Alfred Lautwein

MUTATIONAL AND KINETIC ANALYSES OF THE GTPASE-ACTIVATING PROTEIN (GAP)-P21 INTERACTION - THE C-TERMINAL DOMAIN OF GAP IS NOT SUFFICIENT FOR FULL ACTIVITY

The GTPase-activating protein (GAP) stimulates the GTPase reaction of p21 by 5 orders of magnitude such that the kcat of the reaction is increased to 19 s-1. Mutations of residues in loop L1 (Gly-12 and Gly-13), in loop L2 (Thr-35 and Asp-38), and in loop L4 (Gln-61 and Glu-63) influence the reaction in different ways, but all of these mutant p21 proteins still form complexes with GAP. The C-terminal domain of the human GAP gene product, GAP334, which comprises residues 714 to 1047, is 20 times less active than full-length GAP on a molar basis and has a fourfold lower affinity. This finding indicates that the N terminus of GAP containing the SH2 domains modifies the interaction between the catalytic domain and p21.

STRUCTURAL DIFFERENCES IN THE MINIMAL CATALYTIC-DOMAINS OF THE GTPASE-ACTIVATING PROTEINS P120GAP AND NEUROFIBROMIN

The kinetic properties for the enzymatic stimulation of the GTPase reaction of p21ras by the two GTPase-activating proteins (GAPs) p120GAP and neurofibromin are different. In order to understand these differences and since crystallization attempts have only been successful with truncated fragments, structure/function requirements of the catalytic core of these proteins were investigated. Differences in size of the minimal catalytic domains of these two proteins were found as determined by limited proteolysis. The minimal catalytic domain has a molecular mass of 30 kDa in the case of p120GAP and of 26 kDa in the case of neurofibromin. Both catalytic domains contain the homology boxes as well as the residues perfectly conserved among all Ras GAPs. The C termini of these fragments are identical, whereas the N-terminal part of the minimal p120GAP domain is 47 amino acids longer. These newly identified minimal catalytic fragments were as active in stimulating GTPase activity toward p21ras as the corresponding larger fragments GAP-334 and NF1-333 from which they had been generated via proteolytic digestion. Recently it was postulated that a fragment of 91 amino acids from neurofibromin located outside the conserved domain contains catalytic activity. In our hands this protein is unstable and has no catalytic activity. Thus, we believe that we have defined the true minimal domains of p120GAP (GAP-273, residues Met714-His986) and neurofibromin (NF1-230, residues Asp1248-Phe1477), which can be expressed via LMM fusion vectors in Escherichia coli and isolated in high purity.

The inhibition of the GTPase activating protein-Ha-ras interaction by acidic lipids is due to physical association of the C-terminal domain of the GTPase activating protein with micellar structures.

The effects of fatty acids and phospholipids on the interaction of the full‐length GTPase activating protein (GAP) as well as its isolated C‐terminal domain and the Ha‐ras proto‐oncogene product p21 were studied by various methods, viz. GTPase activity measurements, fluorescence titrations and gel permeation chromatography. It is shown that all fatty acids and acidic phospholipids tested, provided the critical micellar concentration and the critical micellar temperature are reached, inhibit the GAP stimulated p21 GTPase activity. This is interpreted to mean that it is not the molecular structure of acidic lipid molecules per se but rather their physical state of aggregation which is responsible for the inhibitory effect of lipids on the GTPase activity. The relative inhibitory potency of various lipids was measured under defined conditions with mixed Triton X‐100 micelles to follow the order: unsaturated fatty acids greater than saturated acids approximately phosphatidic acids greater than or equal to phosphatidylinositol phosphates much greater than phosphatidylinositol and phosphatidylserine. GTPase experiments with varying concentrations of p21 and constant concentrations of GAP and lipids indicate that the binding of GAP by the lipid micelles is responsible for the inhibition, a finding which was confirmed by fluorescence titrations and gel filtrations which show that the C‐terminal domain of GAP is bound by lipid micelles.

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 P212121with unit cell dimensions of a = 42.2 Å, b = 55.6 Å, c = 142.2 Å, α = β = γ = 90°. Assuming a Matthews parameter of 2.2 Å3/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 Å using X‐rays from a rotating anode generator and to better than 1.8 Å 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.

Crystallization and preliminary X−ray structure analysis of thermally unstable p21H−ras guanosine complexes

p21 is a small guanine nucleotide binding protein that is involved in intracellular signal transduction. Biochemical data suggest that the presence of the beta-phosphate is essential for strong binding of guanine nucleotides to the protein. Guanosine or GMP bind six orders of magnitude more weakly to p21 than GDP or GTP. Moreover, the thermal stability of the protein is dramatically reduced when bound to GMP or guanosine. We have crystallized C-terminally truncated forms of p21(H-ras), with guanosine or GMP bound, in the space groups P4(3)2(1)2, P2(1)2(1)2 and P2(1). The crystals diffract in the range 2.8-2.2 A. Details of the crystallization procedures, the characterization of the crystals and preliminary results of structure determination are described. An unexpected electron-density peak was found close to the position of the beta-phosphate in the phosphate-binding loop.

The Ras-RasGAP Complex: How to Complement an Inefficient Active Site

GTP-hydrolysis as carried out by GTP-binding proteins[1] is intrinsically very slow but can be accelerated by orders of magnitude upon interaction with GTPase Activating Proteins, GAPs, which are specific for the respective GTP-binding proteins. Focusing on p21ras (Ras), a key element in growth control and differentiation, we have used biochemical and structural methods to elucidate the mechanism of GTPase activation. An arginine side chain is supplied into the active site of Ras to contact the nucleotide and stabilize the transition state of the reaction, consistent with mutational analyses. The switch II region of Ras is stabilized by GAP-334 to allow Gln61, the mutation of which activates the oncogenic potential of Ras, to participate in catalysis. The structure provides an explanation how Gly12 and Gln61 mutations might escape regulation by GAPs.

The Three-Dimensional Structure of P21 in the Catalytically Active Conformation and Analysis of Oncogenic Mutants

The three-dimensional crystal structure of the catalytically active, GTP-analogue containing complex of H-ras encoded p21 (aa 1–166) has been determined at 1.35 A resolution. It has the same topology as the G-binding domain of elongation factor Tu. The structure analysis revealed the binding sites of the nucleotide and of the essential cofactor Mg2+ in great detail and made it possible to propose a mechanism for GTP hydrolysis. In addition to the wild-type protein, the structures of several p21 mutants have been solved. While the overall structures of these proteins are not perturbed, there are small, but significant differences at the positions of the mutated amino acids. In the oncogenic mutants (Gly-12→Arg, Gly-12→Val, Gln-61→Leu, Gln-61→His), these mutations interfere with the proposed mechanism of catalysis and thus lead to a reduced rate of GTP hydrolysis.