ABSTRACT
Background. Annexins are a family of structurally related proteins that bind to phospholipid membranes in a Ca²+-dependent manner. Annexins are characterized by highly conserved canonical domains of approximately 70 amino acids. Anexin V contains four such domains. Each of these domains has a highly conserved arginine (R). Methods. To evaluate the role of the conserved arginines in the molecular structure of annexin V, negatively charged amino acids were substituted for arginines at positions R43, R115, R199, and R274 using site-directed mutagenesis. Results. Mutants R199D and R274E were rapidly degraded when expressed in bacteria, and were not further characterized. R43E exhibited an electrophoretic mobility similar to the wild-type protein, while R115E migrated significantly in a slower fashion, suggesting a less compact conformation, R43E and R115E exhibited much grater susceptibility to proteolytic digestion than the wild type. While Ca²+-dependence for phospholipid binding was similar in both mutants (half-maximal 50-80 µ; Ca²+), R43E and R115E exhibited a phospholipid affinities of the annexins, a phospholipid-dependent clotting reaction, the activated partial thromboplastin time (aPTT), was significantly prolonged by the wild-type protein and mutants R115E and R115A. The aPTT was unaffected by R43E. Conclusions. Our data suggest that mutation of these highly conserved arginine residus in each of the four canonical domains of annexin have differential effects on the phospholipid binding tertiary structure, and proteolytic susceptibility of annexin V. The site I mutation , R43E, produced a large decrease in phospholipid affinity associated with an increase in proteolytic susceptibility. The site II mutation, R115E, produced a small change in phospholipid binding but a significant modification of electrophoretic mobility. Our data suggest that highly conserved arginine residues are required to stabilize the tertiary structure of ammexin V by establishing hydrogen bonds and ionic bridges