Enhanced vulnerability of human proteins towards disease-associated inactivation through divergent evolution

Abstract

Human proteins are vulnerable towards disease-associated single amino acid replacements affecting protein stability and function. Interestingly, a few studies have shown that consensus amino acids from mammals or vertebrates can enhance protein stability when incorporated into human proteins. Here, we investigate yet unexplored relationships between the high vulnerability of human proteins towards disease-associated inactivation and recent evolutionary site-specific divergence of stabilizing amino acids. Using phylogenetic, structural and experimental analyses, we show that divergence from the consensus amino acids at several sites during mammalian evolution has caused local protein destabilization in two human proteins linked to disease: cancer-associated NQO1 and alanine:glyoxylate aminotransferase, mutated in primary hyperoxaluria type I. We demonstrate that a single consensus mutation (H80R) acts as a disease suppressor on the most common cancer-associated polymorphism in NQO1 (P187S). The H80R mutation reactivates P187S by enhancing FAD binding affinity through local and dynamic stabilization of its binding site. Furthermore, we show how a second suppressor mutation (E247Q) cooperates with H80R in protecting the P187S polymorphism towards inactivation through long-range allosteric communication within the structural ensemble of the protein. Our results support that recent divergence of consensus amino acids may have occurred with neutral effects on many functional and regulatory traits of wild-type human proteins. However, divergence at certain sites may have increased the propensity of some human proteins towards inactivation due to disease-associated mutations and polymorphisms. Consensus mutations also emerge as a potential strategy to identify structural hot-spots in proteins as targets for pharmacological rescue in loss-of-function genetic diseases.

A mechanism for cancer‐associated inactivation of NQO1 due to P187S and its reactivation by the consensus mutation H80R

“…This integrated analysis completes and refines previous information obtained from high‐resolution structural techniques such as X‐ray crystallography or NMR spectroscopy . We also provide here a detailed characterization of the suppressor effect of the consensus amino acid Arg80, which has recently diverged to His80 along primate evolution (about 30 million years ago), potentially predisposing human NQO1 towards cancer‐associated inactivation . Our work also supports that a detailed understanding of mutational effects on the structure and dynamics of protein conformational ensembles may help to provide a deeper knowledge on the mechanisms used by evolution to shape protein stability and function.…”

“…A ). Therefore, the apo‐state is mostly populated by noncompetent binding states constituting a very flexible and dynamic ensemble (as seen by the large sensitivity of apo‐proteins towards partial proteolysis, and also supported by NMR, MD and SAXS analyses and this work). In contrast, the binding competent states may structurally resemble the protein in the bound conformation (i.e.…”

“…B). These results reveal a particular thermodynamic signature of H80R on FAD binding to P187S with structural implications: the local structural switch caused by H80R in the vicinity of the FAD‐binding site may lead to a less favourable enthalpic contribution likely associated with local structural rearrangements, and entropically counterbalanced by a smaller loss of conformational entropy upon FAD binding due to the dynamic restriction of the FAD‐binding site in the apo‐state .…”

“…To further prove the structural and energetic consequences of the H80R mutation, we have characterized the role of Glu71 and Glu78 by alanine scanning mutagenesis. These two glutamates constitute along with Arg80 a strong electrostatic cluster close to the FAD binding (the EER cluster ) that stabilizes the conformational switch triggered by H80R. As shown in Fig.…”

“…Understanding the mechanisms by which disease‐associated mutations affect protein stability and function, as well as the effects of second‐site suppressor mutations may help to guide the development of novel therapeutic strategies aimed at treating inherited diseases . In this work, we provide a detailed mechanism that unifies the inactivation caused by P187S and its rescue by H80R in the context of a FAD binding mechanism in which ligand binding is strongly coupled to the conformational transition between binding competent and noncompetent states.…”