The impact of long-distance mutations on the Ω-loop conformation in TEM type β-lactamases

Abstract

β-lactamases are hydrolytic enzymes primarily responsible for occurrence and abundance of bacteria resistant to β-lactam antibiotics. TEM type β-lactamases are formed by the parent enzyme TEM-1 and more than two hundred of its mutants. Positions for the known amino acid substitutions cover ∼30% of TEM type enzyme's sequence. These substitutions are divided into the key mutations that lead to changes in catalytic properties of β-lactamases, and the secondary ones, which role is poorly understood. In this study, Residue Interaction Networks were constructed from molecular dynamic trajectories of β-lactamase TEM-1 and its variants with two key substitutions, G238S and E240K, and their combinations with secondary ones (M182T and Q39K). Particular attention was paid to a detailed analysis of the interactions that affect conformation and mobility of the Ω-loop, representing a part of the β-lactamase active site. It was shown that key mutations weakened the stability of contact inside the Ω-loop thus increasing its mobility. Combination of three amino acid substitutions, including the 182 residue, leads to the release of R65 promoting its new contacts with N175 and D176. As a result, Ω-loop is fixed on the protein globule. The second distal mutation Q39K prevents changes in spatial position of R65, which lead to the weakening of the effect of M182T substitution and the recovery of the Ω-loop mobility. Thus, the distal secondary mutations are directed for recovering the mobility of enzyme disturbed by the key mutations responsible for expansion of substrate specificity.

Abbreviations
ESBL	=	extended spectrum beta-lactamases
IR	=	inhibitor resistant beta-lactamases
MD	=	molecular dynamics
RIN	=	residue interaction networks
RMSD	=	root mean square deviation
RMSF	=	root mean square fluctuations.

Communicated by Ramaswamy H. Sarma

Keywords:

• Residue interaction networks
• RINs
• TEM type beta-lactamases
• mutations
• Ω-loop
• molecular dynamics

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Russian Science Foundation (Project 15-14-00014-П).