Interfacial basic cluster in annexin V couples phospholipid binding and trimer formation on membrane surfaces.
Annexin V is an abundant eukaryotic protein that binds phospholipid membranes in a Ca(2+)-dependent manner. In the present studies, site-directed mutagenesis was combined with x-ray crystallography and solution liposome binding assays to probe the functional role of a cluster of interfacial basic residues in annexin V. Four mutants were investigated: R23E, K27E, R61E, and R149E. All four mutants exhibited a significant reduction in adsorption to phospholipid membranes relative to the wild-type protein, and the R23E mutation was the most deleterious. Crystal structures of wild-type and mutant proteins were similar except for local changes in salt bridges involving basic cluster residues. The combined data indicate that Arg(23) is a major determinant for interfacial phospholipid binding and participates in an intermolecular salt bridge that is key for trimer formation on the membrane surface. Together, crystallographic and solution data provide evidence that the interfacial basic cluster is a locus where trimerization is synergistically coupled to membrane phospholipid binding.
Mo
Youde
Y
Department of Physiology and Biophysics, Boston University School of Medicine, Massachusetts 02118, USA.
Campos
Begoña
B
Mealy
Tanya R
TR
Commodore
Lois
L
Head
James F
JF
Dedman
John R
JR
Seaton
Barbara A
BA
D000818:Animals; D017304:Annexin A5 / Q000737:chemistry* / Q000378:metabolism; D001120:Arginine / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D055672:Static Electricity
D017304:Annexin A5; D008081:Liposomes; D010743:Phospholipids; D011994:Recombinant Proteins; D001120:Arginine; D002118:Calcium