Riassunto analitico
Rab GTPases, the largest subgroup in the superfamily of Ras-like GTPases, play regulatory roles in multiple steps of intracellular vesicle trafficking. They are activated by guanine nucleotide exchange factors (GEFs), which catalyze the interconversion of the GDP-bound (inactive) form to the GTP-bound (active) form. Relatively little is known of the mechanisms by which GEFs activate Rabs.
The crystal structures of the GEF domain of Sec2p in complex with its nucleotide-free Rab partner Sec4p (PDB codes: 2EQB and 2OCY [1, 2]) provided insights on the structural deformations occurring in Sec4p upon Sec2p binding.
Like all Ras GTPases, Sec4p holds a αβα three-layer sandwich architecture and a Rossmann fold. The Sec2p GEF domain is a 220 Å long coiled coil, striking in its simplicity and in the use of the coiled-coil motif for catalysis. The structures suggest a mechanism whereby Sec2p induces extensive structural rearrangements in the Sec4p switch regions and phosphate-binding loop that are incompatible with nucleotide binding.
My Master’s thesis concerns extensive structural analysis of sub-microsecond and microsecond molecular dynamics (MD) simulations carried out on the crystal structures of the GDP- (PDB: 1G16) and GTP- (PDB: 1G17) bound states of Sec4p [3] and of the Sec4p-Sec2p complex (PDB: 2EQB [2]). To investigate the early steps of the nucleotide exchange mechanism, microsecond MD simulations were also carried out on a number of predicted complexes between Sec2p extracted from the 2OCY complex and GDP-bound Sec4p.
The main aim is to investigate those structural deformations in the Sec4p protein induced by the Sec2p GEF leading to nucleotide depletion and the determinants of the structural communication between the two proteins.
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