Structure of apo- and monometalated forms of NDM-1--a highly potent carbapenem-hydrolyzing metallo-β-lactamase

Abstract

The New Delhi Metallo-β-lactamase (NDM-1) gene makes multiple pathogenic microorganisms resistant to all known β-lactam antibiotics. The rapid emergence of NDM-1 has been linked to mobile plasmids that move between different strains resulting in world-wide dissemination. Biochemical studies revealed that NDM-1 is capable of efficiently hydrolyzing a wide range of β-lactams, including many carbapenems considered as "last resort" antibiotics. The crystal structures of metal-free apo- and monozinc forms of NDM-1 presented here revealed an enlarged and flexible active site of class B1 metallo-β-lactamase. This site is capable of accommodating many β-lactam substrates by having many of the catalytic residues on flexible loops, which explains the observed extended spectrum activity of this zinc dependent β-lactamase. Indeed, five loops contribute "keg" residues in the active site including side chains involved in metal binding. Loop 1 in particular, shows conformational flexibility, apparently related to the acceptance and positioning of substrates for cleavage by a zinc-activated water molecule.

Figure 1. NDM-1 structure and comparisons with selected carbapenemases.

A. Aerial view of active site (represented by two phosphate ions), the protein is made up of four layers, α/β/β/α, and forms a sandwich. Secondary structure elements and N- and C-termini are labeled. B. Comparison of K. pneumoniae NDM-1 (PDB id 3RKJ – magenta, N and C-termini are labeled) with three MBLs with carbapenemase activity P. aeruginosa VIM-2 (PDB id 2YZ3 - wheat), P. aeruginosa IMP-1 (PDB id 1DD6 - light blue) and P. aeruginosa VIM-4 (PDB id 2WHG - pale green). Loops contributing to the active site are labeled ASL1–5. Zn1 and Zn2 (blue spheres) are from the structure of VIM-2.

Figure 2. Active site comparisons of K. pneumoniae apoNDM-1 (PDB id 3RKJ) (aqua) vs monozinc NDM-1 (PDB id 3SFP) (violet) vs di-zinc NDM-1 PDB id 3Q6X (wheat).

Zn1′ and Zn2′ (wheat) are from the structure of di-zinc NDM-1 and Zn1″ is from the structure of monozinc NDM-1. Conformations of residues coordinating Zn1 (His120, His122 and His189) and Zn2 (Asp124, Cys208 and His250) are shown for all three structures.

Figure 3. Flexibility of the active site loops.

A. Structures of six NDM-1 molecules (identified using PDB ids) were aligned (PDB id 3RKJ (this work) molecule A - blue, PDB id 3RKJ (this work) molecule B - light blue, PDB id 3SBL (this work) molecule A - green, PDB id 3RKK (this work) molecule B – red, PDB id 3SFP (this work) molecule B – violet and PDB id 3Q6X molecule B – wheat), hAmp is a hydrolyzed ampicillin and Zn1′ and Zn2′ are zinc atoms from the structure of NDM-1 (PDB id 3Q6X molecule B) and Zn1″ is from the structure of monozinc NDM-1. B. The structural variability at each residue position in the ASL1 loop is evaluated across six structurally unique loops shown in A. After a structural alignment of the entire molecules, the Euclidean distance between the residues' center-of-mass was measured between all pair combinations at each position. The results are summarized as a boxplot showing the median, quartiles, maximum and minimum distances for each residue. The aligned loops are shown in cartoon putty representation, with the loop radius proportional to residue B-factors (view facing active site, left; top-down view, right).

Figure 4. Active site expansion of NDM-1.

The active site cavity comparisons of K. pneumoniae NDM-1 (gray) and three MBLs: IMP-1 (light brown), VIM-2 (orange) and VIM-4 (magenta). Superposition of the IMP-1, VIM-2 and VIM-4 molecules with NDM-1 reveal highly conserved structural arrangements. The most prominent variations occur between the catalytic sites (shown as surface representations), in which the side chains of IMP-1, VIM-2 and VIM-4 restrict the access to the active site by decreasing the volume of the cavity. The top row depicts NDM-1 and its active site surface (green) in secondary structure cartoon (left) and surface representation (right). The subsequent rows show NDM-1 structural alignments with IMP-1, VIM-2 and VIM-4. Each row highlights the IMP-1, VIM-2 and VIM-4 molecules' respective active sites, shown as colored surfaces. To highlight the greater accessibility to the NDM-1 active site, the aligned NDM-1 molecule is shown in surface representation (right). The obstructing interactions between ASLs restricting access to active site in IMP-1, VIM-2 and VIM-4 are observed as colored protrusions through the NDM-1 molecule and are highlighted for IMP-1, VIM-2 and VIM-4 with black circles.