The crystal structure of the H‐ras oncogene protein p21 complexed to the slowly hydrolysing GTP analogue GppNp has been determined at 1.35 A resolution. 211 water molecules have been built into the electron density. The structure has been refined to a final R‐factor of 19.8% for all data between 6 A and 1.35 A. The binding sites of the nucleotide and the magnesium ion are revealed in high detail. For the stretch of amino acid residues 61‐65, the temperature factors of backbone atoms are four times the average value of 16.1 A2 due to the multiple conformations. In one of these conformations, the side chain of Gln61 makes contact with a water molecule, which is perfectly placed to be the nucleophile attacking the gamma‐phosphate of GTP. Based on this observation, we propose a mechanism for GTP hydrolysis involving mainly Gln61 and Glu63 as activating species for in‐line attack of water. Nucleophilic displacement is facilitated by hydrogen bonds from residues Thr35, Gly60 and Lys16. A mechanism for rate enhancement by GAP is also proposed.

v-Ha-ras encoded p21 protein (p21V), the cellular c-Ha-ras encoded protein (p21C) and its T24 mutant form P21T were produced in Escherichia coli under the control of the tac promoter. Large amounts of the authentic proteins in a soluble form can be extracted and purified without the use of denaturants or detergents. All three proteins are highly active in GDP binding, GTPase and, for p2lv, autokinase activity. Inhibition of [3H]GDP binding to p21C by regio-and stereospecific phosphorothioate analogs of GDP and GTP was investigated to obtain a measure of the relative affinities of the three diphosphate and five triphosphate analogs of guanosine. p21 has a preference for the Sp isomers of GDPaS and GTPaS. It has low specificity for the Sp isomer of GTP,BS. Together with the data for GDP,BS and GTP-yS these results are compared with those obtained for elongation factor (EF)-Tu and transducin. This has enabled us to probe the structural relatedness of these proteins. We conclude that p21 seems to be more closely related to EF-Tu than to transducin.

The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'-(3-thiotriphosphate) in the presence of Mg(2+) are incompatible with a simple bimolecular association process. A substrate-induced conformation change with DeltaG(0)<-24kJ.mol(-1) (i.e. DeltaG(0) could be more negative) at pH8 and 21 degrees C is proposed as the additional step in the binding of ATP. The postulated binding mechanism is M+ATPright harpoon over left harpoonM.ATPright harpoon over left harpoonM*.ATP, where the association constant for the first step, K(1), is 4.5x10(3)m(-1) at I 0.14m and the rate of isomerization is 400s(-1). In the presence of Mg(2+), ADP binds in a similar fashion to ATP, the rate of the conformation change also being 400s(-1), but with DeltaG(0) for that process being -14kJ.mol(-1). The effect of increasing ionic strength is to decrease K(1), the kinetics of the conformation change being essentially unaltered. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, although they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants.

Analysis of three different Rab-RabGEF pairs reveals that RabGEFs contain the minimal targeting machinery for recruiting Rabs to specific membranes.

The Legionella pneumophila protein AnkX that is injected into infected cells by a Type IV secretion system transfers a phosphocholine group from CDP-choline to a serine in the Rab1 and Rab35 GTPase Switch II regions. We show here that the consequences of phosphocholination on the interaction of Rab1/Rab35 with various partner proteins are quite distinct. Activation of phosphocholinated Rabs by GTP/GDP exchange factors (GEFs) and binding to the GDP dissociation inhibitor (GDI) are strongly inhibited, whereas deactivation by GTPase activating proteins (GAPs) and interactions with Rab-effector proteins (such as LidA and MICAL-3) are only slightly inhibited. We show that the Legionella protein lpg0696 has the ability to remove the phosphocholine group from Rab1. We present a model in which the action of AnkX occurs as an alternative to GTP/ GDP exchange, stabilizing phosphocholinated Rabs in membranes in the GDP form because of loss of GDI binding ability, preventing interactions with cellular GTPase effectors, which require the GTP-bound form. Generation of the GTP form of phosphocholinated Rab proteins cannot occur due to loss of interaction with cellular GEFs.

No abstract

We describe here the kinetics of the interaction of GTP and GDP with the small GTP-binding proteins Rab5 and Rab7. It was possible to make use of the intrinsic fluorescence of these proteins, since Rab5 contains two and Rab7 three tryptophan residues, respectively. With both enzymes, there is a significant decrease in fluorescence on binding GTP and an increase on binding GDP. As with the small GTP-binding protein Ha-Ras p21 and with EF-Tu, nucleotide binding occurs in at least two steps and is describable in terms of a relatively weak initial interaction followed by a highly irreversible isomerization of the protein-nucleotide complex, which results in a change in the fluorescence properties. Dissociation of GDP and GTP could be followed in a time-dependent manner using fluorescently labeled GDP (methylanthraniloyl GDP) as displacing agent and taking advantage of substantial fluorescent energy transfer from tryptophan to the nucleotide. Fluorescence techniques could also be used to quantitate the interaction of Mg 2؉ ions with the GTP and GDP forms of Rab7, and it was shown that the metal ion was bound ϳ1000-fold more strongly to the GTP than the GDP form. The rate of GTP cleavage by the two proteins differed by a factor of ϳ20 (2 ؋ 10 ؊3 s ؊1 for Rab5 and 9 ؋ 10 ؊4 s ؊1 for Rab7 at 37°C). Both proteins showed significant discrimination against xanthosine 5-O-diphosphate (K d ϳ10 3 -fold higher than that of GDP) and dramatic discrimination against ADP or ATP (K d ϳ10 6 -fold higher than that of GDP). The results demonstrate a high degree of mechanistic similarity between the Rab proteins and other GTP-binding proteins, which have been examined in detail, including Ha-Ras p21, Ran, and EF-Tu.The Rab proteins are members of the Ras superfamily of small GTP-binding proteins. They are involved in protein trafficking in the cell and have been implicated in the mechanisms by which transport vesicles identify and fuse with their target compartment (1-4). Like the other members of the Ras superfamily, and in common with other classes of GTP-binding proteins, they cycle between the GTP-and GDP-bound forms and appear to share at least some of the general properties of the family, including interaction with nucleotide exchange factors (Refs. 5-8), in some cases (and perhaps in all) with GTPaseactivating proteins (GAPs 1 ; Refs. 9 and 10) and with effector molecules. A class of molecules which appear to be critical for the role of Rab proteins are GDP dissociation inhibitors, which bind Rabs that are prenylated (doubly geranylgeranylated near the C terminus) in a specific manner and deliver them to their respective membranes, as well as retrieving them from these locations (11). A related molecule, the Rab escort protein (REP), is necessary for prenylation of Rabs (catalyzed by a geranylgeranyltransferase) and is also involved in delivery to, and retrieval of Rabs from, membrane compartments (12). Other molecules that interact with Rab proteins have also been identified. These include rabphilin-3A, which is a putative targ...

Chemotactic excitation responses to caged ligand photorelease of rapidly swimming bacteria that reverse (Vibrio alginolyticus) or tumble (Escherichia coli and Salmonella typhimurium) have been measured by computer. Mutants were used to assess the effects of abnormal motility behavior upon signal processing times and test feasibility of kinetic analyses of the signaling pathway in intact bacteria. N-1-(2-Nitrophenyl)ethoxycarbonyl-L-serine and 2-hydroxyphenyl 1-(2-nitrophenyl) ethyl phosphate were synthesized. These compounds are a 'caged' serine and a 'caged' proton and on flash photolysis release serine and protons and attractant and repellent ligands, respectively, for Tsr, the serine receptor. The product quantum yield for serine was 0.65 (+/- 0.05) and the rate of serine release was proportional to [H+] near-neutrality with a rate constant of 17 s-1 at pH 7.0 and 21 degrees C. The product quantum yield for protons was calculated to be 0.095 on 308-nm irradiation but 0.29 (+/- 0.02) on 300-350-nm irradiation, with proton release occurring at > 10(5) s-1. The pH jumps produced were estimated using pH indicators, the pH-dependent decay of the chromophoric aci-nitro intermediate and bioassays. Receptor deletion mutants did not respond to photorelease of the caged ligands. Population responses occurred without measurable latency. Response times increased with decreased stimulus strength. Physiological or genetic perturbation of motor rotation bias leading to increased tumbling reduced response sensitivity but did not affect response times. Exceptions were found. A CheR-CheB mutant strain had normal motility, but reduced response. A CheZ mutant had tumbly motility, reduced sensitivity, and increased response time to attractant, but a normal repellent response. These observations are consistent with current ideas that motor interactions with a single parameter, namely phosphorylated CheY protein, dictate motor response to both attractant and repellent stimuli. Inverse motility motor mutants with extreme rotation bias exhibited the greatest reduction in response sensitivity but, nevertheless, had normal attractant response times. This implies that control of CheY phosphate concentration rather than motor reactions limits responses to attractants.