Structural consequences of the inhibitor-resistant Ser130Gly substitution in TEM beta-lactamase

Beta-lactamase confers resistance to penicillin-like antibiotics by hydrolyzing their beta-lactam bond. To combat these enzymes, inhibitors covalently cross-linking the hydrolytic Ser70 to Ser130 were introduced. In turn, mutant beta-lactamases have emerged with decreased susceptibility to these mechanism-based inhibitors. Substituting Ser130 with glycine in the inhibitor-resistant TEM (IRT) mutant TEM-76 (S130G) prevents the irreversible cross-linking step. Since the completely conserved Ser130 is thought to transfer a proton important for catalysis, its substitution might be hypothesized to result in a nonfunctional enzyme; this is clearly not the case. To investigate how TEM-76 remains active, its structure was determined by X-ray crystallography to 1.40 A resolution. A new water molecule (Wat1023) is observed in the active site, with two configurations located 1.1 and 1.3 A from the missing Ser130 Ogamma; this water molecule likely replaces the Ser130 side-chain hydroxyl in substrate hydrolysis. Intriguingly, this same water molecule is seen in the IRT TEM-32 (M69I/M182T), where Ser130 has moved significantly. TEM-76 shares other structural similarities with various IRTs; like TEM-30 (R244S) and TEM-84 (N276D), the water molecule activating clavulanate for cross-linking (Wat1614) is disordered (in TEM-30 it is actually absent). As expected, TEM-76 has decreased kinetic activity, likely due to the replacement of the Ser130 side-chain hydroxyl with a water molecule. In contrast to the recently determined structure of the S130G mutant in the related SHV-1 beta-lactamase, in TEM-76 the key hydrolytic water (Wat1561) is still present. The conservation of similar accommodations among IRT mutants suggests that resistance arises from common mechanisms, despite the disparate locations of the various substitutions.     

The importance of a critical protonation state and the fate of the catalytic steps in class A beta-lactamases and penicillin-binding proteins

Beta-lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to beta-lactam antibiotics and biosynthetic assembly of cell wall, respectively. Members of these large families of enzymes all experience acylation by their respective substrates at an active site serine as the first step in their catalytic activities. A Ser-X-X-Lys sequence motif is seen in all these proteins, and crystal structures demonstrate that the side-chain functions of the serine and lysine are in contact with one another. Three independent methods were used in this report to address the question of the protonation state of this important lysine (Lys-73) in the TEM-1 beta-lactamase from Escherichia coli. These techniques included perturbation of the pK(a) of Lys-73 by the study of the gamma-thialysine-73 variant and the attendant kinetic analyses, investigation of the protonation state by titration of specifically labeled proteins by nuclear magnetic resonance, and by computational treatment using the thermodynamic integration method. All three methods indicated that the pK(a) of Lys-73 of this enzyme is attenuated to 8.0-8.5. It is argued herein that the unique ground-state ion pair of Glu-166 and Lys-73 of class A beta-lactamases has actually raised the pK(a) of the active site lysine to 8.0-8.5 from that of the parental penicillin-binding protein. Whereas we cannot rule out that Glu-166 might activate the active site water, which in turn promotes Ser-70 for the acylation event, such as proposed earlier, we would like to propose as a plausible alternative for the acylation step the possibility that the ion pair would reconfigure to the protonated Glu-166 and unprotonated Lys-73. As such, unprotonated Lys-73 could promote serine for acylation, a process that should be shared among all active-site serine beta-lactamases and penicillin-binding proteins.     

Miniature protein ligands for EVH1 domains: interplay between affinity, specificity, and cell motility

Dynamic rearrangements of the actin cytoskeleton power cell motility in contexts ranging from intracellular microbial pathogenesis to axon guidance. The Ena/VASP family proteins-Mena, VASP, and Evl-are believed to control cell motility by serving as a direct link between signaling events and the actin cytoskeleton. It has previously been reported that a novel miniature protein, pGolemi, binds with high affinity to the EVH1 domain of Mena (Mena1-112) but not to those of VASP (VASP1-115) or Evl (Evl1-115) and also causes an unusual defect in actin-driven Listeria monocytogenes motility. Here, scanning mutagenesis was used to examine the effects of single amino acid changes within pGolemi on EVH1 domain affinity and specificity, miniature protein secondary structure, and L. monocytogenes motility. The data suggest that pGolemi contains the expected aPP-like fold and binds Mena1-112 in a manner highly analogous to the proline-rich repeat region of L. monocytogenes ActA protein. Residues throughout pGolemi contribute to both EVH1 domain affinity and paralog specificity. Moreover, the affinities of pGolemi variants for Mena1-112 correlate with selectivity against the EVH1 domains of VASP and Evl. In L. monocytogenes motility assays, speed and speed variability correlate strongly with EVH1 paralog specificity, suggesting that the Ena/VASP paralogs do not play equivalent roles in the process of L. monocytogenes actin tail maturation.     

Phosphorylation-Induced Activation of the Response Regulator VraR from Staphylococcus aureus: Insights from Hydrogen Exchange Mass Spectrometry

A two-component system consisting of the histidine kinase vancomycin-resistance-associated sensor and the response regulator vancomycin-resistance-associated regulator (VraR) allows Staphylococcus aureus to sense antibiotic-related cell wall stress and to mount a suitable response. An experimental structure of full-length VraR is not available yet, but previous work points to similarities between VraR and the well-characterized NarL. This work employs hydrogen exchange mass spectrometry to gain insights into the phosphorylation-induced activation of VraR, a process that primes the protein for dimerization and DNA binding. Whereas VraR is highly dynamic, phosphorylated VraR shows less extensive deuteration. This rigidification is most dramatic within the receiver domain, which carries the phosphorylation site D55. Alterations in the DNA-binding domain are much less pronounced. Changes in deuteration within the receiver domain are consistent with a Y-T coupling mechanism. In analogy to NarL, the activation of VraR is thought to involve separation and subsequent reorientation of the two domains, thereby allowing the α8-turn-α9 element to engage in DNA binding. The current work suggests that this structural transition is triggered by a reduction in the effective length of the linker through enhanced hydrogen bonding. In addition, separation of the two domains may be favored by the establishment of noncovalent protein-protein interactions and intradomain contacts at the expense of previously existing interdomain bonds. α9 appears to be packed against the receiver domain in nonactivated VraR. Support is presented for α1 as a dimerization interface in phosphorylated VraR, whereas protein-protein interactions for nonphosphorylated VraR are impeded by extensive disorder in this region.     

Hydrolytic mechanism of OXA-58 enzyme, a carbapenem-hydrolyzing class D β-lactamase from Acinetobacter baumannii

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) represent an emerging antibiotic resistance mechanism encountered among the most opportunistic Gram-negative bacterial pathogens. We report here the substrate kinetics and mechanistic characterization of a prominent CHDL, the OXA-58 enzyme, from Acinetobacter baumannii. OXA-58 uses a carbamylated lysine to activate the nucleophilic serine used for β-lactam hydrolysis. The deacylating water molecule approaches the acyl-enzyme species, anchored at this serine (Ser-83), from the α-face. Our data show that OXA-58 retains the catalytic machinery found in class D β-lactamases, of which OXA-10 is representative. Comparison of the homology model of OXA-58 and the recently solved crystal structures of OXA-24 and OXA-48 with the OXA-10 crystal structure suggests that these CHDLs have evolved the ability to hydrolyze imipenem, an important carbapenem in clinical use, by subtle structural changes in the active site. These changes may contribute to tighter binding of imipenem to the active site and removal of steric hindrances from the path of the deacylating water molecule.     

Diversity of penicillin-binding proteins. Resistance factor FmtA of Staphylococcus aureus

Antibiotic-resistant Staphylococcus aureus is a major concern to public health. Methicillin-resistant S. aureus strains are completely resistant to all beta-lactams antibiotics. One of the main factors involved in methicillin resistance in S. aureus is the penicillin-binding protein, PBP2a. This protein is insensitive to inactivation by beta-lactam antibiotics such as methicillin. Although other proteins are implicated in high and homogeneous levels of methicillin resistance, the functions of these other proteins remain elusive. Herein, we report for the first time on the putative function of one of these proteins, FmtA. This protein specifically interacts with beta-lactam antibiotics forming covalently bound complexes. The serine residue present in the sequence motif Ser-X-X-Lys (which is conserved among penicillin-binding proteins and beta-lactamases) is the active-site nucleophile during the formation of acyl-enzyme species. FmtA has a low binding affinity for beta-lactams, and it experiences a slow acylation rate, suggesting that this protein is intrinsically resistant to beta-lactam inactivation. We found that FmtA undergoes conformational changes in presence of beta-lactams that may be essential to the beta-lactam resistance mechanism. FmtA binds to peptidoglycan in vitro. Our findings suggest that FmtA is a penicillin-binding protein, and as such, it may compensate for suppressed peptidoglycan biosynthesis under beta-lactam induced cell wall stress conditions.     

A close-up view of the VraSR two-component system. A mediator of Staphylococcus aureus response to cell wall damage

Staphylococcus aureus remains a clinical scourge. Recent studies have revealed that S. aureus is capable of mounting a response to antibiotics that target cell wall peptidoglycan biosynthesis, such as beta-lactams and vancomycin. A phosphotransfer-mediated signaling pathway composed of a histidine protein kinase, VraS, and a response regulator protein, VraR, has been linked to the coordination of this response. Herein, we report for the first time on the signal transduction mechanism of the VraSR system. We found that VraS is capable of undergoing autophosphorylation in vitro and its phosphoryl group is rapidly transferred to VraR. In addition, phosphorylated VraR undergoes rapid dephosphorylation by VraS. Evidence is presented that VraR has adopted a novel strategy in regulating the output response of the VraSR-mediated signaling pathway. The VraR effector domain inhibits formation of inactive VraR dimers and, in doing so, it holds the regulatory domain into an intermediate active state. We show that only phosphorylation induces formation of the biological active VraR-dimer species. Furthermore, we propose that damage inflicted to cell wall peptidoglycan could be the main source of the stimuli that VraR responds to due to the tight control that VraS has on the phosphorylation state of VraR. Our findings provide for the first time insights into the molecular basis for the proposed role of VraSR as a "sentinel" system capable of rapidly sensing cell wall peptidoglycan damage and coordinating a response that enhances the resistance phenotype in S. aureus.     

Resistance to beta-lactam antibiotics and its mediation by the sensor domain of the transmembrane BlaR signaling pathway in Staphylococcus aureus

Staphylococci, a leading cause of infections worldwide, have devised two mechanisms for resistance to beta-lactam antibiotics. One is production of beta-lactamases, hydrolytic resistance enzymes, and the other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by beta-lactam antibiotics. The beta-lactam sensor-transducer (BlaR), an integral membrane protein, binds beta-lactam antibiotics on the cell surface and transduces the information to the cytoplasm, where gene expression is derepressed for both beta-lactamase and penicillin-binding protein 2a. The gene for the sensor domain of the sensor-transducer protein (BlaR(S)) of Staphylococcus aureus was cloned, and the protein was purified to homogeneity. It is shown that beta-lactam antibiotics covalently modify the BlaR(S) protein. The protein was shown to contain the unusual carboxylated lysine that activates the active site serine residue for acylation by the beta-lactam antibiotics. The details of the kinetics of interactions of the BlaR(S) protein with a series of beta-lactam antibiotics were investigated. The protein undergoes acylation by beta-lactam antibiotics with microscopic rate constants (k(2)) of 1-26 s(-1), yet the deacylation process was essentially irreversible within one cell cycle. The protein undergoes a significant conformational change on binding with beta-lactam antibiotics, a process that commences at the preacylation complex and reaches its full effect after protein acylation has been accomplished. These conformational changes are likely to be central to the signal transduction events when the organism is exposed to the beta-lactam antibiotic.