0000000000038370
AUTHOR
Mark S. Johnson
showing 18 related works from this author
"RKKH" peptides from the snake venom metalloproteinase of Bothrops jararaca bind near the metal ion-dependent adhesion site of the human integrin alp…
1999
Integrin alpha(1)beta(1) and alpha(2)beta(1) are the major cellular receptors for collagen, and collagens bind to these integrins at the inserted I-domain in their alpha subunit. We have previously shown that a cyclic peptide derived from the metalloproteinase domain of the snake venom protein jararhagin blocks the collagen-binding function of the alpha(2) I-domain. Here, we have optimized the structure of the peptide and identified the site where the peptide binds to the alpha(2) I-domain. The peptide sequence Arg-Lys-Lys-His is critical for recognition by the I-domain, and five negatively charged residues surrounding the "metal ion-dependent adhesion site" (MIDAS) of the I-domain, when mu…
Production, crystallization and preliminary X-ray analysis of the human integrin alpha1 I domain.
1999
Integrin α1β1 is one of the main collagen receptors in many cell types. A fast large-scale production, purification and crystallization method for the integrin α1 I domain is reported here. The α1 I domain was crystallized using the vapour-diffusion method with a reservoir solution containing a mixture of PEG 4000, sodium acetate, glycerol and Tris–HCl buffer. The crystals beong to the C2 space group, with unit-cell parameters a = 74.5, b = 81.9, c = 37.3 Å, α = γ = 90.0, β = 90.8°. The crystals diffract to 2.0 Å and a 94.2% complete data set to 2.2 Å has been collected from a single crystal with an R merge of 5.8%.
alpha 11beta 1 integrin recognizes the GFOGER sequence in interstitial collagens.
2002
The integrins alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1) are referred to as a collagen receptor subgroup of the integrin family. Recently, both alpha(1)beta(1) and alpha(2)beta(1) integrins have been shown to recognize triple-helical GFOGER (where single letter amino acid nomenclature is used, O = hydroxyproline) or GFOGER-like motifs found in collagens, despite their distinct binding specificity for various collagen subtypes. In the present study we have investigated the mechanism whereby the latest member in the integrin family, alpha(11)beta(1), recognizes collagens using C2C12 cells transfected with alpha(11) cDNA and the bacterially expressed recombinant a…
The Major Conformational IgE-binding Epitopes of Hevein (Hev b6.02) Are Identified by a Novel Chimera-based Allergen Epitope Mapping Strategy
2002
A novel approach to localize and reconstruct conformational IgE-binding epitope regions of hevein (Hev b6.02), a major natural rubber latex allergen, is described. An antimicrobial protein (AMP) from the amaranth Amaranthus caudatus was used as an immunologically non-IgE-binding adaptor molecule to which terminal or central parts of hevein were fused. Hevein and AMP share a structurally identical core region but have different N-terminal and C-terminal regions. Only 1 of 16 hevein-allergic patients showed weak IgE binding to purified native or recombinant AMP. Chimeric AMP with the hevein N terminus was recognized by IgE from 14 (88%) patients, and chimeric AMP with the hevein C terminus wa…
Integrin alpha(2)I domain recognizes type I and type IV collagens by different mechanisms.
2000
The collagens are recognized by the alphaI domains of the collagen receptor integrins. A common structural feature in the collagen-binding alphaI domains is the presence of an extra helix, named helix alphaC. However, its participation in collagen binding has not been shown. Here, we have deleted the helix alphaC in the alpha(2)I domain and tested the function of the resultant recombinant protein (DeltaalphaCalpha(2)I) by using a real-time biosensor. The DeltaalphaCalpha(2)I domain had reduced affinity for type I collagen (430 +/- 90 nM) when compared with wild-type alpha(2)I domain (90 +/- 30 nM), indicating both the importance of helix alphaC in type I collagen binding and that the collag…
Construction of hevein (Hev b 6.02) with reduced allergenicity for immunotherapy of latex allergy by comutation of six amino acid residues on the con…
2004
Abstract Recently we have established that IgE Abs bind to conformational epitopes in the N- and C-terminal regions of the major natural rubber latex allergen, hevein (Hev b 6.02). To identify the critical amino acid residues that interact with IgE, the hevein sequence was scanned by using site-specific mutations. Twenty-nine hevein mutants were designed and produced by a baculovirus expression system in insect cells and tested by IgE inhibition-ELISA using sera from 26 latex allergic patients. Six potential IgE-interacting residues of hevein (Arg5, Lys10, Glu29, Tyr30, His35, and Gln38) were identified and characterized further in detail. Based on these six residues, two triple mutants (HΔ…
Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli
2004
Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin–agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray cryst…
Controlling quaternary structure assembly: subunit interface engineering and crystal structure of dual chain avidin.
2006
Dual chain avidin (dcAvd) is an engineered avidin form, in which two circularly permuted chicken avidin monomers are fused into one polypeptide chain. DcAvd can theoretically form two different pseudotetrameric quaternary assemblies because of symmetry at the monomer-monomer interfaces. Here, our aim was to control the assembly of the quaternary structure of dcAvd. We introduced the mutation I117C into one of the circularly permuted domains of dcAvd and scanned residues along the 1-3 subunit interface of the other domain. Interestingly, V115H resulted in a single, disulfide locked quaternary assembly of dcAvd, whereas I117H could not guide the oligomerisation process even though it stabilis…
Functional display of an alpha2 integrin-specific motif (RKK) on the surface of baculovirus particles.
2005
The use of baculovirus vectors shows promise as a tool for gene delivery into mammalian cells. These insect viruses have been shown to transduce a variety of mammalian cell lines, and gene transfer has also been demonstrated in vivo. In this study, we generated two recombinant baculovirus vectors displaying an integrin-specific motif, RKK, as a part of two different loops of the green fluorescent protein (GFP) fused with the major envelope protein gp64 of Autographa californica M nucleopolyhedrovirus. By enzyme linked immunosorbent assays, these viruses were shown to bind a peptide representing the receptor binding site of an α2 integrin, the α2I-domain. However, the interaction was not st…
Chicken Avidin-related Protein 4/5 Shows Superior Thermal Stability when Compared with Avidin while Retaining High Affinity to Biotin
2003
The protein chicken avidin is a commonly used tool in various applications. The avidin gene belongs to a gene family that also includes seven other members known as the avidin-related genes (AVR). We report here on the extremely high thermal stability and functional characteristics of avidin-related protein AVR4/5, a member of the avidin protein family. The thermal stability characteristics of AVR4/5 were examined using a differential scanning calorimeter, microparticle analysis, and a microplate assay. Its biotin-binding properties were studied using an isothermal calorimeter and IAsys optical biosensor. According to these analyses, in the absence of biotin AVR4/5 is clearly more stable (T…
The collagen receptor subfamily of the integrins
2003
The four collagen receptor integrins, alpha1beta1, alpha2beta1, alpha10beta1 and alpha11beta1, form a structurally and functionally distinct subgroup when compared to other members of the integrin family. In this review, we discuss the structures of these receptors and their differences in collagen binding and signalling function.
Jararhagin-derived RKKH Peptides Induce Structural Changes in α1I Domain of Human Integrin α1β1
2003
Integrin alpha(1)beta(1) is one of four collagen-binding integrins in humans. Collagens bind to the alphaI domain and in the case of alpha(2)I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In alpha(2)I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound "open" conformation of alpha(2)I differs significantly from the "closed" conformation seen in the structure of apo-alpha(2)I near MIDAS. Here we show that two peptides,…
Structural and functional analysis of integrin alpha2I domain interaction with echovirus 1.
2004
Integrins are cell surface receptors for several microbial pathogens including echovirus 1 (EV1), a picornavirus. Cryo-electron microscopy revealed that the functional domain (alpha(2)I) of human alpha(2)beta(1) integrin binds to a surface depression on the EV1 capsid. This three-dimensional structure of EV1 bound to alpha(2)I domain provides the first structural details of an integrin interacting with a picornavirus. The model indicates that alpha(2)beta(1) integrin cannot simultaneously bind both EV1 and the physiological ligand collagen. Compared with collagen binding to the alpha(2)I domain, the virus binds with a 10-fold higher affinity but in vitro uncoating of EV1 was not observed as…
Binding Properties of HABA-Type Azo Derivatives to Avidin and Avidin-Related Protein 4
2006
Summary The chicken genome encodes several biotin-binding proteins, including avidin and avidin-related protein 4 (AVR4). In addition to D -biotin, avidin binds an azo dye compound, 4-hydroxyazobenzene-2-carboxylic acid (HABA), but the HABA-binding properties of AVR4 are not yet known. Differential scanning calorimetry, UV/visible spectroscopy, and molecular modeling were used to analyze the binding of 15 azo molecules to avidin and AVR4. Significant differences are seen in azo compound preferences for the two proteins, emphasizing the importance of the loop between strands β3 and β4 for azo ligand recognition; information on these loops is provided by the high-resolution (1.5 A) X-ray stru…
A peptide inhibiting the collagen binding function of integrin alpha2I domain.
1999
Integrin alpha2 subunit forms in the complex with the beta1 subunit a cell surface receptor binding extracellular matrix molecules, such as collagens and laminin-1. It is a receptor for echovirus-1, as well. Ligands are recognized by the special "inserted" domain (I domain) in the integrin alpha2 subunit. Venom from a pit viper, Bothrops jararaca, has been shown to inhibit the interaction of platelet alpha2beta1 integrin with collagen because of the action of a disintegrin/metalloproteinase named jararhagin. The finding that crude B. jararaca venom could prevent the binding of human recombinant ralpha2I domain to type I collagen led us to study jararhagin further. Synthetic peptides represe…
Molecular mechanism of α2β1 integrin interaction with human echovirus 1
2009
Conformational activation increases the affinity of integrins to their ligands. On ligand binding, further changes in integrin conformation elicit cellular signalling. Unlike any of the natural ligands of alpha2beta1 integrin, human echovirus 1 (EV1) seemed to bind more avidly a 'closed' than an activated 'open' form of the alpha2I domain. Furthermore, a mutation E336A in the alpha2 subunit, which inactivated alpha2beta1 as a collagen receptor, enhanced alpha2beta1 binding to EV1. Thus, EV1 seems to recognize an inactive integrin, and not even the virus binding could trigger the conformational activation of alpha2beta1. This was supported by the fact that the integrin clustering by EV1 did …
Selective Binding of Collagen Subtypes by Integrin α1I, α2I, and α10I Domains
2001
Four integrins, namely alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1), form a special subclass of cell adhesion receptors. They are all collagen receptors, and they recognize their ligands with an inserted domain (I domain) in their alpha subunit. We have produced the human integrin alpha(10)I domain as a recombinant protein to reveal its ligand binding specificity. In general, alpha(10)I did recognize collagen types I-VI and laminin-1 in a Mg(2+)-dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When alpha(10)I was tested together with the alpha(1)I and alpha(2)I domains, all three I domains seemed to have their own collag…
Structure and characterization of a novel chicken biotin-binding protein A (BBP-A)
2007
Background. The chicken genome contains a BBP-A gene showing similar characteristics to avidin family genes. In a previous study we reported that the BBP-A gene may encode a biotin-binding protein due to the high sequence similarity with chicken avidin, especially at regions encoding residues known to be located at the ligand-binding site of avidin. Results. Here, we expand the repertoire of known macromolecular biotin binders by reporting a novel biotin-binding protein A (BBP-A) from chicken. The BBP-A recombinant protein was expressed using two different expression systems and purified with affinity chromatography, biochemically characterized and two X-ray structures were solved – in comp…