Crystal structure of New Delhi metallo-β-lactamase reveals molecular basis for antibiotic resistance

Abstract

β-Lactams are the most commonly prescribed class of antibiotics and have had an enormous impact on human health. Thus, it is disquieting that an enzyme called New Delhi metallo-β-lactamase-1 (NDM-1) can confer Enterobacteriaceae with nearly complete resistance to all β-lactam antibiotics including the carbapenams. We have determined the crystal structure of Klebsiella pneumoniae apo-NDM-1 to 2.1-Å resolution. From the structure, we see that NDM-1 has an expansive active site with a unique electrostatic profile, which we propose leads to a broader substrate specificity. In addition, NDM-1 undergoes important conformational changes upon substrate binding. These changes have not been previously observed in metallo-β-lactamase enzymes and may have a direct influence on substrate recognition and catalysis.

Figure 1

Overall structure of apo-NDM-1, NDM-1 localization, and crystallographic dimerization. A: Cartoon of apo-NDM-1 structure (green), active-site zinc ions (gray spheres), zinc ligands (stick, CPK coloring), and active-site loops L3 and L10 (red). Secondary structure designations are labeled. B: Superposition of NDM-1 on apo-VIM-2 (PDB ID 1K03) (magenta). Apo-NDM-1 zinc ligands are colored by atom, and the NDM-1 loops L3, L8, and L10 are highlighted with a red arrow. C: Western blot of a step sucrose gradient of membrane-associated 8xHIS FL-NDM-1. Negative control is E. coli BL21 membranes only. Positive control is outer membrane protein ZirT. D: NDM-1 crystallographic dimer interface for both apo (green) and ampicillinic acid-bound (gray, PDB ID: 3Q6X) forms. The L8, L10, and α3 helix constitute the dimer interface and are labeled on the apo structure (red).

Figure 2

Apo-NDM-1/hydrolyzed ampicillin (hAMP)-bound NDM-1 and apo-NDM-1/apo-VIM-2 active-site comparisons. A: Apo-NDM-1/hAMP NDM-1 active-site overlay: apo-NDM-1 backbone (green), hydrolyzed ampicillin (hAMP) product complex (gray, PDB ID: 3Q6X) with selected active-site residues (sticks) labeled. B: Apo-NDM-1/apo-VIM-2 active-site overlay: apo-NDM-1 backbone (green), selected NDM-1 and VIM-2 active-site residues are shown as green and magenta sticks with cpk atom coloring, respectively. The 2F_o − F_c map at 2σ is shown for zinc ions and coordinating ligands. C: Electrostatic surface representation of apo-NDM-1 and apo-VIM-2 active-site clefts, calculated using APBS software, with a PARSE force field contoured at −3 and +3. D: Zinc coordination: zinc ions and ligands are in green and gray for apo-NDM-1 and hAMP-bound NDM-1. Coordination interactions are blue dashes, and hAMP is in cpk colored stick representation (carbons are magenta).