Combinatorial mutagenesis of the four domains of annexin IV: effects on chromaffin granule binding and aggregating activities.
This study addresses the roles of individual annexin IV domains in calcium-dependent membrane binding and aggregation through an analysis of the activities of mutant annexin IV proteins in which critical residues in one or more domains have been altered. The consensus sequence for high-affinity Ca(2+)-binding pockets obtained from the annexin V crystal structure is GXGT-38 residues-D/E [Huber, R., et al. (1992) J. Mol. Biol. 223, 683-704]. Site-directed mutagenesis was used to change the conserved acidic residues (D/E) in these sequences to alanine residues in each of the four domains of bovine annexin IV, singly or in combinations. Fourteen mutants with one, two, three, or four mutated domains were constructed and expressed in Escherichia coli. Purified recombinant product was evaluated for Ca(2+)-dependent binding to and aggregation of bovine chromaffin granules. Increases in the number of mutated domains resulted in increased Ca2+ requirements for both granule binding and aggregation. Further analysis revealed that mutations in individual domains had preferential effects on the binding or aggregating activities of the protein. For example, mutation of the first or fourth domains had a greater effect on membrane binding than aggregation, while conversely, mutation of the second domain had a more dramatic effect on membrane aggregation. Mutation of the third domain was largely silent in these assays. An additional mutation was made in the fourth domain to substitute a serine for a highly conserved arginine residue (Arg274) present at the C-terminus of the fourth endonexin fold. This mutation increased the calcium requirement for membrane binding 2-fold and for membrane aggregation 3-fold. This mutant protein was found to be an effective inhibitor of membrane aggregation by native annexin IV at intermediate levels of calcium.
Nelson
M R
MR
Department of Pharmacology, University of Virginia, Charlottesville 22908.
Creutz
C E
CE
D000595:Amino Acid Sequence; D000818:Animals; D017314:Annexin A4 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism*; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D017314:Annexin A4; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D002118:Calcium