title: Contribution of conserved lysine residues in the alpha2-antiplasmin C terminus to plasmin binding and inhibition.
abstract: α(2)-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 α(2)-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 α(2)-antiplasmin to kringle domains of plasmin and in accelerating the rate of interaction between α(2)-antiplasmin and plasmin. When the C terminus of α(2)-antiplasmin was removed, the binding affinity for active site-blocked plasmin remained high, suggesting additional exosite interactions between the serpin core and plasmin.
authors.affiliation: Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria 3004, Australia.
authors.forename: Bernadine G C
authors.initials: BG
authors.lastname: Lu
authors.forename: Trifina
authors.initials: T
authors.lastname: Sofian
authors.forename: Ruby H P
authors.initials: RH
authors.lastname: Law
authors.forename: Paul B
authors.initials: PB
authors.lastname: Coughlin
authors.forename: Anita J
authors.initials: AJ
authors.lastname: Horvath
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain / Q000502:physiology; D005341:Fibrinolysin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D010449:Peptide Mapping / Q000379:methods; D011485:Protein Binding / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000979:alpha-2-Antiplasmin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; C534949:SERPINF2 protein, human; D000979:alpha-2-Antiplasmin; D005341:Fibrinolysin