Structure and Dynamics of the G121V Dihydrofolate Reductase Mutant: Lessons from a Transition-State Inhibitor Complex

It is well known that enzyme flexibility is critical for function. This is due to the observation that the rates of intramolecular enzyme motions are often matched to the rates of intermolecular events such as substrate binding and product release. Beyond this role in progression through the reaction cycle, it has been suggested that enzyme dynamics may also promote the chemical step itself. Dihydrofolate reductase (DHFR) is a model enzyme for which dynamics have been proposed to aid in both substrate flux and catalysis. The G121V mutant of DHFR is a well studied form that exhibits a severe reduction in the rate of hydride transfer yet there remains dispute as to whether this defect is caused by altered structure, dynamics, or both. Here we address this by presenting an NMR study of the G121V mutant bound to reduced cofactor and the transition state inhibitor, methotrexate. NMR chemical shift markers demonstrate that this form predominantly adopts the closed conformation thereby allowing us to provide the first glimpse into the dynamics of a catalytically relevant complex. Based on 15N and 2H NMR spin relaxation, we find that the mutant complex has modest changes in ps-ns flexibility with most affected residues residing in the distal adenosine binding domain rather than the active site. Thus, aberrant ps-ns dynamics are likely not the main contributor to the decreased catalytic rate. The most dramatic effect of the mutation involves changes in µs-ms dynamics of the F-G and Met20 loops. Whereas loop motion is quenched in the wild type transition state inhibitor complex, the F-G and Met20 loops undergo excursions from the closed conformation in the mutant complex. These excursions serve to decrease the population of conformers having the correct active site configuration, thus providing an explanation for the G121V catalytic defect

Factor market rivalry and interindustry competitive dynamics

We study competitive dynamics by using 10 years of data from the biotech industry on patent infringement lawsuits because they capture offensive actions and defensive responses. Results suggest that larger patent portfolios, long-term partnerships, and short-term agreements lead to factor market rivalry. Specifically, long-term research partnerships are associated with lower proclivity and vulnerability to attack, while short-term licensing and marketing agreements with increased proclivity and vulnerability to attack. Lastly, although nonbiotech companies attack biotech firms, the proclivity of the latter to launch cross-industry attacks is not significant. We discuss how the results contribute to competitive dynamics research and theory

Glutathione-S-transferase FosA6 of klebsiella pneumoniae origin conferring fosfomycin resistance in ESBL-producing Escherichia coli

Objectives: The objectives of this study were to elucidate the genetic context of a novel plasmid-mediated fosA variant, fosA6, conferring fosfomycin resistance and to characterize the kinetic properties of FosA6.Methods: The genome of fosfomycin-resistant Escherichia coli strain YD786 was sequenced. Homologues of FosA6 were identified through BLAST searches. FosA6 and FosAST258 were purified and characterized using a steady-state kinetic approach. Inhibition of FosA activity was examined with sodium phosphonoformate.Results: Plasmid-encoded glutathione-S-transferase (GST) FosA6 conferring high-level fosfomycin resistance was identified in a CTX-M-2-producing E. coli clinical strain at a US hospital. fosA6 was carried on a self-conjugative, 69 kb IncFII plasmid. The ΔlysR-fosA6-ΔyjiR_1 fragment, located between IS10R and ΔIS26, was nearly identical to those on the chromosomes of some Klebsiella pneumoniae strains (MGH78578, PMK1 and KPPR1). FosA6 shared >99% identity with chromosomally encoded FosAPMK1 in K. pneumoniae of various STs and 98% identity with FosAST258, which is commonly found in K. pneumoniae clonal complex (CC) 258 including ST258. FosA6 and FosAST258 demonstrated robust GST activities that were comparable to each other. Sodium phosphonoformate, a GST inhibitor, reduced the fosfomycin MICs by 6- to 24-fold for K. pneumoniae and E. coli strains carrying fosA genes on the chromosomes and plasmids, respectively.Conclusions: FosA6, probably captured from the chromosome of K. pneumoniae, conferred high-level fosfomycin resistance in E. coli. FosA6 functioned as a GST and inactivated fosfomycin efficiently. K. pneumoniae may serve as a reservoir of fosfomycin resistance for E. coli

Atomic structures of peptide self-assembly mimics

Although the β-rich self-assemblies are a major structural class for polypeptides and the focus of intense research, little is known about their atomic structures and dynamics due to their insoluble and noncrystalline nature. We developed a protein engineering strategy that captures a self-assembly segment in a water-soluble molecule. A predefined number of self-assembling peptide units are linked, and the β-sheet ends are capped to prevent aggregation, which yields a mono-dispersed soluble protein. We tested this strategy by using Borrelia outer surface protein (OspA) whose single-layer β-sheet located between two globular domains consists of two β-hairpin units and thus can be considered as a prototype of self-assembly. We constructed self-assembly mimics of different sizes and determined their atomic structures using x-ray crystallography and NMR spectroscopy. Highly regular β-sheet geometries were maintained in these structures, and peptide units had a nearly identical conformation, supporting the concept that a peptide in the regular β-geometry is primed for self-assembly. However, we found small but significant differences in the relative orientation between adjacent peptide units in terms of β-sheet twist and bend, suggesting their inherent flexibility. Modeling shows how this conformational diversity, when propagated over a large number of peptide units, can lead to a substantial degree of nanoscale polymorphism of self-assemblies