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p33: Structure-Function Predictions from the Crystal Structure
Immunobiol. (2002) 205, pp. 421 – 432 © 2002 Urban & Fischer Verlag http://www.urbanfischer.de/journals/immunobiol
1
Department of Medicine, State University of New York, Stony Brook, N. Y., and 2 Department of Pathology, Weill Medical College of Cornell University, N. Y., USA
gC1q-R/p33: Structure-Function Predictions from the Crystal Structure BERHANE GHEBREHIWET1, JOLYON JESTY1, and ELLINOR I. B. PEERSCHKE2
Abstract Human gC1q-R (p33) is a multicompartmental cellular protein expressed on various types of cells and tissues. Although originally isolated as a receptor for C1q by virtue of its specificity for the globular heads of that molecule, a large body of evidence has now been accumulated which shows that in addition to C1q, gC1q-R can serve as a receptor for diverse ligands including proteins of the intrinsic coagulation/bradykinin forming cascade, as well as antigens of cellular, bacterial, and viral origin. Furthermore, since gC1q-R has been shown to regulate the functions of protein kinase C (PKC), it is postulated that gC1q-R-induced signaling cascade may involve activation of PKC. These data collectively therefore suggest that gC1q-R plays an important role in blood coagulation, inflammation, and infection. However, although significant progress has been made in unraveling the molecular, biochemical, and structural features of this molecule, and data in support of its biological relevance is accumulating, it is still unclear as to how the molecule is anchored on the membrane since its sequence is devoid of a classical transmembrane domain or a glycosylphosphatidylinositol (GPI) anchor. Furthermore, while recombinant gC1q-R can bind to cell surfaces suggesting that it may bind directly to the phospholipid bilayer, our recent experiments show that, at least in vitro, gC1q-R does not bind to unilamellar vesicle preparations of either phosphatidylcholine (PC) or phosphatidylserine: phosphatidylcholine. This work was therefore undertaken to analyze the three-dimensional structure of gC1q-R in order to identify unique structural features that may serve not only to anchor the protein but also to explain its affinity for such a diversity of plasma as well as microbial and viral ligands.
Introduction The gC1q-R molecule is a 33 kDa, widely expressed, highly acidic cellular protein, which was originally identified and characterized (1) as a molecule that binds to the globular ‘heads’ of C1q (Kd = 50–100 nM) under physiologic ionic strength. However, recent evidence (2) shows that gC1q-R can also interact with proteins of the intrinsic coagulation/bradykinin forming cascade including high molecular weight kininogen (HK) (3), fibrinogen (4), thrombin (2) and multimeric vitronectin (5). In general, binding of C1q to cells expressing both cC1q-R and gC1q-R (C1q-Rs) has been shown to modulate a Abbreviations: bis-ANS = 4,4′-bis (1-anilino-8-naphthalene sulfonic acid); HeBS = Hepes buffered saline; gC1q-R = 33 kDa cell protein which binds to the globular heads of C1q. 0171-2985/02/205/04-05-421 $ 15.00/0
422 · B. GHEBREHIWET et al. diversity of cell-specific biologic responses including inositol-tris-phosphate (IP3) production in, and generation of procoagulant activity on, platelets (6) and induction of antiproliferative response on B and T lymphocytes (7–8). On endothelial cells on the other hand, C1q is known to induce activation and expression of the adhesion molecules E-selectin, ICAM-1 and VCAM-1 (9); and production of IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) (10) in a manner that may require at least in part the participation of both cC1q-R and gC1q-R since both are upregulated by inflammatory cytokines (11). Furthermore, gC1q-R, which was shown to be expressed on the surface of endothelial cells (12), can serve as a high-affinity site (Kd = 9 ± 2 nM) for HK (3, 13) in association with u-PAR (14) and cytokeratin 1 (15–16). Interestingly, the binding of HK is inhibited by factor XII, suggesting that the two proteins bind either to the same or overlapping site (17). Binding of HK and/or FXII to gC1q-R on the endothelial cell surface is, therefore, hypothesized to facilitate the assembly and activation of the intrinsic coagulation cascade, kallkrein formation, and the generation of bradykinin (18–19). Bradykinin in turn can induce morphologic changes in the endothelium rendering the subendothelial matrix accessible to blood components leading to infiltration of vascular tissue by proinflammatory cells (18–19). The gC1q-R molecule is synthesized as a pre-pro-protein of 282 residues (1) and is initially targeted to the mitochondria (20). The mature cell surface or cytosolic protein, which is generated by a site-specific cleavage and removal of the 73-residue sequence during post-translational processing (1) is highly acidic with a calculated pI of 4.15. On SDS-PAGE, gC1q-R migrates as a 33 kDa band under both reducing and non-reducing conditions, but it behaves as a homotrimer on gel filtration in non-dissociating conditions (1). Recent evidence from our laboratory also suggests that multimer formation may be a critical process by which the molecule increases its affinity for multivalent ligands such as C1q (2). The gC1q-R amino acid sequence contains three consensus N-glycosylation sites at residues 114 (Asn-Gly-Thr), 136 (Asn-Asn-Ser), and 223 (Asn-Tyr-Thr); a PKC phosphorylation site at residue 207; a tyrosine kinase recognition site at position 268; and a myristylation site at position 252, all of which may be useful in the expression of its biologic functions. It is highly conserved in mammals since comparison of the cDNA-derived amino acid sequences revealed that rat and mouse gC1q-R share a degree of identity of 97.6%, whereas either of the rodent sequences is 89.9% identical to the human sequence (21). The human as well as the mouse gC1q-R genes (22–24) are essentially similar in their exon/intron organization comprising 6 exons and 5 introns, each within a total length of approximately 6 kb of DNA, which is spliced into a 1.5 kb mRNA encoding 282 amino acid residues. The first exon encodes a long stretch of 70 amino acid residues including the putative signal peptide and 4 amino acid residues found in the mature protein. Exons 2–5 encode four hydrophilic domains, whereas exon 6 encodes a domain which is more or less neutral. The human gene is a single copy and is similar in structure to the mouse gene (23–24). They are localized on chromosomes 17p.13.3 and 11 respectively (22–23). The putative binding sites for the ligands C1q (1), cC1q-R (25), and multimeric vitronectin (5) have been identified in non-overlapping regions of the domain encoded by exon 2, whereas the site for HK has been located in a domain encoded by exon 5 and corresponding to residues 204–218 (3). As will be discussed later in more detail, the crystal structure of gC1q-R has also been solved (26) and confirms the homotrimeric nature of gC1q-R.
gC1q-R structure function · 423
Significant progress has been achieved over the past few years in elucidating the molecular, biochemical, and structural features of this molecule. However, tackling the critical issues that would lead towards unraveling its biologic relevance has unfortunately been hampered by the controversy (15, 22) that focused more on its intracellular localization and disregarded its presence on cell membranes (20). However, recent results obtained independently by several laboratories have confirmed the original observation, in that in addition to its intracellular localization, gC1q-R is unequivocally expressed on the extracellular cell surface of endothelial cells, platelets, and T cells (2), thereby giving credence to the postulate that cell surface expressed gC1q-R can serve as a site for a diversity of extracellular ligands. Ligand binding is now postulated to trigger a series of events including, at least on endothelial cells, recruitment of b1-integrin to form a docking/signaling partnership (2). Nevertheless, several critical questions still remain largely unanswered. First, how is gC1q-R anchored and how does it transmit its signal across the membrane on every cell? Second, what unique features in its structure allow it to participate in such a multiplicity of specific interactions? Third, why have pathogenic organisms such as hepatitis C virus (HCV) core protein or Listeria monocytogenes targeted gC1q-R as a tool to promote persistent infection? The purpose of this paper is, therefore, to dissect the three dimensional structure of gC1q-R so as to extract information which may shed some light into the relationship between structure and function. gC1q-R is a receptor for multiple microbial antigens
Several reports have recently appeared in the literature, which collectively indicate that one of the major functions of gC1q-R may be to serve as a receptor for several pathophysiologically important microbial antigens. Platelet gC1q-R for instance, has been shown to bind specifically to the virulence factor, protein A, of S.aureus, but not to the protein A-deficient Wood 46 strain of S. aureus, indicating that the gC1q-R-protein A interaction is specific. Since platelets play a major role in the pathogenesis of S. aureus-mediated endocarditis, it is postulated that platelet gC1q-R may serve as a novel cellular binding site for this pathogen and suggest an additional mechanism for bacterial cell adhesion to sites of vascular injury and thrombosis (27). Listeria monocytogenes is another pathogenic organism, which has been shown to use gC1q-R for cell adhesion and invasion (28). L. monocytogenes is a ubiquitous, gram-positive, rod-shaped, non-spore-forming, facultative aerobic bacillus, which is known to cause severe infections in immunocompromised humans and animals (29–30). Like most intracellular pathogens, L. monocytogenes, has evolved a strategy of cell invasion that involves bacteria/host cell adhesion as a critical first step. In Listeria, adhesion and invasion are mediated largely by two similar but distinct gene products, designated internalin A (InlA) and internalin B (InlB) respectively (31–33). Whereas, InlA uses the cellcell adhesion protein, E-cadherin (32), InlB has been recently shown to use gC1q-R as its cellular partner and promotes cell entry by stimulating tyrosine phosphorylation of the adaptor proteins Gab 1, Cbl, and Shc, and activation of PI-3 kinase (28). The specificity of the InIB–gC1q-R interaction was demonstrated by experiments in which internalin-mediated entry of L. monocytogenes was blocked by either soluble C1q or monoclonal anti-gC1q-R (mAb 60.11), which recognizes the C1q-binding site on the N-terminus of gC1q-R. Interestingly, Internalin shows a structural organization strikingly similar to streptococcal M proteins and, the protein A of S.aureus, and the fibronec-
424 · B. GHEBREHIWET et al. tin-binding protein of streptococci (34). It is therefore tempting to postulate that bacterial antigens such as Protein A and InlB, recognizing the same or overlapping site(s) on gC1q-R may, at least in part, have structural homologues in their respective sequences that permit such interaction. More recently, it was found that hepatitis C virus (HCV) core protein, the first immunosuppressive protein expressed during the early phase of HCV infection, and primarily responsible for the persistent HCV infection, binds to T cells via gC1q-R (35). Previously, we reported that both CD4+ and CD8+ T cells express cC1q-R and gC1q-R on their surface, and that C1q could inhibit mitogen-induced T cell proliferation (8). Similarly, the HCV core protein was found not only to bind to the T cell gC1q-R, but also to mimick the anti-proliferative effects of C1q in vitro. The effect of HCV core protein was specific and could be inhibited by mAb anti-gC1q-R (35). More importantly, recent evidence shows that the anti-proliferative response induced by the interaction of HCV core protein with gC1q-R is linked to the interference of the ERK/MEK mitogentriggered protein kinase activation cascade (36). These results clearly show that the interaction between HCV core protein and the T cell gC1q-R is critical in triggering the early events that lead to blockgage of intracellular events in T cell activation. This in turn would imply that gC1q-R/HCV core protein interaction may play a significant role in the establishment of HCV persistence, especially during the acute phase of viral infection (36). The site for HCV core protein on gC1q-R has been localized to the region spanning residues 188–259 (Table 1), a domain largely encoded by exon 5 (35). Although other binding sites for C1q may be present on gC1q-R, the major binding site recognized by the inhibitory mAb 60.11 is in the N-terminal region, a site clearly distinct from the HCV core-protein binding site. Interestingly, most of the viral antigens which have been reported to bind gC1q-R (Table 1) bind to the domains encoded by exons 5–6, suggesting that this region of the molecule might be a critical “hot” area for ligand interaction.
Table 1. Interactions of gC1q-R/p33 with viral proteins Viral Antigens
gC1q-R site aa residues
Encoding Exon (aa)
EBNA-1 (40) HIV-1 Tat (38)
244–282 244–260**
VI (234–282) VI (234–282)
HIV-1 Rev (37)
196–208
V (193–233)
ORFP of Herpes Simplex virus* (41)
–
–
Core protein V of Adenovirus (39)
–
–
Core protein of Hepatitis C virus (35)
188–259
IV–VI (159–282)
Capsid protein of Rubella virus (42)
212–282
V–VI (193–282)
* ORFP; open reading frame P ** In addition to this core element required for Tat activation additional gC1q-R/P33 elements are provided by flanking sequences, 261–279 and 217–243 (38).
gC1q-R structure function · 425
Non-interaction of gC1q-R with phospholipid vesicles
We have shown previously that gC1q-R is not anchored to the cell surface by a GPI anchor (25). Nevertheless, isolated gC1q-R does bind to cell surfaces, suggesting that it may bind directly to the phospholipid bilayer in a non-integral fashion. To test this hypothesis, two types of binding experiment were done using gel-filtration and detection of (i) phospholipid vesicles by light scattering, and (ii) FITC-labeled gC1q-R by a filter fluorimeter (lex = 486; lem = 530 ± 10 nm). Small unilamellar vesicle preparations of phosphatidylcholine (PC) and phosphatidylserine:phosphatidylcholine (PS:PC, 30:70) were prepared by sonication (25W, 1 min on ice) and then centrifuged at 20,000 × g for 20 min to remove any large vesicle contaminants. Gel filtration of pre-incubated material (Fig. 1A). FITC:gC1q-R, 10 mg/ml, was incubated with either PS:PC or PC vesicles, 1 mg/ml in Hepes-buffered saline containing
Figure 1. Non-interaction of gC1q-R with phospholipid vesicles. FITC-gC1q-R was incubated with either PS:PC or PC as described in the text and analyzed by gel filtration (1A). Figure 1B is a Hummel-Dryer equilibrium gel filtration profile of similarly treated FITC-gC1q-R and shows no detectable binding of gC1q-R to the vesicles.
426 · B. GHEBREHIWET et al. 5 mM CaCl2 and 50 mM ZnCl2 (HBS/Ca/Zn). Samples were applied to a column, 26 × 0.5 cm, of Biogel A15M, run at 0.07 ml/min. Output was passed through both an absorbance monitor (detecting the vesicles by light scattering) and a filter fluorimeter (detecting FITC:gC1q-R) in series. The traces have different polarity so as to distinguish easily between the two. As shown in Fig. 1A, it is clear that no fluorescent protein elutes with the vesicles, and there is thus no evidence of binding. Hummel-Dryer equilibrium gel filtration (Fig. 1B). An identical analysis column was equilibrated with a solution of FITC:gC1q-R, 10 mg/ml in HBS/Ca/Zn. A sample of PS:PC vesicles, 300 ml of 0.1 mg/ml in HBS/Ca/Zn, was then applied and flow restarted, continuing with the FITC:gC1q-R medium (Fig. 1B). Because vesicles are not detectable at this concentration the column was pre-calibrated to determine the vesicle elution time, shown by the middle arrow. Only the fluorescence signal is shown. The early rising limb is the completion of column equilibration with fluorescent protein and is not of interest. There is no evidence of FITC:gC1q-R elution at the time of vesicle elution, showing that under these conditions there is no detectable binding of gC1q-R to the vesicles. The later dip in the signal reflects the elution of the protein-free buffer in which the vesicles were applied. Hummel-Dryer is an equilibrium-binding technique that, unlike the study in panel A, is capable of demonstrating even weak binding affinities. Structure-function predictions from crystal structure The structure-function studies alluded to in the introduction, which previously depended mostly on the use of peptides of likely importance and monoclonal antibodies generated from the gC1q-R, have substantially changed because the crystal structure of gC1qR is now known. In this section we will examine this structure to make initial predictions concerning structure and function of the molecule. Crystal structure
The structure of gC1q-R (p32, p33) was recently determined at 2.25 Å resolution (26). The PDB ID is 1P32, available at www.rcsb.org/pdb. Except for the L → M mutation that was introduced at the amino-terminal position 74, the molecule is identical with the mature form of gC1q-R (residues 74–282) as described earlier (1). The structure is a trimeric doughnut of three identical chains, as shown by the peptide backbone (Fig. 2). The spacefill residues here are the amino-terminal Met (green), carboxy-terminal Gln (red). In each monomer the amino- and carboxy-terminal residues are in contact with each other and with the adjacent monomer. (The termini of each monomer are at the counter-clockwise ends in this view). Trp 233 is also shown, for reference with the view below. The spacefill rendering below (Fig. 3) is more informative. Colors are as follows: ❒ Red: negative – one carboxyl oxygen is shown on each Asp and Glu residue. ❒ Green: positive – one nitrogen is shown on each Lys and Arg residue. (The selection of which carboxyl oxygens and which guanidino nitrogens to color was arbitrary. Showing a single charge per residue allows estimation of charge density by simply counting the red and green atoms.) ❒ Light blue: highly hydrophobic sidechains – Val, Leu, Ile, and Phe.
gC1q-R structure function · 427
Figure 2. Peptide backbone of the structure of gC1q-R. The structure of the mature form of gC1q-R (residues 74–282) is a trimeric doughnut of three identical chains designated A, B, and C (26). The spacefill residues are the N-terminal Met (green), which was substituted for Leu in the mature form (26), and the C-terminal Gln (red). Trp 233 is shown for reference with the spacefill rendering view in Fig. 3.
Figure 3. Faces of gC1q-R: The figure shows the spacefill rendering of gC1q-R provisionally labeled S for the solution face that interacts with ligands (left panel) and M for the face that interacts with the membrane or cell surface (right panel). The middle panel shows a 900 rotation of the molecule revealing the protruding structure made up of residues 189–190 and 197–201 that are partially unlocated in the crystal structure. The color assignments in the spacefill rendering are described in the text.
❒ Orange: Trp sidechains – note particularly Trp 233. ❒ Yellow. These are of major interest. All chains have two stretches of sequence that are partially unlocated in the crystal structure, in the regions 133–163 and 189–201. The yellow atoms are the sidechains in these regions that are located in at least one chain but unlocated in other(s). If we assume that these sequences have approximately the same position in each monomeric unit, then the stretches that are located give a useful indication of where the unlocated sequences lie in the other chains. As can be seen they are entirely on what we call the S face of the molecule (see below). Only two short stretches of sequence are unlocated in all three chains: they are the highly charged 141–146 (PTFDGE) and the 191–196 (DEVGQE). ❒ White: peptide backbone plus all other sidechains.
428 · B. GHEBREHIWET et al. Faces. Because gC1q-R is a molecule associated with the cell surface, the characteristics of its two faces are of major interest, and we have provisionally labeled them S for solution face and M for membrane (or cell-surface) face (Fig. 3). (We think it very unlikely that the molecule binds to the cell surface side-on.) The assignment is based primarily on the large difference in charge between the two faces (compare red vs. green: each colored atom = one charge). The S face is highly acidic, whereas the M face is predominantly basic. The high negative-charge density on the S face would a priori mitigate against interaction with the cell’s sialic-acid-rich surface. Although a number of hydrophobic sidechains are partially exposed on both faces (blue), there are no significant projections into the solvent phase that might be involved in bilayer insertion. This, together with our recent data showing that gC1q-R does not bind to either neutral or acidic phospholipid vesicles (Fig. 1), suggests that binding of the molecule to cell surfaces does not involve hydrophobic interaction with the phospholipid bilayer. Anionic Domains. The short sequences that are unlocated in one chain and located in another (yellow) are restricted totally to the S face, and it is notable that these sequences are extaordinarily acidic. The difficulty of locating them in the crystal suggests high solvent mobility, and indeed two short located sections in the A chain are seen projecting well out into the solvent phase (middle panel). They are included in the very acidic sequence 189-201 (PEDEVGQEDEAES), which is completely unlocated in the other two chains of the trimer. The existence of these highly acidic projections on one face, containing 7 negative charges per monomer, constitutes a major feature that is likely important in the binding of gC1q-R to one or more ligands, particularly basic ones. As discussed above, this area also contains the binding site for Hepatitis C virus core protein. Mapping Epitopes to Structure. Of the monoclonal antibodies prepared against gC1q-R that we have analyzed in detail, two interfere with the binding of specific ligands: C1q (mAb 60.11), and HK (mAb 74.5.2). Since these ligands can bind to gC1q-R when it is bound to the cell surface, they should – if our proposed assignment of the S and M faces of the molecule is correct – not bind to the M face. The peptide epitopes for each are identified (Figs. 4 and 5) in dark blue (side chains only: charges still shown). The C1q-binding epitope (Fig. 4) is almost exactly on the equator of the doughnut, while the HK-binding epitope (Fig. 5) is largely restricted to the inside of the hole. Stereo and side
Figure 4. Location of C1q binding site in gC1q-R. The dark blue colors correspond to a putative C1q site in each gC1q-R monomer (residues 76–93) recognized by mAb 60.11. These sites are apparent in the S face but hidden in the M face of the gC1q-R trimer.
gC1q-R structure function · 429
Figure 5. Location of HK binding site. The dark blue colors are the binding sites for high molecular weight kininogen (HK) in each gC1q-R monomer corresponding to residues 204–218 that are recognized by mAb 74.5.2.
Figure 6. HIV-1 tat binding site. Dark blue colors correspond to residues 224–260 of each gC1q-R monomer postulated to contain a binding site for HIV-1 tat (38).
views (not shown) show that neither epitope is exposed on, or projects from, the M face of the molecule. Interestingly, the HK binding site recognized by mAb 74.5.2 is wholly dependent on the presence of zinc. Therefore, while this epitope is still accessible for ligands and antibody, zinc can induce a conformational change in the molecule resulting in enhanced affinity for ligands such as HK or mAb 74.5.2 (43). Similarly, the regions of the molecule containing the HIV-Tat binding site (Fig. 6) and the HCV core-protein binding site are depicted (Fig. 7) and essentially show that the epitopes are exposed predominantly on the S face of the molecule with little or no exposure on the M face. From the structural analysis of gC1q-R discussed above, we have identified putative sites for zinc and ligand binding and cell-surface association. However, site-directed mutagenesis to modify single or multiple residues in the putative ligand-binding sites will have to be performed in order to verify the functional relevance of these sites. For example, there is a single Cys-SH residue in the molecule, which was initially postulated to play in homotrimerization. However, this is unlikely since it is completely hidden in the crystal structure and preliminary experiments performed in our laboratory show that the
430 · B. GHEBREHIWET et al.
Figure 7. Hepatitis C virus core protein binding site. The putative binding site for HCV core protein (35) in each of the gC1q-R monomer is represented by the dark blue colors. These sites are at residues 188–259 of each gC1q-aR monomer.
oligomerization site resides in the last 22 C-terminal residues of the monomer. Conversely, the high concentration of acidic residues on the S surface of the molecule makes them highly relevant to the binding of putative solution-phase ligands. Acknowledgement
This work was supported in part by grants, RPG-95068-03-CIM and RPG-95068-06 from the American Cancer Society (B. G.), R01 HL5029101 from National Heart Blood and Lung Institute (E. I. B. P. and B. G.) and a generous gift from Larry and Sheila Dalzell.
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432 · B. GHEBREHIWET et al. 25. GHEBREHIWET, B., P. D. LU, W. ZHANG, S. A. KEILBAUGH, L. E. A. LEIGH, P. EGGLETON, K. B. M. REID, and E. I. B. PEERSCHKE. 1997. Evidence that the two C1q-binding membrane proteins gC1q-R and cC1q-R associate to form a metal ion-independent complex. J. Immunol. 159: 1429. 26. JIANG, J., J. Y. ZHANG, A. KRAINER, and R.-M. XU. 1999. Crystal structure of p32, a doughnutshaped acidic mitochondrial matrix protein. Proc. Natl. Acad. Sci. USA. 96: 3572–3577. 27. NGUYEN, T., B. GHEBREHIWET, and E. I. B. PEERSCHKE. 2000. Staphylococcus aureus Protein A recognizes platelet gC1q-R/p33: A novel mechanism for staphylococcus interactions with plateletes. Infect. Immun. 68: 2061–2068. 28. BRAUN, L., L. B. GHEBREHIWET, and P. COSSART. 2000. gC1q-R/p32, a C1q-binding protein, is a novel receptor for Listeria monocytogenes. EMBO J. 19: 1458–1466. 29. COSSART, P., and D. A. PORTNOY. 2001. The cell biology of invasion and intracellular growth by Listeria monocytogenes. In: Fischetti, V. A., Novick, R. P., Ferretti, J. I., Portnoy, D. A., Rood, J. I. eds. Gram positive pathogens. Washington, DC: ASM Press; pp. 507–515. 30. KUHN, M., and W. GOEBEL. 1995. Molecular studies on the virulence of Listeria monocytogenes. Genetic Engineering J. K. Setlow, ed. Vol. 17; Plenum Press, New York. 31. GAILLARD, J.-L, P. BERCHE, C. FREHL, E. GOUIN and P. COSSART. 1991. Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci. Cell 65: 1127–1141. 32. MENGAUD, J., H, OHAYON, P. GOUNON, R. M. MEGE and P. COSSART. 1966. E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes, into epithelial cells. Cell 84: 923–932. 33. DRAMSI, S., I. BISWAS, E. MAGUIN, L. BRAUN, P. MASTROENI, and P. COSSART. 1995. Entry of Listeria monocytogenes into hepatocytes requires expression of InlB, a surface protein of the internalin multigene family. Mol. Microbiol. 16: 251–261. 34. DRAMSI, S., P. DEHOUX, and M. LEBRUN, P. GOOSSENS, and P. COSSART. 1997. Identification of four new members of the internalin multigene family in Listeria monocytogenes strain EGD. Infect.Immun. 65: 1615–1625. 35. KITTLESEN, D. J., K. A. CHIANESE-BULLOCK, Z. Q. YAO, T. J. BRACIALE, and Y. S. HAHN. 2000. Interaction between complement receptor gC1qR and hepatitis C virus core protein inhibits T-lymphocyte proliferation. J. Clin. Immunol. 106: 1239–1249. 36. YAO, Z. Q., D. T. NGUYEN, A. I. HIOTELLIS, and Y. S. HAHN. 2001. Hepatitis C virus core protein inhibits human T lymphocyte responses by a complement-dependent regulatory pathway. J.Immunol. 167: 5264–5272. 37. LUO, Y., H. YU, and P. M. PETERLIN. 1994. Cellular protein modulates effects of human immunodeficiency virus type 1 Rev. J. Virol. 68: 3850–3856. 38. YU, L., P. M. LOEWENSTEIN, Z. ZHANG, and M. GREEN. 1995. In vitro interaction of the human Immunodeficiency Virus Type 1 Tat Transactivator and the general transcription factor TFIIB with the cellular protein TAP. J. Virol. 69: 3017–3023. 39. MATHEWS, D. A., and W. C. RUSSELL. 1998. Adenovirus core protein V interacts with p32 – a protein which is associated with both the mitochondria and the nucleus. J. Gen. Virol. 79: 1677–1685. 40. WANG, Y., J. E. FINNAN, J. M. MIDDELDORP, and S. D. HAYWARD.1997. p32/TAP, a cellular protein that interacts with EBNA-1 of Epstein-Barr virus. Virology 236: 18–29. 41. BRUNI, R., and B. ROIZMAN. 1996. Open reading frame P – a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA. Proc. Natl. Acad. Sci. USA 93: 10423–10427. 42. MOHAN, K. V. K., I. SOM, B. GHEBREHIWET, and C. D. ATTREYA. 2002. Cellular gC1q-R/p33 interacts with rubella virus capsid protein and enhances viral infectivity. Virus Res. 85: 151–161 43. KUMAR, R., E. I. B. PEERSCHKE, and B. GHEBREHIWET. 2002. Zinc induces exposure of hydrophobic sites in the C-terminal domain of gC1q-R/p33. Mol. Immunol. 119: 1–7 Dr. BERHANE GHEBREHIWET, Department of Medicine, State University of New York, Stony Brook, New York 11794-8161. Phone: (631) 444 2493, Fax: (631) 444 2493, E-mail: [email protected]. sunysb.edu
1
Department of Medicine, State University of New York, Stony Brook, N. Y., and 2 Department of Pathology, Weill Medical College of Cornell University, N. Y., USA
gC1q-R/p33: Structure-Function Predictions from the Crystal Structure BERHANE GHEBREHIWET1, JOLYON JESTY1, and ELLINOR I. B. PEERSCHKE2
Abstract Human gC1q-R (p33) is a multicompartmental cellular protein expressed on various types of cells and tissues. Although originally isolated as a receptor for C1q by virtue of its specificity for the globular heads of that molecule, a large body of evidence has now been accumulated which shows that in addition to C1q, gC1q-R can serve as a receptor for diverse ligands including proteins of the intrinsic coagulation/bradykinin forming cascade, as well as antigens of cellular, bacterial, and viral origin. Furthermore, since gC1q-R has been shown to regulate the functions of protein kinase C (PKC), it is postulated that gC1q-R-induced signaling cascade may involve activation of PKC. These data collectively therefore suggest that gC1q-R plays an important role in blood coagulation, inflammation, and infection. However, although significant progress has been made in unraveling the molecular, biochemical, and structural features of this molecule, and data in support of its biological relevance is accumulating, it is still unclear as to how the molecule is anchored on the membrane since its sequence is devoid of a classical transmembrane domain or a glycosylphosphatidylinositol (GPI) anchor. Furthermore, while recombinant gC1q-R can bind to cell surfaces suggesting that it may bind directly to the phospholipid bilayer, our recent experiments show that, at least in vitro, gC1q-R does not bind to unilamellar vesicle preparations of either phosphatidylcholine (PC) or phosphatidylserine: phosphatidylcholine. This work was therefore undertaken to analyze the three-dimensional structure of gC1q-R in order to identify unique structural features that may serve not only to anchor the protein but also to explain its affinity for such a diversity of plasma as well as microbial and viral ligands.
Introduction The gC1q-R molecule is a 33 kDa, widely expressed, highly acidic cellular protein, which was originally identified and characterized (1) as a molecule that binds to the globular ‘heads’ of C1q (Kd = 50–100 nM) under physiologic ionic strength. However, recent evidence (2) shows that gC1q-R can also interact with proteins of the intrinsic coagulation/bradykinin forming cascade including high molecular weight kininogen (HK) (3), fibrinogen (4), thrombin (2) and multimeric vitronectin (5). In general, binding of C1q to cells expressing both cC1q-R and gC1q-R (C1q-Rs) has been shown to modulate a Abbreviations: bis-ANS = 4,4′-bis (1-anilino-8-naphthalene sulfonic acid); HeBS = Hepes buffered saline; gC1q-R = 33 kDa cell protein which binds to the globular heads of C1q. 0171-2985/02/205/04-05-421 $ 15.00/0
422 · B. GHEBREHIWET et al. diversity of cell-specific biologic responses including inositol-tris-phosphate (IP3) production in, and generation of procoagulant activity on, platelets (6) and induction of antiproliferative response on B and T lymphocytes (7–8). On endothelial cells on the other hand, C1q is known to induce activation and expression of the adhesion molecules E-selectin, ICAM-1 and VCAM-1 (9); and production of IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) (10) in a manner that may require at least in part the participation of both cC1q-R and gC1q-R since both are upregulated by inflammatory cytokines (11). Furthermore, gC1q-R, which was shown to be expressed on the surface of endothelial cells (12), can serve as a high-affinity site (Kd = 9 ± 2 nM) for HK (3, 13) in association with u-PAR (14) and cytokeratin 1 (15–16). Interestingly, the binding of HK is inhibited by factor XII, suggesting that the two proteins bind either to the same or overlapping site (17). Binding of HK and/or FXII to gC1q-R on the endothelial cell surface is, therefore, hypothesized to facilitate the assembly and activation of the intrinsic coagulation cascade, kallkrein formation, and the generation of bradykinin (18–19). Bradykinin in turn can induce morphologic changes in the endothelium rendering the subendothelial matrix accessible to blood components leading to infiltration of vascular tissue by proinflammatory cells (18–19). The gC1q-R molecule is synthesized as a pre-pro-protein of 282 residues (1) and is initially targeted to the mitochondria (20). The mature cell surface or cytosolic protein, which is generated by a site-specific cleavage and removal of the 73-residue sequence during post-translational processing (1) is highly acidic with a calculated pI of 4.15. On SDS-PAGE, gC1q-R migrates as a 33 kDa band under both reducing and non-reducing conditions, but it behaves as a homotrimer on gel filtration in non-dissociating conditions (1). Recent evidence from our laboratory also suggests that multimer formation may be a critical process by which the molecule increases its affinity for multivalent ligands such as C1q (2). The gC1q-R amino acid sequence contains three consensus N-glycosylation sites at residues 114 (Asn-Gly-Thr), 136 (Asn-Asn-Ser), and 223 (Asn-Tyr-Thr); a PKC phosphorylation site at residue 207; a tyrosine kinase recognition site at position 268; and a myristylation site at position 252, all of which may be useful in the expression of its biologic functions. It is highly conserved in mammals since comparison of the cDNA-derived amino acid sequences revealed that rat and mouse gC1q-R share a degree of identity of 97.6%, whereas either of the rodent sequences is 89.9% identical to the human sequence (21). The human as well as the mouse gC1q-R genes (22–24) are essentially similar in their exon/intron organization comprising 6 exons and 5 introns, each within a total length of approximately 6 kb of DNA, which is spliced into a 1.5 kb mRNA encoding 282 amino acid residues. The first exon encodes a long stretch of 70 amino acid residues including the putative signal peptide and 4 amino acid residues found in the mature protein. Exons 2–5 encode four hydrophilic domains, whereas exon 6 encodes a domain which is more or less neutral. The human gene is a single copy and is similar in structure to the mouse gene (23–24). They are localized on chromosomes 17p.13.3 and 11 respectively (22–23). The putative binding sites for the ligands C1q (1), cC1q-R (25), and multimeric vitronectin (5) have been identified in non-overlapping regions of the domain encoded by exon 2, whereas the site for HK has been located in a domain encoded by exon 5 and corresponding to residues 204–218 (3). As will be discussed later in more detail, the crystal structure of gC1q-R has also been solved (26) and confirms the homotrimeric nature of gC1q-R.
gC1q-R structure function · 423
Significant progress has been achieved over the past few years in elucidating the molecular, biochemical, and structural features of this molecule. However, tackling the critical issues that would lead towards unraveling its biologic relevance has unfortunately been hampered by the controversy (15, 22) that focused more on its intracellular localization and disregarded its presence on cell membranes (20). However, recent results obtained independently by several laboratories have confirmed the original observation, in that in addition to its intracellular localization, gC1q-R is unequivocally expressed on the extracellular cell surface of endothelial cells, platelets, and T cells (2), thereby giving credence to the postulate that cell surface expressed gC1q-R can serve as a site for a diversity of extracellular ligands. Ligand binding is now postulated to trigger a series of events including, at least on endothelial cells, recruitment of b1-integrin to form a docking/signaling partnership (2). Nevertheless, several critical questions still remain largely unanswered. First, how is gC1q-R anchored and how does it transmit its signal across the membrane on every cell? Second, what unique features in its structure allow it to participate in such a multiplicity of specific interactions? Third, why have pathogenic organisms such as hepatitis C virus (HCV) core protein or Listeria monocytogenes targeted gC1q-R as a tool to promote persistent infection? The purpose of this paper is, therefore, to dissect the three dimensional structure of gC1q-R so as to extract information which may shed some light into the relationship between structure and function. gC1q-R is a receptor for multiple microbial antigens
Several reports have recently appeared in the literature, which collectively indicate that one of the major functions of gC1q-R may be to serve as a receptor for several pathophysiologically important microbial antigens. Platelet gC1q-R for instance, has been shown to bind specifically to the virulence factor, protein A, of S.aureus, but not to the protein A-deficient Wood 46 strain of S. aureus, indicating that the gC1q-R-protein A interaction is specific. Since platelets play a major role in the pathogenesis of S. aureus-mediated endocarditis, it is postulated that platelet gC1q-R may serve as a novel cellular binding site for this pathogen and suggest an additional mechanism for bacterial cell adhesion to sites of vascular injury and thrombosis (27). Listeria monocytogenes is another pathogenic organism, which has been shown to use gC1q-R for cell adhesion and invasion (28). L. monocytogenes is a ubiquitous, gram-positive, rod-shaped, non-spore-forming, facultative aerobic bacillus, which is known to cause severe infections in immunocompromised humans and animals (29–30). Like most intracellular pathogens, L. monocytogenes, has evolved a strategy of cell invasion that involves bacteria/host cell adhesion as a critical first step. In Listeria, adhesion and invasion are mediated largely by two similar but distinct gene products, designated internalin A (InlA) and internalin B (InlB) respectively (31–33). Whereas, InlA uses the cellcell adhesion protein, E-cadherin (32), InlB has been recently shown to use gC1q-R as its cellular partner and promotes cell entry by stimulating tyrosine phosphorylation of the adaptor proteins Gab 1, Cbl, and Shc, and activation of PI-3 kinase (28). The specificity of the InIB–gC1q-R interaction was demonstrated by experiments in which internalin-mediated entry of L. monocytogenes was blocked by either soluble C1q or monoclonal anti-gC1q-R (mAb 60.11), which recognizes the C1q-binding site on the N-terminus of gC1q-R. Interestingly, Internalin shows a structural organization strikingly similar to streptococcal M proteins and, the protein A of S.aureus, and the fibronec-
424 · B. GHEBREHIWET et al. tin-binding protein of streptococci (34). It is therefore tempting to postulate that bacterial antigens such as Protein A and InlB, recognizing the same or overlapping site(s) on gC1q-R may, at least in part, have structural homologues in their respective sequences that permit such interaction. More recently, it was found that hepatitis C virus (HCV) core protein, the first immunosuppressive protein expressed during the early phase of HCV infection, and primarily responsible for the persistent HCV infection, binds to T cells via gC1q-R (35). Previously, we reported that both CD4+ and CD8+ T cells express cC1q-R and gC1q-R on their surface, and that C1q could inhibit mitogen-induced T cell proliferation (8). Similarly, the HCV core protein was found not only to bind to the T cell gC1q-R, but also to mimick the anti-proliferative effects of C1q in vitro. The effect of HCV core protein was specific and could be inhibited by mAb anti-gC1q-R (35). More importantly, recent evidence shows that the anti-proliferative response induced by the interaction of HCV core protein with gC1q-R is linked to the interference of the ERK/MEK mitogentriggered protein kinase activation cascade (36). These results clearly show that the interaction between HCV core protein and the T cell gC1q-R is critical in triggering the early events that lead to blockgage of intracellular events in T cell activation. This in turn would imply that gC1q-R/HCV core protein interaction may play a significant role in the establishment of HCV persistence, especially during the acute phase of viral infection (36). The site for HCV core protein on gC1q-R has been localized to the region spanning residues 188–259 (Table 1), a domain largely encoded by exon 5 (35). Although other binding sites for C1q may be present on gC1q-R, the major binding site recognized by the inhibitory mAb 60.11 is in the N-terminal region, a site clearly distinct from the HCV core-protein binding site. Interestingly, most of the viral antigens which have been reported to bind gC1q-R (Table 1) bind to the domains encoded by exons 5–6, suggesting that this region of the molecule might be a critical “hot” area for ligand interaction.
Table 1. Interactions of gC1q-R/p33 with viral proteins Viral Antigens
gC1q-R site aa residues
Encoding Exon (aa)
EBNA-1 (40) HIV-1 Tat (38)
244–282 244–260**
VI (234–282) VI (234–282)
HIV-1 Rev (37)
196–208
V (193–233)
ORFP of Herpes Simplex virus* (41)
–
–
Core protein V of Adenovirus (39)
–
–
Core protein of Hepatitis C virus (35)
188–259
IV–VI (159–282)
Capsid protein of Rubella virus (42)
212–282
V–VI (193–282)
* ORFP; open reading frame P ** In addition to this core element required for Tat activation additional gC1q-R/P33 elements are provided by flanking sequences, 261–279 and 217–243 (38).
gC1q-R structure function · 425
Non-interaction of gC1q-R with phospholipid vesicles
We have shown previously that gC1q-R is not anchored to the cell surface by a GPI anchor (25). Nevertheless, isolated gC1q-R does bind to cell surfaces, suggesting that it may bind directly to the phospholipid bilayer in a non-integral fashion. To test this hypothesis, two types of binding experiment were done using gel-filtration and detection of (i) phospholipid vesicles by light scattering, and (ii) FITC-labeled gC1q-R by a filter fluorimeter (lex = 486; lem = 530 ± 10 nm). Small unilamellar vesicle preparations of phosphatidylcholine (PC) and phosphatidylserine:phosphatidylcholine (PS:PC, 30:70) were prepared by sonication (25W, 1 min on ice) and then centrifuged at 20,000 × g for 20 min to remove any large vesicle contaminants. Gel filtration of pre-incubated material (Fig. 1A). FITC:gC1q-R, 10 mg/ml, was incubated with either PS:PC or PC vesicles, 1 mg/ml in Hepes-buffered saline containing
Figure 1. Non-interaction of gC1q-R with phospholipid vesicles. FITC-gC1q-R was incubated with either PS:PC or PC as described in the text and analyzed by gel filtration (1A). Figure 1B is a Hummel-Dryer equilibrium gel filtration profile of similarly treated FITC-gC1q-R and shows no detectable binding of gC1q-R to the vesicles.
426 · B. GHEBREHIWET et al. 5 mM CaCl2 and 50 mM ZnCl2 (HBS/Ca/Zn). Samples were applied to a column, 26 × 0.5 cm, of Biogel A15M, run at 0.07 ml/min. Output was passed through both an absorbance monitor (detecting the vesicles by light scattering) and a filter fluorimeter (detecting FITC:gC1q-R) in series. The traces have different polarity so as to distinguish easily between the two. As shown in Fig. 1A, it is clear that no fluorescent protein elutes with the vesicles, and there is thus no evidence of binding. Hummel-Dryer equilibrium gel filtration (Fig. 1B). An identical analysis column was equilibrated with a solution of FITC:gC1q-R, 10 mg/ml in HBS/Ca/Zn. A sample of PS:PC vesicles, 300 ml of 0.1 mg/ml in HBS/Ca/Zn, was then applied and flow restarted, continuing with the FITC:gC1q-R medium (Fig. 1B). Because vesicles are not detectable at this concentration the column was pre-calibrated to determine the vesicle elution time, shown by the middle arrow. Only the fluorescence signal is shown. The early rising limb is the completion of column equilibration with fluorescent protein and is not of interest. There is no evidence of FITC:gC1q-R elution at the time of vesicle elution, showing that under these conditions there is no detectable binding of gC1q-R to the vesicles. The later dip in the signal reflects the elution of the protein-free buffer in which the vesicles were applied. Hummel-Dryer is an equilibrium-binding technique that, unlike the study in panel A, is capable of demonstrating even weak binding affinities. Structure-function predictions from crystal structure The structure-function studies alluded to in the introduction, which previously depended mostly on the use of peptides of likely importance and monoclonal antibodies generated from the gC1q-R, have substantially changed because the crystal structure of gC1qR is now known. In this section we will examine this structure to make initial predictions concerning structure and function of the molecule. Crystal structure
The structure of gC1q-R (p32, p33) was recently determined at 2.25 Å resolution (26). The PDB ID is 1P32, available at www.rcsb.org/pdb. Except for the L → M mutation that was introduced at the amino-terminal position 74, the molecule is identical with the mature form of gC1q-R (residues 74–282) as described earlier (1). The structure is a trimeric doughnut of three identical chains, as shown by the peptide backbone (Fig. 2). The spacefill residues here are the amino-terminal Met (green), carboxy-terminal Gln (red). In each monomer the amino- and carboxy-terminal residues are in contact with each other and with the adjacent monomer. (The termini of each monomer are at the counter-clockwise ends in this view). Trp 233 is also shown, for reference with the view below. The spacefill rendering below (Fig. 3) is more informative. Colors are as follows: ❒ Red: negative – one carboxyl oxygen is shown on each Asp and Glu residue. ❒ Green: positive – one nitrogen is shown on each Lys and Arg residue. (The selection of which carboxyl oxygens and which guanidino nitrogens to color was arbitrary. Showing a single charge per residue allows estimation of charge density by simply counting the red and green atoms.) ❒ Light blue: highly hydrophobic sidechains – Val, Leu, Ile, and Phe.
gC1q-R structure function · 427
Figure 2. Peptide backbone of the structure of gC1q-R. The structure of the mature form of gC1q-R (residues 74–282) is a trimeric doughnut of three identical chains designated A, B, and C (26). The spacefill residues are the N-terminal Met (green), which was substituted for Leu in the mature form (26), and the C-terminal Gln (red). Trp 233 is shown for reference with the spacefill rendering view in Fig. 3.
Figure 3. Faces of gC1q-R: The figure shows the spacefill rendering of gC1q-R provisionally labeled S for the solution face that interacts with ligands (left panel) and M for the face that interacts with the membrane or cell surface (right panel). The middle panel shows a 900 rotation of the molecule revealing the protruding structure made up of residues 189–190 and 197–201 that are partially unlocated in the crystal structure. The color assignments in the spacefill rendering are described in the text.
❒ Orange: Trp sidechains – note particularly Trp 233. ❒ Yellow. These are of major interest. All chains have two stretches of sequence that are partially unlocated in the crystal structure, in the regions 133–163 and 189–201. The yellow atoms are the sidechains in these regions that are located in at least one chain but unlocated in other(s). If we assume that these sequences have approximately the same position in each monomeric unit, then the stretches that are located give a useful indication of where the unlocated sequences lie in the other chains. As can be seen they are entirely on what we call the S face of the molecule (see below). Only two short stretches of sequence are unlocated in all three chains: they are the highly charged 141–146 (PTFDGE) and the 191–196 (DEVGQE). ❒ White: peptide backbone plus all other sidechains.
428 · B. GHEBREHIWET et al. Faces. Because gC1q-R is a molecule associated with the cell surface, the characteristics of its two faces are of major interest, and we have provisionally labeled them S for solution face and M for membrane (or cell-surface) face (Fig. 3). (We think it very unlikely that the molecule binds to the cell surface side-on.) The assignment is based primarily on the large difference in charge between the two faces (compare red vs. green: each colored atom = one charge). The S face is highly acidic, whereas the M face is predominantly basic. The high negative-charge density on the S face would a priori mitigate against interaction with the cell’s sialic-acid-rich surface. Although a number of hydrophobic sidechains are partially exposed on both faces (blue), there are no significant projections into the solvent phase that might be involved in bilayer insertion. This, together with our recent data showing that gC1q-R does not bind to either neutral or acidic phospholipid vesicles (Fig. 1), suggests that binding of the molecule to cell surfaces does not involve hydrophobic interaction with the phospholipid bilayer. Anionic Domains. The short sequences that are unlocated in one chain and located in another (yellow) are restricted totally to the S face, and it is notable that these sequences are extaordinarily acidic. The difficulty of locating them in the crystal suggests high solvent mobility, and indeed two short located sections in the A chain are seen projecting well out into the solvent phase (middle panel). They are included in the very acidic sequence 189-201 (PEDEVGQEDEAES), which is completely unlocated in the other two chains of the trimer. The existence of these highly acidic projections on one face, containing 7 negative charges per monomer, constitutes a major feature that is likely important in the binding of gC1q-R to one or more ligands, particularly basic ones. As discussed above, this area also contains the binding site for Hepatitis C virus core protein. Mapping Epitopes to Structure. Of the monoclonal antibodies prepared against gC1q-R that we have analyzed in detail, two interfere with the binding of specific ligands: C1q (mAb 60.11), and HK (mAb 74.5.2). Since these ligands can bind to gC1q-R when it is bound to the cell surface, they should – if our proposed assignment of the S and M faces of the molecule is correct – not bind to the M face. The peptide epitopes for each are identified (Figs. 4 and 5) in dark blue (side chains only: charges still shown). The C1q-binding epitope (Fig. 4) is almost exactly on the equator of the doughnut, while the HK-binding epitope (Fig. 5) is largely restricted to the inside of the hole. Stereo and side
Figure 4. Location of C1q binding site in gC1q-R. The dark blue colors correspond to a putative C1q site in each gC1q-R monomer (residues 76–93) recognized by mAb 60.11. These sites are apparent in the S face but hidden in the M face of the gC1q-R trimer.
gC1q-R structure function · 429
Figure 5. Location of HK binding site. The dark blue colors are the binding sites for high molecular weight kininogen (HK) in each gC1q-R monomer corresponding to residues 204–218 that are recognized by mAb 74.5.2.
Figure 6. HIV-1 tat binding site. Dark blue colors correspond to residues 224–260 of each gC1q-R monomer postulated to contain a binding site for HIV-1 tat (38).
views (not shown) show that neither epitope is exposed on, or projects from, the M face of the molecule. Interestingly, the HK binding site recognized by mAb 74.5.2 is wholly dependent on the presence of zinc. Therefore, while this epitope is still accessible for ligands and antibody, zinc can induce a conformational change in the molecule resulting in enhanced affinity for ligands such as HK or mAb 74.5.2 (43). Similarly, the regions of the molecule containing the HIV-Tat binding site (Fig. 6) and the HCV core-protein binding site are depicted (Fig. 7) and essentially show that the epitopes are exposed predominantly on the S face of the molecule with little or no exposure on the M face. From the structural analysis of gC1q-R discussed above, we have identified putative sites for zinc and ligand binding and cell-surface association. However, site-directed mutagenesis to modify single or multiple residues in the putative ligand-binding sites will have to be performed in order to verify the functional relevance of these sites. For example, there is a single Cys-SH residue in the molecule, which was initially postulated to play in homotrimerization. However, this is unlikely since it is completely hidden in the crystal structure and preliminary experiments performed in our laboratory show that the
430 · B. GHEBREHIWET et al.
Figure 7. Hepatitis C virus core protein binding site. The putative binding site for HCV core protein (35) in each of the gC1q-R monomer is represented by the dark blue colors. These sites are at residues 188–259 of each gC1q-aR monomer.
oligomerization site resides in the last 22 C-terminal residues of the monomer. Conversely, the high concentration of acidic residues on the S surface of the molecule makes them highly relevant to the binding of putative solution-phase ligands. Acknowledgement
This work was supported in part by grants, RPG-95068-03-CIM and RPG-95068-06 from the American Cancer Society (B. G.), R01 HL5029101 from National Heart Blood and Lung Institute (E. I. B. P. and B. G.) and a generous gift from Larry and Sheila Dalzell.
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