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M Karpusas

Publications and source records attributed to M Karpusas.

At least 19 recordsLinked to original sources
PubMed2001

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.

Amino Acid Sequence↗
PubMed2001

Mapping of IFN-beta 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.

Alanine↗
PubMed2000

Systematic mutational mapping of sites on human interferon-beta-1a that are important for receptor binding and functional activity.

A systematic mutational analysis of human interferon-beta-1a (IFN-beta) was performed to identify regions on the surface of the molecule that are important for receptor binding and for functional activity. The crystal structure of IFN-beta-1a was used to design a panel of 15 mutant proteins, in each of which a contiguous group of 2-8 surface residues was mutated, in most instances to alanine. The mutants were analyzed for activity in vitro in antiviral and in antiproliferation assays, and for their ability to bind to the type I IFN (ifnar1/ifnar2) receptor on Daudi cells and to a soluble ifnar2 fusion protein (ifnar2-Fc). Abolition of binding to ifnar2-Fc for mutants A2, AB1, AB2, and E established that the ifnar2 binding site on IFN-beta comprises parts of the A helix, the AB loop, and the E helix. Mutations in these areas, which together define a contiguous patch of the IFN-beta surface, also resulted in reduced affinity for binding to the receptor on cells and in reductions in activity of 5-50-fold in functional assays. A second receptor interaction site, concluded to be the ifnar1 binding site, was identified on the opposite face of the molecule. Mutations in this region, which encompasses parts of the B, C, and D helices and the DE loop, resulted in disparate effects on receptor binding and on functional activity. Analysis of antiproliferation activity as a function of the level of receptor occupancy allowed mutational effects on receptor activation to be distinguished from effects on receptor binding. The results suggest that the binding energy from interaction of IFN-beta with ifnar2 serves mainly to stabilize the bound IFN/receptor complex, whereas the binding energy generated by interaction of certain regions of IFN-beta with ifnar1 is not fully expressed in the observed affinity of binding but instead serves to selectively stabilize activated states of the receptor.

Alanine↗
PubMed1999

CD154 variants associated with hyper-IgM syndrome can form oligomers and trigger CD40-mediated signals.

X-linked hyper-IgM syndrome is a rare immunodeficiency disorder resulting from mutations in the gene encoding the CD40 ligand (CD154) molecule. These mutations are very heterogeneous, ranging from a single point mutation to a large deletion in the open reading frame. To investigate the molecular mechanisms that are responsible for the functional defect of these mutants, we examined the biochemical properties of 14 hyper-IgM-related CD154 mutant proteins produced by transient expression in COS7 cells. We show that deletion mutants lacking a significant portion of the tumor necrosis factor homologous domain cannot be stably produced. In contrast, point mutants can be detected as oligomers. Surprisingly, gene products of two point mutants, Thr-211 --> Asp and Met-36 --> Arg, can bind to the receptor, CD40. For Thr-211 --> Asp variant, it is comparable to the wild-type protein in its surface expression level, biochemical structure, and functional activities. Thus, it appears that this mutation is a polymorphism of CD154 gene. For Met-36 --> Arg variant, although it is interactive with CD40, it has a much lower surface expression level than wild-type protein. We propose that Met-36 --> Arg mutant represents a prototype of a defective CD154 family whose low cell surface expression of intrinsically active protein is simply insufficient to trigger productive signals through CD40.

Animals↗
PubMed1999

Crystal structure of the alpha1beta1 integrin I-domain: insights into integrin I-domain function.

The alpha1beta1 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 alpha 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 alpha1beta1 integrin at 2.2 A resolution in the absence of divalent cations. The alpha1 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 alpha2beta1 I-domain which also mediates collagen binding. A unique feature of the alpha1 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.

Animals↗
PubMed1999

Induced fit activation mechanism of the exceptionally specific serine protease, complement factor D.

We have investigated the mechanism by which the complement protease, Factor D, achieves its high specificity for the cleavage of Factor B in complex with C3(H2O). Kinetic experiments showed that Factor B and C3(H2O) associate with a KD of >/=2.5 microM and that Factor D acts on this complex with a second-order rate constant of kcat/KM >/= 2 x 10(6) M-1 s-1, close to the rate of a diffusion-controlled reaction for proteins of this size. In contrast, Factor D, which is a member of the trypsin family of serine proteases, was 10(3)-10(4)-fold less active than trypsin toward both thioester and p-nitroanilide substrates containing an arginine at P1. Furthermore, peptides spanning the Factor B cleavage site were not detectably cleaved by Factor D (kcat/KM </= 0.5 M-1 s-1). These results imply that contacts between Factor D and the C3(H2O)B complex, outside the vicinity of the cleavage site in Factor B, generate >/=9 kcal/mol of binding energy to stabilize the transition state for reaction. In support of this, we demonstrate that chemical modification of Factor D at a single lysine residue that is distant from the active site abolishes the activity of the enzyme toward Factor B while not affecting activity toward small synthetic substrates. We propose that Factor D may exemplify a special case of the induced fit mechanism in which the requirement for conformational activation of the enzyme results in a substantial increase in substrate specificity.

Acetylation↗
PubMed1999

An aggressive form of polyarticular arthritis in a man with CD154 mutation (X-linked hyper-IgM syndrome).

Hyper-IgM syndrome (HIM) is a rare immunodeficiency disorder that has been associated with the development of symptoms and clinical features characteristic of rheumatoid arthritis (RA). We describe a patient with HIM and severe erosive arthritis with prominent nodules in the absence of detectable serum rheumatoid factor. Because HIM results from defects in either T cell CD154 (CD40 ligand) expression or abnormal CD40 signaling, the molecular basis of the patient's disease was analyzed. Activated CD4+ T cells failed to express surface CD154 protein, and molecular analysis of CD154 complementary DNA revealed a nucleotide transversion resulting in the nonconservative amino acid substitution G-D at amino acid 257. This case indicates that defective CD154-dependent CD40 signaling can be associated with susceptibility to a severe inflammatory arthritis that has both similarities to and differences from idiopathic RA.

Adult↗
PubMed1998

The role of polar interactions in the molecular recognition of CD40L with its receptor CD40.

CD40 Ligand (CD40L) is transiently expressed on the surface of T-cells and binds to CD40, which is expressed on the surface of B-cells. This binding event leads to the differentiation, proliferation, and isotype switching of the B-cells. The physiological importance of CD40L has been demonstrated by the fact that expression of defective CD40L protein causes an immunodeficiency state characterized by high IgM and low IgG serum levels, indicating faulty T-cell dependent B-cell activation. To understand the structural basis for CD40L/CD40 association, we have used a combination of molecular modeling, mutagenesis, and X-ray crystallography. The structure of the extracellular region of CD40L was determined by protein crystallography, while the CD40 receptor was built using homology modeling based upon a novel alignment of the TNF receptor superfamily, and using the X-ray structure of the TNF receptor as a template. The model shows that the interface of the complex is composed of charged residues, with CD40L presenting basic side chains (K143, R203, R207), and CD40 presenting acidic side chains (D84, E114, E117). These residues were studied experimentally through site-directed mutagenesis, and also theoretically using electrostatic calculations with the program Delphi. The mutagenesis data explored the role of the charged residues in both CD40L and CD40 by switching to Ala (K143A, R203A, R207A of CD40L, and E74A, D84A, E114A, E117A of CD40), charge reversal (K143E, R203E, R207E of CD40L, and D84R, E114R, E117R of CD40), mutation to a polar residue (K143N, R207N, R207Q of CD40L, and D84N, E117N of CD40), and for the basic side chains in CD40L, isosteric substitution to a hydrophobic side chain (R203M, R207M). All the charge-reversal mutants and the majority of the Met and Ala substitutions led to loss of binding, suggesting that charged interactions stabilize the complex. This was supported by the Delphi calculations which confirmed that the CD40/CD40L residue pairs E74-R203, D84-R207, and E117-R207 had a net stabilizing effect on the complex. However, the substitution of hydrophilic side chains at several of the positions was tolerated, which suggests that although charged interactions stabilize the complex, charge per se is not crucial at all positions. Finally, we compared the electrostatic surface of TNF/TNFR with CD40L/CD40 and have identified a set of polar interactions surrounded by a wall of hydrophobic residues that appear to be similar but inverted between the two complexes.

Amino Acid Sequence↗
PubMed1998

The structure of human interferon-beta: implications for activity.

Interferons (IFNs) are potent extracellular protein mediators of host defence and homoeostasis. This article reviews the structure of human IFN-beta (HuIFN-beta), in particular in relation to its activity. The recently determined crystal structure of HuIFN-beta 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-beta, 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-beta.

Crystallography, X-Ray↗
PubMed1998

Structural and functional differences between glycosylated and non-glycosylated forms of human interferon-beta (IFN-beta).

PURPOSE: Two recombinant IFN-beta products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN-beta-1a) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN-beta-1b). The structural basis for activity differences between IFN-beta-1a and IFN-beta-1b, is determined. METHODS: In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN-beta products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences. RESULTS: IFN-beta-1a was 10 times more active than IFN-beta-1b with specific activities in a standard antiviral assay of 20 x 10(7) IU/mg for IFN-beta-1a and 2 x 10(7) IU/mg for IFN-beta-1b. Of the known structural differences between IFN-beta-1a and IFN-beta-1b, only glycosylation affected in vitro activity. Deglycosylation of IFN-beta-1a 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-beta-1b revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 x 10(7) IU/mg). Crystallographic data for IFN-beta-1a 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-beta-1a is due to a stabilizing effect of the carbohydrate on structure.

Adjuvants, Immunologic↗
PubMed1997

The crystal structure of human interferon beta at 2.2-A resolution.

Type I interferons (IFNs) are helical cytokines that have diverse biological activities despite the fact that they appear to interact with the same receptor system. To achieve a better understanding of the structural basis for the different activities of alpha and beta IFNs, we have determined the crystal structure of glycosylated human IFN-beta at 2.2-A resolution by molecular replacement. The molecule adopts a fold similar to that of the previously determined structures of murine IFN-beta and human IFN-alpha2b but displays several distinct structural features. Like human IFN-alpha2b, human IFN-beta contains a zinc-binding site at the interface of the two molecules in the asymmetric unit, raising the question of functional relevance for IFN-beta dimers. However, unlike the human IFN-alpha2b dimer, in which homologous surfaces form the interface, human IFN-beta dimerizes with contact surfaces from opposite sides of the molecule. The relevance of the structure to the effects of point mutations in IFN-beta at specific exposed residues is discussed. A potential role of ligand-ligand interactions in the conformational assembly of IFN receptor components is discussed.

Computer Simulation↗
PubMed1996

Structure of a functional fragment of VCAM-1 refined at 1.9 a resolution.

The crystal structure of the functional amino-terminal two-domain fragment of human vascular cell adhesion molecule 1 (VCAM-1) has been determined at 1.9 A resolution. The crystals contain two copies of the molecule in the asymmetric unit. The structure was solved by multiple isomorphous replacement, using lead and selenium derivatives. Anomalous scattering had to be used to resolve the phase ambiguity of a lead derivative. Since the selenium derivative has very small isomorphous differences, the local scaling algorithm had to be used to obtain an interpretable difference Patterson map. The initial phases were improved by non-crystallographic averaging, solvent flattening and histogram matching. The structure has been refined to a crystallographic R factor of 20.4% (15-1.9 A, F>/= 3sigma) and consists of two Ig domains (D1 and D2). The angle between these domains differs by 12 degrees between the two copies of the molecule in the crystallographic asymmetric unit, demonstrating that some movement is possible at the interface. In the amino-terminal domain D1 there is an 'extra' disulfide bond, in addition to the conserved cross-sheet disulfide bond, at the top of the molecule. This bond, a hallmark of the integrin-binding subclass of Ig superfamily proteins, makes the top of this domain very compact. The feature that projects most prominently from D1 is the CD loop, near the base of the domain. The key residue for integrin binding, Asp40, is located in this loop and is easily accessible.

Journal Article↗
PubMed1996

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 antibody-dependent 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.

B-Lymphocytes↗
PubMed1995

2 A 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.

Amino Acid Sequence↗
PubMed1995

The crystal structure of an N-terminal two-domain fragment of vascular cell adhesion molecule 1 (VCAM-1): a cyclic peptide based on the domain 1 C-D loop can inhibit VCAM-1-alpha 4 integrin interaction.

Vascular cell adhesion molecule 1 (VCAM-1) represents a structurally and functionally distinct class of immunoglobulin superfamily molecules that bind leukocyte integrins and are involved in inflammatory and immune functions. X-ray crystallography defines the three-dimensional structure of the N-terminal two-domain fragment that participates in ligand binding. Residues in domain 1 important for ligand binding reside in the C-D loop, which projects markedly from one face of the molecule near the contact between domains 1 and 2. A cyclic peptide that mimics this loop inhibits binding of alpha 4 beta 1 integrin-bearing cells to VCAM-1. These data demonstrate how crystallographic structural information can be used to design a small molecule inhibitor of biological function.

Amino Acid Sequence↗
PubMed1995

Amino acid residues required for binding of lymphocyte function-associated antigen 3 (CD58) to its counter-receptor CD2.

Efficient activation and regulation of the cellular immune response requires engagement of T cell accessory molecules as well as the antigen-specific T cell receptor. The lymphocyte function-associated antigen (LFA) 3 (CD58)/CD2 accessory pathway, one of the first discovered, has been extensively characterized in terms of structure and function of the CD2 molecule, which is present on all T lymphocytes and natural killer cells of the human immune system. The binding site of human CD2 for LFA-3 has been localized to two epitopes on one face of the first immunoglobulin (Ig)-like domain of this two-domain, Ig superfamily molecule. Human LFA-3 is genetically linked and is 21% identical in amino acid sequence to CD2, suggesting that this adhesive pair may have evolved from a single ancestral molecule. We have aligned the amino acid sequences of LFA-3 and CD2 and mutagenized selected amino acids in the first domain of LFA-3 that are analogous to those implicated in the binding site of CD2. The data show that K30 and K34, in the predicted C-C' loop, and D84, in the predicted F-G loop of LFA-3, are involved in binding to CD2, suggesting that two complementary sites on one face of the first domain of each molecule bind to each other.

Amino Acid Sequence↗
PubMed1994

Crystallization and preliminary crystallographic analysis of the N-terminal two domain fragment of vascular cell adhesion molecule-1 (VCAM-1).

A molecular fragment comprising the first two domains of the human vascular cell adhesion molecule-1 (VCAM-1) has been crystallized by the vapor diffusion method. Two crystal forms have been examined by X-ray analysis: One crystal form belongs to the space group C2 with two molecules in the asymmetric unit and cell parameters: a = 122.1 A, b = 48.9 A, c = 73.4 A, and beta = 117.4 degrees. The other crystal form belongs to the space group P2(1) with one molecule in the asymmetric unit and cell parameters: a = 40.4 A, b = 45.7 A, c = 54.7 A, and beta = 100.5 degrees. Diffraction data up to 1.9 A resolution have been collected for the C2 crystal form.

Amino Acid Sequence↗