Structural insight into the subclass B1 metallo-ß-lactamase AFM-1.

The emergence of drug-resistant bacteria, facilitated by metallo-beta-lactamases (MBLs), presents a significant obstacle to the effective use of antibiotics in the management of clinical drug-resistant bacterial infections. AFM-1 is a MBL derived from Alcaligenes faecalis and shares 86% homology with the NDM-1 family. Both AFM-1 and NDM-1 demonstrate the ability to hydrolyze ampicillin and other ß-lactam antibiotics, however, their substrate affinities vary, and the specific reason for this variation remains unknown. We present the high-resolution structure of AFM-1. The active center of AFM-1 binds two zinc ions, and the conformation of the key amino acid residues in the active center is in accordance with that of NDM-1. However, the substrate-binding pocket of AFM-1 is considerably smaller than that of NDM-1. Additionally, the mutation of amino acid residues in the Loop3 region, as compared to NDM-1, results in the formation of a dense hydrophobic patch comprised of hydrophobic amino acid residues in this area, which facilitates substrate binding. Our findings lay the foundation for understanding the molecular mechanism of AFM-1 with a high affinity for substrates and provide a novel theoretical foundation for addressing the issue of drug resistance caused by B1 MBLs.

Structural basis for GTP-induced dimerization and antiviral function of guanylate-binding proteins.

Guanylate-binding proteins (GBPs) form a family of dynamin-related large GTPases which mediate important innate immune functions. They were proposed to form oligomers upon GTP binding/hydrolysis, but the molecular mechanisms remain elusive. Here, we present crystal structures of C-terminally truncated human GBP5 (hGBP51-486), comprising the large GTPase (LG) and middle (MD) domains, in both its nucleotide-free monomeric and nucleotide-bound dimeric states, together with nucleotide-free full-length human GBP2. Upon GTP-loading, hGBP51-486 forms a closed face-to-face dimer. The MD of hGBP5 undergoes a drastic movement relative to its LG domain and forms extensive interactions with the LG domain and MD of the pairing molecule. Disrupting the MD interface (for hGBP5) or mutating the hinge region (for hGBP2/5) impairs their ability to inhibit HIV-1. Our results point to a GTP-induced dimerization mode that is likely conserved among all GBP members and provide insights into the molecular determinants of their antiviral function.