Crystal Structure of DIM-1, an Acquired Subclass B1 Metallo-β-Lactamase from Pseudomonas stutzeri

Metallo-β-lactamases (MBLs) hydrolyze almost all classes of β-lactam antibiotic, including carbapenems—currently first choice drugs for opportunistic infections by Gram-negative bacterial pathogens. MBL inhibitor development is complicated by the diversity within this group of enzymes, and by the appearance of new enzymes that continue to be identified both as chromosomal genes and on mobile genetic elements. One such newly discovered MBL is DIM-1, a mobile enzyme originally discovered in the opportunist pathogen Pseudomonas stutzeri but subsequently identified in other species and locations. DIM-1 is a subclass B1 MBL more closely related to the TMB-1, GIM-1 and IMP enzymes than to other clinically encountered MBLs such as VIM and NDM; and possesses Arg, rather than the more usual Lys, at position 224 in the putative substrate binding site. Here we report the crystallization and structure determination of DIM-1. DIM-1 possesses a binuclear metal center with a 5 (rather than the more usual 4) co-ordinate tri-histidine (Zn1) site and both 4- and 5-co-ordinate Cys-His-Asp- (Zn2) sites observed in the two molecules of the crystallographic asymmetric unit. These data indicate a degree of variability in metal co-ordination geometry in the DIM-1 active site, as well as facilitating inclusion of DIM-1 in structure-based MBL inhibitor discovery programmes

Cross-class metallo-β-lactamase inhibition by bisthiazolidines reveals multiple binding modes

Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics and are unaffected by clinically available β-lactamase inhibitors (βLIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of βLIs 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 βLIs of all MBL classes in vitro, with Ki sof6-15 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 Ki s (0.26-0.36 μM) than D-BTZs (26-29 μM). Importantly, cell-based time-kill assays show BTZs restore β-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 β-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 β-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.Fil: Hinchliffe, Philip. University of Bristol; Reino UnidoFil: Gonzalez, Javier Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mojica, María. Louis Stokes Cleveland Department of Veterans Affairs Medical Center; Estados Unidos. Case Western Reserve University; Estados UnidosFil: Gonzalez, Javier Marcelo. Universidad Nacional de Santiago del Estero. Instituto de Bionanotecnología del Noa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Bionanotecnología del Noa; ArgentinaFil: Castillo, Valerie. Universidad de la República; UruguayFil: Saiz Garcia, Cecilia. Universidad de la República; UruguayFil: Kosmopoulou, Magda. University of Bristol; Reino UnidoFil: Tooke, Catherine. University of Bristol; Reino UnidoFil: Llarrull, Leticia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mahler, Graciela. Universidad de la República; UruguayFil: Bonomo, Robert. Louis Stokes Cleveland Department of Veterans Affairs Medical Center; Estados Unidos. Case Western Reserve University; Estados UnidosFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Spencer, James. University of Bristol; Reino Unid

Crystal structures of VIM-1 complexes explain active site heterogeneity in VIM-class metallo-β-lactamases

Metallo‐β‐Lactamases (MBLs) protect bacteria from almost all β‐lactam antibiotics. Verona integron‐encoded MBL (VIM) enzymes are among the most clinically important MBLs, with VIM‐1 increasing in carbapenem‐resistant Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae) that are among the hardest bacterial pathogens to treat. VIM enzymes display sequence variation at residues (224 and 228) that in related MBLs are conserved and participate in substrate binding. How they accommodate this variability, while retaining catalytic efficiency against a broad substrate range, has remained unclear. Here, we present crystal structures of VIM‐1 and its complexes with a substrate‐mimicking thioenolate inhibitor, ML302F, that restores meropenem activity against a range of VIM‐1 producing clinical strains, and the hydrolysed product of the carbapenem meropenem. Comparison of these two structures identifies a water‐mediated hydrogen bond, between the carboxylate group of substrate/inhibitor and the backbone carbonyl of the active site zinc ligand Cys221, that is common to both complexes. Structural comparisons show that the responsible Cys221‐bound water is observed in all known VIM structures, participates in carboxylate binding with other inhibitor classes, and thus effectively replicates the role of the conserved Lys224 in analogous complexes with other MBLs. These results provide a mechanism for substrate binding that permits the variation at positions 224 and 228 that is a hallmark of VIM MBLs

Metal:ligand distances (Å).

<p>^aChain A.</p><p>^bFigures in parentheses are for chain B.</p><p>^cRefined as a chloride ion</p><p>Metal:ligand distances (Å).</p

Data Collection and Refinement Statistics.

<p>^a Data for the highest resolution shell are in parentheses.</p><p>^b R_free was calculated with 5% of the reflections omitted.</p><p>^cFigures in parentheses are for chain B.</p><p>Data Collection and Refinement Statistics.</p

Overlay of B1 Metallo-β-Lactamase Active Sites Showing Hydrolyzed β-Lactam Binding.

<p>Figure shows DIM-1, IMP-1 (1DDK [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref023" target="_blank">23</a>], carbon, zinc and waters teal); VIM-2 (1KO3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref044" target="_blank">44</a>], carbon, zinc and waters magenta) and NDM-1:ampicillin complex (3Q6X [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref041" target="_blank">41</a>], carbon, zinc and waters cyan). Interactions of hydrolyzed ampicillin with NDM-1 are shown as dashed lines. Note differing positions of Arg-161 (224) (DIM-1), Lys-224 (IMP-1, NDM-1) and Arg-228 (VIM-2). This Figure was generated using Pymol.</p

Comparison of DIM-1 Active Site with L-Captopril-bound Complexes of Subclass B1 Metallo-β-Lactamases.

<p>A. DIM-1 (green); B. IMP-1 (4C1F, teal); C. VIM-2 (4C1D, magenta); D. NDM-1 (4EXS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref017" target="_blank">17</a>], cyan). This Figure was generated using Pymol.</p

Crystal Structure of DIM-1.

<p>A. Overall fold of the enzyme. Protein backbone is color-ramped from blue (N-) to red (C-terminus). Active site residues are rendered as sticks (carbon atoms in green, other atom colors as standard). Zinc ions (gray) and water molecules (red) are shown as spheres. B. Superposition of B1 MBL structures: DIM-1 (green); BcII (pdb accession 1BC2 ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref043" target="_blank">43</a>], blue); IMP-1 (1DDK [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref023" target="_blank">23</a>], teal); VIM-2 (1KO3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref044" target="_blank">44</a>], magenta); BlaB (1M2X [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref045" target="_blank">45</a>], dark green); CcrA (1ZNB [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref046" target="_blank">46</a>], yellow); NDM-1 (3Q6X [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref041" target="_blank">41</a>], cyan); IND-7 (3L6N [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref047" target="_blank">47</a>], red) and GIM-1 (2YNT [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref048" target="_blank">48</a>], orange). This Figure was generated using Pymol (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>).</p

Active Site Grooves of B1 Metallo-β-Lactamases.

<p>Figure shows surfaces of A. DIM-1; B. GIM-1 (pdb accession 2YNT [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref048" target="_blank">48</a>]) C. IMP-1 (1DDK [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref023" target="_blank">23</a>]) and D. NDM-1 (3Q6X [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref041" target="_blank">41</a>]) colored according to electrostatic potential from red (- 0.5 V) to blue (+ 0.5 V). Hydrolyzed ampicillin bound to NDM-1 is shown as sticks (carbon atoms green, other atom colors as standard). This Figure was generated using CCP4MG [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140059#pone.0140059.ref050" target="_blank">50</a>].</p