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Ernesto Cota,
Celine Jones,
Peter Simpson, Harri Altroff,
Kirstine L Anderson,
Laurence du Merle,
Julie Guignot,
Alain Servin,
Chantal Le Bouguénec,
Helen Mardon,
Stephen Matthews
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ABSTRACT: Afa/Dr family of adhesins are produced by pathogenic Escherichia coli strains that are especially prevalent in chronic diarrhoeal and recurrent urinary tract infections. Most notably, they are found in up to 50% of cystitis cases in children and 30% of pyelonephritis in pregnant women. Afa/Dr adhesins are capped surface fibrils that mediate recognition of the host and subsequent bacterial internalization. Using the newly solved three-dimensional structure of the minimal invasive complex (AfaDE) combined with biochemical and cellular assays, we reveal the architecture of the fibrillar cap and identify a novel mode of synergistic integrin recognition.
Molecular Microbiology 11/2006; 62(2):356-66. · 5.01 Impact Factor
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ABSTRACT: Integrins are an important family of signaling receptors that mediate diverse cellular processes. The binding of the abundant extracellular matrix ligand fibronectin to integrins alpha(5)beta(1) and alpha(v)beta(3) is known to depend upon the Arg-Gly-Asp (RGD) motif on the tenth fibronectin FIII domain. The adjacent ninth FIII domain provides a synergistic effect on RGD-mediated integrin alpha(5)beta(1) binding and downstream function. The precise molecular basis of this synergy remains elusive. Here we have dissected further the function of FIII9 in integrin binding by analyzing the biological activity of the FIII9-10 interdomain interface variants and by determining their structural and dynamic properties in solution. We demonstrate that the contribution of FIII9 to both alpha(5)beta(1) and alpha(v)beta(3) binding and downstream function critically depends upon the interdomain tilt between the FIII9 and FIII10 domains. Our data suggest that modulation of integrin binding by FIII9 may arise in part from its steric properties that determine accessibility of the RGD motif. These findings have wider implications for mechanisms of integrin-ligand binding in the physiological context.
Journal of Biological Chemistry 01/2005; 279(53):55995-6003. · 4.77 Impact Factor
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ABSTRACT: Non-ionic surfactants have been employed as alternatives to PVA for the emulsification-encapsulation of a conformationally labile protein (FIII9'-10) into PLGA microspheres. FIII9'-10 was encapsulated using a w/o/w double emulsification-evaporation technique and the microspheres fabricated were characterized by SEM and CLSM. The peptide backbone integrity of FIII9'-10 was assayed by SDS-PAGE and the degree of unfolding of FIII9'-10 following emulsification-encapsulation was assessed using a fibroblast cell-attachment assay. The encapsulation efficiency for FIII9'-10 was 25% when using PVA, compared to 50-60% when using Igepal CA-630 or Triton-X100, with values below for the other surfactants. FIII9'-10 released from microspheres promoted cell attachment in a concentration-dependent manner, only Igepal CA-630 and Triton X-100 maintaining near-maximal cell attachment, indicating that the conformation of the relatively unstable FIII9' domain was preserved. All non-ionic surfactants reduced microsphere surface porosity, compared to PVA, and an increasing surface rugosity (leading to minor 'ridges') could be correlated with decreasing surfactant HLB. Low surface porosities did not effect the diffusion of FIII9'-10 from the microspheres' internal pores in a 'burst release', as may have been imagined. In summary, non-ionic surfactants should be considered over PVA for the maintenance of biological activity of conformationally labile proteins during encapsulation.
Journal of Controlled Release 04/2004; 95(3):557-66. · 5.73 Impact Factor
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ABSTRACT: The ninth and tenth FIII domains (FIII9-10) of human fibronectin act in synergy to promote cell adhesion via the interaction with integrin receptors. Here we describe the functional and structural properties of a set of recombinant FIII9-10 mutants containing various alanine substitutions within the key synergistic site, DRVPHSRN in FIII9, either alone or in combination with another substitution (Leu(1408) to Pro), on the opposite face of FIII9, that increases stability and the functional capacity of FIII9-10. We show that the introduction of mutations into the synergistic sequence of FIII9-10 has a negative effect on the adhesion of baby hamster kidney fibroblasts and results in reduced ability of these ligands to recognize integrin alpha(5)beta(1). Conformational stability of the FIII9 domain in the synergy site mutants is likewise reduced in comparison with native FIII9. The Leu(1408) to Pro substitution in mutant FIII9-10 proteins carrying substitutions in the synergy site results in a substantial recovery of the adhesive activity of the mutants and affinity to alpha(5)beta(1). In keeping with the enhancement of functional activity, the Leu(1408) to Pro substitution in the FIII9-10 synergy site mutants also causes a significant increase in conformational stability of FIII9. These observations imply a strong positive correlation between the biological activity and conformational stability of the assessed FIII9-10 mutants and suggest that a Leu(1408) to Pro substitution restores the biological activity of the mutants via their ability to restore their conformational stability. We conclude that domain stability may be a major determinant of the synergistic potential of FIII9. Our data underscore the value of using more than one approach in such structure-function studies and the requirement for validating the global structural integrity of protein ligands in which sequences that disrupt function have been perturbed.
Journal of Biological Chemistry 02/2003; 278(1):491-7. · 4.77 Impact Factor
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ABSTRACT: The ninth and tenth type III domains (FIII9-10) in the central cell binding domain of human fibronectin contain integrin receptor binding sites, including RGD in FIII10 and a synergy site, PHSRN, in FIII9. The specific amino acids that contribute to cell binding have been identified by the use of wild-type and mutant fragments of human fibronectin containing the FIII9-10 domain pair. At high concentrations FIII9-10 mimics, to a large extent, the biological activity of the full-length fibronectin molecule. However, FIII9 is conformationally unstable, even in the context of the FIII9-10 pair. Here we report the construction of a series of hybrid mouse-human FIII9-10 pairs that confer varying degrees of conformational stability to FIII9. The conformational stability of the human FIII9 module was increased 2-3-fold by substitution of Leu1408 with Pro. We demonstrate that the biological activity of this mutant is enhanced. The resulting FIII9-10 mutant has good solution properties and will provide a template into which further mutations can be incorporated in order to probe the structure-function relationship of the cell binding module of fibronectin.
Protein engineering 01/2003; 15(12):1021-4.
