Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity.

The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO(2)/bicarbonate, and there is a positive correlation between the CO(2)/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His→Asp) and phosphoablative (His→Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.

Molecular basis for the catalytic specificity of the CTX-M extended-spectrum ß-lactamases.

Extended-spectrum ß-lactamases (ESBLs) pose a threat to public health because of their ability to confer resistance to extended-spectrum cephalosporins such as cefotaxime. The CTX-M ß-lactamases are the most widespread ESBL enzymes among antibiotic resistant bacteria. Many of the active site residues are conserved between the CTX-M family and non-ESBL ß-lactamases such as TEM-1, but the residues Ser237 and Arg276 are specific to the CTX-M family, suggesting that they may help to define the increased specificity for cefotaxime hydrolysis. To test this hypothesis, site-directed mutagenesis of these positions was performed in the CTX-M-14 ß-lactamase. Substitutions of Ser237 and Arg276 with their TEM-1 counterparts, Ala237 and Asn276, had a modest effect on cefotaxime hydrolysis, as did removal of the Arg276 side chain in an R276A mutant. The S237A:R276N and S237A:R276A double mutants, however, exhibited 29- and 14-fold losses in catalytic efficiency for cefotaxime hydrolysis, respectively, while the catalytic efficiency for benzylpenicillin hydrolysis was unchanged. Therefore, together, the Ser237 and Arg276 residues are important contributors to the cefotaximase substrate profile of the enzyme. High-resolution crystal structures of the CTX-M-14 S70G, S70G:S237A, and S70G:S237A:R276A variants alone and in complex with cefotaxime show that residues Ser237 and Arg276 in the wild-type enzyme promote the expansion of the active site to accommodate cefotaxime and favor a conformation of cefotaxime that allows optimal contacts between the enzyme and substrate. The conservation of these residues, linked to their effects on structure and catalysis, imply that their coevolution is an important specificity determinant in the CTX-M family.

Structural plasticity of the coiled-coil domain of rotavirus NSP4.

UNLABELLED: Rotavirus (RV) nonstructural protein 4 (NSP4) is a virulence factor that disrupts cellular Ca(2+) homeostasis and plays multiple roles regulating RV replication and the pathophysiology of RV-induced diarrhea. Although its native oligomeric state is unclear, crystallographic studies of the coiled-coil domain (CCD) of NSP4 from two different strains suggest that it functions as a tetramer or a pentamer. While the CCD of simian strain SA11 NSP4 forms a tetramer that binds Ca(2+) at its core, the CCD of human strain ST3 forms a pentamer lacking the bound Ca(2+) despite the residues (E120 and Q123) that coordinate Ca(2+) binding being conserved. In these previous studies, while the tetramer crystallized at neutral pH, the pentamer crystallized at low pH, suggesting that preference for a particular oligomeric state is pH dependent and that pH could influence Ca(2+) binding. Here, we sought to examine if the CCD of NSP4 from a single RV strain can exist in two oligomeric states regulated by Ca(2+) or pH. Biochemical, biophysical, and crystallographic studies show that while the CCD of SA11 NSP4 exhibits high-affinity binding to Ca(2+) at neutral pH and forms a tetramer, it does not bind Ca(2+) at low pH and forms a pentamer, and the transition from tetramer to pentamer is reversible with pH. Mutational analysis shows that Ca(2+) binding is necessary for the tetramer formation, as an E120A mutant forms a pentamer. We propose that the structural plasticity of NSP4 regulated by pH and Ca(2+) may form a basis for its pleiotropic functions during RV replication. IMPORTANCE: The nonstructural protein NSP4 of rotavirus is a multifunctional protein that plays an important role in virus replication, morphogenesis, and pathogenesis. Previous crystallography studies of the coiled-coil domain (CCD) of NSP4 from two different rotavirus strains showed two distinct oligomeric states, a Ca(2+)-bound tetrameric state and a Ca(2+)-free pentameric state. Whether NSP4 CCD from the same strain can exist in different oligomeric states and what factors might regulate its oligomeric preferences are not known. This study used a combination of biochemical, biophysical, and crystallography techniques and found that the NSP4 CCD can undergo a reversible transition from a Ca(2+)-bound tetramer to a Ca(2+)-free pentamer in response to changes in pH. From these studies, we hypothesize that this remarkable structural adaptability of the CCD forms a basis for the pleiotropic functional properties of NSP4.

Mutagenesis of zinc ligand residue Cys221 reveals plasticity in the IMP-1 metallo-ß-lactamase active site.

Metallo-ß-lactamases catalyze the hydrolysis of a broad range of ß-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 ß-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of ß-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward ß-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 µM. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable.

2-Substituted 4,5-dihydrothiazole-4-carboxylic acids are novel inhibitors of metallo-ß-lactamases.

Bacterial resistance to ß-lactam antibiotics caused by class B metallo-ß-lactamases (MBL), especially for certain hospital-acquired, Gram-negative pathogens, poses a significant threat to public health. We report several 2-substituted 4,5-dihydrothiazole-4-carboxylic acids to be novel MBL inhibitors. Structure activity relationship (SAR) and molecular modeling studies were performed and implications for further inhibitor design are discussed.

Analysis of the functional contributions of Asn233 in metallo-ß-lactamase IMP-1.

Metallo-ß-lactamases, such as IMP-1, are a major global health threat, as they provide for bacterial resistance to a wide range of ß-lactam antibiotics, including carbapenems. Understanding the molecular details of the enzymatic process and the sequence requirements for function are essential aids in overcoming ß-lactamase-mediated resistance. An asparagine residue is conserved at position 233 in approximately 67% of all metallo-ß-lactamases. Despite its conservation, the molecular basis of Asn233 function is poorly understood and remains controversial. It has previously been shown that mutations at this site exhibit context-dependent sequence requirements in that the importance of a given amino acid depends on the antibiotic being tested. To provide a more thorough examination as to the function and sequence requirements at this position, a collection of IMP-1 mutants encoding each of the 19 possible amino acid substitutions was generated. The resistance levels toward four ß-lactam antibiotics were measured for Escherichia coli containing each of these mutants. The sequence requirements at position 233 for wild-type levels of resistance toward two cephalosporins were the most relaxed, while there were more stringent sequence requirements for resistance to ampicillin or imipenem. Enzyme kinetic analysis and determinations of steady-state protein levels indicated that the effects of the substitutions on resistance are due to changes in the kinetic parameters of the enzyme. Taken together, the results indicate that substitutions at position 233 significantly alter the kinetic parameters of the enzyme, but most substituted enzymes are able to provide for a high level of resistance to a broad range of ß-lactams.