Restoring functional neurofibromin by protein transduction.

In Neurofibromatosis 1 (NF1) germ line loss of function mutations result in reduction of cellular neurofibromin content (NF1+/-, NF1 haploinsufficiency). The Ras-GAP neurofibromin is a very large cytoplasmic protein (2818 AA, 319 kDa) involved in the RAS-MAPK pathway. Aside from regulation of proliferation, it is involved in mechanosensoric of cells. We investigated neurofibromin replacement in cultured human fibroblasts showing reduced amount of neurofibromin. Full length neurofibromin was produced recombinantly in insect cells and purified. Protein transduction into cultured fibroblasts was performed employing cell penetrating peptides along with photochemical internalization. This combination of transduction strategies ensures the intracellular uptake and the translocation to the cytoplasm of neurofibromin. The transduced neurofibromin is functional, indicated by functional rescue of reduced mechanosensoric blindness and reduced RasGAP activity in cultured fibroblasts of NF1 patients or normal fibroblasts treated by NF1 siRNA. Our study shows that recombinant neurofibromin is able to revert cellular effects of NF1 haploinsuffiency in vitro, indicating a use of protein transduction into cells as a potential treatment strategy for the monogenic disease NF1.

GTPase activating proteins: structural and functional insights 18 years after discovery.

The conversion of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (P(i)) by guanine nucleotide binding proteins (GNBPs) is a fundamental process in living cells and represents an important timer in intracellular signalling and transport processes. While the rate of GNBP-mediated GTP hydrolysis is intrinsically slow, direct interaction with GTPase activating proteins (GAPs) accelerates the reaction by up to five orders of magnitude in vitro. Eighteen years after the discovery of the first GAP, biochemical and structural research has been accumulating evidence that GAPs employ a much wider spectrum of chemical mechanisms than had originally been assumed, in order to regulate the chemical players on the catalytic protein-protein interaction stage.

The Ras-Byr2RBD complex: structural basis for Ras effector recognition in yeast.

BACKGROUND: The small GTP binding protein Ras has important roles in cellular growth and differentiation. Mutant Ras is permanently active and contributes to cancer development. In its activated form, Ras interacts with effector proteins, frequently initiating a kinase cascade. In the lower eukaryotic Schizosaccharomyces pombe, Byr2 kinase represents a Ras target that in terms of signal-transduction hierarchy can be considered a homolog of mammalian Raf-kinase. The activation mechanism of protein kinases by Ras is not understood, and there is no detailed structural information about Ras binding domains (RBDs) in nonmammalian organisms. RESULTS: The crystal structure of the Ras-Byr2RBD complex at 3 A resolution shows a complex architecture similar to that observed in mammalian homologous systems, with an interprotein beta sheet stabilized by predominantly polar interactions between the interacting components. The C-terminal half of the Ras switch I region contains most of the contact anchors, while on the Byr2 side, a number of residues from topologically distinct regions are involved in complex stabilization. A C-terminal helical segment, which is not present in the known mammalian homologous systems and which is part of the auto-inhibitory region, has an additional binding site outside the switch I region. CONCLUSIONS: The structure of the Ras-Byr2 complex confirms the Ras binding module as a communication element mediating Ras-effector interactions; the Ras-Byr2 complex is also conserved in a lower eukaryotic system like yeast, which is in contrast to other small GTPase families. The extra helical segment might be involved in kinase activation.

Conformational switch and role of phosphorylation in PAK activation.

p21-activated protein kinases (PAKs) are involved in signal transduction processes initiating a variety of biological responses. They become activated by interaction with Rho-type small GTP-binding proteins Rac and Cdc42 in the GTP-bound conformation, thereby relieving the inhibition of the regulatory domain (RD) on the catalytic domain (CD). Here we report on the mechanism of activation and show that proteolytic digestion of PAK produces a heterodimeric RD-CD complex consisting of a regulatory fragment (residues 57 to 200) and a catalytic fragment (residues 201 to 491), which is active in the absence of Cdc42. Cdc42-GppNHp binds with low affinity (K(d) 0.6 microM) to intact kinase, whereas the affinity to the isolated regulatory fragment is much higher (K(d) 18 nM), suggesting that the difference in binding energy is used for the conformational change leading to activation. The full-length kinase, the isolated RD, and surprisingly also their complexes with Cdc42 behave as dimers on a gel filtration column. Cdc42-GppNHp interaction with the RD-CD complex is also of low affinity and does not dissociate the RD from the CD. After autophosphorylation of the kinase domain, Cdc42 binds with high (14 nM) affinity and dissociates the RD-CD complex. Assuming that the RD-CD complex mimics the interaction in native PAK, this indicates that the small G protein may not simply release the RD from the CD. It acts in a more subtle allosteric control mechanism to induce autophosphorylation, which in turn induces the release of the RD and thus full activation.

Crystal structure of the alpha-actinin rod reveals an extensive torsional twist.

BACKGROUND: Alpha-actinin is a ubiquitously expressed protein found in numerous actin structures. It consists of an N-terminal actin binding domain, a central rod domain, and a C-terminal domain and functions as a homodimer to cross-link actin filaments. The rod domain determines the distance between cross-linked actin filaments and also serves as an interaction site for several cytoskeletal and signaling proteins. RESULTS: We report here the crystal structure of the alpha-actinin rod. The structure is a twisted antiparallel dimer that contains a conserved acidic surface. CONCLUSIONS: The novel features revealed by the structure allow prediction of the orientation of parallel and antiparallel cross-linked actin filaments in relation to alpha-actinin. The conserved acidic surface is a possible interaction site for several cytoplasmic tails of transmembrane proteins involved in the recruitment of alpha-actinin to the plasma membrane.

A novel N-ras mutation in malignant melanoma is associated with excellent prognosis.

Mutations in the ras gene are key events in the process of carcinogenesis; in particular, point mutations in codon 61 of exon 2 of the N-ras gene occur frequently in malignant melanoma (MM). We searched for point mutations in the N-ras gene in a large series of primary and metastatic MM from 81 different retrospectively selected patients using the very sensitive denaturing gradient gel electrophoresis technique, followed by sequencing. The classical codon 12 and codon 61 mutations were found in 21 and 17% of the cases, respectively. No codon 13 mutation was found. A novel mutation at codon 18 of exon 1, consisting of a substitution of alanine (GCA) by threonine (ACA), was found in 15% of the primary MMs but in none of the metastatic MMs. All of the other cases were free of mutations. Using microdissected cells from distinctive MM growth phases as source of DNA for mutation analysis, this particular N-ras exon 1 mutation at codon 18 was already present in the radial growth phase and preserved throughout the successive growth phases; it was also found in a dysplastic nevi in continuity with a MM, indicating a clonal relationship between both lesions. Our findings also illustrate the clonal relationship between the distinctive growth phases in MM and suggest the codon 18 mutation to occur early in MM development. The MM in patients with this mutation were significantly thinner than those without a codon 18 mutation (P = 0.0257). Statistical analysis, comparing the group of codon 18 patients with the group of patients with the classical mutations and without mutations, revealed a highly significant difference in overall outcome. The cumulative probability of developing metastasis was significantly lower for the group patients with a codon 18 mutation (P = 0.0130). We can thus conclude that this codon 18 mutation identifies a group of patients with better prognosis than patients with melanoma that harbor wild-type sequence or classical activating point mutations in codon 12 or 61. Preliminary nucleotide binding measurements could not detect a difference between wild-type Ras protein and the mutant Ras(A18T) protein. However, for a precise elucidation of the role of the N-Ras(A18T) mutant in melanoma, additional studies aimed to measure the affinity to guanine nucleotide exchange factors and GTPase-activating proteins are needed.

The Rac-RhoGDI complex and the structural basis for the regulation of Rho proteins by RhoGDI.

Rho family-specific guanine nucleotide dissociation inhibitors (RhoGDIs) decrease the rate of nucleotide dissociation and release Rho proteins such as RhoA, Rac and Cdc42 from membranes, forming tight complexes that shuttle between cytosol and membrane compartments. We have solved the crystal structure of a complex between the RhoGDI homolog LyGDI and GDP-bound Rac2, which are abundant in leukocytes, representing the cytosolic, resting pool of Rho species to be activated by extracellular signals. The N-terminal domain of LyGDI (LyN), which has been reported to be flexible in isolated RhoGDIs, becomes ordered upon complex formation and contributes more than 60% to the interface area. The structure is consistent with the C-terminus of Rac2 binding to a hydrophobic cavity previously proposed as isoprenyl binding site. An inner segment of LyN forms a helical hairpin that contacts mainly the switch regions of Rac2. The architecture of the complex interface suggests a mechanism for the inhibition of guanine nucleotide dissociation that is based on the stabilization of the magnesium (Mg2+) ion in the nucleotide binding pocket.

Guanosine triphosphatase stimulation of oncogenic Ras mutants.

Interest in the guanosine triphosphatase (GTPase) reaction of Ras as a molecular drug target stems from the observation that, in a large number of human tumors, Ras is characteristically mutated at codons 12 or 61, more rarely 13. Impaired GTPase activity, even in the presence of GTPase activating proteins, has been found to be the biochemical reason behind the oncogenicity of most Gly12/Gln61 mutations, thus preventing Ras from being switched off. Therefore, these oncogenic Ras mutants remain constitutively activated and contribute to the neoplastic phenotype of tumor cells. Here, we show that the guanosine 5'-triphosphate (GTP) analogue diaminobenzophenone-phosphoroamidate-GTP (DABP-GTP) is hydrolyzed by wild-type Ras but more efficiently by frequently occurring oncogenic Ras mutants, to yield guanosine 5'-diphosphate-bound inactive Ras and DABP-Pi. The reaction is independent of the presence of Gln61 and is most dramatically enhanced with Gly12 mutants. Thus, the defective GTPase reaction of the oncogenic Ras mutants can be rescued by using DABP-GTP instead of GTP, arguing that the GTPase switch of Ras is not irreversibly damaged. An exocyclic aromatic amino group of DABP-GTP is critical for the reaction and bypasses the putative rate-limiting step of the intrinsic Ras GTPase reaction. The crystal structures of Ras-bound DABP-beta,gamma-imido-GTP show a disordered switch I and identify the Gly12/Gly13 region as the hydrophobic patch to accommodate the DABP-moiety. The biochemical and structural studies help to define the requirements for the design of anti-Ras drugs aimed at the blocked GTPase reaction.

Structural analysis of the GAP-related domain from neurofibromin and its implications.

Neurofibromin is the product of the NF1 gene, whose alteration is responsible for the pathogenesis of neurofibromatosis type 1 (NF1), one of the most frequent genetic disorders in man. It acts as a GTPase activating protein (GAP) on Ras; based on homology to p120GAP, a segment spanning 250-400 aa and termed GAP-related domain (NF1GRD; 25-40 kDa) has been shown to be responsible for GAP activity and represents the only functionally defined segment of neurofibromin. Missense mutations found in NF1 patients map to NF1GRD, underscoring its importance for pathogenesis. X-ray crystallographic analysis of a proteolytically treated catalytic fragment of NF1GRD comprising residues 1198-1530 (NF1-333) of human neurofibromin reveals NF1GRD as a helical protein that resembles the corresponding fragment derived from p120GAP (GAP-334). A central domain (NF1c) containing all residues conserved among RasGAPs is coupled to an extra domain (NF1ex), which despite very limited sequence homology is surprisingly similar to the corresponding part of GAP-334. Numerous point mutations found in NF1 patients or derived from genetic screening protocols can be analysed on the basis of the three-dimensional structural model, which also allows identification of the site where structural changes in a differentially spliced isoform are to be expected. Based on the structure of the complex between Ras and GAP-334 described earlier, a model of the NF1GRD-Ras complex is proposed which is used to discuss the strikingly different properties of the Ras-p120GAP and Ras-neurofibromin interactions.

GTPase-activating proteins: helping hands to complement an active site.

Stimulation of the intrinsic GTPase activity of GTP-binding proteins by GTPase-activating proteins (GAPs) is a basic principle of GTP-binding-protein downregulation. Recently, the molecular mechanism behind this reaction has been elucidated by studies on Ras and Rho, and their respective GAPs. The basic features involve stabilizing the existing catalytic machinery and supplementing it by an external arginine residue. This represents a novel mechanism for enzyme active-site formation.

Selective disactivation of neurofibromin GAP activity in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a common familial tumour syndrome with multiple clinical features such as neurofibromas, café-au-lait spots (CLS), iris Lisch nodules, axillary freckling, optic glioma, specific bone lesions and an increased risk of malignant tumours. It is caused by a wide spectrum of mutations affecting the NF1 gene. Most mutations result in the loss of one allele at the DNA, mRNA or protein level and thus in the loss of any function of the gene product neurofibromin. The idea of the simultaneous loss of several different neurofibromin functions has been postulated to explain the pleiotropic effects of its loss. However, we have identified a novel missense mutation in a family with a classical multi-symptomatic NF1 phenotype, including a malignant schwannoma, that specifically abolishes the Ras-GTPase-activating function of neurofibromin. In this family, Arg1276 had mutated into proline. Based on complex biochemical studies as well as the analysis of the crystal structure of the GTPase-activating protein (GAP) domain of p120GAP in the presence of Ras, we unequivocally identified this amino acid as the arginine finger of the neurofibromin GAP-related domain (GRD)-the most essential catalytic element for RasGAP activity. Here, we present data demonstrating that the mutation R1276P, unlike previously reported missense mutations of the GRD region, does not impair the secondary and tertiary protein structure. It neither reduces the level of cellular neurofibromin nor influences its binding to Ras substantially, but it does completely disable GAP activity. Our findings provide direct evidence that failure of neurofibromin GAP activity is the critical element of NF1 pathogenesis. Thus, therapeutic approaches aimed at the reduction of Ras.GTP levels in neural crest-derived cells can be expected to relieve most of the NF1 symptoms.

Confirmation of the arginine-finger hypothesis for the GAP-stimulated GTP-hydrolysis reaction of Ras.

RasGAPs supply a catalytic residue, termed the arginine finger,into the active site of Ras thereby stabilizing the transition state of the GTPase reaction and increasing the reaction rate by more than one thousand-fold, in good agreement with the structure of the Ras.RasGAP complex.

The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants.

The three-dimensional structure of the complex between human H-Ras bound to guanosine diphosphate and the guanosine triphosphatase (GTPase)-activating domain of the human GTPase-activating protein p120GAP (GAP-334) in the presence of aluminum fluoride was solved at a resolution of 2.5 angstroms. The structure shows the partly hydrophilic and partly hydrophobic nature of the communication between the two molecules, which explains the sensitivity of the interaction toward both salts and lipids. An arginine side chain (arginine-789) of GAP-334 is supplied into the active site of Ras to neutralize developing charges in the transition state. The switch II region of Ras is stabilized by GAP-334, thus allowing glutamine-61 of Ras, mutation of which activates the oncogenic potential, to participate in catalysis. The structural arrangement in the active site is consistent with a mostly associative mechanism of phosphoryl transfer and provides an explanation for the activation of Ras by glycine-12 and glutamine-61 mutations. Glycine-12 in the transition state mimic is within van der Waals distance of both arginine-789 of GAP-334 and glutamine-61 of Ras, and even its mutation to alanine would disturb the arrangements of residues in the transition state.

The interaction of Ras with GTPase-activating proteins.

Ras plays a major role as a molecular switch in many signal transduction pathways which lead to cell growth and differentiation. The GTPase reaction of Ras is of central importance in the function of the switch since it terminates Ras-effector interactions. GTPase-activating proteins (GAPs) accelerate the very slow intrinsic hydrolysis reaction of the GTP-bound Ras by several orders of magnitude and thereby act as presumably negative regulators of Ras action. The GTP hydrolysis of oncogenic mutants of Ras remains unaltered. In this review we discuss recent biochemical and structural findings relating to the mechanism of GAP action, which strengthen the hypothesis that GAP accelerates the actual cleavage step by stabilizing the transition state of the phosphoryl transfer reaction.

The role of the metal ion in the p21ras catalysed GTP-hydrolysis: Mn2+ versus Mg2+.

GTP and ATP hydrolysing proteins have an absolute requirement for a divalent cation, which is usually Mg2+, as a cofactor in the enzymatic reaction. Other phosphoryl transfer enzymes employ more than one divalent ion for the enzymatic reaction. It is shown here for p21ras, a well studied example of GTP hydrolysing proteins, that the GTP-hydrolysis rate is significantly faster if Mg2+ is replaced by Mn2+, both in the presence or absence of its GTPase-activating protein Ras-GAP. This effect is not due to a different stoichiometry of metal ion binding, since one metal ion is sufficient for full enzymatic activity. To determine the role of the metal ion, the crystal structure of p21(G12P). GppCp complexed with Mn2+ was determined and shown to be very similar to the corresponding p21(G12P). GppCp.Mg2+ structure. Especially the coordination sphere around the metal ions is very similar, and no second metal ion binding site could be detected, consistent with the assumption that one metal ion is sufficient for GTP hydrolysis. In order to explain the biochemical differences, we analysed the GTPase reaction mechanism with a linear free energy relationships approach. The result suggests that the reaction mechanism is not changed with Mn2+ but that the transition metal ion Mn2+ shifts the pKa of the gamma-phosphate by almost half a unit and increases the reaction rate due to an increase in the basicity of GTP acting as the general base. This suggests that the intrinsic GTPase reaction could be an attractive target for anti-cancer drug design. By using Rap1A and Ran, we show that the acceleration of the GTPase by Mn2+ appears to be a general phenomenon of GTP-binding proteins.

Crystallization and preliminary X-ray crystallographic study of the Ras-GTPase-activating domain of human p120GAP.

Ras-GTPase-activating proteins (Ras-GAPs) are important regulators of the biological activity of Ras within the framework of intracellular communication where GTP-bound Ras (Ras:GTP) is a key signal transducing molecule (Trahey and McCormick, Science 238:542-545, 1987; Boguski and McCormick, Nature 366:643-654, 1993). By accelerating Ras-mediated GTP hydrolysis, Ras-GAPs provide an efficient means to reset the Ras-GTPase cycle to the GDP-bound 'OFF'-state and terminate the Ras-mediated signal. Here we report the crystallization of the GTPase-activating domain of the human p120GAP. The crystals-belong to the orthorhombic space group symmetry P2(1)2(1)2(1) with unit cell dimensions of a = 42.2 A, b = 55.6 A, c = 142.2 A, alpha = beta = gamma = 90 degrees. Assuming a Matthews parameter of 2.2 A3/Da, there is one molecule per asymmetric unit. Applying micro-seeding techniques, we grew large single crystals that could not be obtained by other routine methods for crystal improvement. They diffracted to a resolution of approximately 3 A using X-rays from a rotating anode generator and to better than 1.8 A in a synchrotron beam. Chemical cross-linking led to reduction of the maximum resolution but to significantly increased stability against mechanical and heavy atom stress.

Crystal structure of the GTPase-activating domain of human p120GAP and implications for the interaction with Ras.

Ras-related GTP-binding proteins function as molecular switches which cycle between GTP-bound 'on'- and GDP-bound 'off'-states. GTP hydrolysis is the common timing mechanism that mediates the return from the 'on' to the 'off'-state. It is usually slow but can be accelerated by orders of magnitude upon interaction with GTPase-activating proteins (GAPs). In the case of Ras, a major regulator of cellular growth, point mutations are found in approximately 30% of human tumours which render the protein unable to hydrolyse GTP, even in the presence of Ras-GAPs. The first structure determination of a GTPase-activating protein reveals the catalytically active fragment of the Ras-specific p120GAP (ref. 2), GAP-334, as an elongated, exclusively helical protein which appears to represent a novel protein fold. The molecule consists of two domains, one of which contains all the residues conserved among different GAPs for Ras. From the location of conserved residues around a shallow groove in the central domain we can identify the site of interaction with Ras x GTP. This leads to a model for the interaction between Ras and GAP that satisfies numerous biochemical and genetic data on this important regulatory process.

Crystal structure of the complex of UMP/CMP kinase from Dictyostelium discoideum and the bisubstrate inhibitor P1-(5'-adenosyl) P5-(5'-uridyl) pentaphosphate (UP5A) and Mg2+ at 2.2 A: implications for water-mediated specificity.

The three-dimensional structure of the UMP/CMP kinase (UK) from the slime mold Dictyostelium discoideum complexed with the specific and asymmetric bisubstrate inhibitor P1-(5'-adenosyl) P5-(5'-uridyl) pentaphosphate (UP5A) has been determined at a resolution of 2.2 A. The structure of the enzyme, which has up to 41% sequence homology with known adenylate kinases (AK), represents a closed conformation with the flexible monophosphate binding domain (NMP site) being closed over the uridyl moiety of the dinucleotide. Two water molecules were found within hydrogen-bonding distance to the uracil base. The key residue for the positioning and stabilization of those water molecules appears to be asparagine 97, a residue that is highly specific for AK-homologous UMP kinases, but is almost invariably a glutamine in adenylate kinases. Other residues in this region are highly conserved among AK-related NMP kinases. The catalytic Mg2+ ion is coordinated with octahedral geometry to four water molecules and two oxygens of the phosphate chain of UP5A but has no direct interactions with the protein. The comparison of the geometry of the UKdicty.UP5A.Mg2+ complex with the previously reported structure of the UKyeast.ADP.ADP complex [Müller-Dieckmann & Schulz (1994) J. Mol. Biol. 236, 361-367] suggests that UP5A in our structure mimics an ADP.Mg.UDP biproduct inhibitor rather than an ATP. MG.UMP bisubstrate inhibitor.

Crystallization and preliminary X-ray analysis of UMP/CMP-kinase from Dictyostelium discoideum with the specific bisubstrate inhibitor P1-(adenosine 5')-P5-(uridine 5')-pentaphosphate (UP5A).

UMP/CMP-kinase (UK) from the slime mold Dictyostelium discoideum has been purified to high homogeneity and co-crystallized with the bisubstrate inhibitor P1-(adenosine 5')-P5-(uridine 5')-pentaphosphate (UP5A). UP5A binds to UK with a dissociation constant (Kd) of 3 +/- 0.5 nM at 25 degrees C and pH 7.5. This is some 50-fold tighter than the binding of P1,P5-(diadenosine 5')-pentaphosphate (AP5A, Kd = 160 +/- 15 nM). AP5A is a bisubstrate inhibitor that is specific for adenylate kinase. The crystals have the symmetry of the tetragonal space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2. The unit cell dimensions are a = b = 78.5 A and c = 101.4 A. The crystals diffract to a Bragg spacing of 2.1 A.

Crystal structure of the nuclear Ras-related protein Ran in its GDP-bound form.

The Ran proteins constitute a distinct branch of the superfamily of Ras-related GTP-binding proteins which function as molecular switches cycling between GTP-bound 'on' and GDP-bound 'off' states. Ran is located predominantly in the nucleus of eukaryotic cells and is involved in the nuclear import of proteins as well as in control of DNA synthesis and of cell-cycle progression. We report here the crystal structure at 2.3 A resolution of human Ran (Mr 24K) complexed with GDP and Mg2+. This structure reveals a similarity with the Ras core (G-domain) but with significant variations in regions involved in GDP and Mg2+ coordination (switch I and switch II regions in Ras), suggesting that there could be major conformational changes upon GTP binding. In addition to the G-domain, an extended chain and an alpha-helix were identified at the carboxy terminus. The amino-terminal (amino-acid residues MAAQGEP) stretch and the acidic tail (DEDDDL) appear to be flexible in the crystal structure.