Biophysical characterization of naturally occurring titin M10 mutations

Abstract

The giant proteins titin and obscurin are important for sarcomeric organization, stretch response, and sarcomerogenesis in myofibrils. The extreme C-terminus of titin (the M10 domain) binds to the N-terminus of obscurin (the Ig1 domain) in the M-band. The high-resolution structure of human M10 has been solved, along with M10 bound to one of its two known molecular targets, the Ig1 domain of obscurin-like. Multiple M10 mutations are linked to limb-girdle muscular dystrophy type 2J (LGMD2J) and tibial muscular dystrophy (TMD). The effect of the M10 mutations on protein structure and function has not been thoroughly characterized. We have engineered all four of the naturally occurring human M10 missense mutants and biophysically characterized them in vitro. Two of the four mutated constructs are severely misfolded, and cannot bind to the obscurin Ig1 domain. One mutation, H66P, is folded at room temperature but unfolds at 37 C, rendering it binding incompetent. The I57N mutation shows no significant structural, dynamic, or binding differences from the wild-type domain. We suggest that this mutation is not directly responsible for muscle wasting disease, but is instead merely a silent mutation found in symptomatic patients. Understanding the biophysical basis of muscle wasting disease can help streamline potential future treatments.

TITINdb—a computational tool to assess titin’s role as a disease gene

“…From the pre-calculated in silico analysis, it can be seen that all these disease associated nsSNVs are predicted to be destabilizing by DUET ( Pires et al , 2014a ). TMD associated nsSNVs are also predicted, by mCSM ( Pires et al , 2014b ), to disrupt the interaction between titin and obscurin, albeit by varying magnitudes; this has been validated experimentally ( Fukuzawa et al , 2008 ; Rudloff et al , 2015 ). Interestingly the I35947N variant is predicted to have the least impact on the titin-obscurin interaction affinity (mCSM score -0.17 kcal/mol) out of all the TMD associated variants; this correlates with in vitro experimental observations where negligible differences have been found between this variant and wild-type titin ( Fukuzawa et al , 2008 ; Rudloff et al , 2015 ).…”

“…TMD associated nsSNVs are also predicted, by mCSM ( Pires et al , 2014b ), to disrupt the interaction between titin and obscurin, albeit by varying magnitudes; this has been validated experimentally ( Fukuzawa et al , 2008 ; Rudloff et al , 2015 ). Interestingly the I35947N variant is predicted to have the least impact on the titin-obscurin interaction affinity (mCSM score -0.17 kcal/mol) out of all the TMD associated variants; this correlates with in vitro experimental observations where negligible differences have been found between this variant and wild-type titin ( Fukuzawa et al , 2008 ; Rudloff et al , 2015 ). Additionally, the majority of HMERF associated nsSNVs are predicted to be deleterious by Condel ( González-Pérez and López-Bigas, 2011 ), whereas only half the TMD associated nsSNVs are predicted to be deleterious.…”

Patient mutations linked to arrhythmogenic cardiomyopathy enhance calpain-mediated desmoplakin degradation

“…Computational modeling was done using the human WT desmoplakin Protein Data Bank number 3R6N (25). The desmoplakin S299R, N375I, S442F, I445V, R451G, N458Y, K470E, and S507F mutation models were generated with the "swap residue" command in the computer program YASARA and allowed to equilibrate for at least 60 ns in explicit solvent at 310 °K, 150 mM NaCl, as previously described (45,46). All hydrogen bonds and electrostatic interactions within 7 Å of the putative calpain target site in the original structure were surveyed every 25 ns, and the data were tabulated using YASARA.…”

A novel FLNC frameshift and an OBSCN variant in a family with distal muscular dystrophy

“…All chemicals were ACS grade or higher and were purchased from Fisher Scientific unless otherwise specified. Recombinant 15 N, 15 N- 13 C, and unlabeled proteins were purified after overexpression in BL21 cells using a pET24a vector system (Novagen, San Diego CA) in a manner similar to [ 26 ]. For purification, all proteins were passed over a G75-sepharose size exclusion column, and all eluted as monomers.…”