The carbapenem-hydrolyzing class D β-lactamases OXA-23 and OXA-24/40 have emerged world-wide as causative agents for β-lactam antibiotic resistance in Acinetobacter species. Many variants of these enzymes have appeared clinically, including OXA-160 and OXA-225, which both contain a P→S substitution at homologous positions in the OXA-24/40 and OXA-23 backgrounds respectively. We purified OXA-160 and OXA-225 and used steady-state kinetic analysis to compare the substrate profiles of these variants to their parental enzymes, OXA-24/40 and OXA-23. OXA-160 and OXA-225 possess greatly enhanced hydrolytic activities against aztreonam, ceftazidime, cefotaxime and ceftriaxone when compared to OXA-24/40 and OXA-23. These enhanced activities are the result of much lower Km values, suggesting that the P→S substitution enhances the binding affinity of these drugs. We have determined the structures of the acylated forms of OXA-160 (with ceftazidime and aztreonam) and OXA-225 (ceftazidime). These structures show that the R1 oxyimino side-chain of these drugs occupies a space near the β5-β6 loop and the omega loop of the enzymes. The P→S substitution found in OXA-160 and OXA-225 results in a deviation of the β5-β6 loop, relieving the steric clash with the R1 side-chain carboxypropyl group of aztreonam and ceftazidime. These results reveal worrying trends in the enhancement of substrate spectrum of class D β-lactamases, but may also provide a map for β-lactam improvement.

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The dynamics characteristics of the currently available structure of Torpedo nicotinic acetylcholine receptor (nAChR), including the extracellular, transmembrane, and intracellular domains (ICDs), were analyzed using the Gaussian Network Model (GNM) and Anisotropic Network Model (ANM). We found that a symmetric quaternary twist motion, reported previously in the literature in a homopentameric receptor (Cheng et al. J Mol Biol 2006;355:310-324; Taly et al. Biophys J 2005;88:3954-3965), occurred also in the heteropentameric Torpedo nAChR. We believe, however, that the symmetric twist alone is not sufficient to explain a large body of experimental data indicating asymmetry and subunit nonequivalence during gating. Here we report our results supporting the hypothesis that a combination of symmetric and asymmetric motions opens the gate. We show that the asymmetric motion involves tilting of the TM2 helices. Furthermore, our study reveals three additional aspects of channel dynamics: (1) loop A serves as an allosteric mediator between the ligand binding loops and those at the domain interface, particularly the linker between TM2 and TM3; (2) the ICD can modulate the pore dynamics and thus should not be neglected in gating studies; and (3) the F loops, which are peculiarly longer and poorly-conserved in non-alpha-subunits, have important dynamical implications.

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The new crystal structures of the product-bound firefly luciferase combined with the previously determined substrate-free structures allow for a detailed analysis of the dynamics basis for the luciferase enzymatic activities. Using the Gaussian network model and the anisotropic network model, we show here that the superposition of the three slowest anisotropic network model modes, consisting of the bending, rotating, and rocking motions of the C-domain, accounts for large rearrangement of domains from the substrate-free (open) to product-bound (closed) conformation and thus constitutes a critical component of the enzyme's functions. The analysis also offers a unique platform to reexamine the molecular mechanism of the anesthetic inhibition of the firefly luciferase. Through perturbing the protein backbone network by introducing additional nodes to represent anesthetics, we found that the presence of two representative anesthetics, halothane and n-decanol, in different regions of luciferase had distinctively different effects on the protein's global motion. Only at the interface of the C- and N-domains did the anesthetics cause the most profound reduction in the overall flexibility of the C-domain and the concomitant increase in the flexibility of the loop, where the substitution of a conserved lysine residue was found experimentally to lead to >2-3 orders of magnitude reduction in activity. These anesthetic-induced dynamics changes can alter the normal function of the protein, appearing as an epiphenomenon of an "inhibition". The implication of the study is that a leading element for general anesthetic action on proteins is to disrupt the modes of motion essential to protein functions.