Kringle 2 Mediates High Affinity Binding of Plasminogen to an Internal Sequence in Streptococcal Surface Protein PAM

Abstract

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 ؋ 10 M ؊1, as determined by surface plasmon resonance, a value close to that (1.6 ؋ 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.The plasma glycoprotein plasminogen (Pg) 1 is a single-chain 92-kDa precursor for the broad spectrum serine proteinase plasmin (1, 2) (see Fig. 1A). In vivo, the tissue-type and urokinase-type plasminogen activators convert the zymogen into the two-chain proteinase by cleavage of a single peptide bond (Arg 561 -Val 562 ). Activation can also be achieved by some bacterial proteins, such as streptokinase from streptococci (1, 2). Plasmin plays a key role in fibrinolysis (1-3) but also participates in several other physiological and pathophysiological processes, including wound healing, tissue penetration of cancer cells, neuronal cell death, and bacterial dissemination (4 -8).The activable serine proteinase domain is located in the COOH-terminal third of Pg. The NH 2 -terminal two-thirds of Pg contains an 8-kDa preactivation peptide and five characteristic kringle domains (K1-K5), each ϳ9 kDa. The kringles mediate interactions with multiple ligands, including fibrin, the primary target of Pg, and ␣ 2 -plasmin inhibitor, its principal regulator (1, 2). The recognition events depend upon interactions between lysine-binding sites in the kringles and exposed COOH-terminal lysines in the ligands. Lysine analogues, such as 6-aminohexanoic acid (6-AHA), mimic COOH-terminal lysines in the interaction with kringles and the structural basis of the interactions between some kringles, particularly K1 and K4, and 6-AHA has been disclosed (9 -12). The affinity of the different kringles for lysine or 6-AHA is...

Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain

“…Thus, binding interactions of VEK-peptides, regardless of their oligomerization states, mediate the transformation of the tight (T) to the relaxed (R) conformations of hPg. Although binding of these peptides are specific for the LBS of K2 hPg (14,38), this does not rule out the possibility that exosites, especially in the longer peptides, interact with other FIGURE 1. Sedimentation equilibrium ultracentrifugation analysis of VEK-peptides.…”

“…It has been demonstrated that PAM binding to K2 hPg is mediated by an internal a1a2 repeat domain in the N terminus of PAM (14,42). The importance of the a1a2 domain of PAM in hPg binding has also been demonstrated in Arp4, an M-like protein, which does not interact with hPg.…”

“…The locus of PAM that interacts with hPg has been identified as the a1a2 N-terminal region (PAM a1a2 ) (14). A small peptide, VEK30 (PAM residues 97-125), which spans the PAM a1a2 domain, has been shown to bind in a unique manner to K2 hPg .…”

Dihydrolipoamide Dehydrogenase of Pseudomonas aeruginosa Is a Surface-Exposed Immune Evasion Protein That Binds Three Members of the Factor H Family and Plasminogen

“…Lpd attaches plasminogen via a major binding site localized in kringle domain 4. Streptococcal surface protein PAM binds human plasminogen via kringle domain 2, and the Streptococcus canis protein SCM binds plasminogen via kringle domain 5 (53,54). Thus, the three bacterial proteins bind to different kringle domains of plasminogen.…”

M protein‐mediated plasminogen binding is essential for the virulence of an invasiveStreptococcus pyogenesisolate

“…Plasminogen binding M proteins have been well characterised in vitro (12,(14)(15)(16)(17)(18)(19)(20), however the direct contribution of these proteins to GAS virulence has not been defined. Whilst the deletion of the PAM gene results in a loss of virulence (7,14), this may be due to a loss of other functions such as protection from phagocytosis or binding of other host molecules attributed to M proteins (21).…”