The structural and mechanistic basis for recycling of Rab proteins between membrane compartments.

Rab proteins are members of the Ras superfamily of GTPases and are key regulators of intracellular vesicular transport. They undergo a cycle of GTPase activity, and this activity is interconnected to a cycle of reversible attachment to membranes. This cycle is mediated by geranylgeranylation of (usually) two C-terminal cysteines, which in turn is effected by Rab geranylgeranyltransferase in concert with REP (Rab escort protein). After delivery to their respective membranes, Rabs are activated by replacement of GDP by GTP, allowing interaction with a wide variety of effector molecules involved in vesicular transport, in particular with docking of transport vesicles to their specific target membranes. After completion of these events and GTP hydrolysis, Rabs are retrieved by GDI (GDP dissociation inhibitor) and delivered to their starting compartment. Here, the structural and mechanistic basis of events occurring in Rab delivery and cycling, and the differences between REP and GDI are discussed on the basis of recent advances in the field.

Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes.

By using single-molecule multiparameter fluorescence detection, fluorescence resonance energy transfer experiments, and newly developed data analysis methods, this study demonstrates directly the existence of three structurally distinct forms of reverse transcriptase (RT):nucleic acid complexes in solution. Single-molecule multiparameter fluorescence detection also provides first information on the structure of a complex not observed by x-ray crystallography. This species did not incorporate nucleotides and is structurally distinct from the other two observed species. We determined that the nucleic acid substrate is bound at a site far removed from the nucleic acid-binding tract observed by crystallography. In contrast, the other two states are identified as being similar to the x-ray crystal structure and represent distinct enzymatically productive stages in DNA polymerization. These species differ by only a 5-A shift in the position of the nucleic acid. Addition of nucleoside triphosphate or of inorganic pyrophosphate allowed us to assign them as the educt and product state in the polymerization reaction cycle; i.e., the educt state is a complex in which the nucleic acid is positioned to allow nucleotide incorporation. The second RT:nucleic acid complex is the product state, which is formed immediately after nucleotide incorporation, but before RT translates to the next nucleotide.

Phosphoisoprenoids modulate association of Rab geranylgeranyltransferase with REP-1.

Rab geranylgeranyltransferase (RabGGTase or GGTase-II) catalyzes the post-translational prenylation of Rab proteins. Rab proteins are recognized as substrates only when they are complexed to Rab Escort Protein (REP). The classical model of prenylation complex assembly assumes initial formation of the Rab.REP binary complex, which subsequently binds to RabGGTase loaded with the isoprenoid donor geranylgeranyl pyrophosphate (GGpp). We demonstrate here that REP-1 can also associate with RabGGTase in the absence of Rab protein and that this interaction is dramatically strengthened by the presence of phosphoisoprenoids such as GGpp. The GGpp-dependent interaction between RabGGTase and REP-1 was observed using affinity precipitations and gel filtration and was quantitated on the basis of fluorescence assays. In the presence of GGpp, REP-1 binds to RabGGTase with a K(d) value of approximately 10 nm, while in its absence the affinity between the two proteins is in the micromolar range. We further demonstrate that binding of Rab7 to the RabGGTase.GGpp.REP-1 complex occurs without prior dissociation of REP-1. Analysis of binding and prenylation rate constants indicate that the RabGGTase.GGpp.REP-1 complex can function as a kinetically competent intermediate of the prenylation reaction. We conclude that, depending on the prevailing concentrations, binding of REP-1 to RabGGTase in the presence of GGpp may serve as an alternative pathway for the assembly of the prenylation machinery in vivo. Implications of these findings for the role of REP-1 in the prenylation reaction are discussed.

Double prenylation by RabGGTase can proceed without dissociation of the mono-prenylated intermediate.

Rab geranylgeranyltransferase (RabGGTase) catalyzes the prenylation of Rab proteins. Despite possessing a single active site, RabGGTase is able to add geranylgeranyl moieties onto each of the two C-terminal cysteine residues of Rab. We have studied the kinetics of Rab double prenylation employing a combination of a novel high pressure liquid chromatography (HPLC)-based in vitro prenylation assay and fluorescence spectroscopy. Transfer of the first geranylgeranyl group proceeds with a k(1) = 0.16 s(-1), while the conversion from singly to double prenylated Rab is 4-fold slower (k(2) = 0.039 s(-1)). We found that following the first transfer reaction, the conjugated lipid is removed from the active site of RabGGTase but mono-prenylated Rab.REP complex remains bound to RabGGTase with a K(d) < 1 nm. In contrast to the doubly prenylated Rab7.REP dissociation of the mono-prenylated species from RabGGTase was only weakly stimulated by phosphoisoprenoid. Based on the obtained rate constants we calculated that at least 72% of mono-prenylated Rab molecules proceed to double prenylation without dissociating from RabGGTase. The obtained data provides an explanation of how RabGGTase discriminates between mono-prenylated intermediate and double prenylated reaction product. It also indicates that the phosphoisoprenoid acts both as a substrate and as a sensor governing the kinetics of protein.protein interactions in the double prenylation reaction.

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.

A sensitive fluorescence monitor for the detection of activated Ras: total chemical synthesis of site-specifically labeled Ras binding domain of c-Raf1 immobilized on a surface.

BACKGROUND: The Ras.GDP-Ras.GTP cycle plays a central role in eukaryotic signaling cascades. Mutations in Ras which stabilize activated Ras.GTP lead to a continuous stimulation of downstream effectors and ultimately to cell proliferation. Ras mutants which increase the steady-state concentration of Ras.GTP are involved in about 30% of all human cancers. It is therefore of great interest to develop a biosensor which is sensitive to Ras.GTP but not to Ras.GDP. RESULTS: The Ras binding domain (RBD) of c-Raf1 was synthesized from two unprotected peptide segments by native chemical ligation. Two fluorescent amino acids with structures based on the nitrobenz-2-oxa-1,3-diazole and coumaryl chromophores were incorporated at a site which is close to the RBD/Ras.GTP binding surface. Additionally, a C-terminal tag consisting of His6 was introduced. The Kd values for binding of the site-specifically modified proteins to Ras.GTP are comparable to that of wild-type RBD. Immobilization of C-terminal His6 tag-modified fluorescent RBD onto Ni-NTA-coated surfaces allowed the detection of Ras.GTP in the 100 nM range. Likewise, Ras.GTP/Q61L (an oncogenic mutant of Ras with very low intrinsic GTP hydrolysis activity) can also be detected in this assay system. Ras.GDP does not bind to the immobilized RBD, thus allowing discrimination between inactive and activated Ras. CONCLUSIONS: The site-specific incorporation of a fluorescent group at a strategic position in a Ras effector protein allows the detection of activated Ras with high sensitivity. This example illustrates the fact that the chemical synthesis of proteins or protein domains makes it possible to incorporate any kind of natural or unnatural amino acid at the position of choice, thereby enabling the facile preparation of specific biosensors, enhanced detection systems for drug screening, or the synthesis of activated proteins, e.g. phosphorylated proteins involved in signaling pathways, as defined molecular species.

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.

Intramolecular interactions in protein tyrosine phosphatase RPTPmu: kinetic evidence.

The receptor-like protein tyrosine phosphatase RPTPmu contains three intracellular domains: the juxtamembrane (JM) and two phosphatase domains (D1 and D2). D1 is catalytically active in vitro. The functional roles of JM and D2 are still unclear. To find out whether and how they modulate the phosphatase activity of D1, we compared the enzymatic characteristics of two constructs, containing a truncated JM and either D1 or both phosphatase domains. p-Nitrophenyl phosphate and two peptide substrates were efficiently dephosphorylated by both constructs. The specificity constant of D1 alone was up to 50% higher. D2 induces (a) decreased K(m) values for peptide substrates, (b) decreased catalytic efficiency for these substrates, (c) shifting of the optimal pH to slightly lower values, and (d) looser binding of competitive inhibitors. These data suggest that the phosphatase activity of D1 is negatively modulated and its ligand binding capacity is sensibly modified by domain D2, having possible functional significance.

Allosteric regulation of substrate binding and product release in geranylgeranyltransferase type II.

GTPases of the Rab family are key components of vesicular transport in eukaryotic cells. Posttranslational attachment of geranylgeranyl moieties is essential for Rab function. Geranylgeranyltransferase type II (GGTase-II) catalyzes the modification of Rab proteins once they are in complex with their escort protein (REP). Upon completion of prenylation, REP and modified Rab leave the enzyme, enabling a new round of catalysis. We have studied the mechanism underlying substrate binding and product release in the geranylgeranylation of Rab proteins. Binding of the Rab7:REP-1 complex to GGTase-II was found to be strongly modulated by geranylgeranyl pyrophosphate (GGpp). The affinity of GGTase-II for the Rab7:REP-1 complex increases from ca. 120 nM to ca. 2 nM in the presence of GGpp. To study the effect of GGpp on interaction of the enzyme with its product, we generated semisynthetic doubly prenylated Rab7 bearing a fluorescent reporter group. Using this novel compound, we demonstrated that the affinity of doubly prenylated Rab7:REP-1 complex for GGTase-II was 2 and 18 nM in the absence and presence of GGpp, respectively. The difference in affinities originates mainly from a difference in the dissociation rates. Thus, binding of the new isoprenoid substrate molecule facilitates the product release by GGTase-II. The affinity of GGpp for the prenylated Rab7:REP-1:GGTase-II was K(d) = 22 nM, with one molecule of GGpp binding per molecule of prenylated ternary complex. We interpreted this finding as an indication that the geranylgeranyl moieties transferred to Rab protein do not occupy the GGpp binding site of the GGTase-II. In summary, these results demonstrate that GGpp acts as an allosteric activator that stabilizes the Rab7:REP-1:GGTase-II complex and triggers product release upon prenylation, preventing product inhibition of the enzyme.

Crystallization and preliminary X-ray diffraction analysis of the Rab escort protein-1 in complex with Rab geranylgeranyltransferase.

Posttranslational prenylation of proteins is a widespread phenomenon and the majority of prenylated proteins are geranylgeranylated members of the Rab GTPase family. Geranylgeranylation is catalyzed by Rab geranylgeranyltransferase (RabGGTase) and is critical for the ability of Rab protein to mediate vesicular docking and fusion of various intracellular vesicles. RabGGTase consists of a catalytic alpha/beta heterodimer and an accessory protein termed Rab escort protein (REP-1) that delivers the newly prenylated Rab proteins to their target membrane. Mutations in the REP-1 gene in humans lead to an X-chromosome-linked defect known as choroideremia--a debilitating disease that inevitably culminates in complete blindness. Here we report in vitro assembly and purification of the stoichiometric ternary complex of RabGGTase with REP-1 stabilized by a hydrolysis-resistant phosphoisoprenoid analog--farnesyl phosphonyl(methyl)phoshonate. The complex formed crystals of extended plate morphology under low ionic-strength conditions. X-ray diffraction data were collected to 2.8 A resolution at the ESRF. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 68.7, b = 197.7, c = 86.1 A, beta = 113.4 degrees. Preliminary structural analysis revealed the presence of one molecule in the asymmetric unit.

Potentiating AZT activation: structures of wild-type and mutant human thymidylate kinase suggest reasons for the mutants' improved kinetics with the HIV prodrug metabolite AZTMP.

The 60-fold reduced phosphorylation rate of azidothymidine (AZT) monophosphate (AZTMP), the partially activated AZT metabolite, by human thymidylate kinase (TMPK) severely limits the efficacy of this anti-HIV prodrug. Crystal structures of different TMPK nucleotide complexes indicate that steric hindrance by the azido group of AZTMP prevents formation of the catalytically active closed conformation of the P-loop of TMPK. The F105Y mutant and a chimeric mutant that contains sequences of the human and Escherichia coli enzyme phosphorylate AZTMP 20-fold faster than the wild-type enzyme. The structural basis of the increased activity is assigned to stabilization of the closed P-loop conformation.

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.

Phosphoisoprenoid binding specificity of geranylgeranyltransferase type II.

Geranylgeranyltransferase type II (GGTase-II) modifies small monomeric GTPases of the Rab family by attaching geranylgeranyl moieties onto two cysteines of their C-terminus. We investigated to what extent GGTase-II discriminates between its native substrate geranylgeranyl pyrophosphate (GGpp) and other phosphoisoprenoids, including farnesyl pyrophosphate (Fpp). On the basis of a novel fluorescent assay, we demonstrated that GGpp binds to GGTase-II with an affinity of 8 +/- 4 nM, while Fpp is bound less strongly (K(d) = 60 +/- 8 nM). Analysis of the binding kinetics of four different phosphoisoprenoids indicated that in all cases association is rapid, with rate constants in the range of 0.15 nM(-1) s(-1). In contrast, the dissociation rates differed greatly, depending on the phosphoisoprenoid used, with weak binding substrates generally displaying an increased rate of dissociation. The affinity of GGpp and Fpp for GGTase-II was also determined in the presence of the Rab7-REP-1 complex. The affinity for GGpp was essentially unaffected by the presence of the complex; Fpp on the other hand bound less strongly to the GGTase-II under these conditions, resulting in a K(d) of 260 +/- 60 nM. In vitro prenylation experiments were used to establish that Fpp not only does bind to GGTase-II but also is transferred with an observed rate constant of 0.082 s(-1) which is very similar to that of GGpp. The implications of the low level of discrimination by GGTase-II for the in vivo specificity of the enzyme and the use of farnesyltransferase inhibitors in anti-cancer therapy are discussed.

Temperature-dependent equilibrium between the open and closed conformation of the p66 subunit of HIV-1 reverse transcriptase revealed by site-directed spin labelling.

X-ray crystallographic studies of human immunodeficiency virus type 1 reverse transcriptase complexed with or without substrates or inhibitors show that the heterodimeric enzyme adopts distinct conformations that differ in the orientation of the so-called thumb subdomain in the large subunit. Site-directed spin labelling of mutated residue positions W24C and K287C is applied here to determine the distances between the fingers and thumb subdomains of liganded and unliganded RT in solution. The inter-spin distances of a DNA/DNA and a pseudoknot RNA complexed reverse transcriptase in solution was found to agree with the respective crystal data of the open and closed conformations. For the unliganded reverse transcriptase a temperature-dependent equilibrium between these two states was observed. The fraction of the closed conformation decreased from 95% at 313 K to 65% at 273 K. The spectral separation between the two structures was facilitated by the use of a perdeuterated ([15)N]nitroxide methane-thiosulfonate spin label.

Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate.

BACKGROUND: Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes the reversible phosphoryltransfer between ATP and TMP to yield ADP and TDP. In addition to its vital role in supplying precursors for DNA synthesis, human TMPK has an important medical role participating in the activation of a number of anti-HIV prodrugs. RESULTS: Crystal structures of human TMPK in complex with TMP and ADP, TMP and the ATP analog AppNHp, TMP with ADP and the phosphoryl analog AlF(3), TDP and ADP, and the bisubstrate analog TP(5)A were determined. The conformations of the P-loop, the LID region, and the adenine-binding loop vary according to the nature of the complex. Substitution of ADP by AppNHp results in partial closure of the P-loop and the rotation of the TMP phosphate group to a catalytically unfavorable position, which rotates back in the AlF(3) complex to a position suitable for in-line attack. In the fully closed state observed in the TP(5)A and the TDP-ADP complexes, Asp15 interacts strongly with the 3'-hydroxyl group of TMP. CONCLUSIONS: The observed changes of nucleotide state and conformation and the corresponding protein structural changes are correlated with intermediates occurring along the reaction coordinate and show the sequence of events occurring during phosphate transfer. The low catalytic activity of human TMPK appears to be determined by structural changes required to achieve catalytic competence and it is suggested that a mechanism might exist to accelerate the activity.

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.

Kinetics of the interaction of translation factor SelB from Escherichia coli with guanosine nucleotides and selenocysteine insertion sequence RNA.

The kinetics of the interaction of GTP and GDP with SelB, the specific translation factor for the incorporation of selenocysteine into proteins, have been investigated using the stopped-flow method. Useful signals were obtained using intrinsic (i.e. tryptophan) fluorescence, the fluorescence of methylanthraniloyl derivatives of nucleotides, or fluorescence resonance energy transfer from tryptophan to the methylanthraniloyl group. The affinities of SelB for GTP (K(d) = 0.74 micrometer) and GDP (K(d) = 13.4 micrometer) were considerably lower than those of other translation factors. Of functional significance is the fact that the rate constant for GDP release from its complex with SelB (15 s(-)(1)) is many orders of magnitude larger than for elongation factor Tu, explaining why a GDP/GTP exchange factor is not required for the action of SelB. In contrast, the rate of release of GTP is 2 orders of magnitude slower and not significantly faster than for elongation factor Tu. Using a fluorescently labeled 17-nucleotide RNA minihelix that represents a binding site for the protein and that is part of the fdhF selenocysteine insertion sequence element positioned immediately downstream of the UGA triplet coding for selenocysteine incorporation, the kinetics of the interaction were studied. The high affinity of the interaction (K(d) approximately 1 nm) appeared to be increased even further when selenocysteyl-tRNA(Sec) was bound to SelB, but to be independent of the presence or nature of the guanosine nucleotide at the active site. These results suggest that the affinity of SelB for its RNA binding site is maximized when charged tRNA is bound and decreases to allow dissociation and reading of codons downstream of the selenocysteine codon after selenocysteine peptide bond formation.

HIV-1 reverse transcriptase-pseudoknot RNA aptamer interaction has a binding affinity in the low picomolar range coupled with high specificity.

Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the identification of small oligonucleotides that bind with high affinity and specificity to target proteins. Such DNAs/RNAs are a new class of potential chemotherapeutics that could block the enzymatic activity of pathologically relevant proteins. We have conducted a detailed biochemical study of the interaction of human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) with a SELEX-derived pseudoknot RNA aptamer. Electron paramagnetic resonance spectroscopy of site-directed spin-labeled RT mutants revealed that this aptamer was selected for binding to the "closed" conformation of the enzyme. Kinetic analysis showed that the RNA inhibitor bound to HIV RT in a two-step process, with association rates similar to those described for model DNA/DNA and DNA/RNA substrates. However, the dissociation of the pseudoknot RNA from RT was dramatically slower than observed for model substrates. Equilibrium binding studies revealed an extraordinarily low K(d), of about 25 pm, for the enzyme-aptamer interaction, presumably a consequence of the slow off-rates. Additionally, this pseudoknot aptamer is highly specific for HIV-1 RT, with the closely related HIV-2 enzyme showing a binding affinity close to 4 orders of magnitude lower.

Semi-synthetic Rab proteins as tools for studying intermolecular interactions.

Rab GTPases play a key role in the regulation of membrane traffic. Posttranslational geranylgeranylation is critical for their biological activity and is conferred by a Rab geranylgeranyl transferase (RabGGTase). To study the interactions between Rab proteins and RabGGTase, we used in vitro ligation methodology to generate a fluorescent semi-synthetic Rab7 protein. The obtained protein was functionally active and was used to demonstrate a micromolar affinity interaction of Rab7 with the RabGGTase in the absence of Rab escort protein (REP). This finding is consistent with an earlier proposed model according to which RabGGTase possesses two independent weak binding sites for REP and Rab proteins.