title: Crystal structure of a defective folding protein.
abstract: Maltose-binding protein (MBP or MalE) of Escherichia coli is the periplasmic receptor of the maltose transport system. MalE31, a defective folding mutant of MalE carrying sequence changes Gly 32-->Asp and Ile 33-->Pro, is either degraded or forms inclusion bodies following its export to the periplasmic compartment. We have shown previously that overexpression of FkpA, a heat-shock periplasmic peptidyl-prolyl isomerase with chaperone activity, suppresses MalE31 misfolding. Here, we have exploited this property to characterize the maltose transport activity of MalE31 in whole cells. MalE31 displays defective transport behavior, even though it retains maltose-binding activity comparable with that of the wild-type protein. Because the mutated residues are in a region on the surface of MalE not identified previously as important for maltose transport, we have solved the crystal structure of MalE31 in the maltose-bound state in order to characterize the effects of these changes. The structure was determined by molecular replacement methods and refined to 1.85 A resolution. The conformation of MalE31 closely resembles that of wild-type MalE, with very small displacements of the mutated residues located in the loop connecting the first alpha-helix to the first beta-strand. The structural and functional characterization provides experimental evidence that MalE31 can attain a wild-type folded conformation, and suggest that the mutated sites are probably involved in the interactions with the membrane components of the maltose transport system.
authors.affiliation: Unité d'Immunologie Structurale, Unité de Repliement et Modélisation des Protéines, Institut Pasteur, CNRS URA 2185, 75724 Paris Cedex 15, France.
authors.forename: Frederick A
authors.initials: FA
authors.lastname: Saul
authors.forename: Michaël
authors.initials: M
authors.lastname: Mourez
authors.forename: Brigitte
authors.initials: B
authors.lastname: Vulliez-Le Normand
authors.forename: Nathalie
authors.initials: N
authors.lastname: Sassoon
authors.forename: Graham A
authors.initials: GA
authors.lastname: Bentley
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Betton
mesh_terms: D001692:Biological Transport; D002451:Cell Compartmentation; D002478:Cells, Cultured; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D002479:Inclusion Bodies; D008320:Maltose / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019897:Periplasm / Q000737:chemistry*; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011510:Proteolipids / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D033902:Periplasmic Binding Proteins; D011510:Proteolipids; C020485:proteoliposomes; D008320:Maltose