Binding of human growth hormone (hGH) to its receptor is required for regulation of normal human growth and development. Examination of the 2.8 angstrom crystal structure of the complex between the hormone and the extracellular domain of its receptor (hGHbp) showed that the complex consists of one molecule of growth hormone per two molecules of receptor. The hormone is a four-helix bundle with an unusual topology. The binding protein contains two distinct domains, similar in some respects to immunoglobulin domains. The relative orientation of these domains differs from that found between constant and variable domains in immunoglobulin Fab fragments. Both hGHbp domains contribute residues that participate in hGH binding. In the complex both receptors donate essentially the same residues to interact with the hormone, even though the two binding sites on hGH have no structural similarity. Generally, the hormone-receptor interfaces match those identified by previous mutational analyses. In addition to the hormone-receptor interfaces, there is also a substantial contact surface between the carboxyl-terminal domains of the receptors. The relative extents of the contact areas support a sequential mechanism for dimerization that may be crucial for signal transduction.

The protein deamidation process involves the conversion of the amide side-chain moieties of asparagine and glutamine residues to carboxyl groups. This conversion is an unusual form of protein modification in that it requires catalysis by an intramolecular reaction where both the substrate (asparagine and glutamine side chains) and "catalytic site" (the peptide nitrogen of the succeeding residue) are constituents of several consecutive residues along the polypeptide chain. The stereochemical factors governing this process were studied with a data base derived from the neutron crystallographic structure of trypsin from which amide groups and oxygen can be unambiguously differentiated because of their different neutron scattering properties. The neutron structure allowed for the direct determination of those residues that were deamidated; 3 of 13 asparagine residues were found to be modified. These modified residues were clearly distinguished by a distinct local conformation and hydrogen-bonding structure in contrast to those observed for the other asparagine residues. No correlation was found between preference to deamidate and the chemical character of residues flanking the site, as had been proposed from previous peptide studies.

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Abstract:A variant of human interferon-gamma (IFN-y) has been created in which the two chains of the homodimeric cytokine were linked N-to C-terminus by an eight residue polypeptide linker. The sequence of this linker was derived from a loop in bira bifunctional protein, and was determined from a structural database search. This "single-chain" variant was used to create an IFN-y molecule that binds only a single copy of the a-chain receptor, rather than the 2 a-chain receptor: 1 IFN-y binding stoichiometry observed for the native hormone. Crystals have been grown of a 1 : 1 complex between this single-chain molecule and the extracellular domain of its a-chain receptor. These crystals diffract beyond 2.0 A, significantly better than the 2.9 8, observed for the native 2: 1 complex.Density calculations suggest these crystals contain two complexes in the asymmetric unit; a self-rotation function confirms this conclusion.Keywords: interferon-gamma; receptor; structure-based design; X-ray crystallography Interferon-y (IFN-y) is a homodimeric cytokine with potent antiviral, antiproliferative, and immunomodulatory activities (Wheelock, 1965;Farrar & Schreiber, 1993). It is expressed as a 143 amino acid polypeptide, with a post-translation modification resulting in a pyroglutamate amino terminus, the addition of carbohydrate, and a heterogeneous carboxyl terminus Rinderknecht et al., 1984;Pan et al., 1987). IFN-y exerts its various activities through receptor aggregation, a general mechanism common to the cytokine family (Sprang & Bazan, 1993). The first step of this process involves IFN-y binding to its high-affinity cell surface receptor, IFN-yRa, a member of the class-2 hematopoietic receptor superfamily (Schreiber & Celada, 1985;Langer & Pestka, 1988). While IFN-y has been shown to induce the dimerization of IFN-yRa (Greenlund et al., 1993) The ability to produce large quantities of fully active recombinant IFN-y (Gray et al., 1982; Tanaka et al., 1983) has made it the focus of numerous mutagenesis and structural studies aimed at establishing the determinants of receptor recognition. The X-ray crystal structure has been determined at 2.9 8, resolution of IFN-y with two copies of the extracellular domain of IFN-yRa bound symmetrically to the homodimer (Walter et al., 1995; S. Ealick, pers. comm.). This structure has provided details of the interaction between IFN-y and its high-affinity receptor, including the observation that the two receptors do not themselves interact. It also shows that the IFN-y residues in contact with the receptor form a discontinuous epitope made from residues on each monomer. One segment consists of helix A, the AB loop, and helix B from monomer one; the other consists of helix F and the C-terminus from the monomer two. Unfortunately, interpretation of structural detail is affected by the relatively limited resolution of this structure. In addition, this structure does not elucidate the role of the positively charged C-terminus of IFN-y, a segment shown by mutagenesis to be essential for re...