Kringle 2 mediates high affinity binding of plasminogen to an internal sequence in streptococcal surface protein PAM.
Many cells express receptors for plasminogen (Pg), although the responsible molecules in most cases are poorly defined. In contrast, the group A streptococcal surface protein PAM contains a domain with two 13-amino acid residue long repeated sequences (a1 and a2) responsible for Pg binding. Here we identify the region in Pg that interacts with PAM. A radiolabeled proteolytic plasminogen fragment containing the first three kringles (K1-K3) interacted with streptococci expressing PAM or a chimeric surface protein harboring the a1a2 sequence. In contrast, plasminogen fragments containing kringle 4 or kringle 5 and the activable serine proteinase domain failed to bind to PAM-expressing group A streptococci. A synthetic and a recombinant polypeptide containing the a1a2 sequence both bound to immobilized recombinant K2 (rK2) but not to rK1 or rK3. The interaction between the a repeat region and rK2 was reversible, and rK2 completely blocked the binding of Pg to the a1a2 region. The binding of the a repeat containing polypeptide to K2 occurred with an equilibrium association constant of 4.5 x 10(7) M-1, as determined by surface plasmon resonance, a value close to that (1.6 x 10(7) M-1) calculated for the a1a2-Pg interaction. Inhibition experiments suggested involvement of the lysine-binding site of K2 in the interaction. These data demonstrate that K2 contains the major Pg-binding site for PAM, providing the first well defined example of an interaction between an internal Pg-binding region in a protein and a single kringle domain.
Wistedt
A C
AC
Department of Laboratory Medicine, University of Lund, S-22362 Lund, Sweden.
Kotarsky
H
H
Marti
D
D
Ringdahl
U
U
Castellino
F J
FJ
Schaller
J
J
Sjöbring
U
U
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C084529:plasminogen-binding protein, bacteria; D010958:Plasminogen