Structural rationale to understand the effect of disease-associated mutations on Myotubularin

Abstract

Myotubularin or MTM1 is a lipid phosphatase that regulates vesicular trafficking in the cell. The MTM1 gene is mutated in a severe form of muscular disease, X-linked myotubular myopathy or XLMTM, affecting 1 in 50,000 newborn males worldwide. There have been several studies on the disease pathology of XLMTM, but the structural effects of missense mutations of MTM1 are underexplored due to the unavailability of a crystal structure. MTM1 consists of three domains- a lipid-binding N-terminal GRAM domain, the phosphatase domain and a coiled-coil domain which aids dimerization of Myotubularin homologs. While most mutations reported to date map to the phosphatase domain of MTM1, the other two domains on the sequence are also frequently mutated in XLMTM. To understand the overall structural and functional effects of missense mutations on MTM1, we curated several missense mutations and performed in silico and in vitro studies. Apart from significantly impaired binding to substrate, abrogation of phosphatase activity was observed for a few mutants. Possible long-range effects of mutations from non-catalytic domains on phosphatase activity were observed as well. Coiled-coil domain mutants have been characterised here for the first time in XLMTM literature. Author SummaryX-linked myotubular myopathy is a rare paediatric disorder and affected males suffer neonatal death or may live on only with ventilatory support. In this study, we employed a range of approaches to understand the molecular level effects of patient-derived mutations on an enzyme directly linked to the congenital muscular disorder. Using three-dimensional modelling and simulations, the effect of these mutations on the structure of the enzyme and its ability to bind its substrate was studied. To complement theoretical observations, experiments were performed with cells expressing this enzyme and its mutants. These studies reveal that each part of the protein may directly or indirectly affect its enzyme activity and most of the patient-derived mutations are expressed insufficiently in the cell. With the advent of genome sequencing technology, identification of congenital mutations is easier; computational studies of molecular consequences of mutations on a protein function such as this will prove immensely useful in understanding the disease and its prognosis.