Molecular mechanism of Ras-related protein Rab-5A and effect of mutations in the catalytically active phosphate-binding loop

Abstract

Ras-related protein (Rab-5a) is primarily involved in the regulation of early endosome fusion during endocytosis and takes part in the budding process. During GTP hydrolysis, Rab5a was spotted in the cytoplasmic side of early endosomes in association with the GTP. Previous study suggested that the substitution of alanine with proline at position 30 of Rab5a reduces the GTPase activity around 12-fold, while, with arginine substitution stimulates the intrinsic GTP hydrolysis by 5-fold. Most of the other substitutions at this position show a little or no effect on the GTPase activity. In this paper, structure analysis and molecular dynamics (MD) simulation studies of human Rab5a and its mutants have been extensively carried out. The effect of binding of a non-hydrolyzable GTP analog guanosine-5'-(β, γ)-imidotriphosphate (GppNHp) with Rab5a and its mutants are described. The objective of the current study is to perform a detailed examination of structural flexibility of Rab5a and its mutants p.Ala30Pro and p.Ala30Arg using MD simulations. Our observations suggest that mutant p.Ala30Arg stabilize the protein molecule when bound to GppNHp which offers additional contacts. Despite an in silico approach, this study provides a deep insight into the impact of mutation on the structure, function, stability, and mechanism of binding of GppNHp to the Rab5a at molecular level.

Structural and Biophysical Characterization of Rab5a from Leishmania Donovani

“…For the latter, it was shown that binding of c-Raf Ras binding domain decreased the flexibility of the P-loop and switch regions (26). The dynamic regions of Rab5 have also been recently explored through molecular dynamics (MD) simulations (27). Although the study was primarily undertaken to explain the differences in the GTPase activities of mutants of the P-loop residue A30 of Rab5a (A30R and A30P), control MD simulations of wt-Rab5a showed higher root mean square fluctuation (RMSF) values for the switch I, switch II, and the interswitch regions (27).…”

Improving phospholipase activity of PLA1 by protein engineering and its effects on oil degumming

“…Therefore, the obtained structure was open and the lid was identical with the open crystal structure of TLL. In the absence of crystal structure, in silico methods can be helpful to study the impact of point mutations on the structure of PLA 1 . Models of PLA 1 mutants were also designed based on generated 3D structure of wild‐type using the Discovery Studio (Accelrys, San Diego, CA) software.…”

Identifying novel sphingosine kinase 1 inhibitors as therapeutics against breast cancer

“…The plots of the 3D models were readied utilising PyMol and VMD (Visual Molecular Dynamics) 56 . The details of MD simulation method were previously described [57][58][59][60][61] .…”