Structural and functional consequences of age-related isomerization in α-crystallins

  1. Ryan R. Julian?3
  1. From the ?Department of Chemistry, University of California, Riverside, Riverside, California 92521,
  2. the §Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, United Kingdom, and
  3. the ?Department of Chemistry, Durham University, South Road, Durham DH1 3LE, United Kingdom
  1. ?2 To whom correspondence may be addressed. E-mail: justin.benesch{at}chem.ox.ac.uk.
  2. ?3 To whom correspondence may be addressed. E-mail: ryan.julian{at}ucr.edu.
  1. ?1 Both authors contributed equally to this work.

  2. Edited by Ursula Jakob

Abstract

Long-lived proteins are subject to spontaneous degradation and may accumulate a range of modifications over time, including subtle alterations such as side-chain isomerization. Recently, tandem MS has enabled identification and 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 examined the impact of isomerization of aspartic acid or epimerization of serine at four sites mapping to crucial oligomeric interfaces in human αA- and αB-crystallin, the most abundant chaperone proteins in the eye lens. To characterize the effect of isomerization on quaternary assembly, we utilized synthetic peptide mimics, enzyme assays, molecular dynamics calculations, and native MS experiments. The oligomerization of recombinant forms of αA- and αB-crystallin that mimic isomerized residues deviated from native behavior in all cases. Isomerization also perturbs recognition of peptide substrates, either enhancing or inhibiting kinase activity. Specifically, epimerization of serine (αASer-162) dramatically weakened inter-subunit binding. Furthermore, phosphorylation of αBSer-59, known to play an important regulatory role in oligomerization, was severely inhibited by serine epimerization and altered by isomerization of nearby αBAsp-62. Similarly, isomerization of αBAsp-109 disrupted a vital salt bridge with αBArg-120, a contact that when broken has previously been shown to yield aberrant oligomerization and aggregation in several disease-associated variants. Our results illustrate how isomerization of amino acid residues, which may seem to be only a minor structural perturbation, can disrupt native structural interactions with profound consequences for protein assembly and activity.

Footnotes

  • This work was supported by National Institutes of Health Grant R01GM107099 from NIGMS (to R. R. J.), Oxford University Press Clarendon Award (to M. P. C.), and Engineering and Physical Sciences Research Council Grants EP/P016499/1 (to M. T. D.) and EP/J01835X/1 (to J. L. P. B.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

  • This article was chosen as one of our Editors' Picks.

  • This article contains Figs. S1–S4, Tables S1–S5, supporting Materials and methods, and supporting Refs. 1–12.

  • Received December 6, 2018.
  • Revision received February 15, 2019.

Author's Choice—Final version open access under the terms of the Creative Commons CC-BY license.

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This Article

  1. The Journal of Biological Chemistry 294, 7546-7555.
  1. Free via Open Access: OA
  2. Free via Creative Commons: CC-BY license
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  4. Author profile: Miranda P. Collier
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  6. All Versions of this Article:
    1. RA118.007052v1
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