NMR Backbone Dynamics of VEK-30 Bound to the Human Plasminogen Kringle 2 Domain

AbstractTo gain insights into the mechanisms for the tight and highly specific interaction of the kringle 2 domain of human plasminogen (K2Pg) with a 30-residue internal peptide (VEK-30) from a group A streptococcal M-like protein, the dynamic properties of free and bound K2Pg and VEK-30 were investigated using backbone amide 15N-NMR relaxation measurements. Dynamic parameters, namely the generalized order parameter, S2, the local correlation time, τe, and the conformational exchange contribution, Rex, were obtained for this complex by Lipari-Szabo model-free analysis. The results show that VEK-30 displays distinctly different dynamic behavior as a consequence of binding to K2Pg, manifest by decreased backbone flexibility, particularly at the binding region of the peptide. In contrast, the backbone dynamics parameters of K2Pg displayed similar patterns in the free and bound forms, but, nonetheless, showed interesting differences. Based on our previous structure-function studies of this interaction, we also made comparisons of the VEK-30/K2Pg dynamics results from different kringle modules complexed with small lysine analogs. The differences in dynamics observed for kringles with different ligands provide what we believe to be new insights into the interactions responsible for protein-ligand recognition and a better understanding of the differences in binding affinity and binding specificity of kringle domains with various ligands

NMDA-receptor antagonist requirements in conantokin-G

AbstractA series of variants of the neuroactive 17-residue γ-carboxyglutamate-(Gla)-containing polypeptide, conantokin-G (con-G), were synthesized with the intention of determining those features that were important for its N-methyl-d-aspartate (NMDA) receptor-targeted antagonist activity and for adoption of its divalent cation-dependent α-helical conformation. Employing the binding of [3H]dizolcipine (MK-801) as an assay for open receptor ion channels in rat brain membranes, which displays inhibition by con-G (IC50=0.48 μM), it was found that replacement by an Ala residue of Gla4 led to complete inactivation of the peptide, whereas a similar replacement of Gla3 resulted in a 20-fold decreased potency. Ala substitutions for Gla10 and Gla14 did not substantially affect [3H]MK-801 binding. This same substitution at Gla7 appeared to slightly enhance binding. Ala replacements of non-Gla residues demonstrated that four of them, viz. Glu2, Leu5, Gln9, and Ile12, possessed at least 200-fold decreases in inhibitory potency, whereas similar replacements at Gly1, Leu11, and Arg13 resulted in peptides with 8- to 12-fold increases in the IC50 values. The remaining amino acid residues tested in the single Ala replacement series showed no significant changes in the inhibitory characteristics of wild-type con-G. Additional studies with carboxyl-terminal truncated peptides revealed that the carboxyl-terminal 4 amino acids were unimportant for this activity. There was no strict correlation of inhibition of [3H]MK-801 binding with the ability of these peptides to form cation-dependent α-helices. Peptides with notably low α-helical content in the presence of these cations were lacking at least one, or both, of Gla10 and Gla14. Con-G[Gla3,4,7,10,14E] and con-G[Gla7,10,14E] were the only peptides that remained in a completely random conformation upon metal ion addition

Role of the Hexapeptide Disulfide Loop in the γ-Carboxyglutamic Acid Domain of Protein C in Ca²⁺-Mediated Structural and Functional Properties^†

The anticoagulant and immunomodulatory effects of protein C (PC) rely on the presence of the N-terminal γ-carboxyglutamic acid (Gla) domain. This domain is strongly conserved among vitamin K-dependent blood proteins and, in addition to a high relative content of Gla, contains a hexapeptide disulfide loop between Cys residues 17 and 22. In the present study, the contribution of the hexapeptide loop toward Gla domain structure and function was evaluated using wild-type and Cys17/Cys22-alkylated synthetic peptide analogues of the 47-residue Gla domain/helical stack of PC. Circular dichroism and intrinsic fluorescence measurements revealed significant differences in the metal ion-dependent conformations of the two peptides. Disruption of the disulfide loop slightly altered the capacity of the peptide to interact with acidic phospholipid (PL) vesicles. The affinity of the alkylated peptide for soluble endothelial protein C receptor (EPCR), as demonstrated by surface plasmon resonance studies, was increased compared with the wild-type species, although total binding was compromised. These results suggest that the disulfide loop of PC contributes to the overall Ca2+-dependent conformation but is not strictly required for PL membrane binding or EPCR recognition