Evolutionary and experimental analyses of inorganic phosphate transporter PiT family reveals two related signature sequences harboring highly conserved aspartic acids critical for sodium-dependent phosphate transport function of human PiT2

The mammalian members of the inorganic phosphate (P(i)) transporter (PiT) family, the type III sodium-dependent phosphate (NaP(i)) transporters PiT1 and PiT2, have been assigned housekeeping P(i) transport functions and are suggested to be involved in chondroblastic and osteoblastic mineralization and ectopic calcification. The PiT family members are conserved throughout all kingdoms and use either sodium (Na+) or proton (H+) gradients to transport P(i). Sequence logo analyses revealed that independent of their cation dependency these proteins harbor conserved signature sequences in their N- and C-terminal ends with the common core consensus sequence GANDVANA. With the exception of 10 proteins from extremophiles all 109 proteins analyzed carry an aspartic acid in one or both of the signature sequences. We changed either of the highly conserved aspartates, Asp28 and Asp506, in the N- and C-terminal signature sequences, respectively, of human PiT2 to asparagine and analyzed P(i) uptake function in Xenopus laevis oocytes. Both mutant proteins were expressed at the cell surface of the oocytes but exhibited knocked out NaP(i) transport function. Human PiT2 is also a retroviral receptor and we have previously shown that this function can be exploited as a control for proper processing and folding of mutant proteins. Both mutant transporters displayed wild-type receptor functions implying that their overall architecture is undisturbed. Thus the presence of an aspartic acid in either of the PiT family signature sequences is critical for the Na+-dependent P(i) transport function of human PiT2. The conservation of the aspartates among proteins using either Na+- or H+-gradients for P(i) transport suggests that they are involved in H+-dependent P(i) transport as well. Current results favor a membrane topology model in which the N- and C-terminal PiT family signature sequences are positioned in intra- and extracellular loops, respectively, suggesting that they are involved in related functions on either side of the membrane. The present data are in agreement with a possible role of the signature sequences in translocation of cations.     

Osteoblast autonomous Pi regulation via Pit1 plays a role in bone mineralization

The complex pathogenesis of mineralization defects seen in inherited and/or acquired hypophosphatemic disorders suggests that local inorganic phosphate (P(i)) regulation by osteoblasts may be a rate-limiting step in physiological bone mineralization. To test whether an osteoblast autonomous phosphate regulatory system regulates mineralization, we manipulated well-established in vivo and in vitro models to study mineralization stages separately from cellular proliferation/differentiation stages of osteogenesis. Foscarnet, an inhibitor of NaP(i) transport, blocked mineralization of osteoid formation in osteoblast cultures and local mineralization after injection over the calvariae of newborn rats. Mineralization was also down- and upregulated, respectively, with under- and overexpression of the type III NaP(i) transporter Pit1 in osteoblast cultures. Among molecules expressed in osteoblasts and known to be related to P(i) handling, stanniocalcin 1 was identified as an early response gene after foscarnet treatment; it was also regulated by extracellular P(i), and itself increased Pit1 accumulation in both osteoblast cultures and in vivo. These results provide new insights into the functional role of osteoblast autonomous P(i) handling in normal bone mineralization and the abnormalities seen in skeletal tissue in hypophosphatemic disorders.     

The central half of Pit2 is not required for its function as a retroviral receptor

The type III sodium-dependent phosphate (NaPi) cotransporter, Pit2, is a receptor for amphotropic murine leukemia virus (A-MuLV) and 10A1 MuLV. In order to determine what is sufficient for Pit2 receptor function, a deletion mutant lacking about the middle half of the protein was made. The mutant supported entry for both viruses, unequivocally narrowing down the identification of the sequence that is sufficient to specify the receptor functions of Pit2 to its N-terminal 182 amino acids and C-terminal 170 amino acids.     

A 13-amino-acid Pit1-specific loop 4 sequence confers feline leukemia virus subgroup B receptor function upon Pit2

Feline leukemia virus subgroup B (FeLV-B) and gibbon ape leukemia virus (GALV) utilize the human protein Pit1 but not the related protein, Pit2, as receptor. A stretch of 9 amino acids, named region A, was identified in the putative fourth extracellular loop of Pit1 (residues 550 through 558) as critical for FeLV-B and GALV receptor function. However, the presence of Pit1 region A did not confer receptor function for FeLV-B upon Pit2, while it did so for GALV. We have here shown that the presence of two Pit1-specific loop 4 residues (tyrosine 546 and valine 548) in addition to Pit1 region A is sufficient to make Pit2 an efficient FeLV-B receptor; that is, a stretch of 13 amino acids encompassing all loop 4 amino acid differences between Pit1 and Pit2 comprises a C-terminal determinant for FeLV-B receptor function. Thus, the same limited receptor region is sufficient to confer receptor function for both viruses upon Pit2.     

Fungal phosphate transporter serves as a receptor backbone for gibbon ape leukemia virus.

Pit1, the receptor for gibbon ape leukemia virus (GALV), is proposed to be an integral membrane protein with five extracellular loops. Chimeras made between Pit1 homologs differing in permissivity for infection and between Pit1 and the related protein Pit2 have shown that the fourth extracellular loop plays a critical role in infection. However, further elucidation of the roles of the extracellular loops in infection is hampered by the high level of sequence similarity among these proteins. The sodium-dependent phosphate transporter, Pho-4, from the filamentous fungus Neurospora crassa is distantly related to Pit1 and -2, showing an amino acid identity of only 35% to Pit1 in the putative extracellular loops. We show here that Pho-4 itself does not function as a receptor for GALV. Introduction of 12 Pit1-specific amino acid residues in the putative fourth extracellular loop of Pho-4 resulted in a functional GALV receptor. Therefore, the presence of a Pit1 loop 4-specific sequence is sufficient to confer receptor function for the mammalian retrovirus GALV on the fungal phosphate transporter Pho-4.     

Pit2 assemblies at the cell surface are modulated by extracellular inorganic phosphate concentration

Pit2 is a type III sodium-dependent phosphate transporter and the cell surface receptor for amphotropic murine leukemia virus. Indirect arguments have previously suggested that retrovirus receptor assembly play a role in triggering membrane fusion. Using CHO cells expressing physiological amounts of functional versions of human Pit2 fused to various tagging epitopes, we provide evidence that Pit2 forms assemblies at the cell surface. Living cells were exposed to cross-linking reagents and protein extracts were treated with trifluoroacetic acid (TFA), a chemical that destroys all protein interactions but covalent links. Assemblies were also detected in the absence of cross-linking and TFA treatment, indicating that they are partially resistant to detergent denaturation. The formation of homo-oligomers was documented by the coimmunoprecipitation of differently tagged molecules. The amounts of Pit2 assemblies detected in the presence or in the absence of cross-linking reagents varied with extracellular inorganic phosphate concentration ([P(i)]). Variation of signal intensity was in the range of twofold, occurred in the absence of de novo protein synthesis and took place at the cell surface. These results indicate that Pit2 assemblies exhibit variable conformations at the surface of living cells. Susceptibility to virus infection and phosphate uptake also vary with extracellular [P(i)]. A model is proposed in which cell surface Pit2 assemblies switch from a compacted to an expanded configuration in response to changes of extracellular [P(i)], and possible relationships with the variation of biological activities are discussed.     

Variable regions A and B in the envelope glycoproteins of feline leukemia virus subgroup B and amphotropic murine leukemia virus interact with discrete receptor domains.

The surface (SU) envelope glycoproteins of feline leukemia virus subgroup B (FeLV-B) and amphotropic murine leukemia virus (A-MLV) are highly related, even in the variable regions VRA and VRB that have been shown to be required for receptor recognition. However, FeLV-B and A-MLV use different sodium-dependent phosphate symporters, Pit1 and Pit2, respectively, as receptors for infection. Pit1 and Pit2 are predicted to have 10 membrane-spanning domains and five extracellular loops. The close relationship of the retroviral envelopes enabled us to generate pseudotype virions carrying chimeric FeLV-B/A-MLV envelope glycoproteins. We found that some of the pseudotype viruses could not use Pit1 or Pit2 proteins but could efficiently utilize specific chimeric Pit1/Pit2 proteins as receptors. By studying Mus dunni tail fibroblasts expressing chimeric Pit1/Pit2 proteins and pseudotype virions carrying chimeric FeLV-B/A-MLV envelopes, we show that FeLV-B and A-MLV VRA and VRB interact in a modular manner with specific receptor domains. Our results suggest that FeLV-B VRA interacts with Pit1 extracellular loops 4 and 5 and that residues Phe-60 and Pro-61 of FeLV-B VRA are essential for receptor choice. However, this interaction is insufficient for infection, and an additional interaction between FeLV-B VRB and Pit1 loop 2 is essential. Similarly, A-MLV infection requires interaction of A-MLV VRA with Pit2 loops 4 and 5 and VRB with Pit2 loop 2, with residues Tyr-60 and Val-61 of A-MLV VRA being critical for receptor recognition. Together, our results suggest that FeLV-B and A-MLV infections require two major discrete interactions between the viral SU envelope glycoproteins and their respective receptors. We propose a common two-step mechanism for interaction between retroviral envelope glycoproteins and cell surface receptors.     

Vesicular glutamate transporter contains two independent transport machineries

Vesicular glutamate transporters (VGLUTs) are responsible for the vesicular storage of l-glutamate and play an essential role in glutamatergic signal transmission in the central nervous system. The molecular mechanism of the transport remains unknown. Here, we established a novel in vitro assay procedure, which includes purification of wild and mutant VGLUT2 and their reconstitution with purified bacterial F(o)F(1)-ATPase (F-ATPase) into liposomes. Upon the addition of ATP, the proteoliposomes facilitated l-glutamate uptake in a membrane potential (DeltaPsi)-dependent fashion. The ATP-dependent l-glutamate uptake exhibited an absolute requirement for approximately 4 mm Cl(-), was sensitive to Evans blue, but was insensitive to d,l-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg(184), His(128), and Glu(191) showed a dramatic loss in l-glutamate transport activity, whereas Na(+)-dependent inorganic phosphate (P(i)) uptake remained comparable to that of the wild type. Furthermore, P(i) transport did not require Cl(-) and was not inhibited by Evans blue. Thus, VGLUT2 appears to possess two intrinsic transport machineries that are independent of each other: a DeltaPsi-dependent l-glutamate uptake and a Na(+)-dependent P(i) uptake.     

A comprehensive approach to mapping the interacting surfaces of murine amphotropic and feline subgroup B leukemia viruses with their cell surface receptors

Because mutations in envelope glycoproteins of retroviruses or in their cell surface receptors can eliminate function by multiple mechanisms, it has been difficult to unambiguously identify sites for their interactions by site-directed mutagenesis. Recently, we developed a gain-of-function approach to overcome this problem. Our strategy relies on the fact that feline leukemia virus subgroup B (FeLV-B) and amphotropic murine leukemia virus (A-MLV) have closely related gp70 surface envelope glycoproteins and use related Na(+)-dependent phosphate symporters, Pit1 and Pit2, respectively, as their receptors. We previously observed that FeLV-B/A-MLV envelope glycoprotein chimeras spliced between the variable regions VRA and VRB were unable to use Pit1 or Pit2 as a receptor but could efficiently use specific Pit1/Pit2 chimeras. The latter study suggested that the VRA of A-MLV and FeLV-B functionally interact with the presumptive extracellular loops 4 and 5 (ECL4 and -5) of their respective receptors, whereas VRB interacts with ECL2. We also found that FeLV-B gp70 residues F60 and P61 and A-MLV residues Y60 and V61 in the first disulfide-bonded loop of VRA were important for functional interaction with the receptor's ECL4 or -5. We have now extended this approach to identify additional VRA and VRB residues that are involved in receptor recognition. Our studies imply that FeLV-B VRA residues F60 and P61 interact with the Pit1 ECL5 region, whereas VRA residues 66 to 78 interact with Pit1 ECL4. Correspondingly, A-MLV VRA residues Y60 and V61 interact with the Pit2 ECL5 region, whereas residues 66 to 78 interact with Pit2 ECL4. Similar studies that focused on the gp70 VRB implicated residues 129 to 139 as contributing to specific interactions with the receptor ECL2. These results identify three regions of gp70 that interact in a specific manner with distinct portions of their receptors, thereby providing a map of the functionally interacting surfaces.     

Effect of substrate residues on the P2' preference of retroviral proteinases.

The substrate sequence requirements for preference toward P2' Glu residue by human immunodeficiency virus type 1 (HIV-1) proteinase were studied in both the matrix protein/ capsid protein (MA/CA) and CA/p2 cleavage site sequence contexts. These sequences represent typical type 1 (-aromatic*Pro-) and type 2 (-hydrophobic* hydrophobic-) cleavage site sequences, respectively. While in the type 1 sequence context, the preference for P2' Glu over Ile or Gln was found to be strongly dependent on the ionic strength and the residues being outside the P2-P2' region of the substrate, it remained preferable in the type 2 substrates when typical type 1 substrate sequence residues were substituted into the outside regions. The pH profile of the specificity constants suggested a lower pH optimum for substrates having P2' Glu in contrast to those having uncharged residues, in both sequence contexts. The very low frequency of P2' Glu in naturally occurring retroviral cleavage sites of various retroviruses including equine infectious anemia virus (EIAV) and murine leukemia virus (MuLV) suggests that such a residue may not have a general regulatory role in the retroviral life cycle. In fact, unlike HIV-1 and HIV-2, EIAV and MuLV proteinases do not favor P2' Glu in either the MA/CA or CA/p2 sequence contexts.     

Role of Glu318 at the putative distal site in the catalytic function of cytochrome P450d.

Most microsomal P450s have a conserved "threonine cluster" composed of three Thrs (Thr319, Thr321, Thr322 for P450d) at a putative distal site. An ionic amino acid at 318 is also well conserved as Glu or Asp for most P450s. To understand the role of these conserved polar amino acids at the putative distal site in the catalytic function of microsomal P450, we studied how mutations at this site of P450d influence the activation of molecular oxygen in the reconstituted system. Catalytic activity (0.02 min-1) toward 7-ethoxycoumarin of the Glu318Ala mutant of P450d was just 6% of that (0.33 min-1) of the wild type, while those of Glu318Asp, Thr319Ala, and Thr322Ala were comparable to or even higher than that of the wild type. Consumption rates of O2 and formation rates of H2O2 of those mutants varied in accord with the catalytic activities. Especially, the efficiency (0.5%) of incorporated oxygen atom to the substrate versus produced H2O2 for the Glu318Ala mutant was much lower than that (3.7%) of the wild type, while that (58.8%) for the mutant Glu318Asp was 16-fold higher than that of the wild type. In addition, the autoxidation [Fe(II)---- Fe(III)] rate (0.074 s-1) of the Glu318Ala mutant was much lower than those (0.374-0.803 s-1) of the wild type and other mutants. Thus, we strongly suggest that Glu318 plays an important role in the catalytic function toward 7-ethoxycoumarin of microsomal P450d.     

Identification of Envelope Determinants of Feline Leukemia Virus Subgroup B That Permit Infection and Gene Transfer to Cells Expressing Human Pit1 or Pit2

The retroviral vector systems that are in common use for gene therapy are designed to infect cells expressing either of two widely expressed phosphate transporter proteins, Pit1 or Pit2. Subgroup B feline leukemia viruses (FeLV-Bs) use the gibbon ape leukemia virus receptor, Pit1, as a receptor for entry. Our previous studies showed that some chimeric envelope proteins encoding portions of FeLV-B could also enter cells by using a related receptor protein, Pit2, which serves as the amphotropic murine leukemia virus receptor (S. Boomer, M. Eiden, C. C. Burns, and J. Overbaugh, J. Virol. 71:8116--8123, 1997). Here we show that an arginine at position 73 within variable region A (VRA) of the FeLV-B envelope surface unit (SU) is necessary for viral entry into cells via the human Pit2 receptor. However, C-terminal SU sequences have a dominant effect in determining human Pit2 entry, even though this portion of the protein is outside known receptor binding domains. This suggests that a combination of specific VRA sequences and C-terminal sequences may influence interactions between FeLV-B SU and the human Pit2 receptor. Binding studies suggest that the C-terminal sequences may affect a postbinding step in viral entry via the Pit2 receptor, although in all cases, binding of FeLV-B SU to human Pit2 was weak. In contrast, neither the arginine 73 nor specific C-terminal sequences are required for efficient binding or infection with Pit1. Taken together, these data suggest that different residues in SU may interact with these two receptors. The specific FeLV-Bs described here, which can enter cells using either human Pit receptor, may be useful as envelope pseudotypes for viruses used in gene therapy.     

Role of the progressive ankylosis gene (ank) in cartilage mineralization

Mineralization of growth plate cartilage is a critical event during endochondral bone formation, which allows replacement of cartilage by bone. Ankylosis protein (Ank), which transports intracellular inorganic pyrophosphate (PP(i)) to the extracellular milieu, is expressed by hypertrophic and, especially highly, by terminally differentiated mineralizing growth plate chondrocytes. Blocking Ank transport activity or ank expression in terminally differentiated mineralizing growth plate chondrocytes led to increases of intra- and extracellular PP(i) concentrations, decreases of alkaline phosphatase (APase) expression and activity, and inhibition of mineralization, whereas treatment of these cells with the APase inhibitor levamisole led to an increase of extracellular PP(i) concentration and inhibition of mineralization. Ank-overexpressing hypertrophic nonmineralizing growth plate chondrocytes showed decreased intra- and extracellular PP(i) levels; increased mineralization-related gene expression of APase, type I collagen, and osteocalcin; increased APase activity; and mineralization. Treatment of Ank-expressing growth plate chondrocytes with a phosphate transport blocker (phosphonoformic acid [PFA]) inhibited uptake of inorganic phosphate (P(i)) and gene expression of the type III Na(+)/P(i) cotransporters Pit-1 and Pit-2. Furthermore, PFA or levamisole treatment of Ank-overexpressing hypertrophic chondrocytes inhibited APase expression and activity and subsequent mineralization. In conclusion, increased Ank activity results in elevated intracellular PP(i) transport to the extracellular milieu, initial hydrolysis of PP(i) to P(i), P(i)-mediated upregulation of APase gene expression and activity, further hydrolysis and removal of the mineralization inhibitor PP(i), and subsequent mineralization.     

Amphotropic murine leukemia virus entry is determined by specific combinations of residues from receptor loops 2 and 4

Pit2 is the human receptor for amphotropic murine leukemia virus (A-MuLV); the related human protein Pit1 does not support A-MuLV entry. Interestingly, chimeric proteins in which either the N-terminal or the C-terminal part of Pit2 was replaced by the Pit1 sequence all retained A-MuLV receptor function. A possible interpretation of these observations is that Pit1 harbors sequences which can specify A-MuLV receptor function when presented in a protein context other than Pit1, e.g., in Pit1-Pit2 hybrids. We reasoned that such Pit1 sequences might be identified if presented in the Neurospora crassa protein Pho-4. This protein is distantly related to Pit1 and Pit2, predicted to have a similar membrane topology with five extracellular loops, and does not support A-MuLV entry. We show here that introduction of the Pit1-specific loop 2 sequence conferred A-MuLV receptor function upon Pho-4. Therefore, we conclude that (i) a functional A-MuLV receptor can be constructed by combining sequences from two proteins each lacking A-MuLV receptor function and that (ii) a Pit1 sequence can specify A-MuLV receptor function when presented in another protein context than that provided by Pit1 itself. Previous results indicated a role of loop 4 residues in A-MuLV entry, and the presence of a Pit2-specific loop 4 sequence was found here to confer A-MuLV receptor function upon Pho-4. Moreover, the introduction of a Pit1-specific loop 4 sequence, but not of a Pit2-specific loop 4 sequence, abolished the A-MuLV receptor function of a Pho-4 chimera harboring the Pit1-specific loop 2 sequence. Together, these data suggest that residues in both loop 2 and loop 4 play a role in A-MuLV receptor function. A-MuLV is, however, not dependent on the specific Pit2 loop 2 and Pit2 loop 4 sequences for entry; rather, the role played by loops 2 and 4 in A-MuLV entry can be fulfilled by several different combinations of loop 2 and loop 4 sequences. We predict that the residues in loops 2 and 4, identified in this study as specifying A-MuLV receptor function, are to be found among those not conserved among Pho-4, Pit1, and Pit2.     

Functional studies on a split type II Na/P(i)-cotransporter

Analysis of rat and mouse proximal tubular brush-border membrane expression of the type IIa Na/P(i)-cotransporter provides evidence for its cleavage in the large extracellular loop (ECL-2). To study functional properties and membrane distribution of this split NaP(i)-IIa transporter we followed two strategies. In one strategy we expressed the transporter as two complementary parts (p40 and p45) in Xenopus laevis oocytes and as another strategy we cleaved the WT protein with trypsin. Both strategies resulted in a split NaP(i)-IIa protein located in the plasma membrane. The two domains were tied together by a disulfide bridge, most likely involving the cysteines 306 and 334. Surface expression of the NaP(i)-IIa fragments was dependent on the presence of both domains. If both domains were coexpressed, the transporter was functional and transport characteristics were identical to those of the WT-NaP(i)-IIa protein. Corresponding to this, the transporter cleaved by trypsin also retains its transport capacity. These data indicate that cleavage of the type IIa Na/P(i)-cotransporter at ECL-2 is compatible with its cotransport function.     

Four conserved cytoplasmic sequence motifs are important for transport function of the Leishmania inositol/H(+) symporter

The protozoan Leishmania donovani has a myo-inositol/proton symporter (MIT) that is a member of a large sugar transporter superfamily. Active transport by MIT is driven by the proton electrochemical gradient across the parasite membrane, and MIT is a prototype for understanding the function of an active transporter in lower eukaryotes. MIT contains two duplicated 6- or 7-amino acid motifs within cytoplasmic loops, which are highly conserved among 50 members of the sugar transporter superfamily and are designated A(1), A(2) ((V)(D/E)(R/K)PhiGR(R/K)), and B(1) (PESPRPhiL), B(2) (VPETKG). In particular, the three acidic residues within these motifs, Glu(187)(B(1)), Asp(300)(A(2)), and Glu(429)(B(2)) in MIT, are highly conserved with 96, 78, and 96% amino acid identity within the analyzed members of this transporter superfamily ranging from bacteria, archaea, and fungi to plants and the animal kingdom. We have used site-directed mutagenesis in combination with functional expression of transporter mutants in Xenopus oocytes and overexpression in Leishmania transfectants to investigate the significance of these three acidic residues in the B(1), A(2), and B(2) motifs. Alteration to the uncharged amides greatly reduced MIT transport function to 23% (E187Q), 1.4% (D300N), and 3% (E429Q) of wild-type activity, respectively, by affecting V(max) but not substrate affinity. Conservative mutations that retained the charge revealed a less pronounced effect on inositol transport with 39% (E187D), 16% (D300E) and 20% (E429D) remaining transport activity. Immunofluorescence microscopy of oocyte cryosections confirmed that MIT mutants were expressed on the oocyte surface in similar quantity to MIT wild type. The proton uncouplers carbonylcyanide-4-(trifluoromethoxy) phenylhydrazone and dinitrophenol inhibited inositol transport by 50-70% in the wild type as well as in E187Q, D300N, and E429Q, despite their reduced transport activities, suggesting that transport in these mutants is still proton-coupled. Furthermore, temperature-dependent uptake studies showed an increased Arrhenius activation energy for the B(1)-E187Q and the B(2)-E429Q mutants, which supports the idea of an impaired transporter cycle in these mutants. We conclude that the conserved acidic residues Glu(187), Asp(300), and Glu(429) are critical for transport function of MIT.     

Characterization of two genetically separable inorganic phosphate transport systems in Escherichia coli.

Inorganic phosphate (Pi) transport by wild-type cells of Escherichia coli grown in excess phosphate-containing media involves two genetically separable transport systems. Cells dependent upon the high affinity-low velocity Pst (phosphate specific transport) system have a Km of 0.43 +/- 0.2 microM Pi and a Vmax of 15.9 +/- 0.3 nmol of Pi (mg [dry weight]-1min-1) and will grow in the presence of arsenate in the medium. However, cells dependent upon the low affinity-high velocity Pit (Pi transport) system have a Km of 38.2 +/- 0.4 microM and a Vmax of 55 +/- 1.9 nmol of Pi (mg [dry weight]-1min-1), and these cells cannot grow in the presence of an arsenate-to-Pi ratio of 10 in the medium. Pi transport by both systems was sensitive to the energy uncoupler 2,4-dinitrophenol and the sulfhydryl reagent N-ethylmaleimide, whereas only the Pst system was very sensitive to sodium cyanide. Evidence is presented that Pi is transported as Pi or a very labile intermediate and that accumulated Pi does not exit through the Pst or Pit systems from glucose-grown cells. Kinetic analysis of Pi transport in the wild-type strain containing both the Pst and Pit transport systems revealed that each system was not operating at full capacity. In addition, Pi transport in the wild-type strain was completely sensitive to sodium cyanide (a characteristic of the Pst system).     

Molecular dynamics simulation of the P2Y14 receptor. Ligand docking and identification of a putative binding site of the distal hexose moiety

A rhodopsin-based homology model of the P2Y14 receptor was inserted into a phospholipid bilayer and refined by molecular dynamics (MD) simulation. The binding modes of several known agonists, namely UDP-glucose and its analogues, were proposed using automatic molecular docking combined with Monte Carlo Multiple Minimum calculations. Compared to other P2Y receptors, the P2Y14 receptor has an atypical binding mode of the nucleobase, ribose, and phosphate moieties. The diphosphate moiety interacts with only one cationic residue, namely Lys171 of EL2, while in other P2Y receptor subtypes three Arg or Lys residues interact with the phosphate chain. Two other conserved cationic residues, namely Arg253 (6.55) and Lys277 (7.35) of the P2Y14 receptor together with two anionic residues (Glu166 and Glu174, located in EL2), are likely involved in interactions with the distal hexose moiety.