Crystallization and preliminary X‐ray analysis of a 1:1 complex between a designed monomeric interferon‐gamma and its soluble receptor

Abstract

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...

Self-assembled Structures from Amphiphilic Peptides

“…[39] In this respect, proven polymersomes concepts, such as targeting, may also lead to a fast development of advanced peptosomes. [40,41] Peptide Beads Peptide beads are spherical particles in the submicrometer range, formed by self-assembly of the amphiphilic peptide Ac-X 3 -gT and its close analogues AcC-X 3 -gT, AcC(sl)-X 3 -gT, and Ac-X 3 -gT-C. [13,14] To obtain information about the underlying structure and formation process, we extensively characterized the beads by microscopic and light scattering (LS) techniques, such as AFM, TEM, SEM, SLS and DLS. In summary, the beads exhibited a particle scattering factor and a ρ-parameter (R g /R h ) that confirm the beads to be solid spheres.…”

Redesigning Protein Cavities as a Strategy for Increasing Affinity in Protein-Protein Interaction: Interferon-γReceptor 1 as a Model

“…Our computational predictions are based on the analysis of crystal structures of complexes between IFN γ and the extracellular part of IFN γ R1, namely, the structures of PDB codes 1fg9 [ 19 ] and 1fyh [ 20 ] that contain four crystallographically independent IFN γ /IFN γ R1 complexes. Throughout the paper, IFN γ R1 residues are numbered as in UniProt entry P15260.…”

“…IFN γ R1 models are based on PDB structures 1fg9 [ 19 ] and 1fyh [ 20 ]. Missing residues in both structures were added using Modeller suite of programs [ 28 ].…”

Protein expression, crystallization and preliminary X-ray crystallographic analysis of chicken interferon-γ receptor α chain

“…It has been reported that IFN-possesses high binding affinity (K d = 10 À8 -10 À10 M) for IFNGR1 (Marsters et al, 1995;Green et al, 1998). The IFN-dimer can also directly interact with IFNGR1 (Ealick et al, 1991;Fountoulakis et al, 1992;Chè ne et al, 1995), as shown by the solution of the crystal structure of their complex Randal & Kossiakoff, 1998;Acebró n et al, 2009). Furthermore, specific interaction of IFN-with its receptors is crucial in the IFN-/signal transducer and activator of transcription (STAT) signalling pathway cascade.…”