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

• Published in: Journal of molecular biology Vol. 432; no. 17; pp. 4799 - 4814
• Main Authors: Qadota, Hiroshi, Moody, Jasmine C., Lesanpezeshki, Leila, Moncrief, Taylor, Kitzler, Deborah, Bhat, Purnima Devaki, Vanapalli, Siva A., Oberhauser, Andres F., Benian, Guy M.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 07.08.2020
• Subjects: Animals; Body Size; C. elegans; Caenorhabditis elegans - physiology; Caenorhabditis elegans Proteins - chemistry; Caenorhabditis elegans Proteins - genetics; Caenorhabditis elegans Proteins - metabolism; Cell Plasticity; giant polypeptides; Locomotion; Muscle Proteins - chemistry; Muscle Proteins - genetics; Muscle Proteins - metabolism; Muscle, Skeletal - physiology; obscurin; Protein Domains; sarcomere; Sarcomeres - metabolism; Sequence Deletion; Single Molecule Imaging; UNC-89; Ig domain; UNC-89; C. elegans; giant polypeptides; SPEG; HA; obscurin; Fn3 domain; sarcomere
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2020.06.024

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. [Display omitted] •By single-molecule force spectroscopy, the ~645-residuenon-domain region lying between the two protein kinase domains of UNC-89 behaves as an elastic random coil.•Nematodes lacking this non-domain region have disorganized sarcomeres and reduced whole animal locomotion and force generation.•UNC-89non-domain region is required for proper assembly or stability of A-bands from thick filaments.