Contribution of conserved lysine residues in the α2- antiplasmin C terminus to plasmin binding and inhibition

Abstract

α2-Antiplasmin is the physiological inhibitor of plasmin and is unique in the serpin family due to N- and C-terminal extensions beyond its core domain. The C-terminal extension comprises 55 amino acids from Asn-410 to Lys-464, and the lysine residues (Lys-418, Lys-427, Lys-434, Lys-441, Lys-448, and Lys-464) within this region are important in mediating the initial interaction with kringle domains of plasmin. To understand the role of lysine residues within the C terminus of α2-antiplasmin, we systematically and sequentially mutated the C-terminal lysines, studied the effects on the rate of plasmin inhibition, and measured the binding affinity for plasmin via surface plasmon resonance. We determined that the C-terminal lysine (Lys-464) is individually most important in initiating binding to plasmin. Using two independent methods, we also showed that the conserved internal lysine residues play a major role mediating binding of the C terminus of α2-antiplasmin to kringle domains of plasmin and in accelerating the rate of interaction between α2-antiplasmin and plasmin. When the C terminus of α2-antiplasmin was removed, the binding affinity for active siteblocked plasmin remained high, suggesting additional exosite interactions between the serpin core and plasmin. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.