Saposin B mobilizes lipids from cholesterol-poor and bis(monoacylglycero)phosphate-rich membranes at acidic pH. Unglycosylated patient variant saposin B lacks lipid-extraction capacity

Sphingolipid activator proteins (SAPs), GM2 activator protein (GM2AP) and saposins (Saps) A-D are small, enzymatically inactive glycoproteins of the lysosome. Despite of their sequence homology, these lipid-binding and -transfer proteins show different specificities and varying modes of action. Water-soluble SAPs facilitate the degradation of membrane-bound glycosphingolipids with short oligosaccharide chains by exohydrolases at the membrane-water interface. There is strong evidence that degradation of endocytosed components of the cell membrane takes place at intraendosomal and intralysosomal membranes. The inner membranes of the lysosome differ from the limiting membrane of the organelle in some typical ways: the inner vesicular membranes lack a protecting glycocalix, and they are almost free of cholesterol, but rich in bis(monoacylglycero)phosphate (BMP), the anionic marker lipid of lysosomes. In this study, we prepared glycosylated Sap-B free of other Saps by taking advantage of the Pichia pastoris expression system. We used immobilized liposomes as a model for intralysosomal vesicular membranes to probe their interaction with recombinantly expressed Sap-B. We monitored this interaction using SPR spectroscopy and an independent method based on the release of radioactively labelled lipids from liposomal membranes. We show that, after initial binding, Sap-B disturbs the membrane structure and mobilizes the lipids from it. Lipid mobilization is dependent on an acidic pH and the presence of anionic lipids, whereas cholesterol is able to stabilize the liposomes. We also show for the first time that glycosylation of Sap-B is essential to achieve its full lipid-extraction activity. Removal of the carbohydrate moiety of Sap-B reduces its membrane-destabilizing quality. An unglycosylated Sap-B variant, Asn215His, which causes a fatal sphingolipid storage disease, lost the ability to extract membrane lipids at acidic pH in the presence of BMP.     

Interaction of saposins, acidic lipids, and glucosylceramidase

Activity of lysosomal glucosylceramidase is stimulated by two small glycoproteins, saposin A and C, which are, together with two other similar glycoproteins, derived from a single precursor protein. This enzyme is also stimulated by naturally occurring acidic lipids, such as phosphatidylserine and gangliosides. Using highly purified glucosylceramidase, saposins, and acidic lipids, the mechanism of enzyme stimulation was studied by investigating complex formation between the three components and by examining effects on activity caused by changing amounts of saposins and acidic lipids, individually or in combination. The results indicated that acidic lipids form a water-soluble complex with glucosylceramidase but not with saposins and that saposins and acidic lipids each bind to the enzyme at two different sites for the activation. Based on these observations, the previously proposed three-binding sites model of glucosylceramidase, activator, and substrate was modified to one composed of four binding sites: one for carbohydrate of the substrate, one for aglycon, one for acidic lipids, and one for saposins.     

Membrane environment alters the conformational structure of the recombinant human prion protein

The prion protein (PrP) in a living cell is associated with cellular membranes. However, all previous biophysical studies with the recombinant prion protein have been performed in an aqueous solution. To determine the effect of a membrane environment on the conformational structure of PrP, we studied the interaction of the recombinant human prion protein with model lipid membranes. The protein was found to bind to acidic lipid-containing membrane vesicles. This interaction is pH-dependent and becomes particularly strong under acidic conditions. Spectroscopic data show that membrane binding of PrP results in a significant ordering of the N-terminal part of the molecule. The folded C-terminal domain, on the other hand, becomes destabilized upon binding to the membrane surface, especially at low pH. Overall, these results show that the conformational structure and stability of the recombinant human PrP in a membrane environment are substantially different from those of the free protein in solution. These observations have important implications for understanding the mechanism of the conversion between the normal (PrP(C)) and pathogenic (PrP(Sc)) forms of prion protein.     

Phospholipid vesicle fusion induced by saposin C

Saposin C is a small Trp-free, multifunctional glycoprotein that enhances the hydrolytic activity of acid beta-glucosidase in lysosomes. Saposin C's functions have been shown to include neuritogenic/neuroprotection effects and membrane fusion induction. Here, the mechanism and kinetics of saposin C's fusogenic activity were evaluated by fluorescence spectroscopic methods including dequenching, fluorescence resonance energy transfer, and stopped-flow analyses. Trp or dansyl groups were introduced as fluorescence reporters into selected sites of saposin C to serve as topological probes for protein-protein and protein-membrane interactions. Saposin C induction of liposomal vesicle enlargement was dependent upon anionic phospholipids and acidic pH. The initial fusion burst was completed in the timeframe of a few seconds to minutes and was dependent upon the unsaturated anionic phospholipid content. Two events were associated with saposin C-membrane interaction: membrane insertion of the saposin C terminal helices and reorientation of its central helical region. The latter conformational change likely exposed a binding site for saposins anchored on vesicles. Addition of selected saposin C peptides prior to intact saposin C in reaction mixtures abolished the liposomal fusion. These results indicated that saposin-membrane and saposin-saposin interactions are needed for the fusion process.     

Crystal structures of saposins A and C

Saposins A and C are sphingolipid activator proteins required for the lysosomal breakdown of galactosylceramide and glucosylceramide, respectively. The saposins interact with lipids, leading to an enhanced accessibility of the lipid headgroups to their cognate hydrolases. We have determined the crystal structures of human saposins A and C to 2.0 Angstroms and 2.4 Angstroms, respectively, and both reveal the compact, monomeric saposin fold. We confirmed that these two proteins were monomeric in solution at pH 7.0 by analytical centrifugation. However, at pH 4.8, in the presence of the detergent C(8)E(5), saposin A assembled into dimers, while saposin C formed trimers. Saposin B was dimeric under all conditions tested. The self-association of the saposins is likely to be relevant to how these small proteins interact with lipids, membranes, and hydrolase enzymes.     

Saposins: structure, function, distribution, and molecular genetics.

Saposins A, B, C, and D are small heat-stable glycoproteins derived from a common precursor protein, prosaposin. These mature saposins, as well as prosaposin, activate several lysosomal hydrolases involved in the metabolism of various sphingolipids. All four saposins are structurally similar to one another including placement of six cysteines, a glycosylation site, and conserved prolines in identical positions. In spite of the structural similarities, the specificity and mode of activation of sphingolipid hydrolases differs among individual saposins. Saposins appear to be lysosomal proteins, exerting their action upon lysosomal hydrolases. Prosaposin is a 70 kDa glycoprotein containing four domains, one for each saposin, placed in tandem. Prosaposin is proteolytically processed to saposins A, B, C and D, apparently within lysosomes. However, prosaposin also exists as an integral membrane protein not destined for lysosomal entry and exists uncleaved in many biological fluids such as seminal plasma, human milk, and cerebrospinal fluid, where it appears to have a different function. The physiological significance of saposins is underlined by their accumulation in tissues of lysosomal storage disease patients and the occurrence of sphingolipidosis due to mutations in the prosaposin gene. This review presents an overview of the occurrence, structure and function of these saposin proteins.     

pH-induced alterations in the fusogenic spike protein of Semliki Forest virus

The spike glycoproteins of Semliki Forest virus mediate membrane fusion between the viral envelope and cholesterol-containing target membranes under conditions of mildly acidic pH (pH less than 6.2). The fusion reaction is critical for the infectious cycle, catalyzing virus penetration from the acidic endosome compartment. To define the role of the viral spike glycoproteins in the fusion reaction, conformational changes in the spikes at acid pH were studied using protease digestion and binding assays to liposomes and nonionic detergent. A method was also developed to prepare fragments of both transmembrane subunit glycopolypeptides of the spike, E1 and E2, which lacked the hydrophobic anchor peptides. Unlike the intact spikes the fragments were monomeric and therefore useful for obtaining information on conformational changes in individual subunits. The results showed that both E1 and E2 undergo irreversible conformational changes at the pH of fusion, that the conformational change of E1 depends, in addition to acidic pH, on the presence of cholesterol, and that no major changes in the solubility properties of the spikes takes place. On the basis of these findings it was concluded that fusion involves both subunits of the spike and that E1 confers the stereo-specific sterol requirement. The results indicated, moreover, that acid-induced fusion of Semliki Forest virus differs in important respects from that of influenza virus, another well-defined model system for protein-mediated membrane fusion.     

The 1-127 HA2 construct of influenza virus hemagglutinin induces cell-cell hemifusion.

Conformational changes in the HA2 subunit of influenza hemagglutinin (HA) are coupled to membrane fusion. We investigated the fusogenic activity of the polypeptide FHA2 representing 127 amino-terminal residues of the ectodomain of HA2. While the conformation of FHA2 both at neutral and at low pH is nearly identical to the final low-pH conformation of HA2, FHA2 still induces lipid mixing between liposomes in a low-pH-dependent manner. Here, we found that FHA2 induces lipid mixing between bound cells, indicating that the "spring-loaded" energy is not required for FHA2-mediated membrane merger. Although, unlike HA, FHA2 did not form an expanding fusion pore, both acidic pH and membrane concentrations of FHA2, required for lipid mixing, have been close to those required for HA-mediated fusion. Similar to what is observed for HA, FHA2-induced lipid mixing was reversibly blocked by lysophosphatidylcholine and low temperature, 4 degrees C. The same genetic modification of the fusion peptide inhibits both HA- and FHA2-fusogenic activities. The kink region of FHA2, critical for FHA2-mediated lipid mixing, was exposed in the low-pH conformation of the whole HA prior to fusion. The ability of FHA2 to mediate lipid mixing very similar to HA-mediated lipid mixing is consistent with the hypothesis that hemifusion requires just a portion of the energy released in the conformational change of HA at acidic pH.     

The effects of pH and temperature on the circular dichroism of human erythrocyte membranes.

The effects of pH and temperature on the structure of human erythrocyte membranes were studied by circular dichroism (CD). The results obtained demonstrate that the membrane CD spectra undergo significant changes when the pH of the solution deviates from its native pH range of 7 to 8. Spectral changes in the acidic pH region include drastic reductions and slight shifts in the CD signal which may reflect a decrease in alpha-helical content of the proteins and/or an increase in optical artifacts, both of which are irreversible. In the alkaline pH region, dramatic increases in ellipticity and blue-shifts in the spectra are observed between pH 8 and 10. In addition, the spectra more closely resemble those reported for membrane samples where the spectral distortions have been removed. The changes in the alkaline region are demonstrated to be only partially reversible and may be due to conformational alterations in the membrane proteins and/or to a reduction in optical distortions. Thermal stability studies reinforce the irreversible behavior of the membrane samples.     

Low pH-Induced Conformational Change in Herpes Simplex Virus Glycoprotein B

Herpesviruses can enter host cells using pH-dependent endocytosis pathways in a cell-specific manner. Envelope glycoprotein B (gB) is conserved among all herpesviruses and is a critical component of the complex that mediates membrane fusion and entry. Here we demonstrate that mildly acidic pH triggers specific conformational changes in herpes simplex virus (HSV) gB. The antigenic structure of gB was specifically altered by exposure to low pH both in vitro and during entry into host cells. The oligomeric conformation of gB was altered at a similar pH range. Exposure to acid pH appeared to convert virion gB into a lower-order oligomer. The detected conformational changes were reversible, similar to those in other class III fusion proteins. Exposure of purified, recombinant gB to mildly acidic pH resulted in similar changes in conformation and caused gB to become more hydrophobic, suggesting that low pH directly affects gB. We propose that intracellular low pH induces alterations in gB conformation that, together with additional triggers such as receptor binding, are essential for virion-cell fusion during herpesviral entry by endocytosis.Herpes simplex virus (HSV) is an important human pathogen, causing significant morbidity and mortality worldwide. HSV enters host cells by fusion of the viral envelope with either an endosomal membrane (38) or the plasma membrane (63). The entry pathway taken is thought to be determined by both virus (17, 45) and host cell (4, 17, 35, 39, 45) factors. Based on experiments with lysosomotropic agents, which elevate the normally low pH of endosomes, acidic pH has been implicated in the endocytic entry of HSV into several cell types, including human epithelial cells (37). Low pH has also recently been implicated in cell infection by several other human and veterinary herpesviruses (1, 21, 26, 47). The mechanistic role of endosomal pH in herpesvirus entry into cells is not known.Herpesviruses are a paradigm for membrane fusion mediated by a complex of several glycoproteins. We have proposed that HSV likely encodes machinery to mediate both pH-dependent and pH-independent membrane fusion reactions. Envelope glycoproteins glycoprotein B (gB) and gD and the heterodimer gH-gL are required for both pH-independent and pH-dependent entry pathways (11, 22, 30, 39, 46). Interaction of gD with one of its cognate receptors is an essential trigger for membrane fusion and entry (13, 52), regardless of the cellular pathway. However, engagement of a gD receptor is not sufficient for fusion, and at least one additional unknown trigger involving gB or gH-gL is likely necessary. gB is conserved among all herpesviruses, and in all cases studied to date, it plays roles in viral entry, including receptor binding and membrane fusion. The crystal structure of an ectodomain fragment of HSV type 1 (HSV-1) gB is an elongated, rod-like structure containing hydrophobic internal fusion loops (28). This structure bears striking architectural homology to the low pH, postfusion form of G glycoprotein from vesicular stomatitis virus (VSV-G) (43). Both the gB and G structures have features of class I and class II fusion proteins and are thus designated class III proteins (57).During entry of the majority of virus families, low pH acts directly on glycoproteins to induce membrane fusion (60). In some cases, the low pH trigger is not sufficient, or it plays an indirect role. For example, host cell proteases, such as cathepsins D and L, require intravesicular low pH to cleave Ebola virus and severe acute respiratory syndrome (SARS) glycoproteins to trigger fusion (14, 51).We investigated the role of low pH in the molecular mechanism of herpesviral entry. The results suggest that mildly acidic pH, similar to that found within endosomes, triggers a conformational change in gB. We propose that, together with other cellular cues such as receptor interaction, intracellular low pH can play a direct activating role in HSV membrane fusion and entry.     

Sphingolipid hydrolase activator proteins and their precursors

Activator proteins for sphingolipid hydrolases (saposins) are small acidic, heat-stable glycoproteins that stimulate the hydrolysis of sphingolipids by lysosomal enzymes. The molecular mass of each stimulator is about 10 kDa, but glycosylated forms of higher mass exist too. The distribution and developmental changes in two saposins and their precursor proteins were studied with the aid of monospecific antibodies against saposin-B and saposin-C. They show a wide distribution in rat organs and forms intermediate between saposin and prosaposin (the precursor protein containing four different saposin units) could be seen. The amount of saposin and the degree of processing from prosaposin are quite different in different tissues. The saposins are the dominant forms in spleen, lung, liver, and kidney, while skeletal muscle, heart, and brain contain mainly precursor forms. In human blood, leukocytes contain mainly saposin, while plasma contains mainly precursor forms and platelets show many forms. Their subcellular distribution was studied using rat liver. The saposins of approximately 20 kDa are dominant in the light mitochondrial, mitochondrial, and microsomal fractions, following the distribution of the activity of a lysosomal marker enzyme. The nuclear fraction exhibits bands corresponding to non-glycosylated saposin. The soluble fraction contained much precursor forms. A developmental study of rat brain showed that the concentration of saposin precursors increased with age.     

Redox components of cytochrome bc-type enzymes in acidophilic prokaryotes. II. The Rieske protein of phylogenetically distant acidophilic organisms.

The Rieske proteins of two phylogenetically distant acidophilic organisms, i.e. the proteobacterium Thiobacillus ferrooxidans and the crenarchaeon Sulfolobus acidocaldarius, were studied by EPR. Redox titrations at a range of pH values showed that the Rieske centers of both organisms are characterized by redox midpoint potential-versus-pH curves featuring a common pK value of 6.2. This pK value is significantly more acidic (by almost 2 pH units) than that of Rieske proteins in neutrophilic species. The orientations of the Rieske center's g tensors with respect to the plane of the membrane were studied between pH 4 and 8 using partially ordered samples. At pH 4, the Sulfolobus Rieske cluster was found in the "typical" orientation of chemically reduced Rieske centers, whereas this orientation changed significantly on going toward high pH values. The Thiobacillus protein, by contrast, appeared to be in the "standard" orientation at both low and high pH values. The results are discussed with respect to the molecular parameters conveying acid resistance and in light of the recently demonstrated long-range conformational movement of the Rieske protein during enzyme turnover in cytochrome bc1 complexes.     

Conformational and amino acid residue requirements for the saposin C neuritogenic effect.

Prosaposin is the precursor of four activator proteins, termed saposins A, B, C, and D, that are required for much of glycosphingolipid hydrolysis. The intact precursor also has neurite outgrowth activity ex vivo and in vivo that is localized to amino acid residues 22-31 of saposin C. Across species, this saposin C region has a high degree of identity and similarity with amino acids in the analogous region of saposin A. Wild-type and mutant saposins C and A from human and mouse were expressed in E. coli. Pure proteins, synthetic peptide analogues, conformation-specific antibodies, and CD spectroscopy were used to evaluate the basis of the ex vivo neuritogenic effect. Wild-type saposin A had no neuritogenic activity whereas reduced and alkylated saposin A did. Introduction of the conserved saposin A Tyr 30 (Y30) into saposin C at the analogous position 31, a conserved Ala(A)/Gly(G)31, diminished neuritogenic activity by 50-60%. Nondenatured saposin A with an introduced A30 acquired substantial neuritogenic activity. Polyclonal antibodies directed against the NH2-terminus of saposin C cross-reacted well with reduced and alkylated saposins C and A, wild-type saposin C, and saposin A [Y30A], poorly with saposin C [A31Y], and not at all with wild-type saposin A. CD spectra of wild-type and mutant saposins C and A, the corresponding neuritogenic region of saposin C, and the analogous region of saposin A showed that more "saposin C-like" molecules had neuritogenic properties. Those with more "saposin A-like" spectra did not. These studies show that the neuritogenic activity of saposin C requires specific placement of amino acids, and that Y30 of saposin A significantly alters local conformation in this critical region and suppresses neuritogenic activity.     

Novel organization and properties of annexin 2-membrane complexes

Annexin 2 belongs to the annexin family of proteins that bind to phospholipid membranes in a Ca(2+)-dependent manner. Here we show that, under mild acidic conditions, annexin 2 binds to and aggregates membranes containing anionic phospholipids, a fact that questions the mechanism of its interaction with membranes via Ca(2+) bridges only. The H(+) sensitivity of annexin 2-mediated aggregation is modulated by lipid composition (i.e. cholesterol content). Cryo-electron microscopy of aggregated liposomes revealed that both the monomeric and the tetrameric forms of the protein form bridges between the liposomes at acidic pH. Monomeric annexin 2 induced two different organizations of the membrane junctions. The first resembled that obtained at pH 7 in the presence of Ca(2+). For the tetramer, the arrangement was different. These bridges seemed more flexible than the Ca(2+)-mediated junctions allowing the invagination of membranes. Time-resolved fluorescence analysis at mild acidic pH and the measurement of Stokes radius revealed that the protein undergoes conformational changes similar to those induced by Ca(2+). Labeling with the lipophilic probe 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine indicated that the protein has access to the hydrophobic part of the membrane at both acidic pH in the absence of Ca(2+) and at neutral pH in the presence of Ca(2+). Models for the membrane interactions of annexin 2 at neutral pH in the presence of Ca(2+) and at acidic pH are discussed.     

Structural requirements for lysosomal targeting of the prosaposin precursor protein

Although the Man-6-P-independent lysosomal sorting of prosaposin, a precursor of four saposins (A, B, C, and D) is not understood, a protein/lipid interaction is considered. Immunocytochemical analysis revealed that each single saposin linked to the C-terminus of prosaposin and to secretory albumin, drives the chimeric protein to lysosomes in COS-7 cells. Quantitative image analysis demonstrated that saposins are targeted with different efficiency (P<0.05) and in a less smooth manner than the precursor. Despite a very close homology, the charge distribution at the surface of 3D comparative models between saposins appeared different. Western blotting monitored prosaposin in cells also as a di- or trimeric form, whereas the chimeric saposins as monomeric. This implies that each amphipathic saposin-like motif may be a part of the overall structural requirements for binding of the precursor to the membrane lipids of transport vesicle. The crystal structure of saposin B demonstrating two dimeric units for lipid binding supports current findings.     

Proteome resolution by two-dimensional gel electrophoresis varies with the commercial source of IPG strips

By facilitating reproducible first dimension separations, commercial immobilized pH gradient (IPG) strips enable high throughput and high-resolution proteomic analyses using two-dimensional gel electrophoresis (2DE). Amersham, Biorad, Invitrogen, and Sigma all market linear pH 3-10 IPG strips. We have applied optimized 2DE protocols with both membrane and soluble brain protein extracts to critically evaluate all four products. Resolved protein spots were quantitatively evaluated after carrying out these protocols using IPG strips from the four companies. Biorad and Amersham IPG strips resolved a high number of membrane and soluble proteins, respectively. Furthermore, Amersham IPG strips eluted the largest amount of protein into the second dimension gels and had the most protein remaining in the strip after 2DE. Biorad and Amersham IPG strips maintained a consistent linear pH 3-10 gradient, whereas those from Invitrogen appeared nonlinear or "compressed" within the central pH region. The gradient range within Sigma IPG strips appeared to be slightly less than pH 3-10, due to one extended pH unit within the gradient. Overall, all four commercially available IPG strips have the ability to resolve both membrane and soluble brain proteomes. The difference is that Amersham and Biorad do so more consistently and with better spot resolution. It appears that the physical/chemical nature of commercially available IPG strips can vary considerably, leading to marked differences in subsequent protein resolution in 2DE. These differences likely reflect variations in the uptake of proteins into the strips, and differences in the focusing and elution of proteins from the first to the second dimension. These differences would appear, in part, to underlie some inter-lab variations in the effective resolution of proteomes.     

Structural and membrane-binding properties of saposin D.

Saposin D is generated together with three similar proteins, saposins A, B and C, from a common precursor, called prosaposin, in acidic organelles such as late endosomes and lysosomes. Although saposin D has been reported to stimulate the enzymatic hydrolysis of sphingomyelin and ceramide, its physiological role has not yet been clearly established. In the present study we examined structural and membrane-binding properties of saposin D. At acidic pH, saposin D showed a great affinity for phospholipid membranes containing an anionic phospholipid such as phosphatidylserine or phosphatidic acid. The binding of saposin D caused destabilization of the lipid surface and, conversely, the association with the membrane markedly affected the fluorescence properties of saposin D. The presence of phosphatidylserine-containing vesicles greatly enhanced the intrinsic tyrosine fluorescence of saposin D, which contains tyrosines but not tryptophan residues. The structural properties of saposin D were investigated in detail using advanced MS analysis. It was found that the main form of saposin D consists of 80 amino acid residues and that the six cysteine residues are linked in the following order: Cys5-Cys78, Cys8-Cys72 and Cys36-Cys47. The disulfide pattern of saposin D is identical with that previously established for two other saposins, B and C, which also exhibit a strong affinity for lipids. The common disulfide structure probably has an important role in the interaction of these proteins with membranes. The analysis of the sugar moiety of saposin D revealed that the single N-glycosylation site present in the molecule is mainly modified by high-mannose-type structures varying from two to six hexose residues. Deglycosylation had no effect on the interaction of saposin D with phospholipid membranes, indicating that the glycosylation site is not related to the lipid-binding site. The association of saposin D with membranes was highly dependent on the composition of the bilayer. Neither ceramide nor sphingomyelin, sphingolipids whose hydrolysis is favoured by saposin D, promoted its binding, while the presence of an acidic phospholipid such as phosphatidylserine or phosphatidic acid greatly favoured the interaction of saposin D with vesicles at low pH. These results suggest that, in the acidic organelles where saposins are localized, anionic phospholipids may be determinants of the saposin D topology and, conversely, saposin D may affect the lipid organization of anionic phospholipid-containing membranes.     

Hepatitis C virus is primed by CD81 protein for low pH-dependent fusion

Hepatitis C virus (HCV) entry into permissive cells is a complex process that involves interactions with at least four co-factors followed by endocytosis and low pH-dependent fusion with endosomes. The precise sequence of receptor engagement and their roles in promoting HCV E1E2 glycoprotein-mediated fusion are poorly characterized. Because cell-free HCV tolerates an acidic environment, we hypothesized that binding to one or more receptors on the cell surface renders E1E2 competent to undergo low pH-induced conformational changes and promote fusion with endosomes. To test this hypothesis, we examined the effects of low pH and of the second extracellular loop (ECL2) of CD81, one of the four entry factors, on HCV infectivity. Pretreatment with an acidic buffer or with ECL2 enhanced infection through changing the E1E2 conformation, as evidenced by the altered reactivity of these proteins with conformation-specific antibodies and stable association with liposomes. However, neither of the two treatments alone permitted direct fusion with the cell plasma membrane. Sequential HCV preincubation with ECL2 and acidic buffer in the absence of target cells resulted in a marked loss of infectivity, implying that the receptor-bound HCV is primed for low pH-dependent conformational changes. Indeed, soluble receptor-pretreated HCV fused with the cell plasma membrane at low pH under conditions blocking an endocytic entry pathway. These findings suggest that CD81 primes HCV for low pH-dependent fusion early in the entry process. The simple triggering paradigm and intermediate conformations of E1E2 identified in this study could help guide future vaccine and therapeutic efforts to block HCV infection.     

Viral fusion mechanisms

The majority of viral fusion proteins can be divided into two classes. The influenza hemagglutinin (HA) belongs to the class I fusion proteins and undergoes a series of conformational changes at acidic pH, leading to membrane fusion. The crystal structures of the prefusion and the postfusion forms of HA have been revealed in 1981 and 1994, respectively. On the basis of these structures, a model for the mechanism of membrane fusion mediated by the conformational changes of HA has been proposed. The flavivirus E and alphavirus E1 proteins belong to the class II fusion proteins and mediate membrane fusion at acidic pH. Their prefusion structures are distinct from that of HA. Last year, however, it has become evident that the postfusion structures of these class I and class II fusion proteins are similar. The paramyxovirus F protein belongs to the class I fusion proteins. In contrast to HA, an interaction between F and its homologous attachment protein is required for F to undergo the conformational changes. Since F mediates fusion at neutral pH, the infected cells can fuse with neighboring uninfected cells. The crystal structures of F and the attachment protein HN have recently been clarified, which will facilitate studies of the molecular mechanism of F-mediated membrane fusion.     

Structural properties of human carbonic anhydrase II at pH 9.5.

The structure of human carbonic anhydrase II at pH 9.5 has been studied by X-ray crystallographic methods to 2.2 A resolution. These studies complement those performed under acidic conditions in which the catalytically-important proton-shuttle group, His-64, exhibits conformational mobility about side-chain torsion angle chi 1. However, no structural changes are observed in the conformation of His-64 at high pH. Therefore, we conclude that the protonation of His-64 (as well as zinc-bound hydroxide) may be a factor which contributes to the predominantly "out" conformation for His-64 observed at low pH.