Observation of an unexpected third receptor molecule in the crystal structure of human interferon-gamma receptor complex

BACKGROUND: Molecular interactions among cytokines and cytokine receptors form the basis of many cell-signaling pathways relevant to immune function. Interferon-gamma (IFN-gamma) signals through a multimeric receptor complex consisting of two different but structurally related transmembrane chains: the high-affinity receptor-binding subunit (IFN-gammaRalpha) and a species-specific accessory factor (AF-1 or IFN-gammaRbeta). In the signaling complex, the two receptors probably interact with one another through their extracellular domains. Understanding the atomic interactions of signaling complexes enhances the ability to control and alter cell signaling and also provides a greater understanding of basic biochemical processes. RESULTS: The crystal structure of the complex of human IFN-gamma with the soluble, glycosylated extracellular part of IFN-gammaRalpha has been determined at 2.9 A resolution using multiwavelength anomalous diffraction methods. In addition to the expected 2:1 complex, the crystal structure reveals the presence of a third receptor molecule not directly associated with the IFN-gamma dimer. Two distinct intermolecular contacts, involving the edge strands of the C-terminal domains, are observed between this extra receptor and the 2:1 receptor-ligand complex thereby forming a 3:1 complex. CONCLUSIONS: The observed interactions in the 2:1 complex of the high-affinity cell-surface receptor with the IFN-gamma cytokine are similar to those seen in a previously reported structure where the receptor chains were not glycosylated. The formation of beta-sheet packing interactions between pairs of IFN-gammaRalpha receptors in these crystals suggests a possible model for receptor oligomerization of Ralpha and the structurally homologous Rbeta receptors in the fully active IFN-gamma signaling complex.     

Design, characterization, and structure of a biologically active single-chain mutant of human IFN-gamma

A mutant form of human interferon-gamma (IFN-gamma SC1) that binds one IFN-gamma receptor alpha chain (IFN-gamma R alpha) has been designed and characterized. IFN-gamma SC1 was derived by linking the two peptide chains of the IFN-gamma dimer by a seven-residue linker and changing His111 in the first chain to an aspartic acid residue. Isothermal titration calorimetry shows that IFN-gamma SC1 forms a 1:1 complex with its high-affinity receptor (IFN-gamma R alpha) with an affinity of 27(+/- 9) nM. The crystal structure of IFN-gamma SC1 has been determined at 2.9 A resolution from crystals grown in 1.4 M citrate solutions at pH 7.6. Comparison of the wild-type receptor-binding domain and the Asp111-containing domain of IFN-gamma SC1 show that they are structurally equivalent but have very different electrostatic surface potentials. As a result, surface charge rather than structural changes is likely responsible for the inability of the His111-->Asp domain of to bind IFN-gamma R alpha. The AB loops of IFN-gamma SC1 adopt conformations similar to the ordered loops of IFN-gamma observed in the crystal structure of the IFN-gamma/IFN-gamma R alpha complex. Thus, IFN-gamma R alpha binding does not result in a large conformational change in the AB loop as previously suggested. The structure also reveals the final six C-terminal amino acid residues of IFN-gamma SC1 (residues 253-258) that have not been observed in any other reported IFN-gamma structures. Despite binding to only one IFN-gamma R alpha, IFN-gamma SC1 is biologically active in cell proliferation, MHC class I induction, and anti-viral assays. This suggests that one domain of IFN-gamma is sufficient to recruit IFN-gamma R alpha and IFN-gamma R beta into a complex competent for eliciting biological activity. The current data are consistent with the main role of the IFN-gamma dimer being to decrease the dissociation constant of IFN-gamma for its cellular receptors.     

The structure and activity of a monomeric interferon-gamma:alpha-chain receptor signaling complex

BACKGROUND: Interferon-gamma (IFN-gamma) is a homodimeric cytokine that exerts its various activities by inducing the aggregation of two different receptors. The alpha chain receptor (IFN-gammaRalpha) is a high affinity receptor that binds to IFN-gamma in a symmetric bivalent manner to form a stable, intermediate 1:2 complex. This intermediate forms a binding template for the subsequent binding of two copies of the second receptor, beta chain receptor (IFN-gammaRbeta), producing the active 1:2:2 signaling complex. RESULTS: A single chain monovalent variant of IFN-gamma (scIFN-gamma) was constructed and complexed to one copy of the extracellular domain (ECD) of IFN-gammaRalpha. The structure of this 1:1 complex was determined and the hormone-receptor interface shown to be characterized by a number of hydrophilic interactions mediated by several highly ordered water networks. The scIFN-gamma interface consists of segments from each of the monomer chains of the homodimer. The principal hydrophobic contact of the receptor involves a tripeptide segment of the receptor having an unusual and high energy conformation. Despite containing only one binding site for IFN-gammaRalpha, the monovalent scIFN-gamma molecule has significant activity in antiviral biological assays. CONCLUSIONS: ScIFN-gamma binds the ECD of IFN-gammaRalpha through a highly hydrated interface with an important set of hormone-receptor contacts mediated through structured waters. Although the interface is highly hydrated, it supports tight binding and has a considerable degree of specificity. The biological activity of scIFN-gamma confirms that the scIFN-gamma:IFN-gammaRalpha complex represents a productive intermediate and that it can effectively recruit the other required component, IFN-gammaRbeta, to signal based on the 1:1:1 complex.     

A soluble recombinant multimeric anti-Rh(D) single-chain Fv/CR1 molecule restores the immune complex binding ability of CR1-deficient erythrocytes

CR1 (CD35, the C3b/C4b receptor) is a widely distributed membrane glycoprotein with a unique cluster conformation on the surface of erythrocytes (E). CR1 on E is responsible for the transport of immune complexes (IC) to liver and spleen. As a cofactor of the C3b cleavage by factor I, CR1 is also a potent inhibitor of C activation and inflammation. In some diseases (systemic lupus erythematosus, hemolytic anemia, AIDS, etc.) an acquired low level of CR1 on E has been observed, leading to an impaired clearance of IC. The aim of this study was to design a heterofunctional molecule that will bind to E and restore a normal or a supranormal CR1 density on E that could mimic the unique distribution pattern of CR1 on normal E. For that purpose a new multimerizing system based on the properties of the C-terminal part of the alpha-chain of the C4 binding protein (C4bp) was used. We first produced a multimeric soluble CR1 that proved to be a better inhibitor of in vitro C activation than the monomeric form of CR1, then a heteromultimeric molecule made of CR1 and single-chain Fv anti-Rh(D) valences able to attach E and providing E with as much as a 10-fold increase in CR1 density with the same CR1 distribution pattern as native E. CR1/single-chain Fv anti-Rh(D)-treated E were able in vitro to attach as many opsonized IC as native E. These data open the way for future use of multimeric and heteromultimeric forms of soluble recombinant CR1 as therapy of IC diseases.     

Crystallization and preliminary X-ray diffraction studies of a complex between interleukin-2 and a soluble form of the p55 component of the high affinity interleukin-2 receptor

Human recombinant interleukin-2 (IL-2) and a soluble recombinant form of the human p55 (Tac antigen) component of the IL-2 receptor (IL-2R) have been cocrystallized in 1.7-1.8 M ammonium sulfate, in the pH range 7.0-8.2. Variously glycosylated forms of both receptor and ligand can be cocrystallized under those conditions. The best crystals of the putative receptor-ligand complex involve the enzymatically desialylated receptor and unglycosylated IL-2. These crystals belong to the trigonal space group P3(1)2(1) or its enantiomorph, with unit cell dimensions a = b = 91 A and c = 119 A, and diffract to 3.5 A resolution. There is one receptor-ligand complex asymmetric unit, with a Matthews coefficient of 2.7, assuming the presence of one IL-2 molecule-receptor molecule. Interestingly, in addition to IL-2 (Mr = 14,000), the p55 IL-2 receptor (Mr = 44,000) and two fragments of the receptor, of apparent Mr = 35,000 and 25,000, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the crystals are enriched in a reducible dimeric form of the desialylated receptor (apparent Mr = 90,000), as compared with protein solution from which the crystals grow. The overall amino acid content in the crystals is consistent with a 1:1 ratio of receptor to ligand. A native data set has been collected on a multiwire area detector and the search for suitable heavy atom derivatives is in progress.     

Multisite mutagenesis of interleukin 5 differentiates sites for receptor recognition and receptor activation

Multisite mutagenesis of single-chain and monomeric forms of human interleukin 5 (IL-5) was performed to investigate mechanistic features of receptor activation and the possibility of differentiating sites of activation from those for receptor interaction. The normally dimeric human IL-5 contains two domains, each containing a four-helix bundle. IL-5 has previously been re-engineered into the monomeric, one-domain GM1 form by introducing an eight-residue linker between the third and fourth helices. In this study, we tested a combination of mutations in a single-chain IL-5 (scIL-5) construct, [(89)SLRGG(92),W(110)/(89)AAAAA(92), A(110)]scIL-5. This mutein was found to retain substantial IL-5 receptor alpha-chain binding but with selectively suppressed proliferation of the IL-5-dependent cell line TF-1.28. This result confirms recent findings that IL-5 receptor alpha-chain recognition can be supported by the (89)SLRGG(92) epitope and that, in contrast, Glu110 is important in receptor activation. On the basis of this result, two mutants of GM1 were constructed with the intent to retain receptor alpha-chain binding while modifying receptor activation epitopes. In the first, [(88)SLRGG(92),W(110)]GM1, the wild-type CD-loop sequence (89)EERRR(92) was converted to the mimotope (89)SLRGG(92), and Glu110 to Trp. In the second, [A(13), A(110)]GM1, wild-type Glu13, and Glu110 were both mutated to Ala. GM1 and mutants were expressed in high yield in Escherichia coli, purified under denaturing conditions from inclusion bodies, and refolded. Monomers were screened for binding to shIL-5Ralpha-Fc using optical biosensor and ELISA and for bioactivity by proliferation of TF-1.28 cells. Both [(88)SLRGG(92),W(110)]GM1 and [A(13),A(110)]GM1 were found to interact with the shIL-5Ralpha-Fc, with affinities of 69-585 nM, 2-15-fold weaker than that of the original GM1. The mutants also were able to compete with IL-5 for binding to shIL-5Ralpha in an ELISA. In contrast, both mutants exhibited a disproportionately decreased capacity to stimulate TF-1. 28 cell proliferation. [A(13),A(110)]GM1 bioactivity was 160-fold lower than that of GM1, while that for the [(88)SLRGG(92),W(110)]GM1 mutant was 2600-fold lower. The largely retained IL-5 receptor alpha-chain binding affinities versus relatively suppressed bioactivities of [A(13),A(110)]GM1 and [(88)SLRGG(92),W(110)]GM1 variants, in particular the latter, point to the existence of separable IL-5 epitopes for receptor binding and activation and establish the potential to design smaller IL-5 mimetic antagonists.     

X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule. A completely inactive analogue.

A crystal structure of a totally inactive insulin molecule has been determined. For this insulin molecule, the first without detectable activity to be characterized, the A and B-chains are linked by a peptide bond between A1 Gly and B29 Lys. The molecule has retained all its normal self-association properties and it can also accommodate the two different conformations designated T and R, as seen in 4Zn native pig insulin crystals. The hexamers of the crosslinked insulin molecule were crystallized using the 4Zn insulin recipe of Schlichtkrull. The structure has been crystallographically refined with data extending to 2 A using restrained least-square methods. Comparison of the B29-A1 peptide crosslink insulin and the 4Zn native insulin reveals close structural similarities with the native dimer. The analysis of the structure confirms the earlier hypothesis that insulin structures in crystals are not in an active conformation and that a separation of N-terminal A-chain and C-terminal B-chain is required for interaction with the insulin receptor.     

Chemotactic Signaling by Single-Chain Chemoreceptors

Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit swapping interactions that will need to be taken into account in experimental applications of single-chain chemoreceptors.     

Enhanced immune presentation of a single-chain major histocompatibility complex class I molecule engineered to optimize linkage of a C-terminally extended peptide

Major histocompatibility complex class I molecules can be expressed as single polypeptides wherein the antigenic peptide, beta2-microglobulin, and heavy chain are attached by flexible linkers. These molecules, single-chain trimers (SCTs), are remarkably stable at the cell surface compared with native (noncovalently attached) class I molecules. In this study, we used a structure-based approach to engineer an F pocket variant SCT of the murine class I molecule Kb that presents the SIINFEKL epitope of ovalbumin. Mutation of heavy chain residue Tyr84 (Y84A) in the SCT resulted in enhanced serological and cytolytic CD8 T cell recognition of the covalently linked peptide due to better accommodation of the linker extending from the C terminus of the peptide. These SCTs exhibit significant cell-surface stability, which we hypothesize is rendered by their ability to continuously and efficiently rebind the covalently attached peptide. In addition, we demonstrate that SCT technology can be applied to tetramer construction using recombinant SCTs expressed in Escherichia coli. SCT-based tetramers could have applications for the enumeration of T and natural killer cells that recognize peptide.class I complexes prone to dissociation.     

IL-8 single-chain homodimers and heterodimers: interactions with chemokine receptors CXCR1, CXCR2, and DARC.

Covalent single-chain dimers of the chemokine interleukin-8 (IL-8) have been designed to mimic the dimeric form of IL-8 in solution and facilitate the production of heterodimer variants of IL-8. Physical studies indicated that use of a simple peptide linker to join two subunits, while allowing receptor binding and activation, led to self-association of the tethered dimers. However, addition of a single disulfide crosslink between the tethered subunits prevented this multimer from forming, yielding a species of dimer molecular weight. Crosslinked single-chain dimers bind to both IL-8 neutrophil receptors CXCR1 and CXCR2 as well as to DARC, as does a double disulfide-linked dimer with no peptide linker. In addition, neutrophil response to these dimers as measured by chemotaxis or beta-glucuronidase release is similar to that elicited by wild-type IL-8, providing evidence that the dissociation of the dimeric species is not required for these biologically relevant activities. Finally, through construction of single-chain heterodimer mutants, we show that only the first subunit's ELR motif is the single-chain variants.     

Purification, crystallization, and properties of F1-ATPase complexes from the thermoalkaliphilic Bacillus sp. strain TA2.A1

Recently, we reported the cloning of the atp operon encoding for the F(1)F(0)-ATP synthase from the extremely thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1. In this study, the genes encoding the F(1) moiety of the enzyme complex were cloned from the atp operon into the vector pTrc99A and expressed in Escherichia coli in two variant complexes, F(1)-wt consisting of subunits alpha(3)beta(3)gammadeltaepsilon and F(1)Deltadelta lacking the entire delta-subunit as a prerequisite for overproduction and crystallization trials. Both F(1)-wt and F(1)Deltadelta were successfully overproduced in E. coli and purified in high yield and purity. F(1)Deltadelta was crystallized by micro-batch screening yielding three-dimensional crystals that diffracted to a resolution of 3.1A using a synchrotron radiation source. After establishing cryo and dehydrating conditions, a complete set of diffraction data was collected from a single crystal. No crystals were obtained with F(1)-wt. Data processing of diffraction patterns showed that F(1)Deltadelta crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell parameters of a=121.70, b=174.80, and c=223.50A, alpha, beta, gamma=90.000. The asymmetric unit contained one molecule of bacterial F(1)Deltadelta with a corresponding volume per protein weight (V(M)) of 3.25A(3) Da(-1) and a solvent content of 62.1%. Silver staining of single crystals of F(1)Deltadelta analyzed by SDS-PAGE revealed four bands alpha, beta, gamma, and epsilon with identical M(r)-values as those found in the native F(1)F(0)-ATP synthase isolated from strain TA2.A1 membranes. ATPase assays of F(1)Deltadelta crystals exhibited latent ATP hydrolytic activity that was highly stimulated by lauryldimethylamine oxide, a hallmark of the native enzyme.     

Ubiquitous soluble Mg(2+)-ATPase complex. A structural study.

We have performed a detailed structural analysis of the soluble Mg(2+)-ATPase complex purified from Xenopus laevis ovary, which is an abundant and ubiquitous homo-oligomeric protein complex located in the nucleus and in the cytoplasm, belonging to a novel multigene-family of putative Mg(2+)-ATPases. Enzyme activity staining after non-denaturing polyacrylamide gel electrophoresis revealed that Mg(2+)-ATPase activity of the native protein is dependent on oligomerization and could not be detected in dissociated subunits. For the native protein a sedimentation coefficient of 15.3 S and a corresponding relative molecular mass of 612,000 was determined by analytical ultracentrifugation and a relative molecular mass of 590,000 was estimated from scanning transmission electron microscopy, supporting our previous conclusion that the oligomer comprises six 97,000 Mr subunits. Conventional electron microscopy of negatively stained specimens revealed the Mg(2+)-ATPase complex to be a hexagonal molecule in its favoured "end-on" projection and a double-banded molecule in its "side-on" projection (approx. 12 nm diameter; approx. 9 nm height). In addition, dimerized complexes could be observed in negatively stained specimens, yielding pronounced hexameric images and four-banded images in their end-on and side-on orientations, respectively (approx. 12 nm diameter; approx. 18.5 nm height). Two-dimensional (2D = mono-molecular) crystals have been produced from the dimerized complexes by the negative staining carbon film technique. Hexagonal crystals with a p6 plane group symmetry were obtained from molecules in their end-on orientation and longitudinal arrays with a p2 symmetry from complexes in their side-on orientation. A low-resolution molecular model of the native protein, derived from averages of these two 2D crystals, is presented. From our results we propose oligomerization as an inherent structural principle of organization for this whole newly defined Mg(2+)-ATPase multigene-family, that includes such seemingly diverse functionally defined proteins as mammalian and yeast "vesicle fusion" and "peroxisome assembly" proteins and the product of the yeast cell cycle gene CDC48.     

Structural basis for FGF receptor dimerization and activation.

The crystal structure of FGF2 bound to a naturally occurring variant of FGF receptor 1 (FGFR1) consisting of immunoglobulin-like domains 2 (D2) and 3 (D3) has been determined at 2.8 A resolution. Two FGF2:FGFR1 complexes form a 2-fold symmetric dimer. Within each complex, FGF2 interacts extensively with D2 and D3 as well as with the linker between the two domains. The dimer is stabilized by interactions between FGF2 and D2 of the adjoining complex and by a direct interaction between D2 of each receptor. A positively charged canyon formed by a cluster of exposed basic residues likely represents the heparin-binding site. A general model for FGF- and heparin-induced FGFR dimerization is inferred from the crystal structure, unifying a wealth of biochemical data.     

Cutting edge: a naturally occurring mutation in CD1e impairs lipid antigen presentation

The human CD1a-d proteins are plasma membrane molecules involved in the presentation of lipid Ags to T cells. In contrast, CD1e is an intracellular protein present in a soluble form in late endosomes or lysosomes and is essential for the processing of complex glycolipid Ags such as hexamannosylated phosphatidyl-myo-inositol, PIM(6). CD1e is formed by the association of beta(2)-microglobulin with an alpha-chain encoded by a polymorphic gene. We report here that one variant of CD1e with a proline at position 194, encoded by allele 4, does not assist PIM(6) presentation to CD1b-restricted specific T cells. The immunological incompetence of this CD1e variant is mainly due to inefficient assembly and poor transport of this molecule to late endosomal compartments. Although the allele 4 of CD1E is not frequent in the population, our findings suggest that homozygous individuals might display an altered immune response to complex glycolipid Ags.     

Signaling by covalent heterodimers of interferon-gamma. Evidence for one-sided signaling in the active tetrameric receptor complex

Interferon-gamma (IFN-gamma) and its receptor complex are dimeric and bilaterally symmetric. We created mutants of IFN-gamma that bind only one IFN-gammaR1 chain per dimer molecule (called a monovalent IFN-gamma) to see if the interaction of IFN-gamma with one-half of the receptor complex is sufficient for bioactivity. Mutating a receptor-binding sequence in either AB loop of a covalent dimer of IFN-gamma yielded two monovalent IFN-gammas, gamma(m)-gamma and gamma-gamma(m), which cross-link to only a single soluble IFN-gammaR1 molecule in solution and on the cell surface. Monovalent IFN-gamma competes fully with wild type IFN-gamma for binding to U937 cells but only at a greater than 100-fold higher concentration than wild type IFN-gamma. Monovalent IFN-gamma had anti-vesicular stomatitis virus activity and antiproliferative activity, and it induced major histocompatibility complex class I and class II (HLA-DR) expression. In contrast, the maximal levels of activated Stat1alpha produced by monovalent IFN-gammas after 15 min were never more than half of those produced by either wild type or covalent IFN-gammas in human cell lines. These data indicate that while monovalent IFN-gamma activates only one-half of a four-chain receptor complex, this is sufficient for Stat1alpha activation, major histocompatibility complex class I surface antigen induction, and antiviral and antiproliferative activities. Thus, while interaction with both halves of the receptor complex is required for high affinity binding of IFN-gamma and efficient signal transduction, interaction with only one-half of the receptor complex is sufficient to initiate signal transduction.     

Crystal structure of a SEA variant in complex with MHC class II reveals the ability of SEA to crosslink MHC molecules

Although the biological properties of staphylococcal enterotoxin A (SEA) have been well characterized, structural insights into the interaction between SEA and major histocompatibilty complex (MHC) class II have only been obtained by modeling. Here, the crystal structure of the D227A variant of SEA in complex with human MHC class II has been determined by X-ray crystallography. SEA(D227A) exclusively binds with its N-terminal domain to the alpha chain of HLA-DR1. The ability of one SEA molecule to crosslink two MHC molecules was modeled. It shows that this SEA molecule cannot interact with the T cell receptor (TCR) while a second SEA molecule interacts with MHC. Because of its relatively low toxicity, the D227A variant of SEA is used in tumor therapy.     

Novel biotin linker with alkyne and amino groups for chemical labelling of a target protein of a bioactive small molecule

We synthesized a novel linker (1) with biotin, alkyne and amino groups for the identification of target proteins using a small molecule that contains an azide group (azide probe). The alkyne in the linker bound the azide probe via an azide-alkyne Huisgen cycloaddition. A protein cross-linker effectively bound the conjugate of the linker and an azide probe with a target protein. The covalently bound complex was detected by western blotting. Linker 1 was applied to a model system using an abscisic acid receptor, RCAR/PYR/PYL (PYL). Cross-linked complexes of linker 1, the azide probes and the target proteins were successfully visualized by western blotting. This method of target protein identification was more effective than a previously developed method that uses a second linker with biotin, alkyne, and benzophenone (linker 2) that acts to photo-crosslink target proteins. The system developed in this study is a method for identifying the target proteins of small bioactive molecules and is different from photo-affinity labelling.     

Efficient folding of the FcepsilonRI alpha-chain membrane-proximal domain D2 depends on the presence of the N-terminal domain D1

Human high affinity receptor for IgE is a membrane glycoprotein multichain complex presenting two extracellular Ig modules in its alpha-chain (D1D2). The receptor IgE binding region is located within the membrane-proximal module D2, while the N-terminal module D1 appears to promote an optimal receptor conformation for IgE binding. To understand the structural relationship between the two modules, we dissected FcepsilonRI alpha-chain into its discrete Ig units and expressed them in mammalian cells. Unexpectedly, D2 was secreted as a disulphide-linked dimer, while D1 was monomeric. Active secretion and full glycosylation of dimeric D2 suggest a native-like conformation of the protein, justifying the escape from the endoplasmic reticulum/Golgi quality control systems. We then propose a domain-swapping model for D2, in which two interdigitated polypeptide chains assume the overall conformation of two Ig modules, as observed for rat CD2 N-terminal domain. Fusion of an unrelated Ig fold moiety at the N terminus of D2 did not interfere with its dimerisation. While D1D2 assumes a correct fold, co-expression of both isolated domains in the same cell did not restore monomeric folding of D2. Thus, D1 appears to assist the appropriate folding of FcepsilonRI alpha-chain, acting as an uncleavable intramolecular chaperone-like block towards D2.     

Clostripain Linker Deletion Variants Yield Active Enzyme in Escherichia coli: A Possible Function of the Linker Peptide as Intramolecular Inhibitor of Clostripain Automaturation

The clostripain core protein is composed of the light and heavy chain subunits linked by a nonapeptide into a single polypeptide chain [Mol. Gen. Genet. 240: 140, 1993]. Linker removal is due to autocatalytic processing yielding active heterodimeric enzyme. We have expressed mutationally altered core protein variants in the heterologous host Escherichia coli to gain further insight into the process of clostripain automaturation. In a mutationally created Cys²³¹→ Ser variant, heterodimer formation was largely impaired, providing molecular evidence that the capacity for automaturation is attributed to the active site cysteine, Cys²³¹, of the native enzyme. Artificially generated deletions of the linker peptide did not prevent the formation of active enzyme. One variant gave rise to a single-chain molecule devoid of the authentic processing sites while retaining enzymatic activity. Experiments performed with linker substitution variants suggested that the efficacy of automaturation depends on a proper configuration of the linker region. According to computerized predictions, the formation of a turn-structured protein loop or hinge with hydrophilic characteristics in the linker region is probably a prerequisite for the interaction of the active site cysteine with the processing sites, Arg¹⁸¹ and Arg¹⁹⁰. We propose that the clostripain linker nonapeptide serves as an important transient intramolecular inhibitor in the cellular self-defense program evolved by the natural host Clostridium histolyticum. Received: 23 January 1996 / Accepted: 22 April 1996