The Role of Rigid Residues in Modulating TEM-1 β-Lactamase Function and Thermostability.

Jeremy H Mills,S Banu Ozkan,Bethany Kolbaba-Kartchner,I Can Kazan

doi:10.3390/ijms22062895

Jeremy H Mills, S Banu Ozkan + Show 2 more

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https://doi.org/10.3390/ijms22062895

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Abstract

The relationship between protein motions (i.e., dynamics) and enzymatic function has begun to be explored in β-lactamases as a way to advance our understanding of these proteins. In a recent study, we analyzed the dynamic profiles of TEM-1 (a ubiquitous class A β-lactamase) and several ancestrally reconstructed homologues. A chief finding of this work was that rigid residues that were allosterically coupled to the active site appeared to have profound effects on enzyme function, even when separated from the active site by many angstroms. In the present work, our aim was to further explore the implications of protein dynamics on β-lactamase function by altering the dynamic profile of TEM-1 using computational protein design methods. The Rosetta software suite was used to mutate amino acids surrounding either rigid residues that are highly coupled to the active site or to flexible residues with no apparent communication with the active site. Experimental characterization of ten designed proteins indicated that alteration of residues surrounding rigid, highly coupled residues, substantially affected both enzymatic activity and stability; in contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Our results provide additional insight into the structure-function relationship present in the TEM family of β-lactamases. Furthermore, the integration of computational protein design methods with analyses of protein dynamics represents a general approach that could be used to extend our understanding of the relationship between dynamics and function in other enzyme classes.

Highlights

Since the 1940s, β-lactam antibiotics, which target a key enzyme in bacterial cell wall biosynthesis, have been the antimicrobial weapon of choice in the war against bacterial infection [1]
As new β-lactam antibiotics enter into clinical use, the remarkable adaptivity of β-lactamases complicates efforts to develop novel antibiotics that are resistant to degradation by this class of enzyme [2]
A possible explanation as to how mutations distal to the active site can still exert influence at a great distance is that they serve to reshape the inherent dynamics of the enzyme [12,13,14,15,16,17,18,19,20]. We explored this hypothesis in the TEM-1 β-lactamase using two in silico, dynamics-based metrics: the dynamic flexibility index [16,21], which measures the mobility of each residue, and the dynamic coupling index [17,22], which assesses the coupling between distant residues [15]

Summary

Introduction

Since the 1940s, β-lactam antibiotics, which target a key enzyme in bacterial cell wall biosynthesis, have been the antimicrobial weapon of choice in the war against bacterial infection [1]. As the use of this class of antibiotics became more widespread, so too did the prevalence of β-lactamase enzymes, which hydrolyze the β-lactam ring and render the antibiotic nonfunctional [1]. The TEM family of β-lactamases has been thoroughly studied to gain insight into the manner in which resistance is achieved [3,4,5,6,7]. Despite these efforts, we currently possess an incomplete understanding of the relationship between sequence and function in this enzyme class

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