Computational analysis of pathogen-borne metallo β-lactamases reveals discriminating structural features between B1 types

Abstract

Background: Genes conferring antibiotic resistance to groups of bacterial pathogens are cause for considerable concern, as many once-reliable antibiotics continue to see a reduction in efficacy. The recent discovery of the metallo β-lactamase blaNDM-1 gene, which appears to grant antibiotic resistance to a variety of Enterobacteriaceae via a mobile plasmid, is one example of this distressing trend. The following work describes a computational analysis of pathogen-borne MBLs that focuses on the structural aspects of characterized proteins.

Results: Using both sequence and structural analyses, we examine residues and structural features specific to various pathogen-borne MBL types. This analysis identifies a linker region within MBL-like folds that may act as a discriminating structural feature between these proteins, and specifically resistance-associated acquirable MBLs. Recently released crystal structures of the newly emerged NDM-1 protein were aligned against related MBL structures using a variety of global and local structural alignment methods, and the overall fold conformation is examined for structural conservation. Conservation appears to be present in most areas of the protein, yet is strikingly absent within a linker region, making NDM-1 unique with respect to a linker-based classification scheme. Variability analysis of the NDM-1 crystal structure highlights unique residues in key regions as well as identifying several characteristics shared with other transferable MBLs.

Conclusions: A discriminating linker region identified in MBL proteins is highlighted and examined in the context of NDM-1 and primarily three other MBL types: IMP-1, VIM-2 and ccrA. The presence of an unusual linker region variant and uncommon amino acid composition at specific structurally important sites may help to explain the unusually broad kinetic profile of NDM-1 and may aid in directing research attention to areas of this protein, and possibly other MBLs, that may be targeted for inactivation or attenuation of enzymatic activity.

Figure 1

Structure-based sequence alignment of NDM-1, IMP-1, VIM-2 and ccrA. Alignments were generated using LGA with NDM-1 (3q6x_A) as the reference. Capitalized amino acids indicate structural residue-residue correspondence, whereas lowercase amino acids indicate regions in MBLs where no structural alignment with NDM-1 can be identified. Regions of interested are denoted by boxes and labeled as the mobile flap (1, blue) (residues 65-73 in NDM-1; 23-31 in IMP-1; 59-67 in VIM-2; and 44-52 in ccrA), the linker region including anchoring regions (2, blue) (residues 141-193 in NDM-1; 98-143 in IMP-1; 138-200 in VIM-2; and 120-166 in ccrA), the non-aligning linker region core (3, red) (residues 163-179 in NDM-1; 120-129 in IMP-1; 174-186 in VIM-2; and 142-152 in ccrA), and the L10 loop (4, blue) (residues 210-227 in NDM-1; 160-174 in IMP-1; 223-240 in VIM-2; and 183-200 in ccrA). Notably, ccrA is the only B1 MBL gap free with respect to NDM-1 outside of the linker region.

Figure 2

Heatmap LGA alignments of MBL library against NDM-1. Parameters for LGA were set using a maximal 4.0 Å distance, under side chain comparison. Coloring was set to > 2 Å (at 2 Å increments) from green to yellow to orange to red to gray. Notable areas of interested are framed using the same ranges as in Figure 1, and include regions of major structural misalignment, such as the L3 mobile flap and the linker region.

Figure 3

Structural abundances of NDM-1 based on MBL structural fragment comparisons. (A) Structural abundance, per residue for NDM-1. The first major decrease in abundance at approximately residue 68 corresponds to the mobile flap found in B1 MBLs. The region of structural disagreement starting at about residue 50 and continuing for 30 residues corresponds to the linker region. The last decrease in abundance, at about residues 210-225 align with a lengthy loop region in other MBLs. The flap, anchored linker region and loop L10 are demarcated using the ranges specified in Figure 2.

Figure 4

StralCP dendrograms and clusters for MBL-like proteins. Structure-based similarity dendrograms based on substructures of the (A) active site, and (B) linker region, constructed using pairwise average-link Euclidean distances. The subtrees are magnified sections of the dendrograms corresponding to the main branch containing the B1 MBL clusters, as determined via StralCP, where coloring correspond to VIM-type (red), IMP-type (green), NDM-1 (purple) and ccrA (blue) structures.

Figure 5

Visual alignment of the linker region between NDM-1 and other B1 MBLs. NDM-1 is shown in red, against varying shades of gray for IMP-1, ccrA, VIM-2 and BlaB. N- and C-terminal regions align well across all B1 MBLs, but for NDM-1 the center of the linker region is distal, indicated by arrows and labeled by their positioning and residues within NDM-1. The shown structures correspond to 120-166 in ccrA, 98-143 in IMP-1, 138-200 in VIM-2, 138-200 in BcII and 141-193 in NDM-1; these ranges include N- and C-terminal anchor sequences that flank the linker region and are generally well conserved. Graphics were generated using PyMol [39].

Figure 6

Estimated pocket volumes of the binding site for ccrA, IMP-1, VIM, NDM-1 and other B1 MBLs. Boxplots show the distribution of volume for various MBL binding sites; while the plasmid-bourne MBLs (IMP-1, NDM-1, VIM) share similar volume sizes, with fluctuation, ccrA and other B1 MBLs have a distinctly smaller site. Estimates were calculated using CASTp [23]. Data for generating these plots are from Additional file 3.

Figure 7

Estimated metal ion distances for ccrA, IMP-1, VIM, NDM-1 and other B1 MBLs. Boxplots show the distribution of inter-zinc distances; ccrA has the tightest inter-zinc distance, though this does not appear to be statistically significant when compared to the other types under an adjusted signed rank test. Data for generating these plots are from Additional file 3.

Figure 8

Structural conformation adjustments within the active sites between bound-unbound structures of NDM-1, IMP-1, VIM-2 and ccrA. Charts were generated as follows: in each case, a bound reference from one of the four B1 MBL types was selected for examination. The bound reference was compared to other bound B1 MBL structures using LGA (maximal distance of 4 Å), and similarly to unbound B1 MBL structures. Residue shifts within a 4 Å distance of either the bound ligand or the dinuclear zincs were drawn on a XY-plot, with the X-axis referring to differences in the bound target and the unbound templates, and Y-axis the bound target against other bound templates, alternatively using NDM-1, IMP-1, VIM-2 and ccrA as a bound representative. The horizontal line shows the line of equal change between bound and unbound comparisons (thus, residues on the top right are residues that deviate in either case). K211/161/167 and R185 (Y181) from NDM-1, IMP-1, ccrA and VIM-2, a ligand binding-associated residue, are highlighted for comparison in red.

Figure 9

LGA_pdblist functional side-chain differences between NDM-1 and other B1 MBLs. Using 3q6x_A as a reference, functional side-chain differences are shown of NDM-1 against IMP-1, VIM-2,4 and ccrA. Measurements are taken only for instances where the residue-residue correspondence for NDM-1 to the other MBLs match, and only then for those with functional sidechains (e.g., alanine, glycine are ignored) [44]. Coloring is from green (< 2Å) to red (< 8Å), with gray indicating no match. Notable regions of B1 MBLs are highlighted, and include the zinc binding region, the L10 ligand-binding loop and the K211 residue noted in Figure 8. Additional annotations show areas where side-chains consistently differ from 3q6x_A and either other NDM-1 structures or other B1 MBLs (e.g., flap region).