Shikimate-3-phosphate binds to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the transfer of the enolpyruvyl moiety from phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P). Mutagenesis and X-ray crystallography data suggest that the active site of the enzyme is in the cleft between its two globular domains; however, they have not defined which residues are responsible for substrate binding and catalysis. Here we attempt to establish the binding of the substrate S3P to the isolated N-terminal domain of EPSP synthase using a combination of NMR spectroscopy and isothermal titration calorimetry. Our experimental results indicate that there is a saturable and stable conformational change in the isolated N-terminal domain upon S3P binding and that the chemical environment of the S3P phosphorus when bound to the isolated domain is very similar to that of S3P bound to EPSP synthase. We also conclude that most of the free energy of S3P binding to EPSP synthase is contributed by the N-terminal domain.     

Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase

The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP synthase-S3P-Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native (31)P in S3P and Glp, biosynthetically (13)C-labeled S3P, specifically (13)C and (15)N labeled Glp, and a variety of protein-(15)N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.     

The roles of conserved amino acids on substrate binding and conformational integrity of ClpB N-terminal domain

Escherichia coli heat shock protein ClpB disaggregates denatured protein in cooperation with the DnaK chaperone system. Several studies showed that the N-terminal domain is essential for the chaperone activity, but its role is still largely unknown. The N-terminal domain contains two structurally similar subdomains, and conserved amino acids Thr7 and Ser84 share the same position in two apparent sequence repeats. T7A and S84A substitutions affected chaperone activity of ClpB without significantly changing the native conformation [Liu, Z. et al. (2002) J. Mol. Biol. 321, 111-120]. In this study, we aimed to better understand the roles of several conserved amino acid residues, including Thr7 and Ser84, in the N-terminal domain. We investigated the effects of mutagenesis on substrate binding and conformational states of ClpB N-terminal domain fragment (ClpBN). Fluorescence polarization analysis showed that the T7A and S84A substitutions enhanced the interaction between ClpBN and protein aggregates. Interestingly, further analyses suggested that the mechanisms by which they do so are quite different. For T7A substitution, the increased substrate affinity could be due to a conformational change in the hydrophobic core as revealed by NMR spectroscopy. In contrast, for S84A, increased substrate binding would be explained by a unique conformational state of this mutant as revealed by pressure perturbation analysis. The thermal transition temperature of the S84A mutant, monitored by DSC, was 6.1 degrees C lower than that of wild-type. Our results revealed that conserved amino acids Thr7 and Ser84 both participated in maintaining the conformational integrity of the ClpB N-terminal domain.     

Engineering and characterization of the isolated C-terminal domain of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the formation of EPSP and inorganic phosphate from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) in the biosynthesis of aromatic amino acids. To delineate the domain-specific function, we successfully isolated the discontinuous C-terminal domain (residues 1-21, linkers, 240-427) of EPSP synthase (427 residues) by site-directed mutagenesis. The engineered C-terminal domains containing no linker (CTD), or with gly-gly (CTD(GG)) and gly-ser-ser-gly (CTD(GSSG)) linkers were purified and characterized as having distinct native-like secondary and tertiary structures. However, isothermal titration calorimetry (ITC), 15N-HSQC, and 31P-NMR revealed that neither its substrate nor inhibitor binds the isolated domain. The isolated domain maintained structural integrity, but did not function as the half of the full-length protein.     

Novel GFP-fused protein probes for detecting phosphatidylinositol-4-phosphate in the plasma membrane

Phosphatidylinositol-4-phosphate (PI4P) plays a crucial role in cellular functions, including protein trafficking, and is mainly located in the cytoplasmic surface of intracellular membranes, which include the trans-Golgi network (TGN) and the plasma membrane. However, many PI4P-binding domains of membrane-associated proteins are localized only to the TGN because of the requirement of a second binding protein such as ADP-ribosylation factor 1 (ARF1) in order to be stably localized to the specific membrane. In this study, we developed new probes that were capable of detecting PI4P at the plasma membrane using the known TGN-targeting PI4P-binding domains. The PI4P-specific binding pleckstrin homology (PH) domain of various proteins including CERT, OSBP, OSH1, and FAPP1 was combined with the N-terminal moderately hydrophobic domain of the short-form of Aplysia phosphodiesterase 4 (S(N30)), which aids in plasma membrane association but cannot alone facilitate this association. As a result, we found that the addition of S(N30) to the N-terminus of the GFP-fused PH domain of OSBP (S(N30)-GFP-OSBP-PH), OSH1 (S(N30)-GFP-OSH1-PH), or FAPP1 (S(N30)-GFP-FAPP1-PH) could induce plasma membrane localization, as well as retain TGN localization. The plasma membrane localization of S(N30)-GFP-FAPP1-PH is mediated by PI4P binding only, whereas those of S(N30)-GFP-OSBP-PH and S(N30)-GFP-OSH1-PH are mediated by either PI4P or PI(4,5)P₂ binding. Taken together, we developed new probes that detect PI4P at the plasma membrane using a combination of a moderately hydrophobic domain with the known TGN-targeting PI4P-specific binding PH domain.     

Identification of sorbitol 3-phosphate and fructose 3-phosphate in normal and diabetic human erythrocytes

Using 31P NMR spectroscopy, we have identified sorbitol 3-phosphate and fructose 3-phosphate in normal human erythrocytes wherein their concentrations are estimated to be 13 mumol/liter cells. Incubation of hemolysates with sorbitol, fructose and ATP suggest that both sorbitol and fructose are phosphorylated separately and directly at the 3-hydroxyl position suggesting the presence in these cells of a novel and specific kinase(s). In addition to sorbitol 3-phosphate and fructose 3-phosphate which were previously identified in the mammalian lens and sciatic nerve, erythrocytes have two extra metabolites resonating at 6.7 and 6.8 ppm in the 31P NMR spectrum. Although not identified in this study, the unusual chemical shifts of these compounds, their low pKa values and the fact that they appear as doublet in proton-coupled 31P NMR spectra, suggest that these phosphomonoesters belong to the same class of metabolites as sorbitol 3-phosphate and fructose 3-phosphate. Preliminary studies of erythrocytes from an unselected group of diabetic subjects showed an overall increase in the concentration of all four metabolites, although an overlap with normal values was noted.     

The N-terminal carbohydrate recognition site of the cation-independent mannose 6-phosphate receptor

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays a critical role in the trafficking of newly synthesized mannose 6-phosphate-containing acid hydrolases to the lysosome. The receptor contains two high affinity carbohydrate recognition sites within its 15-domain extracytoplasmic region, with essential residues for carbohydrate recognition located in domain 3 and domain 9. Previous studies have shown that these two sites are distinct with respect to carbohydrate specificity. In addition, expression of truncated forms of the CI-MPR demonstrated that domain 9 can be expressed as an isolated domain, retaining high affinity (Kd approximately 1 nm) carbohydrate binding, whereas expression of domain 3 alone resulted in a protein capable of only low affinity binding (Kd approximately 1 microm) toward a lysosomal enzyme. In the current report the crystal structure of the N-terminal 432 residues of the CI-MPR, encompassing domains 1-3, was solved in the presence of bound mannose 6-phosphate. The structure reveals the unique architecture of this carbohydrate binding pocket and provides insight into the ability of this site to recognize a variety of mannose-containing sugars.     

Sphingosine 1-phosphate and control of vascular tone

Sphingosine-1-phosphate (S1P) is a platelet-derived lipid mediator that activates the endothelial isoform of nitric oxide synthase (eNOS) in endothelial cells. However, the role of S1P in endothelium-dependent vasodilation and the signaling pathways elicited by S1P in intact vessels are largely unknown. We found that S1P induces dose-dependent transient relaxation of isolated pressurized mesenteric arterioles (EC(50) 10 +/- 3 nM); maximal vasodilation (55 +/- 8%) is seen approximately 2 min after S1P addition and returns to baseline by 5 min. S1P promotes comparable responses in arterioles from wild-type but not eNOS(null) mice. S1P-induced vasodilation is abrogated by removal of endothelium or by the addition of the NOS inhibitor N(omega)-monomethyl-l-arginine but is not affected by the cyclooxygenase inhibitor indomethacin, nor by the blockade of K(+) channels by using 4-aminopyridine. S1P-induced vasodilation is attenuated by pertussis toxin, by the phosphoinositide 3-kinase (PI3-kinase) inhibitor wortmannin, and by the calcium chelator BAPTA. With the use of high-sensitivity protein immunoblots in extracts from single pressurized vessels, we found that S1P, but not bradykinin, promotes the phosphorylation of eNOS at Ser(1179). Maximum S1P-induced eNOS Ser(1179) phosphorylation was reached at the time of maximum vasorelaxation, but enzyme phosphorylation persisted for several minutes after vasodilation had resolved. Thus regulatory pathways distinct from eNOS Ser(1179) dephosphorylation serve to terminate agonist-promoted vasorelaxation. Taken together, our findings demonstrate that S1P, an important intercellular mediator of platelet-vessel wall interactions, is a effective arteriolar vasodilator that acts via G protein-dependent, calcium-sensitive, and PI3-kinase-modulated signaling pathways.     

Increased mobility in the membrane targeting PX domain induced by phosphatidylinositol 3-phosphate

Phosphoinositides (PIs) are concentrated in specific subcellular membranes in order to recruit and regulate cytosolic proteins responsible for vesicular trafficking, cytoskeletal rearrangement, and eukaryotic cell growth, differentiation, and survival. Phox homology (PX) domains are found in proteins that are integral players in endocytic pathways. For example, Vam7p is targeted by its PX domain to phosphatidylinositol 3-phosphate [PtdIns(3)P] in the yeast vacuole, where it interacts with other SNARE proteins and GTPases of the vesicular membrane fusion machinery. Although several PX structures have been solved, the role of dynamics in their interactions with membrane lipids is unclear. Here, we present the first detailed characterization of the backbone dynamics of a PX domain, that of Vam7p, in the presence and absence of its ligand. The structure appears to tumble more rapidly in solution upon binding PtdIns(3)P, revealing a conformational change that includes adjustments in the flexible membrane insertion loop (MIL). The flexibilities of the MIL and domain termini are pronounced in both states, while the alpha1 and alpha2 helices are rigid. Dynamic effects are spread across the binding pocket, with PtdIns(3)P inducing altered mobility of different residues on multiple timescales, including a shift in the MIL to slower timescale motions. The bound state is more dynamic overall, particularly in the beta-sheet lobe, which packs against the ligand's 3-phosphate. Thus, the induced dynamic and structural effects are transduced from the buried heart of the binding pocket in the helical lobe through the beta-sheet lobe to the exposed surface of the bilayer-inserted protein.     

Evaluation of 5-enolpyruvoylshikimate-3-phosphate synthase substrate and inhibitor binding by stopped-flow and equilibrium fluorescence measurements

The binding of substrates and the herbicide N-(phosphonomethyl)glycine (glyphosate) to enolpyruvoylshikimate-3-phosphate (EPSP) synthase was evaluated by stopped-flow and equilibrium fluorescence measurements. Changes in protein fluorescence were observed upon the binding of EPSP and upon the formation of the enzyme-shikimate 3-phosphate-glyphosate ternary complex; no change was seen with either shikimate 3-phosphate (S3P) or glyphosate alone. By fluorescence titrations, the dissociation constants were determined for the formation of the enzyme binary complexes with S3P (Kd,S = 7 +/- 1.2 microM) and EPSP (Kd,EPSP = 1 +/- 0.01 microM). The dissociation constant for S3P was determined by competition with EPSP or by measurements in the presence of a low glyphosate concentration. At saturating concentrations of S3P, glyphosate bound to the enzyme--S3P binary complex with a dissociation constant of 0.16 +/- 0.02 microM. Glyphosate did not bind significantly to free enzyme, so the binding is ordered with S3P binding first: (formula; see text) where S refers to S3P, G refers to glyphosate, and E.S.G. represents the complex with altered fluorescence. The kinetics of binding were measured by stopped-flow fluorescence methods. The rate of glyphosate binding to the enzyme--S3P complex was k2 = (7.8 +/- 0.2) X 10(5) M-1 s-1, from which we calculated the dissociation rate k-2 = 0.12 +/- 0.02 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of phosphonate analogues of the tetrahedral reaction intermediate with 5-enolpyruvylshikimate-3-phosphate synthase in atomic detail

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the penultimate step of the shikimate pathway and is the target of the broad-spectrum herbicide glyphosate. Since the functionality of the shikimate pathway is vital not only for plants but also for microorganisms, EPSPS is considered a prospective target for the development of novel antibiotics. We have kinetically analyzed and determined the crystal structures of Escherichia coli EPSPS inhibited by (R)- and (S)-configured phosphonate analogues of the tetrahedral reaction intermediate. Both diastereomers are competitive inhibitors with respect to the substrates of the EPSPS reaction, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). Remarkably, the (S)-phosphonate (K(iS3P) = 750 nM), whose configuration corresponds to that of the genuine tetrahedral intermediate, is a much weaker inhibitor than the (R)-phosphonate analogue (K(iS3P) = 16 nM). The crystal structures of EPSPS liganded with the (S)- and (R)-phosphonates, at 1.5 and 1.9 A resolution, respectively, revealed that binding of the (R)-phosphonate induces conformational changes of the strictly conserved residues Arg124 and Glu341 within the active site. This appears to give rise to substantial structural alterations in the amino-terminal globular domain of the enzyme. By contrast, binding of the (S)-phosphonate renders the enzyme structure unchanged. Thus, EPSPS may facilitate the tight binding of structurally diverse ligands through conformational flexibility. Molecular docking calculations did not explain why the (R)-phosphonate is the better inhibitor. Therefore, we propose that the structural events during the open-closed transition of EPSPS are altered as a result of inhibitor action.     

The bovine mannose 6-phosphate/insulin-like growth factor II receptor. Localization of mannose 6-phosphate binding sites to domains 1-3 and 7-11 of the extracytoplasmic region.

The extracytoplasmic region of the 270-kDa mannose 6-phosphate/IGF-II receptor is composed of 15 repeating domains and is capable of binding 2 mol of mannose 6-phosphate (Man-6-P). To localize the Man-6-P binding domains, bovine receptor was subjected to partial proteolysis with subtilisin followed by affinity chromatography on pentamannosyl phosphate-agarose. Eleven proteolytic fragments ranging in apparent molecular mass from 53 to 206 kDa were isolated. Sequence analysis of six of the fragments localized their amino termini to either the beginning of domain 1 at the amino terminus of the molecule or the beginning of domain 7, according to the alignment of Lobel et al. (Lobel, P., Dahms, N. M., and Kornfeld, S. (1988) J. Biol. Chem. 263, 2563-2570). The smallest fragment, with an apparent molecular mass of 53 kDa, is predicted to encompass domains 1-3. Another fragment, with an apparent molecular mass of 82 kDa, is predicted to encompass domains 7-10 or 7-11. The Man-6-P binding site contained within domains 1-3 was further defined by expressing truncated forms of the receptor in Xenopus laevis oocytes and assaying their ability to bind phosphomannosyl residues. A soluble polypeptide containing domains 1-3 exhibited binding activity, whereas a polypeptide containing domains 1 and 2 did not. This indicates that domain 3 is a necessary component of one of the Man-6-P binding sites of the receptor.     

Investigation of glyceraldehyde-3-phosphate dehydrogenase from human sperms

Glyceraldehyde-3-phosphate dehydrogenase (GAPDs) was purified from human sperms and properties of the enzyme were investigated. After sonication of sperms, the most part of GAPDs is associated with the insoluble cell fraction. Trypsin treatment results in the cleavage of part of the N-terminal domain of the enzyme yielding a soluble fragment that was purified by hydrophobic chromatography on Phenyl-Sepharose. The isolated fragment was shown to be a tetramer with molecular weight of approximately 150 kD (according to Blue Native PAGE) and composed of subunits of 40 kD (according to SDS-PAGE). The specific activity of the isolated fragment reached 374 U/mg. It is supposed that GAPDs exists in sperms as the tetrameric molecule bound to the fibrous sheath of the flagellum through the N-terminus of one or two subunits. Comparative study of the amino acid sequences of mammalian GAPDs revealed conservative cysteine residues (C21, C94, and C150) that are specific for the sperm isoenzyme and absent in the somatic isoenzyme. Residue C21 can be involved in the formation of the disulfide bond between the N-terminal domain of GAPDs and fibrous sheath proteins.     

High-affinity binding of a FYVE domain to phosphatidylinositol 3-phosphate requires intact phospholipid but not FYVE domain oligomerization.

FYVE domains are small zinc-finger-like domains found in many proteins that are involved in regulating membrane traffic and have been shown to bind specifically to phosphatidylinositol 3-phosphate (PtdIns-3-P). FYVE domains are thought to recruit PtdIns-3-P effectors to endosomal locations in vivo, where these effectors participate in controlling endosomal maturation and vacuolar protein sorting. We have compared the characteristics of PtdIns-3-P binding by the FYVE domain from Hrs-1 (the hepatocyte growth factor-regulated tyrosine kinase substrate) with those of specific phosphoinositide binding by Pleckstrin homology (PH) domains. Like certain PH domains (such as that from phospholipase C-delta(1)), the Hrs-1 FYVE domain specifically recognizes a single phosphoinositide. However, while phosphoinositide binding by highly specific PH domains is driven almost exclusively by interactions with the lipid headgroup, this is not true for the Hrs-1 FYVE domain. The phospholipase C-delta(1) PH domain shows a 10-fold preference for binding isolated headgroup over its preferred lipid (phosphatidylinositol 4,5-bisphosphate) in a membrane, while the Hrs-1 FYVE domain greatly prefers (more than 50-fold) intact lipid in a bilayer over the isolated headgroup (inositol 1,3-bisphosphate). By contrast with reports for certain PH domains, we find that this preference for membrane binding over interaction with soluble lipid headgroups does not require FYVE domain oligomerization.     

Leishmania mexicana glycerol-3-phosphate dehydrogenase showed conformational changes upon binding a bi-substrate adduct

Certain pathogenic trypanosomatids are highly dependent on glycolysis for ATP production, and hence their glycolytic enzymes, including glycerol-3-phosphate dehydrogenase (GPDH), are considered attractive drug targets. The ternary complex structure of Leishmania mexicana GPDH (LmGPDH) with dihydroxyacetone phosphate (DHAP) and NAD(+) was determined to 1.9A resolution as a further step towards understanding this enzyme's mode of action. When compared with the apo and binary complex structures, the ternary complex structure shows an 11 degrees hinge-bending motion of the C-terminal domain with respect to the N-terminal domain. In addition, residues in the C-terminal domain involved in catalysis or substrates binding show significant movements and a previously invisible five-residue loop region becomes well ordered and participates in NAD(+) binding. Unexpectedly, DHAP and NAD(+) appear to form a covalent bond, producing an adduct in the active site of LmGPDH. Modeling a ternary complex glycerol 3-phosphate (G3P) and NAD(+) with LmGPDH identified ten active site residues that are highly conserved among all GPDHs. Two lysine residues, Lys125 and Lys210, that are presumed to be critical in catalysis, were mutated resulting in greatly reduced catalytic activity. Comparison with other structurally related enzymes found by the program DALI suggested Lys210 as a key catalytic residue, which is located on a structurally conserved alpha-helix. From the results of site-directed mutagenesis, molecular modeling and comparison with related dehydrogenases, a catalytic mechanism of LmGPDH and a possible evolutionary scenario of this group of dehydrogenases are proposed.     

Time-resolved Ultrastructural Detection of Phosphatidylinositol 3-phosphate

Phosphatidylinositol 3-phosphate [PtdIns(3)P] plays an important role in recruitment of various effector proteins in the endocytic and autophagic pathways. In an attempt to follow the distribution of PtdIns(3)P at the ultrastructural level, we are using the Fab1, YOTB, Vac1, and EEA1 (FYVE) domain, which is a zinc finger motif specifically binding to PtdIns(3)P. To follow PtdIns(3)P trafficking during a defined time window, here we have used a monomeric dimerizable FYVE probe, which binds with high avidity to PtdIns(3)P only after rapalog-induced dimerization. The probe localized to early and late endocytic compartments according to the time period of dimerization, which indicates that PtdIns(3)P is turned over via the endocytic machinery. In the functional context of epidermal growth factor (EGF) stimulation, we observed that dimerization of the probe led to clustering of mainly early endocytic structures, leaving most of the probe localized to the limiting membrane of endosomes. Interestingly, these clustered endosomes contained coats positive for the PtdIns(3)P-binding protein hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs), indicating that the probe did not displace Hrs binding. We conclude that the dimerizer-inducible probe is useful for the time-resolved detection of PtdIns(3)P at the ultrastructural level, but its effects on endosome morphology after EGF stimulation need to be taken into account. (J Histochem Cytochem 58:1025–1032, 2010)     

Phosphatidylinositol 3-phosphate induces the membrane penetration of the FYVE domains of Vps27p and Hrs

The FYVE domain mediates the recruitment of proteins involved in membrane trafficking and cell signaling to phosphatidylinositol 3-phosphate (PtdIns(3)P)-containing membranes. To elucidate the mechanism by which the FYVE domain interacts with PtdIns(3)P-containing membranes, we measured the membrane binding of the FYVE domains of yeast Vps27p and Drosophila hepatocyte growth factor-regulated tyrosine kinase substrate and their mutants by surface plasmon resonance and monolayer penetration analyses. These measurements as well as electrostatic potential calculation show that PtdIns(3)P specifically induces the membrane penetration of the FYVE domains and increases their membrane residence time by decreasing the positive charge surrounding the hydrophobic tip of the domain and causing local conformational changes. Mutations of hydrophobic residues located close to the PtdIns(3)P-binding pocket or an Arg residue directly involved in PtdIns(3)P binding abrogated the penetration of the FYVE domains into the monolayer, the packing density of which is comparable with that of biological membranes and large unilamellar vesicles. Based on these results, we propose a mechanism of the membrane binding of the FYVE domain in which the domain first binds to the PtdIns(3)P-containing membrane by specific PtdIns(3)P binding and nonspecific electrostatic interactions, which is then followed by the PtdIns(3)P-induced partial membrane penetration of the domain.     

Identification of the NAD(+)-binding fold of glyceraldehyde-3-phosphate dehydrogenase as a novel RNA-binding domain

There is growing evidence that metabolic enzymes may act as multifunctional proteins performing diverse roles in cellular metabolism. Among these functions are the RNA-binding activities of NAD(+)-dependent dehydrogenases. Previously, we have characterized the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an RNA-binding protein with preference to adenine-uracil-rich sequences. In this study, we used GST-GAPDH fusion proteins generated by deletion mutagenesis to search for the RNA binding domain. We established that the N-terminal 43 amino acid residues of GAPDH, which correspond to the first mononucleotide-binding domain of the NAD(+)-binding fold is sufficient to confer RNA-binding. We also provide evidence that this single domain, although it retains most of the RNA-binding activity, loses sequence specificity. Our results suggest a molecular basis for RNA-recognition by NAD(+)-dependent dehydrogenases and (di)nucleotide-binding metabolic enzymes that had been reported to have RNA-binding activity with different specificity. To support this prediction we also identified other members of the family of NAD(+)-dependent dehydrogenases with no previous history of nucleic acid binding as RNA binding proteins in vitro. Based on our findings we propose the addition of the NAD(+)-binding domain to the list of RNA binding domains/motifs.     

Phosphorylation-independent internalisation and desensitisation of the human sphingosine-1-phosphate receptor S1P3

Here we demonstrate that phosphorylation of the sphingosine-1-phosphate (S1P) receptor S1P(3) is increased specifically in response to S1P. Truncation of the receptor's carboxyl-terminal domain revealed that the presence of a serine-rich stretch of residues between Leu332 and Val352 was essential to observe this effect. Although agonist-occupied wild-type (WT) S1P(3) could be phosphorylated in vitro by G-protein-coupled receptor kinase 2 (GRK2), a role of S1P(3) phosphorylation in controlling S1P(3)-G(q/11) coupling was excluded since A) a phosphorylation-resistant S1P(3) mutant desensitised in a manner indistinguishable from the WT receptor and was phosphorylated to a greater extent than the WT receptor by GRK2 in vitro, and B) co-expression with GRK2 or GRK3 failed to potentiate S1P(3) phosphorylation. S1P(3) phosphorylation was also not required for receptor sequestration away from the cell surface. Together, these data suggest that S1P(3) function is not subject to conventional regulation by GRK phosphorylation and that novel aspects of S1P(3) function distinct from classical G-protein coupling and receptor internalisation may be controlled its carboxyl-terminal domain.     

FTY720 (Gilenya) phosphate selectivity of sphingosine 1-phosphate receptor subtype 1 (S1P1) G protein-coupled receptor requires motifs in intracellular loop 1 and transmembrane domain 2

FTY720 phosphate (FTY720P) is a high potency agonist for all the endothelial differentiation gene family sphingosine 1-phosphate (S1P) receptors except S1P receptor subtype 2 (S1P(2)). To map the distinguishing features of S1P(2) ligand recognition, we applied a computational modeling-guided mutagenesis strategy that was based on the high degree of sequence homology between S1P(1) and S1P(2). S1P(2) point mutants of the ligand-binding pocket were characterized. The head group-interacting residues Arg3.28, Glu3.29, and Lys7.34 were essential for activation. Mutation of residues Ala3.32, Leu3.36, Val5.41, Phe6.44, Trp6.48, Ser7.42, and Ser7.46, predicted to interact with the S1P hydrophobic tail, impaired activation by S1P. Replacing individual or multiple residues in the ligand-binding pocket of S1P(2) with S1P(1) sequence did not impart activation by FTY720P. Chimeric S1P(1)/S1P(2) receptors were generated and characterized for activation by S1P or FTY720P. The S1P(2) chimera with S1P(1) sequence from the N terminus to transmembrane domain 2 (TM2) was activated by FTY720P, and the S1P(2)(IC1-TM2)(S1P1) domain insertion chimera showed S1P(1)-like activation. Twelve residues in this domain, distributed in four motifs a-d, differ between S1P(1) and S1P(2). Insertion of (78)RPMYY in motif b alone or simultaneous swapping of five other residues in motifs c and d from S1P(1) into S1P(2) introduced FTY720P responsiveness. Molecular dynamics calculations indicate that FTY720P binding selectivity is a function of the entropic contribution to the binding free energy rather than enthalpic contributions and that preferred agonists retain substantial flexibility when bound. After exposure to FTY720P, the S1P(2)(IC1-TM2)(S1P1) receptor recycled to the plasma membrane, indicating that additional structural elements are required for the selective degradative trafficking of S1P(1).