A subtle dance of atoms influences enzymes

Infinitesimal fluctuations occurring in milli- even nano-seconds within the three-dimensional structure of an enzyme, may be key in explaining protein interactions.

Professor Nicolas Doucet's team at the Natural Sciences and Engineering Research Council of Canada (INRS) has demonstrated that even when certain amino acids are far from the active site of an enzyme, a change in their atomic fluctuations can significantly impact that enzyme's activity.

This phenomenon — which has been underestimated up to now — could explain certain protein engineering failures and help improve the way synthetic functional enzymes are designed.

The article "Perturbation of the conformational dynamics of an active-site loop alters enzyme activity" is published in the journal Structure.

Enzymes are nanomachines exceptionally efficient at accelerating chemical reactions. They play a role in all cell mechanisms.

Like all proteins, they are made up of amino acid chains folded and assembled in precise 3D structures.

Some enzymes, like ribonuclease A, are so efficient they accelerate the transformation of molecules thousands of times per second.

Donald Gagné PhD, a researcher in Professor Doucet's laboratory, analyzed the impact of removing one methyl group located in a loop distant from the reaction site of ribonuclease A — a slight chemical alteration that was presumed to have no effect as the loss did not disturb the 3D shape of the enzyme. But, it created a four-fold reduction in the ability of ribonuclease A to bind to other nucleotide molecules and promote their normal functions. How?

Taking crystallographic images and using nuclear magnetic resonance to measure the enzyme at an atomic level, Gagné compared normal ribonuclease A with this mutated methyl group.

Gagné observed that when ribonuclease A is modified, nucleotides [the structural building blocks of DNA and RNA: adenine, thymine, guanine, and cytosine; plus a molecule of sugar and phosphoric acid] do not position themselves properly. They have a harder time binding to other molecules.

This inability to bind is the result of an increase in vibrations — caused by the elimination of that distant methyl group — that spread throughout the enzyme structure and disrupted the reaction site. Understanding this enzyme dynamic could change our understanding of how proteins and enzymes interact.

While it is a challenge to measure fluctuations at atomic scale, researchers have always studied three-dimensional protein structures to understand how they function.

So, despite the staggering complexity of capturing atomic information, the research now confirms proteins are regulated by the subtle dance of their atoms.

Abstract Highlights
•A long-range mutation affects conformational integrity and binding in RNase A
•The mutation induces major repositioning of the purine ligand but not pyrimidine
•Enhanced dynamics in distal regions are observed on the catalytic timescale
•Significant enhancement in loop dynamics induces major ligand repositioning

Summary
The role of internal dynamics in enzyme function is highly debated. Specifically, how small changes in structure far away from the reaction site alter protein dynamics and overall enzyme mechanisms is of wide interest in protein engineering. Using RNase A as a model, we demonstrate that elimination of a single methyl group located >10 Å away from the reaction site significantly alters conformational integrity and binding properties of the enzyme. This A109G mutation does not perturb structure or thermodynamic stability, both in the apo and ligand-bound states. However, significant enhancement in conformational dynamics was observed for the bound variant, as probed over nano- to millisecond timescales, resulting in major ligand repositioning. These results illustrate the large effects caused by small changes in structure on long-range conformational dynamics and ligand specificities within proteins, further supporting the importance of preserving wild-type dynamics in enzyme systems that rely on flexibility for function.

About the publication
This research was conducted by Donald Gagné (INRS-Institut Armand-Frappier), Rachel L. French (University of Illinois, Chicago), Chitra Naryanan (INRS-Institut Armand-Frappier), Mijan Simonovi? (University of Illinois, Chicago), Pratul K. Agarwal (Oak Ridge National Laboratory), and Nicolas Doucet (INRS-Institut Armand-Frappier). The article "Perturbation of the conformational dynamics of an active-site loop alters enzyme activity" was published in the journal Structure (DOI: http://dx.doi.org/10.1016/j.str.2015.10.011).

Financial support for this research was provided by the National Institutes of Health (United States), the Natural Sciences and Engineering Research Council of Canada, and the Fonds de recherche Québec - Santé.

About INRS
INRS is a graduate-level research and training university and ranks first in Canada for research intensity (average grant funding per faculty member). INRS brings together some 150 professors and close to 700 students and postdoctoral fellows in its four centres located in Montreal, Quebec City, Laval, and Varennes. Its applied and fundamental research is essential to the advancement of science in Quebec and internationally, playing a key role in the development of tangible solutions to the problems faced by our society.

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A seemingly insignificant change on a loop of  the enzyme ribonuclease A
resulted in a 4 fold decrease in that enzyme's ability to function.

Image Credit: Laboratory of Nicolas Doucet PhD