The widespread use of oxyimino-cephalosporin antibiotics drives the evolution of the CTX-M family of ␤-lactamases that hydrolyze these drugs and confer antibiotic resistance. Clinically isolated CTX-M enzymes carrying the P167S or D240G active siteassociated adaptive mutation have a broadened substrate profile that includes the oxyimino-cephalosporin antibiotic ceftazidime. The D240G substitution is known to reduce the stability of CTX-M-14 ␤-lactamase, and the P167S substitution is shown here to also destabilize the enzyme. Proteins are marginally stable entities, and second-site mutations that stabilize the enzyme can offset a loss in stability caused by mutations that enhance enzyme activity. Therefore, the evolution of antibiotic resistance enzymes can be dependent on the acquisition of stabilizing mutations. The A77V substitution is present in CTX-M extendedspectrum ␤-lactamases (ESBLs) from a number of clinical isolates, suggesting that it may be important in the evolution of antibiotic resistance in this family of ␤-lactamases. In this study, the effects of the A77V substitution in the CTX-M-14 model enzyme were characterized with regard to the kinetic parameters for antibiotic hydrolysis as well as enzyme expression levels in vivo and protein stability in vitro. The A77V substitution has little effect on the kinetics of oxyimino-cephalosporin hydrolysis, but it stabilizes the CTX-M enzyme and compensates for the loss of stability resulting from the P167S and D240G mutations. The acquisition of global stabilizing mutations, such as A77V, is an important feature in ␤-lactamase evolution and a common mechanism in protein evolution.T he introduction of new antibiotics into clinical use fuels the evolution of enzymes causing drug resistance. These enzymes gain the ability to inactivate antibiotics more efficiently by acquiring point mutations in and around their active sites that increase catalytic activity (1). However, the introduction of new gain-offunction mutations within highly organized active sites often comes at a stability cost to the enzymes (2-6). Therefore, enzymes can absorb only a limited number of destabilizing mutations before they unfold and lose function (7). This limitation in the evolution of catalytic efficiency is often overcome by the acquisition of global stabilizing mutations that offset the incremental loss in stability due to the primary mutation. This compensation mechanism allows the enzymes to continue a mutational trajectory, resulting in increased resistance.The role of global stabilizing mutations in enzyme evolution has been widely studied (4,8,9). In recent decades, the selection of mutant ␤-lactamase enzymes with expanded substrate specificity has occurred for the plasmid-mediated TEM-and SHV-type ␤-lactamases to give rise to extended-spectrum ␤-lactamases (ESBLs) (10, 11). These enzymes arise due to mutations resulting in substitutions near the active site that increase hydrolysis of oxyimino-cephalosporins such as cefotaxime and ceftazidime. The substitutions that increa...

OXA-163 and OXA-48 are closely related class D β-lactamases that exhibit different substrate profiles. OXA-163 hydrolyzes oxyimino-cephalosporins, particularly ceftazidime, while OXA-48 prefers carbapenem substrates. OXA-163 differs from OXA-48 by one substitution (S212D) in the active site β5-strand and a four-amino acid deletion (214-RIEP-217) in the loop connecting the β5 and β6 strands. Although the structure of OXA-48 has been determined, the structure of OXA-163 is unknown. To further understand the basis for their different substrate specificities we performed enzyme kinetic analysis, inhibition assays, x-ray crystallography, and molecular modeling. The results confirm the carbapenemase nature of OXA-48 and the ability of OXA-163 to hydrolyze the oxyimino-cephalosporin ceftazidime. The crystal structure of OXA-163 determined at 1.72-Å resolution reveals an expanded active site compared to OXA-48, which allows the bulky substrate ceftazidime to be accommodated. The structural differences with OXA-48, which cannot hydrolyze ceftazidime, provide a rationale for the change in substrate specificity between the enzymes. OXA-163 also crystallized in another condition that contained iodide. The crystal structure determined at 2.87-Å resolution revealed iodide in the active site accompanied by several significant conformational changes including a distortion of the β5-strand, decarboxylation of Lys73, and distortion of the substrate-binding site. Further studies showed that both OXA-163 and OXA-48 are inhibited in the presence of iodide. In addition, OXA-10, which is not a member of the OXA-48-like family, is also inhibited by iodide. These findings provide a molecular basis for the hydrolysis of ceftazidime by OXA-163 and, more broadly, show how minor sequence changes can profoundly alter the active site configuration and thereby affect the substrate profile of an enzyme.

A wide range of membrane receptors signal through conformational changes, and the resulting protein conformational flexibility often hinders their structural studies. Because the determinants of membrane receptor conformational stability are still poorly understood, identifying a minimal set of perturbations stabilizing a membrane protein in a given conformation remains a major challenge in membrane protein structure determination. We present a novel approach integrating bioinformatics, computational design and experimental techniques that identifies and stabilizes metastable receptor regions. When applied to the beta1-adrenergic receptor, the method generated 13 novel receptor variants stabilized in the intended inactive state among which two exhibit an apparent thermostability higher than WT and M23 (a receptor variant previously stabilized by extensive scanning mutagenesis) by more than 30°C and 11°C, respectively. Targeted regions involve nonconserved unsatisfied polar residues or exhibit significant packing defects, features found in all class A G protein-coupled receptor structures. These findings suggest that natural G protein-coupled receptor sequences have evolved to be conformationally metastable through the design of suboptimal polar and van der Waals tertiary interactions. Given sufficiently accurate structural models, our approach should prove useful for designing stabilized variants of many uncharacterized membrane receptors.computational protein design | protein conformational stability | membrane protein modeling | signal transduction | synthetic biology M embrane proteins represent around 30% of currently sequenced genomes and are critical in the regulation of cell signaling and cell-cell communication (1, 2). When dysfunctional, however, these proteins can be responsible for serious diseases and constitute more than 60% of current drug targets. Despite their abundance and functional importance, membrane proteins are largely underrepresented in the Protein Data Bank, mainly due to technical difficulties in studying these proteins. A large fraction of these proteins such as G protein-coupled receptors (GPCRs) transduces signals across membranes through conformational changes (3-6). The resulting protein conformational heterogeneity and flexibility is a major factor hindering crystallization. An additional obstacle to structural determination is the poor stability of membrane proteins in detergent solution required by traditional crystallization approaches (7,8).In recent years, several approaches to stabilize membrane proteins have been explored (9). These methods include development and optimization of solubilizing detergents (10), reconstitution of proteins into lipidic bicelles (11, 12), cleavage of flexible protein regions (13-18), co-crystallization with stabilizing ligands, antibodies, and fusion with soluble proteins (e.g., T4 lysozyme) (13-15, 17, 19-23). Several of these modifications, however, disrupt important functional regions and interactions with either natural ligands or downstre...

Norovirus (NoV) is the most common agent of nonbacterial epidemic gastroenteritis and is estimated to cause 21 million cases of the disease in the United States annually. The antigen enzyme-linked immunosorbent assays (ELISAs) currently available for NoV diagnosis detect only certain strains and are approved for use in the United States only in epidemics where NoV is suspected. There is a clear need for simpler, more rapid, and more reliable diagnostic tools for the detection of NoV. In this study, phage display technology was used to screen a library of phage displaying random 12-mer peptides for those that bind to Norwalk virus virus-like particles (NV VLPs). Three phage clones displaying unique peptides were identified, and both the peptidedisplaying phages and the peptides were confirmed to bind specifically to NV VLPs. The peptide displayed on phage clone NV-N-R5-1 was determined to bind to the protruding domain of the VP1 capsid protein. This phage also bound to NV VLPs seeded into NoV-negative stool with a limit of detection of 1.56 ng NV VLP. This value was comparable to monoclonal antibody (MAb) 3912, which is currently used in commercially available assays. Furthermore, the NV-N-R5-1 phage exhibited high specificity by detecting NV only in previously characterized NV-positive stool samples in contrast to no detection in NV-negative stool samples. These data demonstrate that the further development of NV-N-R5-1 phage as a diagnostic reagent is possible and might offer several distinct advantages over antibodies, such as decreases in the time and cost of production and ease of isolating phage against other epidemic strains currently circulating as well as those that are emerging.

The b-lactamase inhibitory proteins (BLIPs) are a model system for examining molecular recognition in protein-protein interactions. BLIP and BLIP-II are structurally unrelated proteins that bind and inhibit TEM-1 b-lactamase. Both BLIPs share a common binding interface on TEM-1 and make contacts with many of the same TEM-1 surface residues. BLIP-II, however, binds TEM-1 over 150-fold tighter than BLIP despite the fact that it has fewer contact residues and a smaller binding interface. The role of eleven TEM-1 amino acid residues that contact both BLIP and BLIP-II was examined by alanine mutagenesis and determination of the association (k on ) and dissociation (k off ) rate constants for binding each partner. The substitutions had little impact on association rates and resulted in a wide range of dissociation rates as previously observed for substitutions on the BLIP side of the interface. The substitutions also had less effect on binding affinity for BLIP than BLIP-II. This is consistent with the high affinity and small binding interface of the TEM-1-BLIP-II complex, which predicts per residue contributions should be higher for TEM-1 binding to BLIP-II versus BLIP. Two TEM-1 residues (E104 and M129) were found to be hotspots for binding BLIP while five (L102, Y105, P107, K111, and M129) are hotspots for binding BLIP-II with only M129 as a common hotspot for both. Thus, although the same TEM-1 surface binds to both BLIP and BLIP-II, the distribution of binding energy on the surface is different for the two target proteins, that is, different binding strategies are employed.