Structure and Function Characterization of the a1a2 Motifs of Streptococcus pyogenes M Protein in Human Plasminogen Binding.

Plasminogen (Plg)-binding M protein (PAM) is a group A streptococcal cell surface receptor that is crucial for bacterial virulence. Previous studies revealed that, by binding to the kringle 2 (KR2) domain of host Plg, the pathogen attains a proteolytic microenvironment on the cell surface that facilitates its dissemination from the primary infection site. Each of the PAM molecules in their dimeric assembly consists of two Plg binding motifs (called the a1 and a2 repeats). To date, the molecular interactions between the a1 repeat and KR2 have been structurally characterized, whereas the role of the a2 repeat is less well defined. Here, we report the 1.7-Å x-ray crystal structure of KR2 in complex with a monomeric PAM peptide that contains both the a1 and a2 motifs. The structure reveals how the PAM peptide forms key interactions simultaneously with two KR2 via the high-affinity lysine isosteres within the a1a2 motifs. Further studies, through combined mutagenesis and functional characterization, show that a2 is a stronger KR2 binder than a1, suggesting that these two motifs may play discrete roles in mediating the final PAM-Plg assembly.

Defining the interaction of perforin with calcium and the phospholipid membrane.

Following its secretion from cytotoxic lymphocytes into the immune synapse, perforin binds to target cell membranes through its Ca(2+)-dependent C2 domain. Membrane-bound perforin then forms pores that allow passage of pro-apoptopic granzymes into the target cell. In the present study, structural and biochemical studies reveal that Ca(2+) binding triggers a conformational change in the C2 domain that permits four key hydrophobic residues to interact with the plasma membrane. However, in contrast with previous suggestions, these movements and membrane binding do not trigger irreversible conformational changes in the pore-forming MACPF (membrane attack complex/perforin-like) domain, indicating that subsequent monomer-monomer interactions at the membrane surface are required for perforin pore formation.

The N terminus of the serpin, tengpin, functions to trap the metastable native state.

Serpins fold to a metastable native state and are susceptible to undergoing spontaneous conformational change to more stable conformers, such as the latent form. We investigated conformational change in tengpin, an unusual prokaryotic serpin from the extremophile Thermoanaerobacter tengcongensis. In addition to the serpin domain, tengpin contains a functionally uncharacterized 56-amino-acid amino-terminal region. Deletion of this domain creates a variant--tengpinDelta51--which folds past the native state and readily adopts the latent conformation. Analysis of crystal structures together with mutagenesis studies show that the N terminus of tengpin protects a hydrophobic patch in the serpin domain and functions to trap tengpin in its native metastable state. A 13-amino-acid peptide derived from the N terminus is able to mimick the role of the N terminus in stabilizing the native state of tengpinDelta51. Therefore, the function of the N terminus in tengpin resembles protein cofactors that prevent mammalian serpins from spontaneously adopting the latent conformation.