Abstract The Gram-negative bacterium Burkholderia pseudomallei causes the severe disease melioidosis. β-Lactams, including carbapenems, are the primary treatment for B. pseudomallei infections, but are susceptible to chromosomal serine β-lactamases (SBLs), including the class D enzyme OXA-57. Here we show that recombinant OXA-57 is active toward penicillins and first-generation cephalosporins, slowly hydrolyzes carbapenems, including imipenem and meropenem, but is inactive toward oxyimino-cephalosporins (e.g., ceftazidime). Unlike many OXA enzymes, OXA-57 is sensitive to the mechanism-based SBL inhibitor clavulanic acid, but less so to the diazabicyclooctane avibactam and not to the cyclic boronate vaborbactam. Crystal structures of covalent OXA-57:avibactam and OXA-57:meropenem complexes reveal limited hydrogen-bonding interactions with bound ligands. In molecular dynamics simulations, the bound meropenem acyl-enzyme is mobile, with the 6α-hydroxyethyl group able to sample different orientations, while the water molecule necessary for deacylation is bound only transiently. These observations are consistent with the low level of meropenem turnover, supporting proposals that limited carbapenemase activity is a general property of OXA β-lactamases, and highlight the potential importance of OXA-57 in B. pseudomallei β-lactam resistance.
Abstract Fuzzy interactions enable specific biomolecular recognition while maintaining conformational freedom of the interacting partners. Here, we propose that fuzziness can be exploited for allosteric regulation to provide context-specific functional outputs. Using receptor signaling as an illustrative example, we demonstrate how heterogeneous binding modes with ligands can be fine-tuned through transient interactions. Fuzzy interactions interfere with alternative positions or modulate the mobility to mimic the landscapes of specific functional states that are linked with certain environmental conditions. Furthermore, using allosteric drugs targeting the ABL1 tyrosine kinase, KRAS oncogene, and IDH2 enzyme as examples, we demonstrate how fuzzy interaction mechanisms can affect allosteric couplings in a functionally meaningful manner through rewiring heterogeneous contact patterns. These notions could be helpful for drug development, targeting dynamic conformational ensembles and biasing them toward context-specific functional states.
Abstract It is of interest to develop new approaches to probing protein conformational changes and protein–protein interactions. In this study, we covalently labeled human neuroglobin (Ngb) with a 19F-probe containing a Ph-OCF3 group at Cys120 and by introducing cysteine residues at specific positions in the C120S Ngb mutant. 19F NMR provides valuable insights into its conformational changes upon cleavage of the intramolecular disulfide bond of Cys46-Cys55, as well as its interactions with protein partners such as cytochrome c (Cyt c) through the formation of the Ngb-Cyt c complex. This study presents a convenient approach to investigating the dynamic properties of heme globins regulated by disulfide bonds and protein–protein interactions.
Abstract Cre, a site-specific tyrosine DNA recombinase, enables targeted manipulation of genetic material without the production of cytotoxic double-strand DNA breaks. Though widely deployed in biotechnology, an incomplete understanding of how Cre recognizes its cognate loxP target limits its broader application in human health. Cre is highly specific for its cognate loxP sites, comprising an inverted pair of recombinase binding elements (RBEs) and an asymmetric spacer. Structural studies have shown that Cre protomers make few direct base-specific contacts to each RBE, suggesting that specificity arises through indirect readout, wherein noncognate sites are unable to adopt favorable binding-coupled conformations. We designed a set of loxP half-site variants predicted to alter local DNA shape characteristics, including minor-groove geometry and base-step parameters, and measured the thermodynamics of Cre binding by isothermal titration calorimetry. Variable-temperature thermograms and nuclear magnetic resonance spectra reveal that unfavorable mutations at noncontacted positions in the RBE reduce signatures of binding-coupled conformational changes. Moreover, mass photometry of Cre binding to oligonucleotides containing two properly spaced RBEs revealed that noncontacted positions in the intervening 8-base pair spacer influence cooperative dimerization of two Cre protomers at loxP sites and their synapsis to form catalytically active Cre4-loxP2 complexes. These results demonstrate that noncontacted positions contribute to specificity by encoding favorable DNA mechanics, offering new design principles for engineering Cre variants that target alternative DNA sequences.
Abstract The ability of migratory birds to orient using the Earth’s magnetic field has been proposed to rely on a radical-pair mechanism operating within Cryptochrome 4, a blue-light flavoprotein. In this perspective, we review how computational approaches have shaped our molecular understanding of cryptochrome-based magnetoreception. We organize the discussion around the three steps that connect light absorption to a cellular signal: the photochemical step, in which light absorption by FAD drives a hole-hopping electron-transfer cascade along a conserved tryptophan tetrad; the spin-dependent step, in which the resulting radical pair interconverts between singlet and triplet states under the influence of the magnetic field; and the structural step, in which this transient information is converted into a long-lived signaling state. The early electron-transfer events are now described with growing confidence and appear conserved across avian orthologs, although the relative stability of the final radical-pair states remains debated. The spin-dependent step remains more problematic: once the fluctuations of the hyperfine couplings and a realistic number of nuclear spins are included, the predicted magnetic sensitivity at geomagnetic field intensities becomes very small, and explaining the magnitude of the effect is still a central open problem. The postactivation conformational dynamics, involving intrinsically disordered regions, remains the principal challenge. We suggest that atomistic multiscale modeling, integrated with artificial intelligence-based approaches, has become indispensable for connecting the conserved photochemistry to this downstream process.
