Evidence that the conformation of unliganded human plasminogen is maintained via an intramolecular interaction between the lysine-binding site of kringle 5 and the N-terminal peptide.
Human Glu-plasminogen adopts at least three conformations that provide a means for regulating the specificity of its activation in vivo. It has been proposed previously that the closed (alpha) conformation of human Glu-plasminogen is maintained through physical interaction of the kringle 5 domain and a lysine residue within the N-terminal peptide (NTP). To examine this hypothesis, site-directed mutagenesis was used to generate variant proteins containing substitutions either for aspartic acid residues within the anionic centre of the kringle 5 domain or for conserved lysine residues within the NTP. Size-exclusion HPLC and rates of plasminogen activation by urokinase-type plasminogen activator were used to determine the conformational states of these variants. Variants with substitutions within the kringle 5 lysine-binding site demonstrated extended conformations, as did variants with alanine substitutions for Lys50 and Lys62. In contrast, molecules in which NTP residues Lys20 or Lys33 were replaced were shown to adopt closed conformations. We conclude that the lysine-binding site of kringle 5 is involved in maintaining the closed conformation of human Glu-plasminogen via an interaction with the NTP, probably through Lys50 and/or Lys62. These conclusions advance the current model for the initial stages of fibrinolysis during which fibrin is thought to compete with the NTP for the kringle 5 lysine-binding site.
Cockell
C S
CS
University of Oxford, Laboratory of Molecular Biophysics, Department of Biochemistry, Rex Richards Building, South Parks Road, Oxford OX1 3QU, U.K.
Marshall
J M
JM
Dawson
K M
KM
Cederholm-Williams
S A
SA
Ponting
C P
CP
D019943:Amino Acid Substitution; D015119:Aminocaproic Acid / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D018082:Kringles; D008024:Ligands; D008239:Lysine / Q000378:metabolism*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D008024:Ligands; D011994:Recombinant Proteins; D018698:Glutamic Acid; D010958:Plasminogen; D014568:Urokinase-Type Plasminogen Activator; D008239:Lysine; D015119:Aminocaproic Acid