Mutation of highly conserved arginine residues disrupts the structure and function of annexin V

Background. Annexins are a family of structurally related proteins that bin d to phospholipid membranes in a Ca2+-dependent manner. Annexins are charac terized by highly conserved canonical domains of approximately 70 amino aci ds. Annexin V contains four such domains. Each of these domains has a highl y conserved arginine (R). Methods. To evaluate the role of the conserved arginines in the molecular s tructure of annexin V, negatively charged amino acids were substituted for arginines at positions R43, R115, R199, and R274 using site-directed mutage nesis, Results. Mutants R199D and R274E were rapidly degraded when expressed in ba cteria, and were not further characterized. R43E exhibited an electrophoret ic mobility similar to the wild-type protein, while R115E migrated signific antly in a slower fashion, suggesting a less compact conformation. R43E and R115E exhibited much greater susceptibility to proteolytic digestion than the wild type. While Ca2+-dependence for phospholipid binding was similar i n both mutants (half-maximal 50-80 mu M Ca2+), R43E and R115E exhibited a 6 - and 2-fold decrease in phospholipid affinity, respectively. Consistent wi th the different phospholipid affinities of the annexins, a phospholipid-de pendent clotting reaction, the activated partial thromboplastin time (aPTT) , was significantly prolonged by the wild-type protein and mutants R115E an d R115A. The aPTT was unaffected by R43E. Conclusions. Our data suggest that mutation of these highly conserved argin ine residues in each of the four canonical domains of annexin have differen tial effects on the phospholipid binding, tertiary structure, and proteolyt ic susceptibility of annexin V. The site I mutation, R43E, produced a large decrease in phospholipid affinity associated with an increase in proteolyt ic susceptibility. The site II mutation, R115E, produced a small change in phospholipid binding but a significant modification of electrophoretic mobi lity. Our data suggest that highly conserved arginine residues are required to stabilize the tertiary structure of annexin V by establishing hydrogen bonds and ionic bridges. (C) 1999 IMSS, Published by Elsevier Science Inc.