F1 Subunits Research Articles

Structural and functional characterization of FoF1-ATP synthase on the extracellular surface of rat hepatocytes

Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-FoF1-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F1 (α/β and γ) and Fo (FoI-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF1 is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H+ and are inhibited by F1 and Fo-targeting inhibitors. We hypothesise that ecto-FoF1-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.

S1.26 Structure of the C-terminal domain of the ε subunit of chloroplast-type F1

S1.26 Structure of the C-terminal domain of the ε subunit of chloroplast-type F1

ATP synthases: bioinformatic based insights into how their electrochemically driven motor comprised of subunits a and c might serve as a drug target

ATP synthases, widely distributed in bacteria, eukaryotic mitochondria and chloroplasts, are highly conserved multi-subunit complexes. Although the conserved acidic residue in the transmembrane helix of the c subunit functions in H+ transport, the surrounding residues differ among species. Such divergence could lead to different regulatory modes since pH-dependent H+ transport has been demonstrated in E. coli with a c subunit carrying an additional acidic residue in the helix. There is further divergence in the number of c subunits that form the ring structure which is determined by the higher ordered structure. Recently, it was suggested that certain chemicals recognize the a and c subunits of pathogenic bacterial F0. Since there may be structural divergence even in well-conserved ATP synthases, the c subunit-ring as well as the a subunit in F0 could be targets for drugs for specific bacterial species.

Yersinia pestis caf1Variants and the Limits of Plague Vaccine Protection

Yersinia pestis, the highly virulent agent of plague, is a biological weapon. Strategies that prevent plague have been sought for centuries, and immunization with live, attenuated (nonpigmented) strains or subunit vaccines with F1 (Caf1) antigen is considered effective. We show here that immunization with live, attenuated strains generates plague-protective immunity and humoral immune responses against F1 pilus antigen and LcrV. Y. pestis variants lacking caf1 (F1 pili) are not only fully virulent in animal models of bubonic and pneumonic plague but also break through immune responses generated with live, attenuated strains or F1 subunit vaccines. In contrast, immunization with purified LcrV, a protein at the tip of type III needles, generates protective immunity against the wild-type and the fully virulent caf1 mutant strain, in agreement with the notion that LcrV can elicit vaccine protection against both types of virulent plague strains.

The proximal N-terminal amino acid residues are required for the coupling activity of the bovine heart mitochondrial factor B

Treatment of the recombinant bovine factor B with trypsin yielded a fragment (amino acid residues 62–175) devoid of coupling activity. Removal of the N-terminal Trp2–Gly3–Trp4 peptide resulted in a significant loss of coupling activity in the FBΔW2−W4 deletion mutant. Sucrose density gradient centrifugation demonstrated co-sedimentation of recombinant factor B with the ADP/ATP carrier, which is present in preparations of H+-translocating F0F1-ATPase, but not in preparations of complex V. The N-terminally truncated factor B mutant FBΔW2−W4 did not co-sediment with the ADP/ATP carrier. Recombinant factor B co-sedimented with partially purified membrane sector F0, extracted from F1-stripped bovine submitochondrial particles with n-dodecyl-β-d-maltoside. Factor B inhibited the passive proton conductance catalyzed by F0 reconstituted into asolectin liposomes. A factor B mutant, bearing a photoreactive unnatural amino acid pbenzoyl-l-phenylalanine (pBpa) substituted for Trp2, cross-linked with F0 subunits e and g as well as the ADP/ATP carrier. These results suggest that the N-terminal domain and, in particular, the proximal N-terminal amino acids are important for the coupling activity and protein–protein interactions of bovine factor B.

BASIC FEATURES OF HUMAN METAPNEUMOVIRUS CHINESE ISOLATE PROTEINS

INTRODUCTION: Human metapneumovirus (hMPV), initially described in 2001, is an enveloped RNA virus of the genus Metapneumovirus, subfamily Pneumovirinae, family Paramyxoviridae. OBJECTIVE: We sought to clarify the basic features of hMPV proteins. METHODS: Rabbits were immunized with inactivated virions of hMPV Chinese isolate, CHN05-01, to yield anti-hMPV antiserum. Antiserum was used as primary antibody to detect hMPV proteins by Western blotting. NetNglyc 1.0 server, NetOglyc 3.1 server, and the NetPhos 2.0 server were applied for predicting potential glycosylation and phosphorylation sites of proteins of prototype virus of subtype A, CAN97-83. RESULTS: The highest reactive titer of the antiserum with hMPV antigens reached 1:500 in enzyme-linked immunosorbent assay. Potential glycosylation sites of G protein and phosphorylation sites of P protein were greatest among all hMPV proteins. G protein was shown as a narrow band with molecular weight between 55 and 72 kd (∼68 kd), indicating that its glycosylation level is consistent and remarkably different from that of CAN99-80 and CAN99-81. F1 subunit of fusion protein displayed molecular weight between 40 and 55 kd (∼48 kd), which is consistent with previous reports. CONCLUSIONS: Basic features of 2 major membrane proteins of Chinese hMPV isolate were clarified, which will benefit future studies on protein function and the pathogenesis of this virus.

Role of the ϵ Subunit of Thermophilic F1-ATPase as a Sensor for ATP

The epsilon subunit of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the epsilon subunit remains to be determined. To address this question, 11 mutants of the epsilon subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the alpha(3)beta(3)gamma complex of TF(1). The ATPase activity of the resulting alpha(3)beta(3)gammaepsilon complexes was measured, and the extent of inhibition by the mutant epsilon subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the epsilon subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF(1) by fixing the epsilon subunit into the folded conformation.

Role of the Asymmetry of the Homodimeric b2 Stator Stalk in the Interaction with the F1 Sector of Escherichia coli ATP Synthase

The b subunit dimer in the peripheral stator stalk of Escherichia coli ATP synthase is essential for enzyme assembly and the rotational catalytic mechanism. Recent protein chemical evidence revealed the dimerization domain of b to contain a novel two-stranded right-handed coiled coil with offset helices. Here, the existence of this structure in more complete constructs of b containing the C-terminal domain, and therefore capable of binding to the peripheral F1-ATPase, was supported by the more efficient formation of intersubunit disulfide bonds between cysteine residues that are proximal only in the offset arrangement and by the greater thermal stabilities of cross-linked heterodimers trapped in the offset configuration as opposed to homodimers with the helices trapped in-register. F1-ATPase binding analyses revealed the offset heterodimers to bind F1 more tightly than in-register homodimers. Mutations near the C terminus of b were incorporated specifically into either the N-terminally or the C-terminally shifted polypeptide, bN or bC, respectively, to determine the contribution of each position to F1 binding. Deletion of the last four residues of bN substantially weakened F1 binding, whereas the effect of the deletion in bC was modest. Similarly, benzophenone maleimide introduced at the C terminus of bN, but not bC, mediated cross-linking to the delta subunit of F1. These results imply that the polypeptide in the bN position is more important for F1 binding than the one in the bC position and illustrate the significance of the asymmetry of the b dimer in the enzyme.

An important role for the F2 subunit of Paramyxovirus fusion proteins

An important role for the F2 subunit of Paramyxovirus fusion proteins

Absence of N-linked glycans from the F2 subunit of the major baculovirus envelope fusion protein F enhances fusogenicity

The F protein is the major glycoprotein present in the envelopes of budded virus (BV) of members of the family Baculoviridae. The F protein mediates low-pH-activated fusion with insect cell membranes. Baculovirus F proteins are synthesized as a precursor (F(0)) and cleaved post-translationally into two disulfide-bonded subunits, F(1) (C-terminal, large subunit) and F(2) (N-terminal, small subunit). Recently, N-linked glycosylation of the F(1) and F(2) subunits of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was demonstrated [Long, G., Westenberg, M., Wang, H., Vlak, J. M. & Hu, Z. (2006). J Gen Virol 87, 839-846]. Sequence analysis frequently predicts that one or more N-linked glycosylation sites are present in the F(2) subunit of baculovirus F proteins. N-glycans on envelope fusion proteins are usually required for proper conformational integrity and biological function, such as infectivity. This study examined the importance of N-linked glycosylation of the F(2) subunit of HearNPV by site-directed mutagenesis. The only putative N-linked glycosylation site in F(2) was eliminated by mutating asparagine (N(104)) to glutamine (Q), resulting in the mutant HearNPV(fN104Q). When inserted into an f-null HearNPV and a gp64-null bacmid of Autographa californica multiple nucleopolyhedrovirus, infectious BV could be retrieved that contained unglycosylated F(2). The virulence of HearNPV(fN104Q) was enhanced, as BV was produced earlier after infection and yielded larger plaques than f-null HearNPV repaired with the wild-type f gene. HearNPV(fN104Q) BV also induced much more efficient low-pH-activated syncytium formation. These results indicate that N-linked glycosylation of the HearNPV baculovirus F(2) subunit is not essential for viral infectivity and suggest that it is involved in BV production and fusogenicity.

Assembly of the Stator in Escherichia coli ATP Synthase. Complexation of α Subunit with Other F1 Subunits Is Prerequisite for δ Subunit Binding to the N-Terminal Region of α

Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, was competent in nucleotide binding, and had normal N-terminal sequence. In F1 subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator delta subunit and the N-terminal residue 1-22 region of alpha occurred normally when pure alpha was complexed with other F1 subunits. On the other hand, three different types of experiments showed that no interaction occurred between pure delta and isolated alpha subunit. Unlike in F1, the N-terminal region of isolated alpha was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. We suggest that the N-terminal 1-22 residues of alpha are sequestered in isolated alpha until released by binding of beta to alpha subunit. This prevents 1/1 delta/alpha complexes from forming and provides a satisfactory explanation of the stoichiometry of one delta per three alpha seen in the F1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one delta to the alpha3/beta3 hexagon. The cytoplasmic fragment of the b subunit (bsol) did not bind to isolated alpha. It might also be that complexation of alpha with beta subunits is prerequisite for direct binding of stator b subunit to the F1-sector.

Novel features of the rotary catalytic mechanism revealed in the structure of yeast F1 ATPase

The crystal structure of yeast mitochondrial F(1) ATPase contains three independent copies of the complex, two of which have similar conformations while the third differs in the position of the central stalk relative to the alpha(3)beta(3) sub-assembly. All three copies display very similar asymmetric features to those observed for the bovine enzyme, but the yeast F(1) ATPase structures provide novel information. In particular, the active site that binds ADP in bovine F(1) ATPase has an ATP analog bound and therefore this structure does not represent the ADP-inhibited form. In addition, one of the complexes binds phosphate in the nucleotide-free catalytic site, and comparison with other structures provides a picture of the movement of the phosphate group during initial binding and subsequent catalysis. The shifts in position of the central stalk between two of the three copies of yeast F(1) ATPase and when these structures are compared to those of the bovine enzyme give new insight into the conformational changes that take place during rotational catalysis.

Bovine respiratory syncytial virus lacking the virokinin or with a mutation in furin cleavage site RA(R/K)R109 induces less pulmonary inflammation without impeding the induction of protective immunity in calves

The BRSV fusion (F) protein is cleaved at two furin consensus sequence sites, resulting in the generation of disulphide-linked F1 and F2 subunits and the release of an intervening peptide of 27 amino acids (pep27), which is converted into a biologically active tachykinin (virokinin). The role of the virokinin and the importance of one of the furin cleavage sites, FCS-2 [RA(R/K)R109], in the pathogenesis of BRSV infection and in the subsequent development of immunity was studied in gnotobiotic calves infected with a recombinant BRSV (rBRSV) lacking pep27 (rBRSVdelta p27) or with rBRSV108/109, which contains two amino acid substitutions in FCS-2 (RANN109). Although replication of the mutant viruses and the parental wild-type (WT) rBRSV in the lungs was similar, the extent of gross and microscopic lesions induced by the mutant viruses was less than that induced by WT rBRSV. Furthermore, the numbers of eosinophils in the lungs of calves infected with the mutant viruses were significantly less than that in calves infected with WT virus. These observations suggest a role for the virokinin in the pathogenesis of BRSV infection. Following mucosal immunization with rBRSVdelta p27, the levels of BRSV-specific serum antibodies were similar to those induced by WT virus. In contrast, the level of neutralizing antibodies induced by rBRSV108/109 was 10-fold lower than that induced by WT virus. Nevertheless, resistance to BRSV challenge induced by the mutant and WT viruses was similar, suggesting that neither pep27 nor FCS-2 plays a major role in the induction of protective immunity.

Contribution of cysteine residues in the extracellular domain of the F protein of human respiratory syncytial virus to its function

The mature F protein of all known isolates of human respiratory syncytial virus (HRSV) contains fifteen absolutely conserved cysteine (C) residues that are highly conserved among the F proteins of other pneumoviruses as well as the paramyxoviruses. To explore the contribution of the cysteines in the extracellular domain to the fusion activity of HRSV F protein, each cysteine was changed to serine. Mutation of cysteines 37, 313, 322, 333, 343, 358, 367, 393, 416, and 439 abolished or greatly reduced cell surface expression suggesting these residues are critical for proper protein folding and transport to the cell surface. As expected, the fusion activity of these mutations was greatly reduced or abolished. Mutation of cysteine residues 212, 382, and 422 had little to no effect upon cell surface expression or fusion activity at 32°C, 37°C, or 39.5°C. Mutation of C37 and C69 in the F2 subunit either abolished or reduced cell surface expression by 75% respectively. None of the mutations displayed a temperature sensitive phenotype.

Hydroelectric contributions to mechanisms of mitochondrial oxidative phosphorylation

Solvent fluctuations have been observed by electron microscopy in isolated and mitochondria in situ, in heart and liver under controlled metabolic states. Furthermore, when using mitochondrial particles, ATPase activity is demonstrated in patches on the membranous surface exposed to solvent. These observations have been extended to the molecular level using Protein Data Bank structures. The F1 model presented by Abrahams et al. (Nature 370:621–628) has less solvent content in the βe subunit when compared to the other five subunits of F1. Solvent and proton fluctuations occur in the ATP phosphorylase during ATP synthesis and the experimental ATPase states. Condensation of the mitochondrial matrix is observed during activation of the electron transport chain and ATP synthesis and may be required to obtain solvent proton fluctuations and aggregation of the protein structure. (NIH and University of Wisconsin-Madison Graduate School supported)

A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L

The Nipah virus fusion (F) protein is proteolytically processed to F1 + F2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form.

Both Rotor and Stator Subunits Are Necessary for Efficient Binding of F1 to F0 in Functionally Assembled Escherichia coli ATP Synthase

In F1F0-ATP synthase, the subunit b2delta complex comprises the peripheral stator bound to subunit a in F0 and to the alpha3beta3 hexamer of F1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F0 subunits to the binding of F1 within the assembled holoenzyme, the isolated subunit b dimer, ab2 subcomplex, and fully assembled F0 complex were specifically labeled with tetramethylrhodamine-5-maleimide at bCys64 and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F1 (at gammaCys106 and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F1 dissociation constants of 2.7 nm for the binding of F0 and 9-10 nm for both the ab2 subcomplex and subunit b dimer. This indicates that both rotor and stator components of F0 contribute to F1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F1 to F0, whereas subunit a does not contribute significantly.

Envelope Targeting: Hemagglutinin Attachment Specificity Rather than Fusion Protein Cleavage-Activation Restricts Tupaia Paramyxovirus Tropism

To engineer a targeting envelope for gene and oncolytic vector delivery, we characterized and modified the envelope proteins of Tupaia paramyxovirus (TPMV), a relative of the morbilli- and henipaviruses that neither infects humans nor has cross-reactive relatives that infect humans. We completed the TPMV genomic sequence and noted that the predicted fusion (F) protein cleavage-activation site is not preceded by a canonical furin cleavage sequence. Coexpression of the TPMV F and hemagglutinin (H) proteins induced fusion of Tupaia baby fibroblasts but not of human cells, a finding consistent with the restricted TPMV host range. To identify the factors restricting fusion of non-Tupaia cells, we initially analyzed F protein cleavage. Even without an oligo- or monobasic protease cleavage sequence, TPMV F was cleaved in F1 and F2 subunits in human cells. Edman degradation of the F1 subunit yielded the sequence IFWGAIIA, placing the conserved phenylalanine in position 2, a novelty for paramyxoviruses but not the cause of fusion restriction. We then verified whether the lack of a TPMV H receptor limits fusion. Toward this end, we displayed a single-chain antibody (scFv) specific for the designated receptor human carcinoembryonic antigen on the TPMV H ectodomain. The H-scFv hybrid protein coexpressed with TPMV F mediated fusion of cells expressing the designated receptor, proving that the lack of a receptor limits fusion and that TPMV H can be retargeted. Targeting competence and the absence of antibodies in humans define the TPMV envelope as a module to be adapted for ferrying ribonucleocapsids of oncolytic viruses and gene delivery vectors.

732. Defining Tropism of Oncolytic Vectors by Protease Availability: Measles Viruses Selectively Fusing Matrix-Metalloproteinase Expressing Cells

Measles virus (MV) is oncolytic: wild type MV infection occasionally induces lymphoma regression in humans, and the MV vaccine strain has been used to eliminate different types of human cancer xenografts in mice. Towards enhancing the MV specificity for different tumor types we are operating at different levels, including selective viral activation by tumor-specific matrix metalloproteinases (MMPs). We report here the engineering of a recombinant MV which fusion (F) protein is selectively cleaved and activated by a MMP. The F protein of wild type and vaccine MV is produced as an inative precursor F0 that is cleaved into an amino-terminal F2 subunit and a membrane-anchored F1 subunit bearing a fusion peptide. MV F0 is activated intracellularly by furin, but we have previously engineered a F protein activated by the extracellular protease trypsin (J. Gen. Virol. 81, 441-9, 2000).

An Inhibitor of the F1 Subunit of ATP Synthase (IF1) Modulates the Activity of Angiostatin on the Endothelial Cell Surface

Angiostatin binds to endothelial cell (EC) surface F(1)-F(0) ATP synthase, leading to inhibition of EC migration and proliferation during tumor angiogenesis. This has led to a search for angiostatin mimetics specific for this enzyme. A naturally occurring protein that binds to the F1 subunit of ATP synthase and blocks ATP hydrolysis in mitochondria is inhibitor of F1 (IF1). The present study explores the effect of IF1 on cell surface ATP synthase. IF1 protein bound to purified F(1) ATP synthase and inhibited F(1)-dependent ATP hydrolysis consistent with its reported activity in studies of mitochondria. Although exogenous IF1 did not inhibit ATP production on the surface of EC, it did conserve ATP on the cell surface, particularly at low extracellular pH. IF1 inhibited ATP hydrolysis but not ATP synthesis, in contrast to angiostatin, which inhibited both. In cell-based assays used to model angiogenesis in vitro, IF1 did not inhibit EC differentiation to form tubes and only slightly inhibited cell proliferation compared with angiostatin. From these data, we conclude that inhibition of ATP synthesis is necessary for an anti-angiogenic outcome in cell-based assays. We propose that IF1 is not an angiostatin mimetic, but it can serve a protective role for EC in the tumor microenvironment. This protection may be overridden in a concentration-dependent manner by angiostatin. In support of this hypothesis, we demonstrate that angiostatin blocks IF1 binding to ATP synthase and abolishes its ability to conserve ATP. These data suggest that there is a relationship between the binding sites of IF1 and angiostatin on ATP synthase and that IF1 could be employed to modulate angiogenesis.