RESUMEN
Protein stability is an essential property for biological function. In contrast to the vast knowledge on protein stability in vitro, little is known about the factors governing in-cell stability. Here we show that the metallo-ß-lactamase (MBL) New Delhi MBL-1 (NDM-1) is a kinetically unstable protein on metal restriction that has evolved by acquiring different biochemical traits that optimize its in-cell stability. The nonmetalated (apo) NDM-1 is degraded by the periplasmic protease Prc that recognizes its partially unstructured C-terminal domain. Zn(II) binding renders the protein refractory to degradation by quenching the flexibility of this region. Membrane anchoring makes apo-NDM-1 less accessible to Prc and protects it from DegP, a cellular protease degrading misfolded, nonmetalated NDM-1 precursors. NDM variants accumulate substitutions at the C terminus that quench its flexibility, enhancing their kinetic stability and bypassing proteolysis. These observations link MBL-mediated resistance with the essential periplasmic metabolism, highlighting the importance of the cellular protein homeostasis.
Asunto(s)
Péptido Hidrolasas , beta-Lactamasas , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Estabilidad Proteica , Proteolisis , Péptido Hidrolasas/metabolismo , Antibacterianos , Pruebas de Sensibilidad MicrobianaRESUMEN
Carbapenem-resistant Enterobacteriaceae threaten human health, since carbapenems are last resort drugs for infections by such organisms. Metallo-ß-lactamases (MßLs) are the main mechanism of resistance against carbapenems. Clinically approved inhibitors of MBLs are currently unavailable as design has been limited by the incomplete knowledge of their mechanism. Here, we report a biochemical and biophysical study of carbapenem hydrolysis by the B1 enzymes NDM-1 and BcII in the bi-Zn(II) form, the mono-Zn(II) B2 Sfh-I and the mono-Zn(II) B3 GOB-18. These MßLs hydrolyse carbapenems via a similar mechanism, with accumulation of the same anionic intermediates. We characterize the Michaelis complex formed by mono-Zn(II) enzymes, and we identify all intermediate species, enabling us to propose a chemical mechanism for mono and binuclear MßLs. This common mechanism open avenues for rationally designed inhibitors of all MßLs, notwithstanding the profound differences between these enzymes' active site structure, ß-lactam specificity and metal content.Carbapenem-resistant bacteria pose a major health threat by expressing metallo-ß-lactamases (MßLs), enzymes able to hydrolyse these life-saving drugs. Here the authors use biophysical and computational methods and show that different MßLs share the same reaction mechanism, suggesting new strategies for drug design.
Asunto(s)
Carbapenémicos/metabolismo , Zinc/metabolismo , beta-Lactamasas/química , beta-Lactamasas/metabolismo , Carbapenémicos/química , Hidrólisis , Imipenem/química , Imipenem/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Simulación de Dinámica Molecular , Espectroscopía de Absorción de Rayos XRESUMEN
The synthesis of new oxazolidinylthiazolidines bicycles, oxygen analogues of bisthiazolidines, also known as metallo-ß-lactamase inhibitors is described. The reaction of ß-aminoalcohols and 2,5-dihydroxy-1,4-dithiane led to oxazolidinylthiazolidines and/or dithia-azabicycles as the main products. The distribution pattern depends mainly on the aminoalcohol substituents. In a one-pot reaction, four new bonds are formed in good yields and with high atom efficiency. When the oxazolidinylthiazolidines are formed, two stereogenic centres are generated with high enantiospecificity. The reaction mechanism is discussed based on crystallographic data and interconversion studies. Two oxazolidinylthiazolidines were evaluated as inhibitors of the potent lactamase NDM-1 and compound 4f displayed competitive inhibition with Ki = 1.6 ± 0.6 µM.
RESUMEN
Metallo-ß-lactamases (MBLs) hydrolyze almost all ß-lactam antibiotics and are unaffected by clinically available ß-lactamase inhibitors (ßLIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of ßLIs effective against all enzymes. Bisthiazolidines (BTZs) are carboxylate-containing, bicyclic compounds, considered as penicillin analogs with an additional free thiol. Here, we show both l- and d-BTZ enantiomers are micromolar competitive ßLIs of all MBL classes in vitro, with Kis of 6-15 µM or 36-84 µM for subclass B1 MBLs (IMP-1 and BcII, respectively), and 10-12 µM for the B3 enzyme L1. Against the B2 MBL Sfh-I, the l-BTZ enantiomers exhibit 100-fold lower Kis (0.26-0.36 µM) than d-BTZs (26-29 µM). Importantly, cell-based time-kill assays show BTZs restore ß-lactam susceptibility of Escherichia coli-producing MBLs (IMP-1, Sfh-1, BcII, and GOB-18) and, significantly, an extensively drug-resistant Stenotrophomonas maltophilia clinical isolate expressing L1. BTZs therefore inhibit the full range of MBLs and potentiate ß-lactam activity against producer pathogens. X-ray crystal structures reveal insights into diverse BTZ binding modes, varying with orientation of the carboxylate and thiol moieties. BTZs bind the di-zinc centers of B1 (IMP-1; BcII) and B3 (L1) MBLs via the free thiol, but orient differently depending upon stereochemistry. In contrast, the l-BTZ carboxylate dominates interactions with the monozinc B2 MBL Sfh-I, with the thiol uninvolved. d-BTZ complexes most closely resemble ß-lactam binding to B1 MBLs, but feature an unprecedented disruption of the D120-zinc interaction. Cross-class MBL inhibition therefore arises from the unexpected versatility of BTZ binding.
Asunto(s)
Antibacterianos/química , Proteínas Bacterianas/química , Tiazolidinas/química , Inhibidores de beta-Lactamasas/química , beta-Lactamasas/química , Dominio Catalítico , Diseño de Fármacos , Hidrólisis , Cinética , Modelos MolecularesRESUMEN
The understanding of protein evolution depends on the ability to relate the impact of mutations on molecular traits to organismal fitness. Biological activity and robustness have been regarded as important features in shaping protein evolutionary landscapes. Conformational dynamics, which is essential for protein function, has received little attention in the context of evolutionary analyses. Here we employ NMR spectroscopy, the chief experimental tool to describe protein dynamics at atomic level in solution at room temperature, to study the intrinsic dynamic features of a metallo- Β: -lactamase enzyme and three variants identified during a directed evolution experiment that led to an expanded substrate profile. We show that conformational dynamics in the catalytically relevant microsecond to millisecond timescale is optimized along the favored evolutionary trajectory. In addition, we observe that the effects of mutations on dynamics are epistatic. Mutation Gly262Ser introduces slow dynamics on several residues that surround the active site when introduced in the wild-type enzyme. Mutation Asn70Ser removes the slow dynamics observed for few residues of the wild-type enzyme, but increases the number of residues that undergo slow dynamics when introduced in the Gly262Ser mutant. These effects on dynamics correlate with the epistatic interaction between these two mutations on the bacterial phenotype. These findings indicate that conformational dynamics is an evolvable trait, and that proteins endowed with more dynamic active sites also display a larger potential for promoting evolution.
Asunto(s)
beta-Lactamasas/química , beta-Lactamasas/genética , Proteínas Portadoras , Dominio Catalítico , Evolución Molecular Dirigida/métodos , Epistasis Genética , Evolución Molecular , Genotipo , Mutación , Resonancia Magnética Nuclear Biomolecular/métodos , Fenotipo , Conformación Proteica , Pliegue de Proteína , Relación Estructura-Actividad , beta-Lactamasas/metabolismoRESUMEN
Pathogenic Gram-negative bacteria resistant to almost all ß-lactam antibiotics are a major public health threat. Zn(II)-dependent or metallo-ß-lactamases (MBLs) produced by these bacteria inactivate most ß-lactam antibiotics, including the carbapenems, which are "last line therapies" for life-threatening Gram-negative infections. NDM-1 is a carbapenemase belonging to the MBL family that is rapidly spreading worldwide. Regrettably, inhibitors of MBLs are not yet developed. Here we present the bisthiazolidine (BTZ) scaffold as a structure with some features of ß-lactam substrates, which can be modified with metal-binding groups to target the MBL active site. Inspired by known interactions of MBLs with ß-lactams, we designed four BTZs that behave as in vitro NDM-1 inhibitors with Ki values in the low micromolar range (from 7 ± 1 to 19 ± 3 µM). NMR spectroscopy demonstrated that they inhibit hydrolysis of imipenem in NDM-1-producing Escherichia coli. In vitro time kill cell-based assays against a variety of bacterial strains harboring blaNDM-1 including Acinetobacter baumannii show that the compounds restore the antibacterial activity of imipenem. A crystal structure of the most potent heterocycle (L-CS319) in complex with NDM-1 at 1.9 Å resolution identified both structural determinants for inhibitor binding and opportunities for further improvements in potency.