Functional determinants of the Epstein-Barr virus protease.

Herpesvirus proteases are essential for the production of progeny virus. They cleave the assembly protein that fills the immature capsid in order to make place for the viral DNA. The recombinant protease of the human gamma-herpesvirus Epstein-Barr virus (EBV) was expressed in Escherichia coli and purified. Circular dichroism indicated that the protein was properly folded with a secondary structure content similar to that of other herpesvirus proteases. Gel filtration and sedimentation analysis indicated a fast monomer-dimer equilibrium of the protease with a K(d) of about 60 microM. This value was not influenced by glycerol but was lowered to 1.7 microM in the presence of 0.5 M sodium citrate. We also developed an HPLC-based enzymatic assay using a 20 amino acid residue synthetic peptide substrate derived from one of the viral target sequences for the protease. We found that conditions that stabilised the dimer also led to a higher enzymatic activity. Through sequential deletion of amino acid residues from either side of the cleavage site, the minimal peptide substrate for the protease was determined as P5-P2'. This minimal sequence is shorter than that for other herpesvirus proteases. The implications of our findings are discussed with reference to the viral life-cycle. These results are the first ever published on the EBV protease and represent a first step towards the development of protease inhibitors.

Crystal structure of the catalytic domain of human complement c1s: a serine protease with a handle.

C1s is the highly specific modular serine protease that mediates the proteolytic activity of the C1 complex and thereby triggers activation of the complement cascade. The crystal structure of a catalytic fragment from human C1s comprising the second complement control protein (CCP2) module and the chymotrypsin-like serine protease (SP) domain has been determined and refined to 1.7 A resolution. In the areas surrounding the active site, the SP structure reveals a restricted access to subsidiary substrate binding sites that could be responsible for the narrow specificity of C1s. The ellipsoidal CCP2 module is oriented perpendicularly to the surface of the SP domain. This arrangement is maintained through a rigid module-domain interface involving intertwined proline- and tyrosine-rich polypeptide segments. The relative orientation of SP and CCP2 is consistent with the fact that the latter provides additional substrate recognition sites for the C4 substrate. This structure provides a first example of a CCP-SP assembly that is conserved in diverse extracellular proteins. Its implications in the activation mechanism of C1 are discussed.

Solution structure of the epidermal growth factor (EGF)-like module of human complement protease C1r, an atypical member of the EGF family.

The calcium-dependent interaction between C1r and C1s, the two homologous serine proteases of the first component of human complement C1, is mediated by their N-terminal regions. The latter comprise an epidermal growth factor (EGF)-like module exhibiting the consensus sequence characteristic of Ca(2+)-binding EGF modules, surrounded by two CUB modules. Due to its Ca2+ binding ability, the C1r EGF-like module (C1r-EGF) is supposed to participate in the C1r-C1s interaction. An additional interesting feature of C1r-EGF is the unusually large loop connecting the first two conserved cysteine residues. The solution structure of synthetic C1r-EGF (residues 123-175) has been determined using nuclear magnetic resonance and combined simulated annealing-restrained molecular dynamics calculations. The resulting family of 19 structures is characterized by a well-ordered C-terminal part (residues Cys 144-Ala174) with a backbone rmsd of 0.7 A and a disordered N-terminal, including the large loop between the first two cysteines (Cys129 and Cys144). This loop is known to be surface exposed and may be expected to participate in domain-domain or protein-protein interactions. In its C-terminal part, C1r-EGF possesses the characteristic EGF fold with a major and a minor beta-sheet. The latter comprises a beta-bulge, and comparison with other EGF-like modules reveals the existence of two distinct structural and sequential motifs in the bulged part. Additional experiments in the presence of 80 mM Ca2+ did not show significant structural variation of C1r-EGF, in keeping with previous observations on blood-clotting factors IX and X.

Chemical synthesis and characterization of the epidermal growth factor-like module of human complement protease C1r.

C1r is one of the two serine proteases of C1, the first component of complement, in which it is associated in a calcium-dependent manner to the homologous serine protease C1s. This interaction is mediated by the N-terminal region of C1r, which comprises a single epidermal growth factor (EGF)-like module containing the consensus sequence required for calcium binding, surrounded by two CUB modules. With a view to determine the structure of the EGF-like module of C1r and evaluate its contribution to calcium binding, this module [C1r(123-175)] was synthesized by automated solid-phase methodology using the Boc strategy. A first synthesis using the Boc-His(Z) derivative gave very low yield, due to partial deprotection of His residues leading to chain termination by acetylation, and to insertion of glycine residues. This could be circumvented by using the Boc-His(DNP) derivative and by condensation of appropriate glycine-containing segments. The synthetic peptide was efficiently folded under redox conditions to the species with three correct disulfide bridges, as determined by mass spectrometry and N-terminal sequence analyses of thermolytic fragments. The homogeneity of the synthetic peptide was assessed by reversed-phase HPLC and electrospray mass spectrometry. One-dimensional 1H NMR spectroscopic analysis provided evidence that the EGF-like module had a well defined structure, and was able to bind calcium with an apparent Kd of 10 mM. This value, comparable to that found for the isolated EGF-like modules of coagulation factors IX and X, is much higher than that measured for native C1r. As already proposed for factors IX and X, it is suggested that neighbouring module(s), most probably the N-terminal CUB module, contribute(s) to the calcium binding site.

NMR structures of a mitochondrial transit peptide from the green alga Chlamydomonas reinhardtii.

The 26-amino-acid pre-sequence of the ATP synthase beta subunit that directs the protein from the cytosol to mitochondria in the unicellular green alga Chlamydomonas reinhardtii has been synthesised and analysed using NMR spectroscopy/circular dichroism and compared to a chloroplast transit peptide from the same organism. The results demonstrate that the peptide, though mainly unstructured in water, undergoes a strong conformational change in a 36% water/64% 2,2,2-trifluoroethanol mixture. In this solvent condition, an alpha-helix was characterised by NMR from residue 2 to 26. Structure calculations under NMR restraints lead to a population of models of which 60% are kinked at position 9-10. Structural analysis indicates two hydrophobic sectors on the models with a discontinuity at the 9-10 kink level. The structures suggest a different interaction mode with the mitochondrial membrane compared to the chloroplast transit peptide.

Structure of the catalytic region of human complement protease C1s: study by chemical cross-linking and three-dimensional homology modeling.

C1s is a multidomain serine protease that is responsible for the enzymatic activity of C1, the first component of the classical pathway of complement. Its catalytic region (gamma-B) comprises two contiguous complement control protein (CCP) modules, IV and V (about 60 residues each), a 15-residue intermediary segment, and the B chain (251 residues), which is the serine protease domain. With a view to identify domain-domain interactions within this region, the gamma-B fragment of C1s, obtained by limited proteolysis with plasmin, was chemically cross-linked with the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; then cross-linked peptides were isolated after CNBr cleavage and thermolytic digestion. N-Terminal sequence and mass spectrometry analyses allowed us to identify two cross-links between Lys 405 of module V and Glu 672 of the B chain and between Glu 418 of the intermediary segment and Lys 608 of the B chain. Three-dimensional modeling of the CCP modules IV and V and of the catalytic B chain was also carried out on the basis of their respective homology with the 16th and 5th CCP modules of complement factor H and type I serine proteases. The information provided by both the chemical cross-linking studies and the homology modeling enabled us to construct a three-dimensional model for the assembly of the C-terminal part of the gamma-B region, comprising module V, the intermediary segment, and the B chain. This model shows that module V interacts with the serine protease B chain on the side opposite to both the activation site and the catalytic site. Functional implications of this interaction are discussed in terms of the possible role of module V in the specific recognition and positioning of C4, one of the two substrates of C1s.

The envelope glycoprotein of HIV-1 gp120 and human complement protein C1q bind to the same peptides derived from three different regions of gp41, the transmembrane glycoprotein of HIV-1, and share antigenic homology.

gp41, the transmembrane glycoprotein of HIV-1, has been shown to be non-covalently associated with gp120. We have shown that it also binds human C1q. To analyze the interaction site(s) of gp41 with these two molecules, we established an enzyme-linked immunosorbent assay (ELISA) system using recombinant soluble gp41 [amino acids (aa) 539-684] and peptides thereof. In the cell-external part of gp41 three sites (aa 526-538, aa 590-613 and aa 625-655) were found to bind both gp120 and C1q. That gp120 and C1q use the same sites was evidenced by the fact that these proteins competed with each other for the same sites in recombinant soluble gp41 and gp41 peptides. It could be demonstrated by ELISA, that rabbit antibodies against human C1q recognized gp120, and rabbit antibodies against gp120 cross-reacted with C1q. Rabbit anti-gp120, HIV-1-positive human sera and anti-gp120 obtained from such sera agglutinated sensitized sheep erythrocytes with human C1q (EAC1q). These data suggest that in addition to functional homology between C1q and gp120 structural homology between these two molecules exists. This molecular mimicry might become the basis for immunologically relevant autoimmune phenomena.

Interaction of C1 with HIV-1.

In contrast to animal retroviruses such as murine leukemia virus, HIV-1 is not lysed by human complement. Nevertheless, HIV-1 activates complement via the classical pathway independently of antibody. Evidence is provided for activation of the reconstituted C1 complex by the virus, resulting from direct interaction between C1q and the external part of the viral transmembrane envelope protein (sgp41). Using C1q fragments and synthetic peptides covering the putative interaction regions in C1q and sgp41, we obtain evidence that the C1q/HIV-1 interaction involves: A site on C1q that appears to be located in the intermediary region between the collagen-like and the globular regions of C1q, and which may be conformational, involving two or more C1q chains. A site on gp41 located between residues 601 and 613 (gp160 nomenclature), i.e. within the immunodominant domain of HIV-1. This site shares homology with the corresponding region of HIV-2.

Recombinant human complement subcomponent C1s lacking beta-hydroxyasparagine, sialic acid, and one of its two carbohydrate chains still reassembles with C1q and C1r to form a functional C1 complex.

In contrast to the human serum protein which is approximately one-half erythro-beta-hydroxyasparagine at asparagine 134 [Theilens et al. (1990) Biochemistry 29, 3570-3578], recombinant C1s expressed by insect cells after infection with recombinant baculovirus entirely lacks posttranslational modification at asparagine 134. It is also incompletely glycosylated, lacking, at least, sialic acid. Site-directed mutagenesis of one of the two sites of carbohydrate attachment (Asn 159 to Gln 159) yields a faster migrating recombinant C1s still abundantly secreted. Furthermore, the mutated protein displays good hemolytic activity when reassembled with C1q and either human serum or recombinant C1r, demonstrating that these posttranslational modifications are not critical for any of the multiple interactions between C1s and C1q, C1r, C2, and C4 required for reassembly of the C1 complex, activation, and initiation of the classical complement pathway. The 4.0S recombinant C1s dimerizes to yield 5.6S C1s2 in the presence of Ca2+ and forms the 9.1S C1s-C1r-C1r-C1s tetramer upon the addition of human serum C1r and the 15.6S C1 complex upon the addition of C1q to the tetramer. The recombinant C1s and human serum C1s have identical N-terminal amino acid sequences, indicating proper recognition by the insect signal peptidase. The recombinant C1s is secreted and isolated as the unactivated zymogen, and it may be activated by human serum C1r which cleaves at Arg422-Ile423 to yield the characteristic heavy and light chains. A very tight complex is formed between C1-inhibitor and the light chain of recombinant C1s.(ABSTRACT TRUNCATED AT 250 WORDS)

Human immunodeficiency virus type 1 activates the classical pathway of complement by direct C1 binding through specific sites in the transmembrane glycoprotein gp41.

Human immunodeficiency virus type 1 (HIV-1), in contrast to animal retroviruses such as murine leukemia virus, is not lysed by human complement. Nevertheless, HIV-1 activates complement via the classical pathway independent of antibody, and C3b deposition facilitates infection of complement receptor-bearing cells. Using gel exclusion chromatography on Sephacryl S-1000, purified virions were found to bind 125I-labeled C1q, but not 125I-labeled dimeric proenzyme C1s. Virions activated the C1 complex, reconstituted from C1q, proenzyme C1r, and 125I-labeled proenzyme C1s, to an extent comparable with that obtained with immunoglobulin G-ovalbumin immune complexes. To determine the activating viral component, recombinant viral proteins were used: in the solid phase, soluble gp41 (sgp41) (the outer membrane part of gp41, residues 539-684 of gp160) bound C1q, but not dimeric proenzyme C1s, while gp120 was ineffective. In the fluid phase, sgp41 activated the C1 complex in a dose- and time-dependent manner, more efficiently than aggregated Ig, but less efficiently than immune complexes. To localize the C1 activating site(s) in gp41, synthetic peptides (15-residue oligomers spanning amino acids 531-695 of gp160) were used. Peptides covering positions 591-605 and 601-620 and, to a lesser extent, positions 561-575, had both the ability to bind C1q and to induce C3 deposition. These data provide the first experimental evidence of a direct interaction between the C1 complex and HIV-1, and indicate that C1 binding and activation are mediated by specific sites in gp41.

Structure and function of C1r and C1s: current concepts.

C1r and C1s, the constituent proteins of C1s-C1r-C1r-C1s, the Ca2+ -dependent catalytic unit of C1, are homologous serine proteinases that share a common activation pattern and have similar structural organizations at the monomeric level. In both cases, activation occurs through cleavage of a single Arg-Ile bond, which converts the single-chain proenzymes into active proteinases comprising two chains linked by a single disulphide bridge. Both NH2-terminal A chains are sub-divided into five structural units (I-V) including a single copy of an Epidermal Growth Factor-like segment (II) and two different pairs of internal repeats (I/III and IV/V). Regions I and III have no equivalent in other proteins, whereas regions IV and V are homologous to short consensus repeats found, in particular, in complement proteins C2, B, H, C4b-binding protein and CR1. The COOH-terminal B chains are homologous to the catalytic chains of serine proteinases, but lack the "histidine-loop", a disulphide bridge common to all other known mammalian serine proteinases. Overall sequence comparison of C1r and C1s reveals 40% amino acid identity and conservation of all cysteine residues. In contrast, C1r and C1s widely differ from each other by their glycosylation patterns: both proteins contain Asn-linked carbohydrates, but four glycosylation sites are present on C1r, and only two on C1s.(ABSTRACT TRUNCATED AT 250 WORDS)

Complete amino acid sequence of the A chain of human complement-classical-pathway enzyme C1r.

The amino acid sequence of human C1r A chain was determined, from sequence analysis performed on fragments obtained from C1r autolytic cleavage, cleavage of methionyl bonds, tryptic cleavages at arginine and lysine residues, and cleavages by staphylococcal proteinase. The polypeptide chain has an N-terminal serine residue and contains 446 amino acid residues (Mr 51,200). The sequence data allow chemical characterization of fragments alpha (positions 1-211), beta (positions 212-279) and gamma (positions 280-446) yielded from C1r autolytic cleavage, and identification of the two major cleavage sites generating these fragments. Position 150 of C1r A chain is occupied by a modified amino acid residue that, upon acid hydrolysis, yields erythro-beta-hydroxyaspartic acid, and that is located in a sequence homologous to the beta-hydroxyaspartic acid-containing regions of Factor IX, Factor X, protein C and protein Z. Sequence comparison reveals internal homology between two segments (positions 10-78 and 186-257). Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 108 and 204. Combined with the previously determined sequence of C1r B chain [Arlaud & Gagnon (1983) Biochemistry 22, 1758-1764], these data give the complete sequence of human C1r.

Primary structure of the A chain of human complement-classical-pathway enzyme C1r. N-terminal sequences and alignment of autolytic fragments and CNBr-cleavage peptides.

Activated human complement-classical-pathway enzyme C1r has previously been shown to undergo autolytic cleavages occurring in the A chain [Arlaud, Villiers, Chesne & Colomb (1980) Biochim. Biophys. Acta 616, 116-129]. Chemical analysis of the autolytic products confirms that the A chain undergoes two major cleavages, generating three fragments, which have now been isolated and characterized. The N-terminal alpha fragment (approx. 210 residues long) has a blocked N-terminus, as does the whole A chain, whereas N-terminal sequences of fragments beta and gamma (approx. 66 and 176 residues long respectively) do not, and their N-terminal sequences were determined. Fragments alpha, beta and gamma, which are not interconnected by disulphide bridges, are located in this order within C1r A chain. Fragment gamma is disulphide-linked to the B chain of C1r, which is C-terminal in the single polypeptide chain of precursor C1r. CNBr cleavage of C1r A chain yields seven major peptides, CN1b, CN4a, CN2a, CN1a, CN3, CN4b and CN2b, which were positioned in that order, on the basis of N-terminal sequences of the methionine-containing peptides generated from tryptic cleavage of the succinylated (3-carboxypropionylated) C1r A chain. About 60% of the sequence of C1r A chain (440-460 residues long) was determined, including the complete sequence of the C-terminal 95 residues. This region shows homology with the corresponding parts of plasminogen and chymotrypsinogen and, more surprisingly, with the alpha 1 chain of human haptoglobin 1-1, a serine proteinase homologue.

Complete amino acid sequence of the catalytic chain of human complement subcomponent C1-r.

The amino acid sequence of human C1-r b chain hs been determined, from sequence analysis performed on fragments obtained by CNBr cleavage, dilute acid hydrolysis, tryptic cleavage of the succinylated protein, and subcleavages by staphylococcal protease. The polypeptide chain contains 242 amino acids (Mr 27 096), and the sequence shows strong homology with other mammalian serine proteases. The histidine, aspartic acid, and serine residues of the active site (His-57, Asp-102, and Ser-195 in bovine chymotrypsinogen) are located at positions 39, 94, and 191, respectively. The chain which lacks the "histidine-loop" disulfide bridge, contains five half-cystine residues, of which four (positions 157-176 and 187-217) are homologous to residues involved in disulfide bonds generally conserved in serine proteases, whereas the half-cystine residue at position 114 is likely to be involved in the single disulfide bridge connecting the catalytic b chain to the n-terminal a chain. Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 51 and 118.