Spastin oligomerizes into a hexamer and the mutant spastin (E442Q) redistribute the wild‐type spastin into filamentous microtubule

Abstract

Spastin, a member of the ATPases associated with various cellular activities (AAA) family of proteins, is the most frequently mutated in hereditary spastic paraplegia. The defining feature of the AAA proteins is a structurally conserved AAA domain which assembles into an oligomer. By chemical cross‐linking and gel filtration chromatography, we show that spastin oligomerizes into a hexamer. Furthermore, to gain a comprehensive overview of the oligomeric structure of spastin, we generated a structural model of the AAA domain of spastin using template structure of VPS4B and p97/VCP. The generated model of spastin provided us with a framework to classify the identified missense mutations in the AAA domain from hereditary spastic paraplegia patients into different structural/functional groups. Finally, through co‐localization studies in mammalian cells, we show that E442Q mutant spastin acts in a dominant negative fashion and causes redistribution of both wild‐type spastin monomer and spastin interacting protein, RTN1 into filamentous microtubule bundles.

Expansion of mutation spectrum, determination of mutation cluster regions and predictive structural classification of SPAST mutations in hereditary spastic paraplegia

“…Beside a few exceptions, almost all the missense mutations in spastin are located in the AAA domain and recent studies suggest that these missense mutations might exert a dominant-negative effect on the molecular function of spastin. 35,36 Utilization of a recently modeled structure of the AAA domain of spastin, 22 as a framework, enabled us to classify the identified missense mutations from our cohort into different functional groups such as active site, protomer -protomer interaction, pore loop and unknown structural group of mutations. The functional categorization of the novel missense mutations, based upon the structural model of spastin will enable us in future to predict any identified sequence variant in a HSP-SPAST patient as disease-causing mutation with greater level of certainty.…”

“…The overall fit of the sequence to the template was checked using Verify 3D. 22 The copies of modeled tertiary structures were assembled to form a hexameric quaternary assembly on the basis of the hexameric template (1s3s). This modeled quaternary structure was energy-minimized using SYBYL.…”

“…This modeled quaternary structure was energy-minimized using SYBYL. 22 The interfacial residues between the monomers were extracted using the Contacts of Structural Units program 22 and the crucial residues were short listed by manual inspection and used for further analysis.…”

“…Labeling of mutated missense residues in the structural model of spastin We used a previously generated structural model of spastin, 22 for labeling of the missense mutations identified in the AAA domain in our HSP cohort. In brief, the tertiary structure of the ATPase domain of spastin was modeled on the basis of the tertiary structures of two templates (PDB codes: 1xwi and 1s3s) and the quaternary structure was modeled on the basis of the hexameric template (1s3s).…”

“…We attempted to classify these 10 missense mutations identified in the AAA domain into different structural/functional groups based upon a previously generated structural model of spastin (PMBD id: PM0074982 and PM0074984). 22 We could consign all these missense mutations in the three dimensional space of the spastin structure into four categories namely, active site, pore loop structure, protomer interface residues and other mutations (Table S3 and Figure S1A-K).…”