Michael Karpusas

Structural and Functional Differences Between Glycosylated and Non-glycosylated Forms of Human Interferon-β (IFN-β)

AbstractPurpose. Two recombinant IFN-β products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN-β-la) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN-β-lb). The structural basis for activity differences between IFN-β-la and IFN-β-lb, is determined. Methods. In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN-β products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences. Results. IFN-β- la was 10 times more active than IFN-β- Ib with specific activities in a standard antiviral assay of 20 × 107 lU/mg for IFN-β-la and 2 × 107 lU/mg for IFN-β-lb. Of the known structural differences between IFN-β-la and IFN-β-lb, only glycosylation affected in vitro activity. Deglycosylation of IFN-β-la produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate. Deglycosylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN-β-lb revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 × 107 lU/mg). Crystallographic data for IFN-β-la revealed that the glycan formed H-bonds with the peptide backbone and shielded an uncharged surface from solvent exposure. Conclusions. Together these results suggest that the greater biological activity of IFN-β-la is due to a stabilizing effect of the carbohydrate on structure.

2 å crystal structure of an extracellular fragment of human CD40 ligand

BACKGROUND The CD40 ligand (CD40L) is a member of the tumor necrosis factor (TNF) family of proteins and is transiently expressed on the surface of activated T cells. The binding of CD40L to CD40, which is expressed on the surface of B cells, provides a critical and unique pathway of cellular activation resulting in antibody isotype switching, regulation of apoptosis, and B cell proliferation and differentiation. Naturally occurring mutations of CD40L result in the clinical hyper-IgM syndrome, characterized by an inability to produce immunoglobulins of the IgG, IgA and IgE isotypes. RESULTS We have determined the crystal structure of a soluble extracellular fragment of human CD40L to 2 A resolution and with an R factor of 21.8%. Although the molecule forms a trimer similar to that found for other members of the TNF family, such as TNF alpha and lymphotoxin-alpha, and exhibits a similar overall fold, there are considerable differences in several loops including those predicted to be involved in CD40 binding. CONCLUSIONS The structure suggests that most of the hyper-IgM syndrome mutations affect the folding and stability of the molecule rather than the CD40-binding site directly. Despite the fact that the hyper-IgM syndrome mutations are dispersed in the primary sequence, a large fraction of them are clustered in space in the vicinity of a surface loop, close to the predicted CD40-binding site.

THE STRUCTURE OF HUMAN INTERFERON-BETA : IMPLICATIONS FOR ACTIVITY

Abstract. Interferons (IFNs) are potent extracellular protein mediators of host defence and homoeostasis. This article reviews the structure of human IFN-β (HuIFN-β), in particular in relation to its activity. The recently determined crystal structure of HuIFN-β provides a framework for understanding of the mechanism of differentiation of type I IFNs by their common receptor. Insights are generated by comparison with the structures of other type I IFNs and from the interpretation of existing mutagenesis data. The details of the observed carbohydrate structure, together with biochemical data, implicate the glycosylation of HuIFN-β, which is uncommon among type I IFNs, as an important factor in the solubility, stability and, consequently, activity of the protein. Finally, these structural implications are discussed in the context of the clinical use of HuIFN-β.

Crystal structure of the α1β1 integrin I-domain: insights into integrin I-domain function

The α1β1 integrin is a major cell surface receptor for collagen. Ligand binding is mediated, in part, through a ∼200 amino acid inserted ‘I’‐domain contained in the extracellular part of the integrin α chain. Integrin I‐domains contain a divalent cation binding (MIDAS) site and require cations to interact with integrin ligands. We have determined the crystal structure of recombinant I‐domain from the rat α1β1 integrin at 2.2 Å resolution in the absence of divalent cations. The α1 I‐domain adopts the dinucleotide binding fold that is characteristic of all I‐domain structures that have been solved to date and has a structure very similar to that of the closely related α2β1 I‐domain which also mediates collagen binding. A unique feature of the α1 I‐domain crystal structure is that the MIDAS site is occupied by an arginine side chain from another I‐domain molecule in the crystal, in place of a metal ion. This interaction supports a proposed model for ligand‐induced displacement of metal ions. Circular dichroism spectra determined in the presence of Ca2+, Mg2+ and Mn2+ indicate that no changes in the structure of the I‐domain occur upon metal ion binding in solution. Metal ion binding induces small changes in UV absorption spectra, indicating a change in the polarity of the MIDAS site environment.

Structure of CD40 ligand in complex with the Fab fragment of a neutralizing humanized antibody.

BACKGROUND CD40 ligand (CD40L or CD154), a member of the tumor necrosis factor (TNF) family, plays a critical role in both humoral and cellular immune responses and has been implicated in biological pathways involving epithelial cells, fibroblasts, and platelets. Such a pathway is T cell-mediated B cell activation, a process that occurs through the interaction of CD40L with CD40 receptor expressed on B cells. It results in various B cell responses, including immunoglobulin isotype switching and B cell differentiation and proliferation. These responses can be inhibited by the monoclonal antibody 5c8, which binds with high affinity to CD40L. RESULTS To understand the structural basis of the inhibition, we determined the crystal structure of the complex of the extracellular domain of CD40L and the Fab fragment of humanized 5c8 antibody. The structure shows that the complex has the shape of a three-bladed propeller with three Fab fragments bound symmetrically to a CD40L homotrimer. To further study the nature of the antibody-antigen interface, we assessed the ability of 23 site-directed mutants of CD40L to bind to 5c8 and CD40 and analyzed the results in the context of the crystal structure. Finally, we observed via confocal microscopy that 5c8 binding to CD40L on the cell surface results in the formation of patches of clustered complexes. CONCLUSIONS The structure reveals that 5c8 neutralizes CD40L function by sterically blocking CD40 binding. The antigenic epitope is localized in a region of the surface that is likely to be structurally perturbed as a result of genetic mutations that cause hyper-IgM syndrome. The symmetric trimeric arrangement of the Fab fragments in the complex results in a geometry that facilitates the formation of large clusters of complexes on the cell surface.

Analysis of imatinib and sorafenib binding to p38alpha compared with c-Abl and b-Raf provides structural insights for understanding the selectivity of inhibitors targeting the DFG-out form of protein kinases.

Protein kinases c-Abl, b-Raf, and p38alpha are recognized as important targets for therapeutic intervention. c-Abl and b-Raf are major targets of marketed oncology drugs Imatinib (Gleevec) and Sorafenib (Nexavar), respectively, and BIRB-796 is a p38alpha inhibitor that reached Phase II clinical trials. A shared feature of these drugs is the fact that they bind to the DFG-out forms of their kinase targets. Although the discovery of this class of kinase inhibitors has increased the level of emphasis on the design of DFG-out inhibitors, the structural determinants for their binding and stabilization of the DFG-out conformation remain unclear. To improve our understanding of these determinants, we determined cocrystal structures of Imatinib and Sorafenib with p38alpha. We also conducted a detailed analysis of Imatinib and Sorafenib binding to p38alpha in comparison with BIRB-796, including binding kinetics, binding interactions, the solvent accessible surface area (SASA) of the ligands, and stabilization of key structural elements of the protein upon ligand binding. Our results yield an improved understanding of the structural requirements for stabilizing the DFG-out form and a rationale for understanding the genesis of ligand selectivity among DFG-out inhibitors of protein kinases.

Crystal Structure of the α1β1 Integrin I Domain in Complex with an Antibody Fab Fragment

The α1β1 (VLA-1) integrin is a cell-surface receptor for collagen and laminin and has been implicated in biological pathways involved in several pathological processes. These processes may be inhibited by the monoclonal antibody AQC2, which binds with high affinity to human α1β1 integrin. To understand the structural basis of the inhibition we determined the crystal structure of the complex of a chimeric rat/human I domain of the α1β1 integrin and the Fab fragment of humanized AQC2 antibody. The structure of the complex shows that the antibody blocks the collagen binding site of the I domain. An aspartate residue, from the CDR3 loop of the antibody heavy chain, coordinates the MIDAS metal ion in a manner similar to that of a glutamate residue from collagen. Substitution of the aspartate residue by alanine or arginine results in significant reduction of antibody binding affinity. Interestingly, although the mode of metal ion coordination resembles that of the open conformation, the I domain maintains an overall closed conformation previously observed only for unliganded I domains.

Mapping of IFN-β Epitopes Important for Receptor Binding and Biologic Activation: Comparison of Results Achieved Using Antibody-Based Methods and Alanine Substitution Mutagenesis

The epitopes important for receptor binding and activation of human interferon-beta1a (IFN-beta1a) were mapped with monoclonal antibodies (mAb), grouped on the basis of their specificity and ability to neutralize biologic activity, and alanine scanning mutagenesis (ASM). The binding properties of nine mAb were defined, using ASM-IFN-beta mutants having alanine substituted at targeted, surface-exposed residues. The results were correlated with the mAb neutralizing potency. Of six mAb that bound either at or adjacent to the IFNAR-2 receptor chain binding site defined by the ASM epitopes, only three had measurable neutralizing activity. Two of these inhibited IFN-beta/IFNAR-2 complex formation, suggesting that steric hindrance of receptor binding constitutes their mechanism of neutralization. However, two mAb that bound to sites remote from the IFNAR-2 binding site on IFN-beta also inhibited IFN-beta/IFNAR-2 complex formation and demonstrated potent neutralizing activity. Thus, neutralizing mAb may employ mechanisms other than steric blockade to inhibit directly the binding of receptor by cytokine, limiting their usefulness as tools to define precise receptor-ligand interaction sites.

The central role of the CD40-ligand and CD40 pathway in T-lymphocyte-mediated differentiation of B lymphocytes.

This review summarizes recent findings concerning the role of CD40‐ligand and CD40 interactions in B‐cell differentiation. CD40‐ligand on helper CD4+ T lymphocytes interacts with CD40 on B cells and directs the selection and differentiation of clones of B lymphocytes to generate specific antibodydependent immune responses. CD40‐ligand is necessary for normal B‐cell differentiation and plays several distinctive roles in this multistage process. The CD40 signaling pathway that normally regulates B‐cell death appears to be usurped by the Epstein‐Barr virus to mediate B‐cell transformation.

Prednisolone exerts late mitogenic and biphasic effects on resistant acute lymphoblastic leukemia cells: Relation to early gene expression

Resistance or sensitivity to glucocorticoids is considered to be of crucial importance for disease prognosis in childhood acute lymphoblastic leukemia. Prednisolone exerted a delayed biphasic effect on the resistant CCRF-CEM leukemic cell line, necrotic at low doses and apoptotic at higher doses. At low doses, prednisolone exerted a pre-dominant mitogenic effect despite its induction on total cell death, while at higher doses, prednisolones mitogenic and cell death effects were counterbalanced. Early gene microarray analysis revealed notable differences in 40 genes. The mitogenic/biphasic effects of prednisolone are of clinical importance in the case of resistant leukemic cells. This approach might lead to the identification of gene candidates for future molecular drug targets in combination therapy with glucocorticoids, along with early markers for glucocorticoid resistance.