bioRxiv2018
Long-lived proteins are subject to spontaneous degradation and may accumulate a range of modifications over time, including subtle alterations such as isomerization. Recently, tandem-mass spectrometry approaches have enabled the identification and detailed characterization of such peptide isomers, including those differing only in chirality. However, the structural and functional consequences of these perturbations remain largely unexplored. Here we examine the site-specific impact of isomerization of aspartic acid and epimerization of serine in human A- and B-crystallin. From a total of 81 sites of modification identified in aged eye lenses, four (BSer59, ASer162, BAsp62, BAsp109) map to crucial oligomeric interfaces. To characterize the effect of isomerization on quaternary assembly, molecular dynamics calculations and native mass spectrometry experiments were performed on recombinant forms of A- and B-crystallin that incorporate, or mimic, isomerized residues. In all cases, oligomerization is significantly affected, with epimerization of a single serine residue (ASer162) sufficing to weaken inter-subunit binding dramatically. Furthermore, phosphorylation of BSer59, known to play an important regulatory role in oligomerization, is severely inhibited by serine epimerization and altered by isomerization of nearby BAsp62. Similarly, isomerization of BAsp109 disrupts a vital salt-bridge with BArg120, a loss previously shown to yield aberrant oligomerization and aggregation in several disease variants. Our results illustrate how isomerization of amino-acid residues, which may seem like a minor structural perturbation, can have profound consequences on protein assembly and activity by disrupting specific hydrogen bonds and salt bridges.\n\nSignificance StatementProteins play numerous critical roles in our bodies but suffer damage with increasing age. For example, isomerization is a spontaneous post-translational modification that alters the three-dimensional connectivity of an amino acid, yet remains invisible to traditional proteomic experiments. Herein, radical-based fragmentation was used for isomer identification while molecular dynamics and native mass spectrometry were utilized to assess structural consequences. The results demonstrate that isomerization disrupts both oligomeric assembly and phosphorylation in the -crystallins, which are long-lived proteins in the lens of the eye. The loss of function associated with these modifications is likely connected to age-related diseases such as cataract and neurodegenerative disorders, while the methodologies we present represent a framework for structure-function studies on other isomerized proteins.