Crystal structure of a xylulose 5-phosphate phosphoketolase. Insights into the substrate specificity for xylulose 5-phosphate.

Phosphoketolases (PK) are TPP-dependent enzymes which play essential roles in carbohydrate metabolism of numerous bacteria. Depending on the substrate specificity PKs can be subdivided into xylulose 5-phosphate (X5P) specific PKs (XPKs) and PKs which accept both X5P and fructose 6-phosphate (F6P) (XFPKs). Despite their key metabolic importance, so far only the crystal structures of two XFPKs have been reported. There are no reported structures for any XPKs and for any complexes between PK and substrate. One of the major unknowns concerning PKs mechanism of action is related to the structural determinants of PKs substrate specificity for X5P or F6P. We report here the crystal structure of XPK from Lactococcus lactis (XPK-Ll) at 2.1 Šresolution. Using small angle X-ray scattering (SAXS) we proved that XPK-Ll is a dimer in solution. Towards better understanding of PKs substrate specificity, we performed flexible docking of TPP-X5P and TPP-F6P on crystal structures of XPK-Ll, two XFPKs and transketolase (TK). Calculated structure-based binding energies consistently support XPK-Ll preference for X5P. Analysis of structural models thus obtained show that substrates adopt moderately different conformation in PKs active sites following distinct networks of polar interactions. Based on the here reported structure of XPK-Ll we propose the most probable amino acid residues involved in the catalytic steps of reaction mechanism. Altogether our results suggest that PKs substrate preference for X5P or F6P is the outcome of a fine balance between specific binding network and dissimilar catalytic residues depending on the enzyme (XPK or XFPK) - substrate (X5P or F6P) couples.

Inhibition of endosome fusion by wortmannin persists in the presence of activated Rab5.

Rab5-dependent endosome fusion is sensitive to the phosphoinositide 3-kinase inhibitor, wortmannin. It has been proposed that phosphoinositide 3-kinase activity may be required for activation of rab5 by influencing its nucleotide cycle such as to promote its active GTP state. In this report we demonstrate that endosome fusion remains sensitive to wortmannin despite preloading of endosomes with stimulatory levels of a GTPase-defective mutant rab5(Q79L) or of a xanthosine triphosphate-binding mutant, rab5(D136N), in the presence of the nonhydrolysable analogue XTPgammaS. These results suggest that activation of rab5 cannot be the principal function of the wortmannin-sensitive factor on the endosome fusion pathway. This result is extrapolated to all GTPases by demonstrating that endosome fusion remains wortmannin sensitive despite prior incubation with the nonhydrolysable nucleotide analogue GTPgammaS. Consistent with these results, direct measurement of clathrin-coated vesicle-stimulated nucleotide dissociation from exogenous rab5 was insensitive to the presence of wortmannin. A large excess of rab5(Q79L), beyond levels required for maximal stimulation of the fusion assay, afforded protection against wortmannin inhibition, and partial protection was also observed with an excess of wild-type rab5 independent of GTPgammaS.

The pre-hydrolysis state of p21(ras) in complex with GTP: new insights into the role of water molecules in the GTP hydrolysis reaction of ras-like proteins.

BACKGROUND: In numerous biological events the hydrolysis of guanine triphosphate (GTP) is a trigger to switch from the active to the inactive protein form. In spite of the availability of several high-resolution crystal structures, the details of the mechanism of nucleotide hydrolysis by GTPases are still unclear. This is partly because the structures of the proteins in their active states had to be determined in the presence of non-hydrolyzable GTP analogues (e.g. GppNHp). Knowledge of the structure of the true Michaelis complex might provide additional insights into the intrinsic protein hydrolysis mechanism of GTP and related nucleotides. RESULTS: The structure of the complex formed between p21(ras) and GTP has been determined by X-ray diffraction at 1.6 A using a combination of photolysis of an inactive GTP precursor (caged GTP) and rapid freezing (100K). The structure of this complex differs from that of p21(ras)-GppNHp (determined at 277K) with respect to the degree of order and conformation of the catalytic loop (loop 4 of the switch II region) and the positioning of water molecules around the gamma-phosphate group. The changes in the arrangement of water molecules were induced by the cryo-temperature technique. CONCLUSIONS: The results shed light on the function of Gln61 in the intrinsic GTP hydrolysis reaction. Furthermore, the possibility of a proton shuffling mechanism between two attacking water molecules and an oxygen of the gamma-phosphate group can be proposed for the basal GTPase mechanism, but arguments are presented that render this protonation mechanism unlikely for the GTPase activating protein (GAP)-activated GTPase.

High-resolution crystal structure of S. cerevisiae Ypt51(DeltaC15)-GppNHp, a small GTP-binding protein involved in regulation of endocytosis.

Ypt/Rab proteins are membrane-associated small GTP-binding proteins which play a central role in the coordination, activation and regulation of vesicle-mediated transport in eukaryotic cells. We present the 1.5 A high-resolution crystal structure of Ypt51 in its active, GppNHp-bound conformation. Ypt51 is an important regulator involved in the endocytic membrane traffic of Saccharomyces cerevisiae. The structure reveals small but significant structural differences compared with H-Ras p21. The effector loop and the catalytic loop are well defined and stabilized by extensive hydrophobic interactions. The switch I and switch II regions form a well-defined epitope for hypothetical effector protein binding. Sequence comparisons between the different isoforms Ypt51, Ypt52 and Ypt53 provide the first insights into determinants for specific effector binding and for fine-tuning of the intrinsic GTP-hydrolysis rate.

Crystal structure of PTP-SL/PTPBR7 catalytic domain: implications for MAP kinase regulation.

Protein tyrosine phosphatases PTP-SL and PTPBR7 are isoforms belonging to cytosolic membrane-associated and to receptor-like PTPs (RPTPs), respectively. They represent a new family of PTPs with a major role in activation and translocation of MAP kinases. Specifically, the complex formation between PTP-SL and ERK2 involves an unusual interaction leading to the phosphorylation of PTP-SL by ERK2 at Thr253 and the inactivating dephosphorylation of ERK2 by PTP-SL. This interaction is strictly dependent upon a kinase interaction motif (KIM) (residues 224-239) situated at the N terminus of the PTP-SL catalytic domain. We report the first crystal structure of the catalytic domain for a member of this family (PTP-SL, residues 254-549, identical with residues 361-656 of PTPBR7), providing an example of an RPTP with single cytoplasmic domain, which is monomeric, having an unhindered catalytic site. In addition to the characteristic PTP-core structure, PTP-SL has an N-terminal helix, possibly orienting the KIM motif upon interaction with the target ERK2. An unusual residue in the catalytically important WPD loop promotes formation of a hydrophobically and electrostatically stabilised clamp. This could induce increased rigidity to the WPD loop and therefore reduced catalytic activity, in agreement with our kinetic measurements. A docking model based on the PTP-SL structure suggests that, in the complex with ERK2, the phosphorylation of PTP-SL should be accomplished first. The subsequent dephosphorylation of ERK2 seems to be possible only if a conformational rearrangement of the two interacting partners takes place.

X-ray crystal structure analysis of the catalytic domain of the oncogene product p21H-ras complexed with caged GTP and mant dGppNHp.

The X-ray structures of the 1:1 complexes formed between p21H-ras (residues 1 to 166) and the nucleotides P3-1-(2-nitrophenyl)ethyl guanosine triphosphate ("caged GTP"; pure R- and S-diastereomers) and 3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-(beta, gamma-imido)-triphosphate ("mant dG-ppNHp"), have been refined to an R-factor of 21.4% (R-caged GTP, 1.85 A resolution), 18.9% (S-caged GTP, 2.5 A resolution) and 17.6% (mant dGppNHp, 2.7 A resolution), respectively. Details of the structure determination, refinement and the structures themselves are presented. The overall structures of the complexes are identical in terms of the general organization of their secondary structure elements and are also identical to that reported for the analogous complex of p21H-ras with GppNHp. The binding of the GTP part is not significantly affected by the additional aromatic group (cage and mant, respectively) in contrast to the original observation on p21:caged GTP using the racemic mixture of R- and S-caged GTP. The main differences in the structures are observed in the region of loop L2 (residues Glu31 to Thr35) where the additional aromatic group attached to the nucleotide comes very close to the side-chain of Tyr32, including backbone displacements of 2.6 A, 2.2 A and 0.3 A for the residues from Glu31 to Thr35 for R-caged, S-caged GTP and mant dGppNHp, respectively. The refined structures provide additional data for the design of new nucleotide analogs and the importance of their stereochemistry as well as for the design of new mutant forms of p21H-ras for further biochemical investigations. The binding mode of mant dGppNHp reveals significant features for the understanding of the fluorescence signals observed in solution.

Vps9, Rabex-5 and DSS4: proteins with weak but distinct nucleotide-exchange activities for Rab proteins.

The activities of three Rab-specific factors with GDP/GTP exchange activity, Vps9p, Rabex-5 and DSS4, with their cognate GTPases, Ypt51p, Rab5 and Ypt1p, have been analysed quantitatively. In contrast to other exchange factors examined and to DSS4, Vps9p, and by analogy probably Rabex-5, have considerably lower affinity than GDP to the respective GTPases. In keeping with this, they are relatively weak exchangers, with a maximal rate constant for GDP release from the ternary complex between exchange factor, GTPase and GDP of ca 0.01 s(-1), which is several orders of magnitude lower than for other exchange factors examined. If interaction with these proteins is a mandatory aspect of the Rab cycle, this suggests that the overall rate of cycling might be controlled at this point of the cycle. Surprisingly, DSS4, which has the thermodynamic potential to displace GDP effectively from Ypt1p, also does this very slowly, again with a maximal rate constant of ca 0.01 s(-1). An additional, and based on present knowledge, unique, feature of the Ypt1p.DSS4 complex, is that the association of GTP (or GDP) is more than 10(3)-fold slower than to Ypt1p, thus leading to a long life-time of the binary complex between the two proteins, even at the high nucleotide concentrations that prevail in the cell. This leads to the conclusion that the protein-protein complex is likely to have an important biological significance in addition to its probable role in GTP/GDP exchange.

Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): engineering of inhibitors with altered specificities.

The crystal structures of the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) complexed to bovine chymotrypsin (C-APPI) and trypsin (T-APPI) and basic pancreatic trypsin inhibitor (BPTI) bound to chymotrypsin (C-BPTI) have been solved and analyzed at 2.1 A, 1.8 A, and 2.6 A resolution, respectively. APPI and BPTI belong to the Kunitz family of inhibitors, which is characterized by a distinctive tertiary fold with three conserved disulfide bonds. At the specificity-determining site of these inhibitors (P1), residue 15(I)4 is an arginine in APPI and a lysine in BPTI, residue types that are counter to the chymotryptic hydrophobic specificity. In the chymotrypsin complexes, the Arg and Lys P1 side chains of the inhibitors adopt conformations that bend away from the bottom of the binding pocket to interact productively with elements of the binding pocket other than those observed for specificity-matched P1 side chains. The stereochemistry of the nucleophilic hydroxyl of Ser 195 in chymotrypsin relative to the scissile P1 bond of the inhibitors is identical to that observed for these groups in the trypsin-APPI complex, where Arg 15(I) is an optimal side chain for tryptic specificity. To further evaluate the diversity of sequences that can be accommodated by one of these inhibitors, APPI, we used phage display to randomly mutate residues 11, 13, 15, 17, and 19, which are major binding determinants. Inhibitors variants were selected that bound to either trypsin or chymotrypsin. As expected, trypsin specificity was principally directed by having a basic side chain at P1 (position 15); however, the P1 residues that were selected for chymotrypsin binding were His and Asn, rather than the expected large hydrophobic types. This can be rationalized by modeling these hydrophilic side chains to have similar H-bonding interactions to those observed in the structures of the described complexes. The specificity, or lack thereof, for the other individual subsites is discussed in the context of the "allowed" residues determined from a phage display mutagenesis selection experiment.

2'Halo-ATP and -GTP analogues: rational phasing tools for protein crystallography.

The solution of the crystallographic macromolecular phase problem requires incorporation of heavy atoms into protein crystals. Several 2'-halogenated nucleotides have been reported as potential universal phasing tools for nucleotide binding proteins. However, only limited data are available dealing with the effect of 2'-substitution on recognition by the protein. We have determined equilibrium dissociation constants of 2'-halogenated ATP analogues for the ATP binding proteins UMP/CMP kinase and the molecular chaperone DnaK. Whereas the affinities to UMP/CMP kinase are of the same order of magnitude as for unsubstituted ATP, the affinities to DnaK are drastically decreased to undetectable levels. For 2'-halogenated GTP analogues, the kinetics of interaction were determined for the small GTPases p21ras(Y32W) (fluorescent mutant) and RabS. The rates of association were found to be within about one order of magnitude of those for the nonsubstituted nucleotides, whereas the rates of dissociation were accelerated by factors of approximately 100 (p21ras) or approximately 10(5) (Rab5), and the resulting equilibrium dissociation constants are in the nm or microM range, respectively. The data demonstrate that 2'halo-ATP and -GTP are substrates or ligands for all proteins tested except the chaperone DnaK. Due to the very high affinities of a large number of GTP binding proteins to guanine nucleotides, even a 10(5)-fold decrease in affinity as observed for Rab5 places the equilibrium dissociation constant in the microM range, so that they are still well suited for crystallization of the G-protein:nucleotide complex.

Moderate discrimination of REP-1 between Rab7 x GDP and Rab7 x GTP arises from a difference of an order of magnitude in dissociation rates.

The kinetics of the interaction of Rab7 with REP-1 have been investigated using the fluorescence of GDP and GTP analogs at the active site of Rab7. The results show that REP-1 has higher affinity for the GDP bound form of Rab7 (Kd=1 nM) than for the GTP bound form (Kd=20 nM). Both affinities should still be sufficient for the formation of stable complexes in the cell. The association reaction proceeds in two steps for the GDP bound form. The initial step is fast (k+1 = ca. 10[7] M[-1] s[-1]) and concentration dependent while the second represents a slow equilibration (k+2 + k-2 = 3.5 s[-1]) which has little effect on the overall equilibrium. The difference in affinity of the two nucleotide bound forms arises from a difference in dissociation rates (0.012 s[-1] for Rab7 x GDP and 0.2 s[-1] for Rab7 x GTP).

Structure of the N6-adenine DNA methyltransferase M.TaqI in complex with DNA and a cofactor analog.

The 2.0 A crystal structure of the N6-adenine DNA methyltransferase M.TaqI in complex with specific DNA and a nonreactive cofactor analog reveals a previously unrecognized stabilization of the extrahelical target base. To catalyze the transfer of the methyl group from the cofactor S-adenosyl-l-methionine to the 6-amino group of adenine within the double-stranded DNA sequence 5'-TCGA-3', the target nucleoside is rotated out of the DNA helix. Stabilization of the extrahelical conformation is achieved by DNA compression perpendicular to the DNA helix axis at the target base pair position and relocation of the partner base thymine in an interstrand pi-stacked position, where it would sterically overlap with an innerhelical target adenine. The extrahelical target adenine is specifically recognized in the active site, and the 6-amino group of adenine donates two hydrogen bonds to Asn 105 and Pro 106, which both belong to the conserved catalytic motif IV of N6-adenine DNA methyltransferases. These hydrogen bonds appear to increase the partial negative charge of the N6 atom of adenine and activate it for direct nucleophilic attack on the methyl group of the cofactor.

Time-resolved FTIR studies of the GTPase reaction of H-ras p21 reveal a key role for the beta-phosphate.

FTIR difference spectroscopy has been established as a new tool to study the GTPase reaction of H-ras p21 (Ras) in a time-resolved mode at atomic resolution without crystallization. The phosphate vibrations were analyzed using site specifically 18O-labeled caged GTP isotopomers. One nonbridging oxygen per nucleotide was replaced for an 18O isotope in the alpha-, beta-, or gamma-position of the phosphate chain. In photolysis experiments with free caged GTP, strong vibrational coupling was observed among all phosphate groups. The investigation of Ras*caged GTP photolysis and the subsequent hydrolysis reaction of Ras*GTP showed that the phosphate vibrations are largely decoupled by interaction with the protein in contrast to free GTP. The characteristic isotope shifts allow band assignments to isolated alpha-, beta-, and gamma-phosphate vibrations of caged GTP, GTP, and the liberated inorganic phosphate. The unusually low frequency of the beta (PO2-) vibration of Ras-bound GTP, as compared to free GTP, indicates a large decrease in the P-O bond order. The bond order decrease reveals that the oxygen atoms of the beta (PO2-) group interact much more strongly with the protein environment than the gamma-oxygen atoms. Thereby, electrons are withdrawn from the beta-phosphorus, and thus also from the beta/gamma-bridging oxygen. This leads to partial bond breakage or at least weakening of the bond between the beta/gamma-bridging oxygen and the gamma-phosphorus atom as a putative early step of the GTP hydrolysis. Based on these results, we propose a key role of the beta-phosphate for GTP hydrolysis. The assignments of phosphate bands provide a crucial marker for further time-resolved FTIR studies of the GTPase reaction of Ras.

Binding of nucleotides to guanylate kinase, p21(ras), and nucleoside-diphosphate kinase studied by nano-electrospray mass spectrometry.

The binding of nucleotides to three different nucleotide-binding proteins and to a control protein was studied by means of nano-electrospray mass spectrometry applied to aqueous nondenaturing solutions. The method leads to unambiguous identification of enzyme complexes with substrates and products but does not allow the determination of dissociation constants or even stoichiometries relevant to the binding in solution. For guanylate kinase (EC 2.7.4. 8), the transfer of HPO(3) between nucleotides was observed whenever a ternary complex with adenylate or guanylate nucleotides was formed. Guanosine 5'-tetraphosphate was generated after prolonged incubation with GDP or GTP. Mg(2+) binding was considerably enhanced in functional high affinity complexes, such as observed between guanylate kinase and its bisubstrate inhibitor P(1)-(5'-guanosyl)-P(5)-(5'-adenosyl) pentaphosphate or with the tight nucleotide-binding protein p21(ras) and GDP. Nucleoside-diphosphate kinase (EC 2.7.4.6) itself was phosphorylated in accordance to its known ping-pong mechanism. All nucleotide-binding proteins were shown to bind sulfate (SO(4)(2-)) with presumably high affinity and slow exchange rate. The binding of phosphate (PO(4)(3-)) could be inferred indirectly from competition with SO(4)(2-).

Three-dimensional structure and properties of wild-type and mutant H-ras-encoded p21.

Ras (or p21) is the product of the ras proto-oncogene and is believed to be involved in growth-promoting signal transduction. The structure of the guanine nucleotide-binding domain of H-Ras (or p21H-ras) in the triphosphate conformation was determined at very high resolution (1.4 A). All the binding interactions between protein and Gpp[NH]p and Mg2+ can be resolved in great detail. The region around amino acids 61-65 is flexible and exists in two conformations, one of which seems to be important for catalysis. The properties and structures of several oncogenic and non-oncogenic mutant forms of Ras have also been determined. Since the structure of the GDP-bound form is also known, the nature of the conformational change from the GTP-bound to the GDP-bound form can be inferred from the 3-D structure. A mechanism for the intrinsic GTP hydrolysis has been proposed. Its implications for the GAP-stimulated GTPase reaction is discussed in the light of recent kinetic and mutational experiments.

Towards structural determination of the water-splitting enzyme. Purification, crystallization, and preliminary crystallographic studies of photosystem II from a thermophilic cyanobacterium.

A photosystem II preparation from the thermophilic cyanobacterium Synechococcus elongatus, which is especially suitable for three-dimensional crystallization in a fully active form was developed. The efficient purification method applied here yielded 10 mg of protein of a homogenous dimeric complex of about 500 kDa within 2 days. Detailed characterization of the preparation demonstrated a fully active electron transport chain from the manganese cluster to plastoquinone in the Q(B) binding site. The oxygen-evolving activity, 5000-6000 micromol of O(2)/(h.mg of chlorophyll), was the highest so far reported and is maintained even at temperatures as high as 50 degrees C. The crystals obtained by the vapor diffusion method diffracted to a resolution of 4.3 A. The space group was determined to be P2(1)2(1)2(1) with four photosystem II dimers per unit cell. Analysis of the redissolved crystals revealed that activity, supramolecular organization, and subunit composition were maintained during crystallization.

Characterization of the ternary complex between Rab7, REP-1 and Rab geranylgeranyl transferase.

Geranylgeranylation is a post-translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein (REP) and are presented to the Rab geranylgeranyl transferase (RabGGTase) which covalentely modifies the Rab protein with two geranylgeranyl moieties. After prenylation, the Rab protein remains in complex with REP and is delivered to the target membrane by the latter. In this work, we show that RabGGTase can form a stable complex with Rab7-REP in the absence of its lipid substrate geranylgeranyl pyrophosphate. In order to characterize this interaction, we developed three fluorescence assays reporting on the interaction of RabGGTase with the Rab7-REP complex. For this interaction we determined a Kd value of about 120 nM. Association of RabGGTase with the Rab7-REP complex occurs with a rate constant of approximately 108 M-1 x s-1. We demonstrate that the state of the nucleotide bound to Rab7 does not influence the affinity of RabGGTase for the Rab7-REP-1 complex. Finally, we address the issue of substrate specificity of RabGGTase. Titration experiments demonstrate that, in contrast with farnesyl transferase, RabGGTase does not recognize a defined C-terminal sequence motif. Experiments using Rab7 mutants in which the last 16 amino acids were either mutated or truncated revealed that the distal part of the C-terminus makes only a limited contribution to the binding affinity between RabGGTase and the Rab7-REP-1 complex. This demonstrates the functional dissimilarity between RabGGTase and geranylgeranyl transferase I and farnesyl transferase, which interact specifically with the C-terminus of their substrates. Based on these experiments, we propose that RabGGTase recognizes the overall structure arising from the association of Rab and REP and then 'scans' the flexible C-terminus to position the proximal cysteines into the active site.

Kinetic analysis of four HIV-1 reverse transcriptase enzymes mutated in the primer grip region of p66. Implications for DNA synthesis and dimerization.

The highly conserved primer grip region in the p66 subunit of HIV-1 reverse transcriptase (RT) is formed by the beta12-beta13 hairpin (residues 227-235). It has been proposed to play a role in aligning the 3'-OH end of the primer in a position for nucleophilic attack on an incoming dNTP. To analyze the importance of the primer grip for RT function, mutant RTs were used that contain single alanine substitutions of residues Trp229, Met230, Gly231, and Tyr232 in the p66 subunit of the heterodimeric p66/51 enzyme. Steady-state and pre-steady-state kinetic analyses of the enzymes were performed. All mutant enzymes revealed reduced polymerase activity. Mutation of Y232A showed the smallest effect on polymerase function. Equilibrium fluorescence titrations demonstrated that the affinity of the mutants for tRNA was only slightly affected. However, the affinity for primer-template DNA was reduced 27-fold for mutant p66(W229A)/51 and 23-fold for mutant p66(G231A)/51, and the maximal pre-steady-state rate of nucleotide incorporation, kpol, was reduced 27-fold for p66(W229A)/51 and 70-fold for p66(G231A)/51, respectively. Mutant p66(M230A)/51 revealed no reduced affinity for primer-template but showed a 71-fold reduced affinity for dTTP. Additionally, the mutations Trp229 and Gly231 affected the stability of the RT heterodimer.

Crystal structure of the GAP domain of Gyp1p: first insights into interaction with Ypt/Rab proteins.

We present the 1.9 A resolution crystal structure of the catalytic domain of Gyp1p, a specific GTPase activating protein (GAP) for Ypt proteins, the yeast homologues of Rab proteins, which are involved in vesicular transport. Gyp1p is a member of a large family of eukaryotic proteins with shared sequence motifs. Previously, no structural information was available for any member of this class of proteins. The GAP domain of Gyp1p was found to be fully alpha-helical. However, the observed fold does not superimpose with other alpha-helical GAPs (e.g. Ras- and Cdc42/Rho-GAP). The conserved and catalytically crucial arginine residue, identified by mutational analysis, is in a comparable position to the arginine finger in the Ras- and Cdc42-GAPs, suggesting that Gyp1p utilizes an arginine finger in the GAP reaction, in analogy to Ras- and Cdc42-GAPs. A model for the interaction between Gyp1p and the Ypt protein satisfying biochemical data is given.