aside randomly.21.221.718.91-a Values in parentheses are for the highest resolution shell.1-b Rfree was calculated with 10% of reflections set aside randomly.1-c r.m.s.d., root mean square deviation. Open table in a new tab In the TEM-30 structure, two water molecules, Wat63 and Wat139, were modeled into the cavity created by the Arg-244 → Ser substitution using an Fo − Fc electron density map (Fig.4A). With the exception of the Arg-244 → Ser substitution, other catalytic residues are located in positions similar to those seen in WT* (Table II). On the other hand, the well ordered water that is observed to hydrogen bond to Arg-244 in most WT structures (e.g. Wat294 in Protein Data Bank accession number structure 1FQG (34Strynadka N.C. Adachi H. Jensen S.E. Johns K. Sielecki A. Betzel C. Sutoh K. James M.N. Nature. 1992; 359: 700-705Crossref PubMed Scopus (529) Google Scholar)) is not observed in the TEM-30 structure (Fig. 5A).FIG. 4Simulated annealing omit electron density maps, contoured at 2.5 ς (A, B, and D), as well as an overlay of 2Fo −Fc and Fo −Fc electron density maps (C), in the regions where the major changes are observed in the mutant enzymes. Carbon, nitrogen, oxygen, and sulfur atoms are coloredyellow, blue, red, and green, respectively. A, TEM-30 in the Ser-244 region. B, TEM-32 in the Ser-130 region. C, 2Fo − Fc electron density map (blue at 1.0 ς) and Fo −Fc electron density map (green at 2.0 ς and red at −2.0 ς) in the Val-69 and Ser-70 region of TEM-34 before multiple conformations of Ser-70 were modeled. The stick model represents the final model of TEM-34. D, TEM-32 in the Ile-69 and Ser-70 region. Space group was P212121 for all crystals.View Large Image Figure ViewerDownload (PPT)Table IISelected distances in TEM-30, TEM-32, and TEM-34 as compared with WT*DistanceTEM-30TEM-32TEM-34WT*2-aPDB 1JWP (27).ÅÅÅÅCatalytic water2-bThe catalytic water is numbered Wat6 in TEM-30, TEM-32, and TEM-34, and Wat57 in WT*., Ser-70 Oγ2.92.82.72.8Catalytic water, Glu-166 Oɛ22.42.52.52.7Catalytic water, Asn-170 Nδ2.72.72.62.6Oxyanion hole water2-cThe oxyanion hole water is numbered Wat5 in TEM-30, TEM-32, and TEM-34, and Wat196 in WT*., Ser-70 Oγ2.83.02.82.7Oxyanion hole water, Ser-70 N2.72.93.12.8Oxyanion hole water, Ala-237 N3.03.23.03.1Oxyanion hole water, Ala-237 O2.92.62.42.8C3 water2-dThis water, expected to interact with C3 carboxylate of β-lactam, is numbered Wat7 in TEM-32 and TEM-34, and Wat99A in WT*., Arg-244 NH1NP2-eNot present.2.92.92.8C3 water, Ser-235 OγNP3.12.92.8Ser-70 Oγ, Lys-73 Nζ2.83.13.22.8Ser-70 Oγ, Ser-130 Oγ3.55.53.13.2Ser-70 Oγ(B)2-fMeasured from the alternative conformations seen in TEM-32 and TEM-34., Lys-73 NζNP4.34.1NPSer-70 Oγ(B), Ser-130 OγNP5.53.4NPSer-130 Oγ, Lys-73 Nζ4.25.43.23.8Ser-130 Oγ, Lys-234 Nζ2.53.32.82.8Lys-73 Nζ, Asn-132 Oδ13.03.02.93.02-a PDB 1JWP (27Wang X. Minasov G. Shoichet B.K. J. Mol. Biol. 2002; 320: 85-95Crossref PubMed Scopus (368) Google Scholar).2-b The catalytic water is numbered Wat6 in TEM-30, TEM-32, and TEM-34, and Wat57 in WT*.2-c The oxyanion hole water is numbered Wat5 in TEM-30, TEM-32, and TEM-34, and Wat196 in WT*.2-d This water, expected to interact with C3 carboxylate of β-lactam, is numbered Wat7 in TEM-32 and TEM-34, and Wat99A in WT*.2-e Not present.2-f Measured from the alternative conformations seen in TEM-32 and TEM-34. Open table in a new tab FIG. 5Comparing the IRT mutant structure with that of WT and WT* TEM-1. Mutant structures are colored as in Fig. 4.A, comparison of the C3 carboxylate region of the penicillin G complex structure (purple, Protein Data Bank accession number 1FQG (34Strynadka N.C. Adachi H. Jensen S.E. Johns K. Sielecki A. Betzel C. Sutoh K. James M.N. Nature. 1992; 359: 700-705Crossref PubMed Scopus (529) Google Scholar)) and of TEM-30. Water 294 in the acyl complex structure is labeled as Wat′294. The three hydrogen bond interactions observed in the penicillin G acyl complex structure are represented as dashed lines. B, stereo view of the superposition of the Ser-130 region of TEM-32 and WT* (purple). C, stereo view of the superposition of the Ser-130 regions of TEM-34 and WT* (purple). Thedashed lines represented the hydrogen bond interactions between Ser-130, Lys-73, and Lys-234.View Large Image Figure ViewerDownload (PPT) In the TEM-32 and TEM-34 structures, Met-69 is substituted to isoleucine and valine, respectively, and these residues were fit into Fo − Fc electron density maps (Fig. 4, B–D). Most active site residues in TEM-32 and TEM-34 are in locations similar to those seen in WT* (Table II). There are two exceptions to this: in TEM-32, Ser-130, which has a major role in reacting with inhibitors (Fig. 2), adopts a different conformation in the active site, rotating by 64o about χ1(Fig. 5B). In TEM-34, Ser-130 rotates by 27oabout χ1 (Fig. 5C). In both TEM-32 and TEM-34, two conformations of catalytic residue Ser-70 were modeled based onFo − Fc electron density maps (Fig. 4, C and D). When only the canonical conformation of Ser-70 was modeled, the B-factor of the Ser-70 Oγ atom was twice as high as that of the Ser-70 Cβ, which is, in turn, in the range of those of nearby residues (data not shown). Also, a positive electron density peak was observed adjacent to the canonical position of the Oγ, and a negative peak was observed overlapping the c" @default.
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• W2171970664 title "The Structural Bases of Antibiotic Resistance in the Clinically Derived Mutant β-Lactamases TEM-30, TEM-32, and TEM-34" @default.
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