Unraveling the mechano-chemical processes involved in human MxA antiviral activity

Abstract

The Myxovirus resistance (Mx) proteins of vertebrates restrict replication of a diverse range of viruses. The Mx proteins are closely related to the fission GTPases such as dynamin. These two groups of proteins are able to oligomerize into helical or ring structures that are capable of constriction through GTPase activity. Our interest lies in one of the two Mx proteins found in humans: human MxA. While the exact antiviral mechanism of MxA is currently unknown, previous studies have tied the GTPase activity of MxA to its antiviral function. Therefore, we hope that a clearer understanding of the mechano-chemical activity of MxA will give us further insight into the nature of its antiviral activity. We constructed an assembly deficient MxA variant, unable to form helical or ring structures. This variant was intended to allow us to tractably characterize the mechano-chemical cycle. We present experimental small-angle X-ray scattering (SAXS) data of this assembly deficient MxA when in the presence of product- and substrate-analogues. These experiments intended to show the conformational change of MxA in solution when in the product and substrate-bound states. In silico modeling was conducted to simulate these conformational changes and determine the quality of SAXS data required to detect the change experimentally. While the data suggest Mx undergoes conformational change, associated with GTP hydrolysis, they do not unambiguously establish the nature of conformational transition, We report the preliminary purification results of an MxA variant containing a disulfide bond positioned to prevent cycling between conformational states. The purpose of this was to investigate the nature of coupling between the GTPase activity of the catalytic domain and the conformational changes in the neighboring domains. Finally, we describe an MxA variant protein consisting of only the catalytic GTPase domain. This construct was intended to examine the effects of the neighboring domains on GTPase activity. However, during purification it proved to form large insoluble aggregates, preventing further analysis.