Mark Stuart Johnson

Selective Binding of Collagen Subtypes by Integrin α1I, α2I, and α10I Domains

Four integrins, namely α1β1, α2β1, α10β1, and α11β1, 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 α subunit. We have produced the human integrin α10I domain as a recombinant protein to reveal its ligand binding specificity. In general, α10I did recognize collagen types I–VI and laminin-1 in a Mg2+-dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When α10I was tested together with the α1I and α2I domains, all three I domains seemed to have their own collagen binding preferences. The integrin α2I domain bound much better to fibrillar collagens (I–III) than to basement membrane type IV collagen or to beaded filament-forming type VI collagen. Integrin α1I had the opposite binding pattern. The integrin α10I domain was similar to the α1I domain in that it bound very well to collagen types IV and VI. Based on the previously published atomic structures of the α1I and α2I domains, we modeled the structure of the α10I domain. The comparison of the three I domains revealed similarities and differences that could potentially explain their functional differences. Mutations were introduced into the αI domains, and their binding to types I, IV, and VI collagen was tested. In the α2I domain, Asp-219 is one of the amino acids previously suggested to interact directly with type I collagen. The corresponding amino acid in both the α1I and α10I domains is oppositely charged (Arg-218). The mutation D219R in the α2I domain changed the ligand binding pattern to resemble that of the α1I and α10I domains and, vice versa, the R218D mutation in the α1I and α10I domains created an α2I domain-like ligand binding pattern. Thus, all three collagen receptors appear to differ in their ability to recognize distinct collagen subtypes. The relatively small structural differences on their collagen binding surfaces may explain the functional specifics.

A Peptide Inhibiting the Collagen Binding Function of Integrin α2I Domain

Integrin α2 subunit forms in the complex with the β1 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 α2 subunit. Venom from a pit viper,Bothrops jararaca, has been shown to inhibit the interaction of platelet α2β1 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 rα2I domain to type I collagen led us to study jararhagin further. Synthetic peptides representing hydrophilic and charged sequences of jararhagin, including the RSECD sequence replacing the well known RGD motif in the disintegrin-like domain, were synthesized. Although the disintegrin-like domain derived peptides failed to inhibit rα2I domain binding to collagen, a basic peptide from the metalloproteinase domain proved to be functional. In an in vitro assay, the cyclic peptide, CTRKKHDNAQC, was shown to bind strongly to human recombinant α2I domain and to prevent its binding to type I and IV collagens and to laminin-1. Mutational analysis indicated that a sequence of three amino acids, arginine-lysine-lysine (RKK), is essential for rα2I domain binding, whereas the mutation of the other amino acids in the peptide had little if any effect on its binding function. Importantly, the peptide was functional only in the cyclic conformation and its affinity was strictly dependent on the size of the cysteine-constrained loop. Furthermore, the peptide could not bind to α2I domain in the absence of Mg2+, suggesting that the conformation of the I domain was critical, as well. Cells could attach to the peptide only if they expressed α2β1integrin, and the attachment was inhibited by anti-integrin antibodies.

AMPA receptors and bacterial periplasmic amino acid-binding proteins share the ionic mechanism of ligand recognition

In order to identify key structural determinants for ligand recognition, we subjected the ligand‐binding domain of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA)‐selective glutamate receptor GluR‐D subunit to site‐directed mutagenesis. Based on the analysis of the [3H]AMPA‐binding properties of the mutated binding sites, we constructed a revised three‐dimensional model of the ligand‐binding site, different in many respects from previously published models. In particular, our results indicate that the residues Arg507 and Glu727 represent the structural and functional correlates of Arg77 and Asp161 in the homologous bacterial lysine/ornithine/arginine‐binding protein and histidine‐binding protein, and directly interact with the α‐carboxyl and α‐amino group of the bound ligand, respectively. In contrast, Glu424, implicated previously in ionic interactions with the α‐amino group of the agonist, is unlikely to have such a role in ligand binding. Our results indicate that glutamate receptors share with the bacterial polar amino acid‐binding proteins the fundamental mechanism of amino acid recognition.

Chicken Avidin-related Protein 4/5 Shows Superior Thermal Stability when Compared with Avidin while Retaining High Affinity to Biotin

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 (Tm = 107.4 ± 0.3 °C) than avidin (Tm = 83.5 ± 0.1 °C) or bacterial streptavidin (Tm = 75.5 °C). AVR4/5 also exhibits a high affinity for biotin (Kd ≈ 3.6 × 10-14 m) comparable to that of avidin and streptavidin (Kd ≈ 10-15 m). Molecular modeling and site-directed mutagenesis were used to study the molecular details behind the observed high thermostability. The results indicate that AVR4/5 and its mutants have high potential as new improved tools for applications where exceptionally high stability and tight biotin binding are needed.

Jararhagin-derived RKKH Peptides Induce Structural Changes in α1I Domain of Human Integrin α1β1

Integrin α1β1 is one of four collagen-binding integrins in humans. Collagens bind to the αI domain and in the case of α2I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In α2I, 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 α2I differs significantly from the “closed” conformation seen in the structure of apo-α2I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to α1I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of α1I with Mg2+ and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-α1I (“closed” conformation) and a model structure of the α1I (“open” conformation) based on the closely related structure of α2I reveals that the binding site is partially blocked to ligands by Glu255 and Tyr285 in the “closed” structure, whereas in the “open” structure helix C is unwound and these residues are shifted, and the “RKKH” peptides fit well when docked. The “open” conformation of α2I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of α1I and shown experimentally for α1I using a fluorescent hydrophobic probe.

“RKKH” Peptides from the Snake Venom Metalloproteinase ofBothrops jararaca Bind Near the Metal Ion-dependent Adhesion Site of the Human Integrin α2 I-domain

Integrin α1β1and α2β1 are the major cellular receptors for collagen, and collagens bind to these integrins at the inserted I-domain in their α 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 α2 I-domain. Here, we have optimized the structure of the peptide and identified the site where the peptide binds to the α2 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 mutated, show significantly impaired binding of the peptide. Removal of helix αC, located along one side of the MIDAS and suggested to be involved in collagen-binding in these I-domains, does not affect peptide binding. This study supports the notion that the metalloproteinase initially binds to the α2 I-domain at a location distant from the active site of the protease, thus blocking collagen binding to the adhesion molecule in the vicinity of the MIDAS, while at the same time leaving the active site free to degrade nearby proteins, the closest being the β1 subunit of the α2β1cell-surface integrin itself.