Structure of a Ran-binding domain complexed with Ran bound to a GTP analogue: implications for nuclear transport

Abstract

The protein Ran is a small GTP-binding protein that binds to two types of effector inside the cell: Ran-binding proteins, which have a role in terminating export processes from the nucleus to the cytoplasm, and importin-beta-like molecules that bind cargo proteins during nuclear transport. The Ran-binding domain is a conserved sequence motif found in several proteins that participate in these transport processes. The Ran-binding protein RanBP2 contains four of these domains and constitutes a large part of the cytoplasmic fibrils that extend from the nuclear-pore complex. The structure of Ran bound to a non-hydrolysable GTP analogue (Ran x GppNHp) in complex with the first Ran-binding domain (RanBD1) of human RanBP2 reveals not only that RanBD1 has a pleckstrin-homology domain fold, but also that the switch-I region of Ran x GppNHp resembles the canonical Ras GppNHp structure and that the carboxy terminus of Ran is wrapped around RanBD1, contacting a basic patch on RanBD1 through its acidic end. This molecular 'embrace' enables RanBDs to sequester the Ran carboxy terminus, triggering the dissociation of Ran x GTP from importin-beta-related transport factors and facilitating GTP hydrolysis by the GTPase-activating protein ranGAP. Such a mechanism represents a new type of switch mechanism and regulatory protein-protein interaction for a Ras-related protein.

Random Mutagenesis and Functional Analysis of the Ran-binding Protein, RanBP1

“…Likewise, at the position equivalent to Asp-93 of mouse RanBP1, there is an acidic residue (Asp or Glu) in nearly all other known RanBDs, but, again, neither a D93A nor a D93N mutation was able to abrogate binding of RanBP1 to Ran⅐GTP. However, the residues in RanBD1 equivalent to Ala-100 and Asp-93 in RanBP1 do not make direct contacts with Ran in the crystal structure (34). Ala-100 is buried within the ␤-barrel, near the end of ␤-strand 4.…”

“…2A, upper panel). Surprisingly, Glu-37 is not a side chain that contributes any contacts at the Ran⅐RanBD interface, as deduced from the crystal structure of the RanBP2 RanBD1⅐Ran⅐GppNHp complex (34), but it resides in the vicinity of conserved residues that provide hydrophobic interactions with Ran (Fig. 3).…”

“…RanBD1 does not bind to Ran⅐GppNHp so as to occlude the nucleotide-binding site (34) but rather appears to preferentially stabilize the effector loops of Ran in their GTP-binding conformation. If so, it might be predicted that only those RanBP1 mutants able to bind Ran⅐GTP with high affinity should be capable of inhibiting RCC1 activity.…”

“…A related protein in the nematode, Caenorhabditis elegans, Ranup96, contains 2 RanBDs (27). The crystal structure of the first RanBD (RanBD1) from Nup358, in a complex with Ran bound to a non-hydrolyzable GTP analog (GppNHp), has been solved (34). RanBD1 possesses a ␤-barrel fold, similar to those of pleckstrin homology, phosphotyrosine binding, and Wiskott-Aldrich syndrome protein homology-1 domains.…”

Nano-encapsulation of a novel anti-Ran-GTPase peptide for blockade of regulator of chromosome condensation 1 (RCC1) function in MDA-MB-231 breast cancer cells

“…Ran acts as a molecular switch through a GTP-GDP cycle (Rush et al, 1996;Sazer, 1996) in which the conversion between GTP-bound and GDP-bound conformations controls its interaction with different effectors (Scheffzek et al, 1995;Vetter et al, 1999). Ran-GTP is formed inside the nucleus by interaction of Ran with its specific guanine nucleotide exchange factor (GEF), termed RCC1, which catalyses the exchange of GDP for GTP (and vice versa) on the nucleotide binding pocket of Ran (Bischoff and Ponstingl, 1991a;Bischoff and Ponstingl, 1991b;Ohtsubo et al, 1989;Renault et al, 2001).…”