Structure-based identification of CaMKII alpha-interacting MUPP1 PDZ domains and rational design of peptide ligands to target such interaction in human fertilization

摘要

The recognition and association between Ca2+/calmodulin-activated protein kinase II-alpha (CaMKII alpha) and multi-PDZ domain protein 1 (MUPP1) plays an important role in sperm acrosome reaction and human fertilization, which is mediated by the binding of CaMKII alpha's C-terminal tail to one or more PDZ domains of the scaffolding protein MUPP1. In this study, we attempt to identify the CaMKII alpha-interacting MUPP1 PDZ domains and to design peptide ligands that can potently target and then competitively disrupt such interaction. Here, a synthetic biology approach was proposed to systematically characterize the structural basis, energetic property, dynamic behavior and biological implication underlying the intermolecular interactions between the C-terminal peptide of CaMKII alpha and all the 13 PDZ domains of MUPP1. These domains can be grouped into four clusters in terms of their sequence, structure and physiochemical profile; different clusters appear to recognize different classes of PDZ-binding motifs. The cluster 3 includes two members, i.e. MUPP1 PDZ 5 and 11 domains, which were suggested to bind class II motif I broken vertical bar-X-I broken vertical bar(-COOH) of the C-terminal peptide SGAPSV(-COOH) of CaMKII alpha. Subsequently, the two domains were experimentally measured as the moderate- and high-affinity binders of the peptide by using fluorescence titration (dissociation constants K (d) = 25.2 +/- 4.6 and 0.47 +/- 0.08 A mu M for peptide binding to PDZ 5 and 11, respectively), which was in line with theoretical prediction (binding free energies Delta G (total) = -7.6 and -9.2 kcal/mol for peptide binding to PDZ 5 and 11, respectively). A systematic mutation of SGAPSV(-COOH) residues suggested few favorable amino acids at different residue positions of the peptide, which were then combined to generate a number of potent peptide mutants for PDZ 11 domain. Consequently, two peptides (SIAPNV(-COOH) and SIVMNV-COOH) were identified to have considerably improved affinity with K (d) increase by similar to tenfold relative to wild type peptide. Thus, the two peptides are considered as promising lead entities to develop therapeutic molecular agents with high efficacy and specificity to target CaMKII alpha-MUPP1 interaction. Other five designed peptides (SILPSV-COOH, SGLPNV(-COOH), SIVMSV-COOH, SIVPNV-COOH and SIAMNV(-COOH)) possessed comparable affinity with the wild type, and they may be further optimized to obtain higher potency.