Structure and mutational analysis of Rab GDP-dissociation inhibitor

Abstract

The crystal structure of the bovine alpha-isoform of Rab GDP-dissociation inhibitor (GDI), which functions in vesicle-membrane transport to recycle and regulate Rab GTPases, has been determined to a resolution of 1.81 A. GDI is constructed of two main structural units, a large complex multisheet domain I and a smaller alpha-helical domain II. The structural organization of domain I is surprisingly closely related to FAD-containing monooxygenases and oxidases. Sequence-conserved regions common to GDI and the choroideraemia gene product, which delivers Rab to catalytic subunits of Rab geranylgeranyltransferase II, are clustered on one face of the molecule. The two most sequence-conserved regions, which form a compact structure at the apex of GDI, are shown by site-directed mutagenesis to play a critical role in the binding of Rab proteins.

A New Functional Domain of Guanine Nucleotide Dissociation Inhibitor (α‐GDI) Involved in Rab Recycling

“…We have previously determined the crystal structure of bovine a-GDI involved in recognition of Rab3 GTPases that regulate neurotransmitter release in the synapse (6,7) at 1.81 A , resolution (8). We found that a-GDI is constructed of two main structural units, a large multi-sheet domain I containing residues from both the N-and C-terminus of the protein, and a smaller entirely a-helical domain II insert (residues 119-219) found at the base of a-GDI.…”

Identification and characterization of a lower plant Ypt/Rab guanosine dissociation inhibitor (GDI)

“…Many if not all small GTPases of the Ypt/Rab family within an organism can be regulated by the same Ypt/Rab-GDI. First insights into the specificity of GDI-Rab interaction have been reported [4,35], but detailed mechanisms remain to be determined. Rab-gdi genes have been cloned from animal and yeast cells so far.…”

“…Algal GDIVlp is nearly equally similar to mammalian, insect, worm, and fungal Rab-GDI, but a rice EST-derived sequence shares greater similarity with GDIVlp than with the remaining sequences (Table 1). Interestingly, GDIVlp shows unique deviations within three 'sequence-conserved regions' (SCRs), which have recently been defined as the best conserved structural and functional RabfYpt-GDI cores [4]. Two of the deviations are conservative (Leu-Ile in SCR1A, Gly-Ala in SCR3B), but one introduces an additional positive charge (Gly-Lys in SCR1 B; Fig.…”

“…Residues conserved in all sequences are given at the bottom (consensus 100%). Three structurally and functionally defined SCRs [4] are indicated by bars, and their consensus sequences shown above the alignment; residues in individual GDIs deviating from the SCRs are boxed. Positions from which PCR primers (this work) were derived are marked by arrows.…”

“…Within a subfamily, a single GDI can interact with many G protein members [2]. Ypt/Rab-GDI is the best characterized molecule with respect to function [3] and structure [4], and is known in animals and yeast so far. Mammalian cells often contain more than one Ypt/Rab-GDI homologue [5].…”