Timing of Events, Reset Mechanism, and Allostery Innate to the GTP Hydrolysis Cycle of Atlastin
O'Donnell, John P
Homotypic membrane fusion of the smooth endoplasmic reticulum (ER) is catalyzed by atlastin (ATL), a member of the dynamin superfamily. Maintaining proper membrane architecture and dynamics in this highly compartmentalized organelle is accomplished in part through ATL-mediated fusion of ER tubules to form a highly interconnected reticular system of three-way junctions. Mechanistically, ATL couples the energy from GTP hydrolysis to a series of conformational changes and homodimerization events that result in membrane fusion. Higher eukaryotes express three ATL isoforms. Two of these isoforms, human ATL1 and ATL3 have been implicated in neurological disorders, namely Hereditary Spastic Paraplegia (HSP) and Hereditary Sensory Neuropathy (HSN) where axon function is impaired. Disease causing mutations in atl1 and atl3 are dominant-negative and cause the exchange of reticular ER for long unbranched membrane tubules. The underlying mechanistic differences between human isoforms and disease-associated mutations are poorly understood. Here we identify and give order to discrete temporal steps in the catalytic cycle for ATL1 and ATL3. A comparative study demonstrates that isoforms sample the same series of events but do so at different timescales. We also establish a conserved mechanism for the displacement of the catalytic magnesium ion post GTP hydrolysis. Investigation into mechanisms of disease pathogenesis yields structural and functional defects of an atypical HSP variant. This HSP mutant, ATL1-F151S, is impaired in its GTP hydrolysis cycle but is capable of sampling the high affinity homodimer when bound to a transition state analog. These findings along with additional structural information from mutant proteins define a mechanism for allosteric coupling where F151 is the central residue in a hydrophobic interaction network connecting the active-site to an inter-domain interface responsible for nucleotide loading.
membrane fusion; allosteric regulation; dynamin-related proteins; enzyme mechanism; enzyme structure; GTPase; Biochemistry; Biophysics
Lin, Hening; Cerione, Richard A.; Sevier, Carolyn S.
Ph. D., Pharmacology
Doctor of Philosophy
dissertation or thesis