title: Structural and dynamic changes in human annexin VI induced by a phosphorylation-mimicking mutation, T356D.
abstract: Phosphorylation of some members of the annexin family of proteins may play a significant role in controlling their calcium-dependent interactions with membranes. Recent electron microscopic studies of annexin VI revealed that the protein's two core domains exhibit a great degree of flexibility and are able to undergo a relative conformational change that could potentially initiate contacts between membranes [Avila-Sakar, A. J., et al. (2000) J. Struct. Biol. 130, 54-62]. To assess the possibility of a regulatory role of phosphorylation in this behavior, the crystal structure of a phosphorylation-mimicking mutant (T356D in the flexible connector region of human annexin VI) was determined to 2.65 A resolution. When the mutant is compared to the wild-type annexin VI, subtle differences are seen at the site of the mutation, while larger changes are evident in one of the calcium-binding loops and in the presence of five calcium ions. Furthermore, biochemical studies provide evidence for additional conformational differences between the T356D and wild-type solution structures. Fluorescence emission and acrylamide quenching suggest a higher level of solvent exposure of Trp-343 in the connector region of T356D in the presence of calcium. Comparisons of retardation coefficients in native gel electrophoresis reveal that T356D has a more extended shape, while proteolytic studies show a greater accessibility of a trypsin cleavage site inside the linker region, indicating a conformation more open than the wild-type form. These data provide insights into a possible regulatory mechanism leading to a higher degree of flexibility and possibly a higher calcium binding affinity of annexin VI upon phosphorylation.
authors.affiliation: Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA. cf9c@virginia.edu
authors.forename: Caroline
authors.initials: C
authors.lastname: Freye-Minks
authors.forename: Robert H
authors.initials: RH
authors.lastname: Kretsinger
authors.forename: Carl E
authors.initials: CE
authors.lastname: Creutz
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D017317:Annexin A6 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D008081:Liposomes / Q000737:chemistry; D018716:Molecular Mimicry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013912:Threonine / Q000235:genetics* / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry
chemical_list: D017317:Annexin A6; D008081:Liposomes; D012997:Solvents; D013912:Threonine; D001224:Aspartic Acid; D014357:Trypsin; D002118:Calcium