A Region of UNC-89 (Obscurin) Lying between Two Protein Kinase Domains Is a Highly Elastic Spring Required for Proper Sarcomere Organization

Hiroshi Qadota, Jasmine C. Moody, Leila Lesanpezeshki, Taylor Moncrief, Deborah Kitzler, Purnima Devaki Bhat, Siva A. Vanapalli, Andres F. Oberhauser, Guy M. Benian

Research output: Contribution to journalArticle

Abstract

In Caenorhabditis elegans, unc-89 encodes a set of giant multi-domain proteins (up 8081 residues) localized to the M-lines of muscle sarcomeres and required for normal sarcomere organization and whole-animal locomotion. Multiple UNC-89 isoforms contain two protein kinase domains. There is conservation in arrangement of domains between UNC-89 and its two mammalian homologs, obscurin and SPEG: kinase, a non-domain region of 647–742 residues, Ig domain, Fn3 domain and a second kinase domain. In all three proteins, this non-domain “interkinase region” has low sequence complexity, has high proline content, and lacks predicted secondary structure. We report that a major portion of this interkinase (571 residues out of 647 residues) when examined by single molecule force spectroscopy in vitro displays the properties of a random coil and acts as an entropic spring. We used CRISPR/Cas9 to create nematodes carrying an in-frame deletion of the same 571-residue portion of the interkinase. These animals display severe disorganization of all portions of the sarcomere in body wall muscle. Super-resolution microscopy reveals extra, short-A-bands lying close to the outer muscle cell membrane and between normally spaced A-bands. Nematodes with this in-frame deletion show defective locomotion and muscle force generation. We designed our CRISPR-generatedin-frame deletion to contain an HA tag at the N terminus of the large UNC-89 isoforms. This HA tag results in normal organization of body wall muscle, but approximately half the normal levels of the giant UNC-89 isoforms, dis-organization of pharyngeal muscle, small body size, and reduced muscle force, likely due to poor nutritional uptake.

Original languageEnglish (US)
Pages (from-to)4799-4814
Number of pages16
JournalJournal of Molecular Biology
Volume432
Issue number17
DOIs
StatePublished - Aug 7 2020

Keywords

  • C. elegans
  • UNC-89
  • giant polypeptides
  • obscurin
  • sarcomere

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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