Defining the architecture of KPC-2 Carbapenemase: identifying allosteric networks to fight antibiotics resistance

Abstract

The rise of multi-drug resistance in bacterial pathogens is one of the grand challenges facing medical science. A major concern is the speed of development of β-lactamase-mediated resistance in Gram-negative species, thus putting at risk the efficacy of the most recently approved antibiotics and inhibitors, including carbapenems and avibactam, respectively. New strategies to overcome resistance are urgently required, which will ultimately be facilitated by a deeper understanding of the mechanisms that regulate the function of β-lactamases such as the Klebsiella Pneumoniae carbapenemases (KPCs). Using enhanced sampling computational methods together with site-directed mutagenesis, we report the identification of two "hydrophobic networks" in the KPC-2 enzyme, the integrity of which has been found to be essential for protein stability and corresponding resistance. Present throughout the structure, these networks are responsible for the structural integrity and allosteric signaling. Disruption of the networks leads to a loss of the KPC-2 mediated resistance phenotype, resulting in restored susceptibility to different classes of β-lactam antibiotics including carbapenems and cephalosporins. The "hydrophobic networks" were found to be highly conserved among class-A β-lactamases, which implies their suitability for exploitation as a potential target for therapeutic intervention.

Figure 1

(A) Structure of KPC-2 β-lactamase. The three loops that surround the active site, i.e. the Ω loop, the hinge region and the loop between α3 and α4 helices are depicted in pink, orange and yellow respectively. (B) Details of the enzyme active site. Residues involved in ligand binding (gray) and the structure of the PSR-3-226 drug (orange), covalently bound to residue S70 have been illustrated as sticks. (C) Engineered loss-of-function variants of KPC-2 disrupting the α-network. The spatial positions of A77, L102, I108, L138, L199 (red sticks) residues that have been mutated are illustrated. Five single and one double variant have been engineered (A77N, L102T, I108N, L138N, L199R and L102T/I108N). It is rationalized that the hydrophobic interactions between residues of the α-network (represented in green) will be disrupted upon mutation.

Figure 2

Conservation of “hydrophobic networks” in class-A β-lactamases. (A) The residues constituting the β-network (green) and the β-network (lilac) have been highlighted and identified with single letter amino acid code for KPC-2, TEM-1, and SHV-1 structures. Residues not conserved among the three β-lactamases have been colored red. The spatial position of the mutated residues (A77N, L102T, I108N, L138N, L199R) has been illustrated as red dots on KPC-2. (B) KPC-2 sequence and secondary structure nomenclature used in the text. The numbering has been kept consistent with the 2OV5 PDB structure. The logo plot highlights the conservation of residues in the hydrophobic networks in class-A β-lactamase family. The conservation has been derived from an alignment of >80 family members. The height of the letter is proportional to the conservation of the residue. The α-network (green) and the β-network (lilac) residues have been colored distinctly.

Figure 3

Free-energy surfaces (FES) plots of the WT, KPC-2 single variants L102T, I108N and double variant L102T/I108N. The FES have been derived from the metadynamics simulations and have been reconstructed along the two variables, CV1 (distance between the center of mass of the indole ring of the side chain of W105 and C_β of L167) and CV2 (distance between the center of mass of the indole ring of the side chain of W105 and C_β of T216). Structures extracted from their corresponding basins have been illustrated. The mutated residues are shown in red sticks, while residues that have been reported to be important for the catalysis are shown in pink sticks.