The Structural Dynamics of Engineered \u03b2-Lactamases Vary Broadly on Three Timescales yet Sustain Native Function

Sophie M C Gobeil,Jürgen Pleiss,Joelle N Pelletier,Maximillian C C J C Ebert,Nicolas Doucet,Donald Gagné,Jaeok Park,Albert M Berghuis

doi:10.1038/s41598-019-42866-8

Abstract

Understanding the principles of protein dynamics will help guide engineering of protein function: altering protein motions may be a barrier to success or may be an enabling tool for protein engineering. The impact of dynamics on protein function is typically reported over a fraction of the full scope of motional timescales. If motional patterns vary significantly at different timescales, then only by monitoring motions broadly will we understand the impact of protein dynamics on engineering functional proteins. Using an integrative approach combining experimental and in silico methodologies, we elucidate protein dynamics over the entire span of fast to slow timescales (ps to ms) for a laboratory-engineered system composed of five interrelated β-lactamases: two natural homologs and three laboratory-recombined variants. Fast (ps-ns) and intermediate (ns-µs) dynamics were mostly conserved. However, slow motions (µs-ms) were few and conserved in the natural homologs yet were numerous and widely dispersed in their recombinants. Nonetheless, modified slow dynamics were functionally tolerated. Crystallographic B-factors from high-resolution X-ray structures were partly predictive of the conserved motions but not of the new slow motions captured in our solution studies. Our inspection of protein dynamics over a continuous range of timescales vividly illustrates the complexity of dynamic impacts of protein engineering as well as the functional tolerance of an engineered enzyme system to new slow motions.

Highlights

It is tempting to assume that there is one relationship between dynamics and function, but multiple influences that are specific to distinct timescales
Chimera cTEM-19m includes both segments of PSE-4 present in either of the other chimeras, resulting in 19 substitutions on two active-site walls (S70 and Ω-loop walls) relative to TEM-1
Despite differences observed in slow dynamics, such as observations made for residue Tyr[105], the engineered chimeras are highly functional, their kinetic parameters ranging near those of the native TEM-1 and PSE-4

Summary

Introduction

It is tempting to assume that there is one relationship between dynamics and function, but multiple influences that are specific to distinct timescales. To examine a continuous spectrum of motions, here we have applied an integrative approach to determine the structural dynamics of the native class A TEM-1 and PSE-4 β-lactamases We have elucidated their protein dynamics over timescales ranging from ps to ms by combining previously acquired results of NMR relaxation, crystallographic and steady-state kinetic data[17,27,28,29,30,31] with new molecular dynamics simulations. We further expand this investigation to engineered enzyme variants by examining the same range of motions in a closely interrelated system composed of three recombined and functionally selected variants of TEM-1 and PSE-4, or ‘chimeras’ (Fig. 1). The three engineered chimeras displayed a remarkable conservation of dynamics on the timescale of the fast and intermediate motions examined (ps to ns and ns to μs) and were generally similar to the native TEM-1 and www.nature.com/scientificreports β-lactamase variant TEM-1a cTEM-17ma TEM-1(M69T) cTEM-18m(M69T) cTEM-2m cTEM-19m PSE-4a

Methods

Results

Conclusion