Contributions of individual kringle domains toward maintenance of the chloride-induced tight conformation of human glutamic acid-1 plasminogen.
The roles of each of the three omega-amino acid-binding kringles (K) of Glu1-Pg, viz., [K1Pg], [K4Pg], and [K5Pg], in engendering the Cl(-)-induced alteration to its tight (T) conformation and in effecting the epsilon-aminocaproic acid (EACA)-mediated change to the relaxed (R) protein conformation have been investigated by mutagenesis strategies wherein the omega-amino acid ligand-binding energies in the individual kringles in recombinant (r)-Glu1-Pg were greatly reduced. This was accomplished in the most conservative manner possible by altering a critical Asp residue in each relevant kringle to Asn. The particular mutations chosen were r-[D139N]Glu1-Pg, r-[D413N]Glu1-Pg, and r-[D518N]Glu1-Pg, in which a conserved Asp residue at a homologous sequence position in each of the three kringle domains is eliminated. These changes also lead to a great reduction of the EACA-binding strength of [K1Pg], [K4Pg], and [K5Pg], respectively. The s0(20,w) of wild-type (wt) r-Glu1-Pg in the presence of levels of Cl(-)-sufficient to fully occupy its binding sites on this protein was 5.9 S, a value reduced to 4.9 S as a result of addition of saturating concentrations of EACA to the Cl-/Glu1-Pg complex. Neither Cl- nor EACA substantially altered the s0(20,w) value of 5.2 S for r-[D139N]Glu1-Pg (4.8 S) or r-[D413N]Glu1-Pg (4.5 S). On the other hand, the s0(20,w) value of 5.2 S for r-[D518N]Glu1-Pg at saturating levels of Cl- is slightly reduced to 4.8 S upon addition of binding maximal concentrations of EACA.(ABSTRACT TRUNCATED AT 250 WORDS)
McCance
S G
SG
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Castellino
F J
FJ
D000595:Amino Acid Sequence; D015119:Aminocaproic Acid / Q000494:pharmacology; D001483:Base Sequence; D002712:Chlorides / Q000494:pharmacology*; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid; D006801:Humans; D018082:Kringles; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D011487:Protein Conformation / Q000187:drug effects*; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification
D002712:Chlorides; D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D018698:Glutamic Acid; D010958:Plasminogen; D015119:Aminocaproic Acid