Structure-function relationships in a bacterial DING protein

A recombinant DING protein from Pseudomonas fluorescens has been previously shown to have a phosphate-binding site, and to be mitogenic for human cells. Here we report the three-dimensional structure of the protein, confirming a close similarity to the "Venus flytrap" structure seen in other human and bacterial phosphate-binding proteins. Site-directed mutagenesis confirms the role of a key residue involved in phosphate binding, and that the mitogenic activity is not dependent on this property. Deletion of one of the two hinged domains that constitute the Venus flytrap also eliminates phosphate binding whilst enhancing mitogenic activity.     

Functional attributes of the phosphate group binding cup of pyridoxal phosphate-dependent enzymes

Twenty-four structures of pyridoxal-5'-phosphate (PLP)-dependent enzymes that represent five different folds are shown to share a common recognition pattern for the phosphate group of their PLP-ligands. All atoms that interact with the phosphate group of PLP in these proteins are organized within a two-layer structure so that the first interacting layer contains from five to seven atoms and parallel with this is a second layer containing from three to seven interacting atoms. In order to identify features of the phosphate-binding site common to PLP-dependent enzymes, a simple procedure is described that assigns relative positions to all interacting atoms unambiguously, such that the networks of interactions for different proteins can be compared. On the basis of these diagrams for 24 enzyme-cofactor complexes, a detailed comparison of the two-layer structures of PLP-dependent enzymes, with both similar and different folds, was made. A majority of the structurally defined PLP-dependent proteins use the same atom types in analogous "key" positions to bind their PLP-ligands. In some instances, proteins use water molecules when a key position is unoccupied. A similar two-layer recognition pattern extends to protein recognition of at least one other, non-PLP ligand, glucosamine 6-phosphate. We refer to this three-dimensional recognition pattern as the phosphate-binding cup. In general, the phosphate-binding cup provides a very stable anchoring point for PLP. When numerous water molecules occur within the cup, however, then the phosphate group of PLP participates directly in the enzymatic reactions with inorganic phosphate replacing the water molecules of the cup. With glucosamine-6-phosphate synthase, the water molecules of the phosphate-binding cup facilitate the entry of substrate and the exit of product.     

Mechanism of elongation factor (EF)-Ts-catalyzed nucleotide exchange in EF-Tu. Contribution of contacts at the guanine base.

Nucleotide exchange in elongation factor Tu (EF-Tu) is catalyzed by elongation factor Ts (EF-Ts). Similarly to other GTP-binding proteins, the structural changes in the P loop and the Mg(2+) binding site are known to be important for nucleotide release from EF-Tu. In the present paper, we determine the contribution of the contacts between helix D of EF-Tu at the base side of the nucleotide and the N-terminal domain of EF-Ts to the catalysis. The rate constants of the multistep reaction between Escherichia coli EF-Tu, EF-Ts, and GDP were determined by stopped-flow kinetic analysis monitoring the fluorescence of either Trp-184 in EF-Tu or mant-GDP. Mutational analysis shows that contacts between helix D of EF-Tu and the N-terminal domain of EF-Ts are important for both complex formation and the acceleration of GDP dissociation. The kinetic results suggest that the initial contact of EF-Ts with helix D of EF-Tu weakens binding interactions around the guanine base, whereas contacts of EF-Ts with the phosphate binding side that promotes the release of the phosphate moiety of GDP appear to take place later. This "base-side-first" mechanism of guanine nucleotide release resembles that found for Ran x RCC1 and differs from mechanisms described for other GTPase x GEF complexes where interactions at the phosphate side of the nucleotide are released first.     

Model of the ran-RCC1 interaction using biochemical and docking experiments.

RCC1, the regulator of chromosome condensation, is the guanine nucleotide exchange factor (GEF) for the nuclear Ras-like GTP-binding protein Ran. Its structure was solved by X-ray crystallography and revealed a seven-bladed beta-propeller, one side of which was proposed to be the interaction site with Ran. To gain more insight into this interaction, alanine mutagenesis studies were performed on conserved residues on the surface of the structure. Purified mutant proteins were analysed by steady-state kinetic analysis of their GEF activities towards Ran. A number of residues were identified whose mutation affected either the KMor kcatof the overall reaction, or had no effect. Mutants were further analysed by plasmon surface resonance in order to get more information on individual steps of the complex reaction pathway. Ran-GDP was coupled to the sensor chip and reacted with RCC1 mutants to categorise them into different groups, demonstrating the usefulness of plasmon surface resonance in the study of complex multi-step kinetic processes. A docking solution of Ran-RCC1 structures in combination with sequence analysis allows prediction of the site of interaction between RCC1 and Ran and proposes a model for the Ran-RCC1 structure which corresponds to and extends the biochemical data. Three invariant residues which most severely affect the kcatof the reaction, D128, D182 and H304, are located in the centre of the Ran-RCC1 interface and interfere with switch II and the phosphate binding area. The structural model suggests that different guanine nucleotide exchange factors use a similar interaction site on their respective GTP-binding proteins, but that the molecular mechanisms for the release of nucleotides are likely to be different.     

The GTP-binding peptide of beta-tubulin. Localization by direct photoaffinity labeling and comparison with nucleotide-binding proteins

The binding site of the guanine moiety of GTP on beta-tubulin was located within the peptide consisting of residues 63-77, AILVDLEPGTMDSVR. The result was obtained using direct photoaffinity labeling, peptide sequencing, and limited proteolysis. Peptides were identified by end-labeling with a monoclonal antibody against beta-tubulin whose epitope was located between 3 and 4 kDa from the C terminus. The sequence of the GTP-binding site is consistent with predictions from other GTP-binding proteins such as elongation factor Tu or ras p21.     

Transient kinetic investigation of GTP hydrolysis catalyzed by interferon-gamma-induced hGBP1 (human guanylate binding protein 1)

Within the family of large GTP-binding proteins, human guanylate binding protein 1 (hGBP1) belongs to a subgroup of interferon-inducible proteins. GTP hydrolysis activity of these proteins is much higher compared with members of other GTPase families and underlies mechanisms that are not understood. The large GTP-binding proteins form self-assemblies that lead to stimulation of the catalytic activity. The unique result of GTP hydrolysis catalyzed by hGBP1 is GDP and GMP. We investigated this reaction mechanism by transient kinetic methods using radioactively labeled GTP as well as fluorescent probes. Substrate binding and formation of the hGBP1 homodimer are fast as no lag phase is observed in the time courses of GTP hydrolysis. Instead, multiple turnover experiments show a rapid burst of P(i) formation prior to the steady state phase, indicating a rate-limiting step after GTP cleavage. Both molecules are catalytically active and cleave off a phosphate ion in the first step. Then bifurcation into catalytic inactivation, probably by irreversible dissociation of the dimer, and into GDP hydrolysis is observed. The second cleavage step is even faster than the first step, implying a rapid rearrangement of the nucleotide within the catalytic center of hGBP1. We could also show that the release of the products, including the phosphate ions, is fast and not limiting the steady state activity. We suggest that slow dissociation of the GMP-bound homodimer gives rise to the burst behavior and controls the steady state. The assembled forms of the GDP- and GMP-bound states of hGBP1 are accessible only through GTP binding and hydrolysis and achieve a lifetime of a few seconds.     

Phosphate binding sites identification in protein structures

Nearly half of known protein structures interact with phosphate-containing ligands, such as nucleotides and other cofactors. Many methods have been developed for the identification of metal ions-binding sites and some for bigger ligands such as carbohydrates, but none is yet available for the prediction of phosphate-binding sites. Here we describe Pfinder, a method that predicts binding sites for phosphate groups, both in the form of ions or as parts of other non-peptide ligands, in proteins of known structure. Pfinder uses the Query3D local structural comparison algorithm to scan a protein structure for the presence of a number of structural motifs identified for their ability to bind the phosphate chemical group. Pfinder has been tested on a data set of 52 proteins for which both the apo and holo forms were available. We obtained at least one correct prediction in 63% of the holo structures and in 62% of the apo. The ability of Pfinder to recognize a phosphate-binding site in unbound protein structures makes it an ideal tool for functional annotation and for complementing docking and drug design methods. The Pfinder program is available at http://pdbfun.uniroma2.it/pfinder.     

DING proteins; novel members of a prokaryotic phosphate-binding protein superfamily which extends into the eukaryotic kingdom

PstS proteins are the cell-bound phosphate-binding elements of the ubiquitous bacterial ABC phosphate uptake mechanisms. Primary and tertiary structures, characteristic of pstS proteins, are conserved in proteins, which are expressed in secretory operons and induced by phosphate deprivation, in Pseudomonas species. There are two subsets of these proteins; AP proteins, which are alkaline phosphatases, and DING proteins, named for their N-terminal sequence, which are phosphate-binding proteins. Both form elements of a proposed phosphate-scavenging system in pseudomonads. DING proteins have also been isolated from many eukaryotic sources, and are associated with both normal and pathological functions in mammals. Their phosphate-binding function suggests a role in biomineralization, but the ability to bind other ligands may be related to signal transduction in eukaryotes. Though it has been claimed that all such proteins may originate from pseudomonads, many eukaryotic DING proteins have unique features which are incompatible with a bacterial origin.     

Towards a structural classification of phosphate binding sites in protein-nucleotide complexes: an automated all-against-all structural comparison using geometric matching

A method is described for the rapid comparison of protein binding sites using geometric matching to detect similar three-dimensional structure. The geometric matching detects common atomic features through identification of the maximum common sub-graph or clique. These features are not necessarily evident from sequence or from global structural similarity giving additional insight into molecular recognition not evident from current sequence or structural classification schemes. Here we use the method to produce an all-against-all comparison of phosphate binding sites in a number of different nucleotide phosphate-binding proteins. The similarity search is combined with clustering of similar sites to allow a preliminary structural classification. Clustering by site similarity produces a classification of binding sites for the 476 representative local environments producing ten main clusters representing half of the representative environments. The similarities make sense in terms of both structural and functional classification schemes. The ten main clusters represent a very limited number of unique structural binding motifs for phosphate. These are the structural P-loop, di-nucleotide binding motif [FAD/NAD(P)-binding and Rossman-like fold] and FAD-binding motif. Similar classification schemes for nucleotide binding proteins have also been arrived at independently by others using different methods.     

R-loop-dependent hypernegative supercoiling in Escherichia coli topA mutants preferentially occurs at low temperatures and correlates with growth inhibition

hGBP1 is a GTPase with antiviral activity encoded by an interferon- activated human gene. Specific binding of hGBP1 to guanine nucleotides has been established although only two classical GTP-binding motifs were found in its primary sequence. The unique position of hGBP1 amongst known GTPases is further demonstrated by the hydrolysis of GTP to GDP and GMP. Although subsequent cleavage of orthophosphates rather than pyrophosphate was demonstrated, GDP coming from bulk solution cannot serve as a substrate. The relation of guanine nucleotide binding and hydrolysis to the antiviral function of hGBP1 is unknown. Here we show similar binding affinities for all three guanine nucleotides and the ability of both products, GDP and GMP, to compete with GTP binding. Fluorimetry and isothermal titration calorimetry were applied to prove that only one nucleotide binding site is present in hGBP1. Furthermore, we identified the third canonical GTP-binding motif and verified its role in nucleotide recognition by mutational analysis. The high guanine nucleotide dissociation rates measured by stopped-flow kinetics are responsible for the weak affinities to hGBP1 when compared to other GTPases like Ras or Galpha. By means of fluorescence and NMR spectroscopy it is demonstrated that aluminium fluoride forms a complex with hGBP1 only in the GDP state, presumably mimicking the transition state of GTP hydrolysis. Tentatively, the involvement of a GAP domain in hGBP1 in GTP hydrolysis is suggested. These results will serve as a basis for the determination of the differential biological functions of the three nucleotide states and for the elucidation of the unique mechanism of nucleotide hydrolysis catalysed by hGBP1.     

Tryptophan-free human PNP reveals catalytic site interactions

Human purine nucleoside phosphorylase (PNP) is a homotrimer, containing three nonconserved tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The Trp residues were replaced with Tyr to produce Trp-free PNP (Leuko-PNP). Leuko-PNP showed near-normal kinetic properties. It was used (1) to determine the tautomeric form of guanine that produces strong fluorescence when bound to PNP, (2) for thermodynamic binding analysis of binary and ternary complexes with substrates, (3) in temperature-jump perturbation of complexes for evidence of multiple conformational complexes, and (4) to establish the ionization state of a catalytic site tyrosine involved in phosphate nucleophile activation. The (13)C NMR spectrum of guanine bound to Leuko-PNP, its fluorescent properties, and molecular orbital electronic transition analysis establish that its fluorescence originates from the lowest singlet excited state of the N1H, 6-keto, N7H guanine tautomer. Binding of guanine and phosphate to PNP and Leuko-PNP are random, with decreased affinity for formation of ternary complexes. Pre-steady-state kinetics and temperature-jump studies indicate that the ternary complex (enzyme-substrate-phosphate) forms in single binding steps without kinetically significant protein conformational changes as monitored by guanine fluorescence. Spectral changes of Leuko-PNP upon phosphate binding establish that the hydroxyl of Tyr88 is not ionized to the phenolate anion when phosphate is bound. A loop region (residues 243-266) near the purine base becomes highly ordered upon substrate/inhibitor binding. A single Trp residue was introduced into the catalytic loop of Leuko-PNP (Y249W-Leuko-PNP) to determine effects on catalysis and to introduce a fluorescence catalytic site probe. Although Y249W-Leuko-PNP is highly fluorescent and catalytically active, substrate binding did not perturb the fluorescence. Thermodynamic boxes, constructed to characterize the binding of phosphate, guanine, and hypoxanthine to native, Leuko-, and Y249W-Leuko-PNPs, establish that Leuko-PNP provides a versatile protein scaffold for introduction of specific Trp catalytic site probes.     

Comparison of the computed structures for the phosphate-binding loop of the p21 protein containing the oncogenic site Gly 12 with the X-ray crystallographic structures for this region in the p21 protein and EFtu. A model for the structure of the p21 protein in its oncogenic form

The GTP-binding p21 protein encoded by the ras-oncogene can be activated to cause malignant transformation of cells by substitution of a single amino acid at critical positions along the polypeptide chain. Substitution of any non-cyclic L-amino acid for Gly 12 in the normal protein results in a transforming protein. This substitution occurs in a hydrophobic sequence (residues 6-15) which is known to be involved in binding the phosphate moities of GTP (and GDP). We find, using conformational energy calculations, that the 6-15 segment of the normal protein (with Gly 12) adopts structures that contain a bend at residues 11 and 12 with the Gly in the D* conformation, not allowed energetically for L-amino acids. Substitution of non-cyclic L-amino acids for Gly 12 results in shifting this bend to residues 12 and 13. We show that many computed structures for the Gly 12-containing phosphate binding loop, segment 9-15, are superimposable on the corresponding segment of the recently determined X-ray crystallographic structure for residues 1-171 of the p21 protein. All such structures contain bends at residues 11 and 12 and most of these contain Gly 12 in the C* or D* conformational state. Other computed conformations for the 9-15 segment were superimposable on the structure of the corresponding 18-23 segment of EFtu, the bacterial chain elongation factor having structural similarities to the p21 protein in the phosphate-binding regions. This segment contains a Val residue where a Gly occurs in the p21 protein. As previously predicted, all of these superimposable conformations contain a bend at positions 12 and 13, not 11 and 12. If these structures that are superimposable on EFtu are introduced into the p21 protein structure, bad contacts occur between the sidechain of the residue (here Val) at position 12 and another phosphate binding loop region around position 61. These bad contacts between the two segments can be removed by changing the conformation of the 61 region in the p21 protein to the corresponding position of the homologous region in EFtu. In this new conformation, a large site becomes available for the binding of phosphate residues. In addition, such phenomena as autophosphorylation of the p21 protein by GTP can be explained with this new model structure for the activated protein which cannot be explained by the structure for the non-activated protein.     

Prevention of the agonist binding to gamma-aminobutyric acid B receptors by guanine nucleotides and islet-activating protein, pertussis toxin, in bovine cerebral cortex. Possible coupling of the toxin-sensitive GTP-binding proteins to receptors

Using the membranes treated with Triton X-100, we studied the interaction between gamma-aminobutyric acid (GABA)B receptors and the GTP-binding proteins which are the substrates for ADP-ribosylation by the islet-activating protein (IAP), pertussis toxin. The addition of guanine nucleotides to the membranes markedly decreased the binding of GABA to GABAB receptors. Preincubation of the membranes with IAP plus NAD caused ADP-ribosylation of the 41,000- and 39,000-Da proteins selectively and decreased GABA binding to GABAB receptors in a time- and dose-dependent manner. This decrease of binding appeared to be due to the reduction of receptor affinity for agonist. The GTP-binding proteins which are ADP-ribosylated by IAP were purified from the membrane fraction of bovine cerebral cortex. The addition of the purified GTP-binding proteins to IAP-treated membranes restored the high affinity binding of GABA to GABAB receptor. The two GTP-binding proteins which were resolved by octyl-Sepharose column chromatography showed similar efficacy in restoring GABA binding. Thus, GABAB receptors are coupled to GTP-binding proteins, IAP-specific substrates, in the brain membranes.     

Alanine scan mutagenesis of the switch I domain of the Caulobacter crescentus CgtA protein reveals critical amino acids required for in vivo function

The Caulobacter crescentus CgtA protein is a member of the Obg/GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP and displays rapid exchange rate constants for either nucleotide, indicating that the guanine nucleotide-binding and exchange properties of CgtA are different from those of the well-characterized Ras-like GTP-binding proteins. The Obg/GTP1 proteins share sequence similarity along the putative effector-binding domain. In this study, we examined the functional consequences of altering amino acid residues within this conserved domain, and identified that T193 was critical for CgtA function. The in vitro binding, exchange and GTP hydrolysis of the T192A, T193A and T192AT193A mutant proteins was examined using fluorescent guanine nucleotide analogues (mant-GDP and mant-GTP). Substitution of either T192 and/or T193 for alanine modestly reduced binding to GDP and significantly reduced the binding affinity for GTP. Furthermore, the T193A mutant protein was more severely impaired for binding GTP than the T192A mutant. The T193A mutation appeared to account solely for the impaired GTP binding of the T192AT193A double mutation. This is the first report that demonstrates that a confirmed defect in guanine nucleotide binding and GTP hydrolysis of an Obg-like protein results in the lack of function in vivo.     

Reconstitution of the muscarinic acetylcholine receptor. Guanine nucleotide-sensitive high affinity binding of agonists to purified muscarinic receptors reconstituted with GTP-binding proteins (Gi and Go)

Muscarinic acetylcholine receptors purified from porcine brain were reconstituted with two kinds of GTP-binding proteins (Gi and Go). The binding of agonists was affected by guanine nucleotides when the receptor was reconstituted with either Gi or Go, but not in the absence of one of the GTP-binding proteins. The displacement curves with agonists for the [3H]quinuclidinyl benzylate [( 3H]QNB) binding were explained by assuming there are two sites with different affinities for a given agonist. The proportion of the high affinity site increased with increasing concentrations of the GTP-binding proteins, and the maximum value represented 50-70% of the total [3H]QNB-binding sites. Reconstitution of the receptor with both Gi and Go did not increase the proportion any further. These results indicate that Gi and Go interact with the same site, which rules out the possibility that there are two kinds of muscarinic receptors, one interacting with Gi and the other with Go. GDP as well as GTP decreased the affinity for the agonists of the muscarinic receptors reconstituted with Gi or Go. The conversion of GDP to GTP during the incubation was less than 1%, indicating that the effect of GDP is not due to its conversion to GTP, and that the binding of either GTP or GDP with the GTP-binding proteins suppresses their interaction with the receptor.     

The P-loop--a common motif in ATP- and GTP-binding proteins

Many ATP- and GTP-binding proteins have a phosphate-binding loop (P-loop), the primary structure of which typically consists of a glycine-rich sequence followed by a conserved lysine and a serine or threonine. The three-dimensional structures of several ATP- and GTP-binding proteins containing P-loops have now been solved. In this review current knowledge of P-loops is discussed with the additional aim of illustrating the fascinating relationship between protein sequence, structure and function.     

The VPS1 protein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with two functionally separable domains

The product of the VPS1 gene, Vps1p, is required for the sorting of soluble vacuolar proteins in the yeast Saccharomyces cerevisiae. We demonstrate here that Vps1p, which contains a consensus tripartite motif for guanine nucleotide binding, is capable of binding and hydrolyzing GTP. Vps1p is a member of a subfamily of large GTP-binding proteins whose members include the vertebrate Mx proteins, the yeast MGM1 protein, the Drosophila melanogaster shibire protein, and dynamin, a bovine brain protein that bundles microtubules in vitro. Disruption of microtubules did not affect the fidelity or kinetics of vacuolar protein sorting, indicating that Vps1p function is not dependent on microtubules. Based on mutational analyses, we propose a two-domain model for Vps1p function. When VPS1 was treated with hydroxylamine, half of all mutations isolated were found to be dominant negative with respect to vacuolar protein sorting. All of the dominant-negative mutations analyzed further mapped to the amino-terminal half of Vps1p and gave rise to full-length protein products. In contrast, recessive mutations gave rise to truncated or unstable protein products. Two large deletion mutations in VPS1 were created to further investigate Vps1p function. A mutant form of Vps1p lacking the carboxy-terminal half of the protein retained the capacity to bind GTP and did not interfere with sorting in a wild-type background. A mutant form of Vps1p lacking the entire GTP-binding domain interfered with vacuolar protein sorting in wild-type cells. We suggest that the amino-terminal domain of Vps1p provides a GTP-binding and hydrolyzing activity required for vacuolar protein sorting, and the carboxy-terminal domain mediates Vps1p association with an as yet unidentified component of the sorting apparatus.     

Nucleolar trafficking of nucleostemin family proteins: common versus protein-specific mechanisms

The nucleolus has begun to emerge as a subnuclear organelle capable of modulating the activities of nuclear proteins in a dynamic and cell type-dependent manner. It remains unclear whether one can extrapolate a rule that predicts the nucleolar localization of multiple proteins based on protein sequence. Here, we address this issue by determining the shared and unique mechanisms that regulate the static and dynamic distributions of a family of nucleolar GTP-binding proteins, consisting of nucleostemin (NS), guanine nucleotide binding protein-like 3 (GNL3L), and Ngp1. The nucleolar residence of GNL3L is short and primarily controlled by its basic-coiled-coil domain, whereas the nucleolar residence of NS and Ngp1 is long and requires the basic and the GTP-binding domains, the latter of which functions as a retention signal. All three proteins contain a nucleoplasmic localization signal (NpLS) that prevents their nucleolar accumulation. Unlike that of the basic domain, the activity of NpLS is dynamically controlled by the GTP-binding domain. The nucleolar retention and the NpLS-regulating functions of the G domain involve specific residues that cannot be predicted by overall protein homology. This work reveals common and protein-specific mechanisms underlying the nucleolar movement of NS family proteins.