Qadota, H. | bioRxiv Science

bioRxiv2020

A Region of UNC-89 (obscurin) Lying Between Two Protein Kinase Domains is a Highly Elastic Spring Required for Proper Sarcomere Assembly

In C. elegans, unc-89 encodes a set of giant multi-domain proteins (up 8,081 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, 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 express normal levels of giant internally deleted UNC-89 proteins, and yet show 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-generated in-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 dis-organization of pharyngeal muscle, small body size, and reduced muscle force, likely due to poor nutritional uptake. HighlightsO_LIThe giant muscle proteins UNC-89 and its mammalian homologs have an [~]700 aa non-domain region lying between two protein kinase domains C_LIO_LIBy single molecule force spectroscopy UNC-89 non-domain region is an elastic random coil C_LIO_LINematodes lacking this non-domain region have disorganized sarcomeres and reduced whole animal locomotion C_LIO_LIUNC-89 non-domain region is required for proper assembly of A-bands from thick filaments C_LI

cell biology↗

bioRxiv2020

Mutational analysis of the structure and function of the chaperone domain of UNC-45B

UNC-45B is a multidomain molecular chaperone that is essential for the proper folding and assembly of myosin into muscle thick filaments in vivo. We have previously demonstrated that its UCS domain is responsible for the chaperone-like properties of UNC-45B. In order to better understand the chaperoning function of the UCS domain we engineered mutations designed to: i) disrupt chaperone-client interactions by removing and altering the structure of the putative client-interacting loop and ii) disrupt chaperone-client interactions by changing highly conserved residues in the putative client-binding groove. We tested the effect of these mutations by using a novel combination of complementary biophysical (circular dichroism, intrinsic tryptophan fluorescence, chaperone activity, and SAXS) and in vivo tools (C. elegans sarcomere structure). Removing the client-holding loop had a pronounced effect on the secondary structure, thermal stability, solution conformation and chaperone function of the UCS domain. These results are consistent with previous in vivo findings that this mutation neither rescue the defect in C. elegans sarcomere organization nor bind to myosin. We found that mutating several conserved residues in the client-binding groove do not affect UCS domain secondary structure or structural stability but reduced its chaperoning activity. We found that these groove mutations also significantly altered the structure and organization of the worm sarcomeres. We also tested the effect of R805W, a mutation distant from the client-binding region. Our in vivo data show that, to our surprise, the R805W mutation appeared to have the most drastic effect on the structure and organization of the worm sarcomeres. In humans, the R805W mutation segregates with human congenital/infantile cataract, indicating a crucial role of R805 in UCS domain stability and/or client interaction. Hence, our experimental approach combining biophysical and biological tools facilitates the study of myosin/chaperone interactions in mechanistic detail. Statement of SignificanceThe folding of myosin and the assembly of a functional sarcomere requires the chaperone UNC-45B. The molecular mechanism(s) for how UNC-45B assist in this assembly process or prevent stress-induced aggregation states are presently unknown. Answering this question is a problem at the core of muscle development and function. Here we developed a novel approach that combines biophysical and biological tools to study UNC-45B/myosin interactions in mechanistic detail. Our approach may provide critical insights into the molecular nature of the pathogenesis of many muscle disorders stemming from mutations in sarcomeric proteins including skeletal myopathies and cardiomyopathies, and possibly the age-associated decline in muscle mass and function found in the elderly known as sarcopenia.

biophysics↗