The X-ray crystal structure of the complex between the Ras-related protein Rap1A in the GTP-analogue (GppNHp) form and the Ras-binding domain (RBD) of the Ras effector molecule c-Raf1, a Ser/Thr-specific protein kinase, has been solved to a resolution of 2.2 A. It shows that RBD has the ubiquitin superfold and that the structure of Rap1A is very similar to that of Ras. The interaction between the two proteins is mediated by an apparent central antiparallel beta-sheet formed by strands B1-B2 from RBD and strands beta 2-beta 3 from Rap1A. Complex formation is mediated by main-chain and side-chain interactions of the so-called effector residues in the switch I region of Rap1A.

We have investigated the dynamic properties of the switch I region of the GTP-binding protein Ras by using mutants of Thr-35, an invariant residue necessary for the switch function. Here we show that these mutants, previously used as partial loss-of-function mutations in cell-based assays, have a reduced affinity to Ras effector proteins without Thr-35 being involved in any interaction. The structure of Ras(T35S)⅐GppNHp was determined by x-ray crystallography. Whereas the overall structure is very similar to wildtype, residues from switch I are completely invisible, indicating that the effector loop region is highly mobile. 31 P-NMR data had indicated an equilibrium between two rapidly interconverting conformations, one of which (state 2) corresponds to the structure found in the complex with the effectors. 31 P-NMR spectra of Ras mutants (T35S) and (T35A) in the GppNHp form show that the equilibrium is shifted such that they occur predominantly in the nonbinding conformation (state 1). On addition of Ras effectors, Ras(T35S) but not Ras(T35A) shift to positions corresponding to the binding conformation. The structural data were correlated with kinetic experiments that show two-step binding reaction of wildtype and (T35S)Ras with effectors requires the existence of a rate-limiting isomerization step, which is not observed with T35A. The results indicate that minor changes in the switch region, such as removing the side chain methyl group of Thr-35, drastically affect dynamic behavior and, in turn, interaction with effectors. The dynamics of the switch I region appear to be responsible for the conservation of this threonine residue in GTP-binding proteins.A n estimated 60-100 different GTP-binding proteins of the Ras superfamily belonging to different subfamilies have been identified and shown to regulate a diverse array of signal transduction reactions and͞or transport processes. These small GTP-binding proteins, but also the larger G␣ proteins or the protein synthesis elongation factors, contain conserved signature motifs in the primary sequence. Accordingly, the structures of many of these proteins show a common structural core called the G domain, an ␣͞␤ fold consisting of six ␤ strands and five ␣ helices (1). The common structural core is modified by insertions and͞or additional structural elements. The structures of several proteins have been solved in both the GDP-and GTP-bound form, which showed that the conformational change is mostly confined to the loop L2-␤2 (by using the Ras nomenclature) and the ␤3͞␣2 regions, which have accordingly been called switches I and II (2). It turned out that the active site of the GDP-bound conformations shows large variations, whereas the triphosphate structures are very similar (3). It was also deduced from these studies that the conformational change is triggered when two hydrogen bonds to the ␥-phosphate from switches I and II, involving invariant Thr and Gly residues, respectively, are released after GTP hydrolysis (3). NMR structural studies have shown that the swit...

31P NMR revealed that the complex of p21ras with the GTP analog GppNHp.Mg2+ exists in two conformational states, states 1 and 2. In wild-type p21ras the equilibrium constant K1(12) between the two states is 1.09. The population of these states is different for various mutants but independent of temperature. The activation enthalpy delta H ++ and activation entropy delta S ++ for the conformational transitions were determined by full-exchange matrix analysis for wild-type p21ras and p21ras(S65P). For the wild-type protein one obtains delta H ++ = 89 +/- 2 kJ mol-1 and delta S ++ = 102 +/- 20 J mol-1 K-1 and for the mutant protein delta H ++ = 93 +/- 7 kJ mol-1 and delta S ++ = 138 +/- 30 J mol-1 K-1. The study of various p21ras mutants suggests that the two states correspond to different conformations of loop L2, with Tyr-32 in two different positions relative to the bound nucleotide. High-field EPR at 95 GHz suggest that the observed conformational transition does not directly influence the coordination sphere of the protein-bound metal ion. The influence of this transition on loop L4 was studied by 1H NMR with mutants E62H and E63H. There was no indication that L4 takes part in the transition described in L2, although a reversible conformational change could be induced by decreasing the pH value. The exchange between the two states is slow on the NMR time scale (< 10 s-1): at approximately pH 5 the population of the two states is equal. The interaction of p21ras-triphosphate complexes with the Ras-binding domain (RBD) of the effector protein c-Raf-1, Raf-RBD, and with the GTPase activating protein GAP was studied by 31P NMR spectroscopy. In complex with Raf-RBD the second conformation of p21ras (state 2) is stabilized. In this conformation Tyr-32 is located in close proximity to the phosphate groups of the nucleotide, and the beta-phosphate resonance is shifted upfield by 0.7 ppm. Spectra obtained in the presence of GAP suggest that in the ground state GAP does not interact directly with the nucleotide bound to p21ras and does not induce larger conformational changes in the neighborhood of the nucleotide. The experimental data are consistent with a picture where GAP accelerates the exchange process between the two states and simultaneously increases the population of state 1 at higher temperature.

Polyphosphate (polyP) occurs ubiquitously in cells, but its functions are poorly understood and its synthesis has only been characterized in bacteria. Using x-ray crystallography, we identified a eukaryotic polyphosphate polymerase within the membrane-integral vacuolar transporter chaperone (VTC) complex. A 2.6 angstrom crystal structure of the catalytic domain grown in the presence of adenosine triphosphate (ATP) reveals polyP winding through a tunnel-shaped pocket. Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains. Mutational analysis of the transmembrane domain indicates that VTC may integrate cytoplasmic polymer synthesis with polyP membrane translocation. Identification of the polyP-synthesizing enzyme opens the way to determine the functions of polyP in lower eukaryotes.