Paul P. Trotta

Zinc mediated dimer of human interferon-α2b revealed by X-ray crystallography

BACKGROUND The human alpha-interferon (huIFN-alpha) family displays broad spectrum antiviral, antiproliferative and immunomodulatory activities on a variety of cell types. The diverse biological activities of the IFN-alphas are conveyed to cells through specific interactions with cell-surface receptors. Despite considerable effort, no crystal structure of a member of this family has yet been reported, because the quality of the protein crystals have been unsuitable for crystallographic studies. Until now, structural models of the IFN-alphas have been based on the structure of murine IFN-beta (muIFN-beta). These models are likely to be inaccurate, as the amino acid sequence of muIFN-beta differs significantly from the IFN-alphas at proposed receptor-binding sites. Structural information on a huIFN-alpha subtype would provide an improved basis for modeling the structures of the entire IFN-alpha family. RESULTS The crystal structure of recombinant human interferon-alpha 2b (huIFN-alpha 2b) has been determined at 2.9 A resolution. HuIFN-alpha 2b exists in the crystal as a noncovalent dimer, which associates in a novel manner. Unlike other structurally characterized cytokines, extensive interactions in the dimer interface are mediated by a zinc ion (Zn2+). The overall fold of huIFN-alpha 2b is most similar to the structure of muIFN-beta. Unique to huIFN-alpha 2b is a 3(10) helix in the AB loop which is held to the core of the molecule by a disulfide bond. CONCLUSIONS The structure of huIFN-alpha 2b provides an accurate model for analysis of the > 15 related type 1 interferon molecules. HuIFN-alpha 2b displays considerable structural similarity with muIFN-beta, interleukin-10 and interferon-gamma, which also bind related class 2 cytokine receptors. From these structural comparisons and numerous studies on the effects of mutations on biological activity, we have identified protein surfaces that appear to be important in receptor activation. This study also reveals the potential biological importance of the huIFN-alpha 2b dimer.

Three-dimensional structure of recombinant human granulocyte-macrophage colony-stimulating factor.

The crystal structure of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) has been determined at 2.8 A resolution using multiple isomorphous replacement techniques. There are two molecules in the crystallographic asymmetric unit, which are related by an approximate non-crystallographic 2-fold axis. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhGM-CSF is a four alpha-helix bundle, which represents approximately 42% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within the connections is a two-stranded antiparallel beta-sheet. The tertiary structure of rhGM-CSF has a topology similar to that of porcine growth factor and interferon-beta. Most of the proposed critical regions for receptor binding are located on a continuous surface at one end of the molecule that includes the C terminus.

Application of electrospray mass spectrometry in probing protein-protein and protein-ligand noncovalent interactions.

A novel mass spectrometry-based methodology using electrospray ionization (ESI) is described for the detection of protein-protein [interferon (IFN)-γ dimer] and protein-ligand [ras-guanosine diphosphate (GDP)] noncovalent interactions. The method utilizes ESI from aqueous solution at appropriate pH. The presence of the noncovalent complex of the IFN-γ dimer was confirmed by the observed average molecular weight of 33,819 Da. The key to the detection of the IFN-γ dimer is the use of an alkaline solution (pH ≈ 9) for sample preparation and for mass spectrornetry analysis. The effect of the declustering energy in the region of the ion sampling orifice and focusing quadrupole on the preservation of the gas-phase noncovalent complex (IFN-γ dimer) was also studied. The effect of the declustering energy on complex dissociation was further extended to probe the noncovalent protein-ligand association of ras-GDP. It was found that little energy is required to dissociate the IFN-γ dimer, whereas a substantial amount of energy is required to dissociate the gas-phase ras-GDP complex.

Cytokines: An Overview

ABSTRACT: Cytokines are a broad, heterogeneous group of proteins and polypeptides that regulate intercellular communication. Examples of cytokines include interleukins, interferons, colony‐stimulating factors, and a variety of growth factors. The preparation of large quantities of highly purified recombinant cytokines has provided a basis for their biological and physicochemical characterization. The pleiotropic biological effects of these factors are expressed through binding to specific, high‐affinity cell‐surface receptors. Although they are different in amino acid sequence, cytokines have a number of biological and physicochemical properties in common.

Structural analysis of biologically active peptides and recombinant proteins and their modified counterparts by mass spectrometry.

The structural characterization of the Escherichia coli-expressed human interferon alpha-2b (rh-IFN alpha-2b) was carried out by employing the fast atom bombardment (FAB) and plasma desorption (PD) mapping methods. The mass spectral data of the rh-IFN alpha-2b and the trypsin-generated peptide mixture allowed rapid and facile confirmation of the cDNA-derived sequence and determination of the existing disulfide pattern in the protein molecule. The same PD/FAB mapping approach was successfully employed in the structural determination of the iodination reaction product of rh-IFN alpha-2b and the potent vasoconstrictor peptide endothelin.

Crystallization and preliminary X-ray investigation of recombinant human interleukin 4☆

Crystals of recombinant human interleukin 4 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space-group P4(1)2(1)2 or P4(3)2(1)2; the unit cell axes are a = 92.2(1) A and c = 46.4(1) A. The crystals are stable to X-rays for at least three days and diffract beyond 2.8 A resolution. The crystals contain approximately 63% solvent, assuming there is one molecule in the asymmetric unit.

Comparative studies of natural and recombinant proteins by californium-252 plasma desorption and cesium ion liquid secondary ion mass spectrometry

Abstract The relative molecular masses of five proteins in the 10–25 kDa range were successfully determined by 252 Cf plasma desorption mass spectrometry (PDMS) and 35-keV Cs + liquid secondary ion mass spectrometry (LSIMS). Both techniques were comparable in demonstrating high mass accuracy and sensitivity, although LSIMS required a shorter analysis time than PDMS. The sensitivity of these methods was excellent, requiring only low-nanomole amounts of protein. The determination of relative molecular mass by the plasma desorption nitrocellulose method was less affected by the salt and buffer content of the sample, as these components could be effectively removed by washing.

Multiple forms of recombinant murine interleukin-4 expressed in COS-7 monkey kidney cells

Recombinant murine interleukin-4 (muIL-4) expressed in COS-7 monkey kidney cells was purified to homogeneity by sequential CM-Sepharose, Sephadex G-100 chromatography and mono-S FPLC to a specific activity of 6.10(7) units per mg of protein based on an in vitro HT-2 cell proliferation assay. Two electrophoretic variants, designated a and b, which migrated on SDS-PAGE as a closely spaced doublet with Mr 19,000, were present in the final product. Gas phase sequencing of the purified protein revealed the presence of an N-terminus corresponding to the mature protein predicted from the cDNA sequence and sequencing of a cyanogen bromide digest confirmed 75 of the 120 predicted amino acids. Elution behavior on gel filtration corresponded to that of a monomer of Mr 19,000. Since there are three potential sites of N-glycosylation predicted by the cDNA sequence, the contribution of glycosylation to the observed heterogeneity was examined by treatment with endoglycosidases. Variant b was digested by either endo-beta-N-acetylglucosaminidase H (endo H) or endo-beta-N-acetylglucosaminidase F (endo F) to protein of Mr 15,000 on SDS-PAGE but was unaffected by treatment with endo-beta-N-acetylglucosaminidase D (endo D), thus indicating the presence of high mannose type of N-glycan. In contrast, variant a was resistant to endo H, F and D. Complete conversion of a mixture of variants a and b to a single protein of Mr 15,000 on SDS-PAGE was obtained only after treatment with N-glycanase. Both variants were resistant to neuraminidase and O-glycanase treatment. These data show that the microheterogeneity observed in purified muIL-4 preparations is due to differences in the nature of the N-linked oligosaccharides. The availability of purified recombinant muIL-4 and a methodology for both total and selective deglycosylation provides a basis for the initiation of structure-function studies of this novel T-cell lymphokine.

Purification and properties of a novel recombinant human hybrid interferon, σ-4 α2/α1

The human interferon (huIFN) delta-4 alpha 2(5-62)/alpha 1(64-166) is a genetically engineered hybrid that consists of residues 5-62 of huIFN alpha 2 and residues 64-166 of huIFN alpha 1. This variant contains four cysteine residues at positions 29, 86, 99 and 139, but does not contain the cysteine at position 1 that is characteristic of naturally occurring huIFN alpha subtypes. This novel recombinant hybrid was purified from Escherichia coli to greater than 95% homogeneity. The purification was based on ethanol extraction of a trichloroacetic acid precipitate and Matrex Gel Blue A chromatography followed by either a selective precipitation or DEAE-Sepharose chromatography. The purified protein that was treated with 2-mercaptoethanol exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 17,800 and 17,100, both of which exhibited antiviral activity. Electrophoresis performed without prior reduction with 2-mercaptoethanol indicated only a minor extent of intermolecular disulfide bonding. The purified protein exhibited a high specific antiviral activity of 7 x 10(7) units/mg when assayed on human fibroblast cells and, in distinction to the parental huIFN alpha 2, it also demonstrated antiviral activity on human fibroblast cells and, in distinction to the parental huIFN alpha 2, it also demonstrated antiviral activity on murine L929 cells. The level of antiproliferative activity of huIFN delta-4 alpha 2(5-62)/alpha 1(64-166) on various cell lines of different histological origin appeared to be more comparable to that of huIFN alpha 1 than huIFN alpha 2. The data suggest that huIFN delta-4 alpha 2(5-62)/alpha 1(64-166) hybrid may be a useful tool for understanding huIFN structure-function relations.

Interferon: Current concepts of mechanisms of action

Almost 30 years ago Isaacs and Lindenmann [1] discovered that the supernatant obtained from cells incubated with heat-inactivated influenza virus contained a substance capable of preventing the growth of active virus. This substance, named interferon (IFN), was later shown to be composed of a system of structurally related proteins that act directly on the target cells, not on the virus, and are produced by many types of animal cells in response to various external stimuli (e.g., viruses, certain types of double-stranded RNA, antigens, or mitogens). Three classes of IFNs have been described that differ in their amino acid sequences, as well as immunochemical and physicochemical properties: alpha (leukocyte), beta (fibroblast), and gamma (immune) IFN. Alpha and beta IFNs have also been designated as type I (acid-stable) and gamma IFN as type II (acid-labile). The alpha IFNs, which are produced by a variety of different cells including macrophages, null cells (non-B- and non-T-lymphocytes) and transformed B-cell lines, are a family of at least 14 species that share a 75% or greater amino acid sequence homology. Beta IFN, which shares ca30% amino acid sequence homology with alpha IFNs, is produced mainly by fibroblasts and epithelial cells, but may also be produced by human tumor cells derived from leukocytes (e.g., the Namalwa cell line). Gamma IFN is predominantly a product of T-cells and natural killer (NK) cells and shares little homology with type I IFNs.