Active-Site Conformational Fluctuations Promote the Enzymatic Activity of NDM-1

Abstract

β-Lactam antibiotics are the mainstay for the treatment of bacterial infections. However, elevated resistance to these antibiotics mediated by metallo-β-lactamases (MBLs) has become a global concern. New Delhi metallo-β-lactamase-1 (NDM-1), a newly added member of the MBL family that can hydrolyze almost all β-lactam antibiotics, has rapidly spread all over the world and poses serious clinical threats. Broad-spectrum and mechanism-based inhibitors against all MBLs are highly desired, but the differential mechanisms of MBLs toward different antibiotics pose a great challenge. To facilitate the design of mechanism-based inhibitors, we investigated the active-site conformational changes of NDM-1 through the determination of a series of 15 high-resolution crystal structures in native form and in complex with products and by using biochemical and biophysical studies, site-directed mutagenesis, and molecular dynamics computation. The structural studies reveal the consistency of the active-site conformations in NDM-1/product complexes and the fluctuation in native NDM-1 structures. The enzymatic measurements indicate a correlation between enzymatic activity and the active-site fluctuation, with more fluctuation favoring higher activity. This correlation is further validated by structural and enzymatic studies of the Q123G mutant. Our combinational studies suggest that active-site conformational fluctuation promotes the enzymatic activity of NDM-1, which may guide further mechanism studies and inhibitor design.

Structural and biochemical analysis of the metallo‐β‐lactamase L1 from emerging pathogen Stenotrophomonas maltophilia revealed the subtle but distinct di‐metal scaffold for catalytic activity

“…One important note here is that in NDM-1, the distance between zinc ions depends on the pH of the crystallization conditions and varies between 3.5 Å at a high pH and 4.6 Å at a lower pH. 12,37 In all reported L1 structures, the crystallizations were done in narrow pH ranges of 6.5-8.0 and the resulting Zn-Zn distances fluctuate between 3.5 and 3.9 Å.…”

Metallo-β-Lactamase Inhibitors Inspired on Snapshots from the Catalytic Mechanism

“…This structure disclosed for the first time a direct interaction of Zn2 in a binuclear enzyme with the N atom and the C4 carboxylate of cephalosporins after bond cleavage, as well as the binding of the C8 carboxylate in the product with Zn1. Information about product binding on B1 enzymes dates back to 2011 with a series of structures of NDM-1 bound to different hydrolyzed substrates [ 69 , 101 , 135 , 139 , 216 , 222 , 223 ]. All these structures show a similar interaction of the β-lactam ring with the active site, but with the R 1 groups adopting different conformations.…”

X-ray Crystallography Deciphers the Activity of Broad-Spectrum Boronic Acid β-Lactamase Inhibitors

“…Two molecules per asymmetric units are present in our NDM-1 experimental models, as observed in other NDM-1 structures. 26,27 Indeed, the two protein chains are present as unique elements in the crystal a.s.u., and the main features of the active site as well as compound binding orientations are conserved along complexes, except for cpd 3. The residues defining the active site undergo minor conformational changes, involving the intrinsically flexible loops L3 and L10.…”