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 respon sible molecules in most cases are poorly defined. In contrast, the gro up A streptococcal surface protein PAM contains a domain with two 13-a mino 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, p lasminogen fragments containing kringle 4 or kringle 5 and the activab le serine proteinase domain failed to bind to PAM expressing group A s treptococci. A synthetic and a recombinant polypeptide containing the a1a2 sequence both bound to immobilized recombinant K2 (rK2) but not t o 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 r egion. The binding of the a repeat containing polypeptide to K2 occurr ed with an equilibrium association constant of 4.5 x 10(7) M-1, as det ermined 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 interac tion. These data demonstrate that K2 contains the major Pg-binding sit e for PAM, providing the first well defined example of an interaction between an internal Pg-binding region in a protein and a single kringl e domain.