title: Structure-based identification of CaMKIIα-interacting MUPP1 PDZ domains and rational design of peptide ligands to target such interaction in human fertilization.
abstract: The recognition and association between Ca(2+)/calmodulin-activated protein kinase II-α (CaMKIIα) 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 CaMKIIα's C-terminal tail to one or more PDZ domains of the scaffolding protein MUPP1. In this study, we attempt to identify the CaMKIIα-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 CaMKIIα 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 Φ-X-Φ(-COOH) of the C-terminal peptide SGAPSV(-COOH) of CaMKIIα. 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 µM for peptide binding to PDZ 5 and 11, respectively), which was in line with theoretical prediction (binding free energies ΔG 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 ~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 CaMKIIα-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.
authors.affiliation: Reproductive Medical Center, The First Affiliated Hospital of Zhengzhou University, 1 East Jianshe Road, Zhengzhou, 450052, China.
authors.forename: Yi-Le
authors.initials: YL
authors.lastname: Zhang
authors.affiliation: Department of Burn and Reconstruction Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China.
authors.forename: Zhao-Feng
authors.initials: ZF
authors.lastname: Han
authors.affiliation: Reproductive Medical Center, The First Affiliated Hospital of Zhengzhou University, 1 East Jianshe Road, Zhengzhou, 450052, China. luna020996@126.com.
authors.forename: Ying-Pu
authors.initials: YP
authors.lastname: Sun
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D005306:Fertilization; D006801:Humans; D008565:Membrane Proteins; D056004:Molecular Dynamics Simulation; D054731:PDZ Domains; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C529569:CAMK2N1 protein, human; D002352:Carrier Proteins; C111442:MPDZ protein, human; D008565:Membrane Proteins; D010455:Peptides; D011506:Proteins
keywords: Ca2+/calmodulin-activated protein kinase II-α; Domain–peptide interaction; Human fertilization; Multi-PDZ domain protein 1; PDZ domain; Peptide ligand