Louis Renault

The 1.7 A crystal structure of the regulator of chromosome condensation (RCC1) reveals a seven-bladed propeller.

The gene encoding the regulator of chromosome condensation (RCC1) was cloned by virtue of its ability to complement the temperature-sensitive phenotype of the hamster cell line tsBN2, which undergoes premature chromosome condensation or arrest in the G1 phase of the cell cycle at non-permissive temperatures. RCC1 homologues have been identified in many eukaryotes, including budding and fission yeast. Mutations in the gene affect pre-messenger RNA processing and transport,, mating, initiation of mitosis and chromatin decondensation, suggesting that RCC1 is important in the control of nucleo-cytoplasmic transport and the cell cycle. Biochemically, RCC1 is a guanine-nucleotide-exchange factor for the nuclear Ras homologue Ran; it increases the dissociation of Ran-bound GDP by 105-fold (ref. 9). It may also bind to DNA via a protein–protein complex. Here we show that the structure of human RCC1, solved to 1.7-Å resolution by X-ray crystallography, consists of a seven-bladed propeller formed from internal repeats of 51–68 residues per blade. The sequence and structure of the repeats differ from those of WD40-domain proteins, which also form seven-bladed propellers and include the β-subunits of G proteins. The nature of the structure explains the consequences of a wide range of known mutations. The region of the protein that is involved in guanine-nucleotide exchange is located opposite the region that is thought to be involved in chromosome binding.

Arf, Arl, Arp and Sar proteins: a family of GTP‐binding proteins with a structural device for ‘front–back’ communication

Arf proteins are important regulators of cellular traffic and the founding members of an expanding family of homologous proteins and genomic sequences. They depart from other small GTP‐binding proteins by a unique structural device, which we call the ‘interswitch toggle’, that implements front–back communication from the N‐terminus to the nucleotide binding site. Here we define the sequence and structural determinants that propagate information across the protein and identify them in all of the Arf family proteins other than Arl6 and Arl4/Arl7. The positions of these determinants lead us to propose that Arf family members with the interswitch toggle device are activated by a bipartite mechanism acting on opposite sides of the protein. The presence of this communication device might provide a more useful basis for unifying Arf homologs as a family than do the cellular functions of these proteins, which are mostly unrelated. We review available genomic sequences and functional data from this perspective, and identify a novel subfamily that we call Arl8.

Structural snapshots of the mechanism and inhibition of a guanine nucleotide exchange factor

Small GTP-binding (G) proteins are activated by GDP/GTP nucleotide exchange stimulated by guanine nucleotide exchange factors (GEFs). Nucleotide dissociation from small G protein–GEF complexes involves transient GDP-bound intermediates whose structures have never been described. In the case of Arf proteins, small G proteins that regulate membrane traffic in eukaryotic cells, such intermediates can be trapped either by the natural inhibitor brefeldin A or by charge reversal at the catalytic glutamate of the Sec7 domain of their GEFs. Here we report the crystal structures of these intermediates that show that membrane recruitment of Arf and nucleotide dissociation are separate reactions stimulated by Sec7. The reactions proceed through sequential rotations of the Arf·GDP core towards the Sec7 catalytic site, and are blocked by interfacial binding of brefeldin A and unproductive stabilization of GDP by charge reversal. The structural characteristics of the reaction and its modes of inhibition reveal unexplored ways in which to inhibit the activation of small G proteins.

Structure of human guanylate-binding protein 1 representing a unique class of GTP-binding proteins.

Interferon-γ is an immunomodulatory substance that induces the expression of many genes to orchestrate a cellular response and establish the antiviral state of the cell. Among the most abundant antiviral proteins induced by interferon-γ are guanylate-binding proteins such as GBP1 and GBP2 (refs 1, 2). These are large GTP-binding proteins of relative molecular mass 67,000 with a high-turnover GTPase activity and an antiviral effect. Here we have determined the crystal structure of full-length human GBP1 to 1.8 Å resolution. The amino-terminal 278 residues constitute a modified G domain with a number of insertions compared to the canonical Ras structure, and the carboxy-terminal part is an extended helical domain with unique features. From the structure and biochemical experiments reported here, GBP1 appears to belong to the group of large GTP-binding proteins that includes Mx and dynamin, the common property of which is the ability to undergo oligomerization with a high concentration-dependent GTPase activity.

Structural Basis for Guanine Nucleotide Exchange on Ran by the Regulator of Chromosome Condensation (RCC1)

RCC1 (regulator of chromosome condensation), a beta propeller chromatin-bound protein, is the guanine nucleotide exchange factor (GEF) for the nuclear GTP binding protein Ran. We report here the 1.8 A crystal structure of a Ran*RCC1 complex in the absence of nucleotide, an intermediate in the multistep GEF reaction. In contrast to previous structures, the phosphate binding region of the nucleotide binding site is perturbed only marginally, possibly due to the presence of a polyvalent anion in the P loop. Biochemical experiments show that a sulfate ion stabilizes the Ran*RCC1 complex and inhibits dissociation by guanine nucleotides. Based on the available structural and biochemical evidence, we present a unified scenario for the GEF mechanism where interaction of the P loop lysine with an acidic residue is a crucial element for the overall reaction.

The complex of Arl2-GTP and PDEδ: from structure to function

Arf‐like (Arl) proteins are close relatives of the Arf regulators of vesicular transport, but their function is unknown. Here, we present the crystal structure of full‐length Arl2‐GTP in complex with its effector PDEδ solved in two crystal forms (Protein Data Bank codes 1KSG, 1KSH and 1KSJ). Arl2 shows a dramatic conformational change from the GDP‐bound form, which suggests that it is reversibly membrane associated. PDEδ is structurally closely related to RhoGDI and contains a deep empty hydrophobic pocket. Further experiments show that H‐Ras, Rheb, Rho6 and Gαi1 interact with PDEδ and that, at least for H‐Ras, the intact C‐terminus is required. We suggest PDEδ to be a specific soluble transport factor for certain prenylated proteins and Arl2‐GTP a regulator of PDEδ‐mediated transport.

How guanylate-binding proteins achieve assembly-stimulated processive cleavage of GTP to GMP.

Interferons are immunomodulatory cytokines that mediate anti-pathogenic and anti-proliferative effects in cells. Interferon-γ-inducible human guanylate binding protein 1 (hGBP1) belongs to the family of dynamin-related large GTP-binding proteins, which share biochemical properties not found in other families of GTP-binding proteins such as nucleotide-dependent oligomerization and fast cooperative GTPase activity. hGBP1 has an additional property by which it hydrolyses GTP to GMP in two consecutive cleavage reactions. Here we show that the isolated amino-terminal G domain of hGBP1 retains the main enzymatic properties of the full-length protein and can cleave GDP directly. Crystal structures of the N-terminal G domain trapped at successive steps along the reaction pathway and biochemical data reveal the molecular basis for nucleotide-dependent homodimerization and cleavage of GTP. Similar to effector binding in other GTP-binding proteins, homodimerization is regulated by structural changes in the switch regions. Homodimerization generates a conformation in which an arginine finger and a serine are oriented for efficient catalysis. Positioning of the substrate for the second hydrolysis step is achieved by a change in nucleotide conformation at the ribose that keeps the guanine base interactions intact and positions the β-phosphates in the γ-phosphate-binding site.

The Crystal Structure of rna1p: A New Fold for a GTPase-Activating Protein

rna1p is the Schizosaccharomyces pombe ortholog of the mammalian GTPase-activating protein (GAP) of Ran. Both proteins are essential for nuclear transport. Here, we report the crystal structure of rna1p at 2.66 A resolution. It contains 11 leucine-rich repeats that adopt the nonglobular shape of a crescent, bearing no resemblance to RhoGAP or RasGAP. The invariant residues of RanGAP form a contiguous surface, strongly indicating the Ran-binding interface. Alanine mutations identify Arg-74 as a critical residue for GTP hydrolysis. In contrast to RasGAP and RhoGAP, Arg-74 could be substituted by lysine and contributed significantly to the binding of Ran. Therefore, we suggest a GAP mechanism for rna1p, which constitutes a variation of the arginine finger mechanism found for Ras GAP and RhoGAP.

Triphosphate structure of guanylate‐binding protein 1 and implications for nucleotide binding and GTPase mechanism

The interferon‐γ‐induced guanylate‐binding protein 1 (GBP1) belongs to a special class of large GTP‐ binding proteins of 60–100 kDa with unique characteristics. Here we present the structure of human GBP1 in complex with the non‐hydrolysable GTP analogue GppNHp. Basic features of guanine nucleotide binding, such as the P‐loop orientation and the Mg2+ co‐ordination, are analogous to those of Ras‐related and heterotrimeric GTP‐binding proteins. However, the glycosidic bond and thus the orientation of the guanine base and its interaction with the protein are very different. Furthermore, two unique regions around the base and the phosphate‐binding areas, the guanine and the phosphate caps, respectively, give the nucleotide‐binding site a unique appearance not found in the canonical GTP‐binding proteins. The phosphate cap, which constitutes the region analogous to switch I, completely shields the phosphate‐binding site from solvent such that a potential GTPase‐activating protein cannot approach. This has consequences for the GTPase mechanism of hGBP1 and possibly of other large GTP‐binding proteins.

Spire and Cordon-bleu: multifunctional regulators of actin dynamics

WASP-homology 2 (WH2) domains, which were first identified in the WASP/Scar (suppressor of cAMP receptor)/WAVE (WASP-family verprolin homologous protein) family of proteins, are multifunctional regulators of actin assembly. Two recently discovered actin-binding proteins, Spire and Cordon-bleu (Cobl), which have roles in axis patterning in developmental processes, use repeats of WH2 domains to generate a large repertoire of novel regulatory activities, including G-actin sequestration, actin-filament nucleation, filament severing and barbed-end dynamics regulation. We describe how these multiple functions selectively operate in a cellular context to control the dynamics of the actin cytoskeleton. In vivo, Spire and Cobl can synergize with other actin regulators. As an example, we outline potential methods to gain insight into the functional basis for reported genetic interactions among Spire, profilin and formin.