Detailed Regulatory Mechanism of the Interaction between ZO-1 PDZ2 and Connexin43 Revealed by MD Simulations

The gap junction protein connexin43 (Cx43) binds to the second PDZ domain of Zonula occludens-1 (ZO-1) through its C-terminal tail, mediating the regulation of gap junction plaque size and dynamics. Biochemical study demonstrated that the very C-terminal 12 residues of Cx43 are necessary and sufficient for ZO-1 PDZ2 binding and phosphorylation at residues Ser (-9) and Ser (-10) of the peptide can disrupt the association. However, only a crystal structure of ZO-1 PDZ2 in complex with a shorter 9 aa peptide of connexin43 was solved experimentally. Here, the interactions between ZO-1 PDZ2 and the short, long and phosphorylated Cx43 peptides were studied using molecular dynamics (MD) simulations and free energy calculation. The short peptide bound to PDZ2 exhibits large structural variations, while the extension of three upstream residues stabilizes the peptide conformation and enhanced the interaction. Phosphorylation at Ser(-9) significantly weakens the binding and results in conformational flexibility of the peptide. Glu210 of ZO-1 PDZ2 was found to be a key regulatory point in Cx43 binding and phosphorylation induced dissociation

The Conformational Transition Pathways of ATP-Binding Cassette Transporter BtuCD Revealed by Targeted Molecular Dynamics Simulation

BtuCD is a member of the ATP-binding cassette transporters in Escherichia coli that imports vitamin B12 into the cell by utilizing the energy of ATP hydrolysis. Crystal structures of BtuCD and its homologous protein HI1470/1 in various conformational states support the ‘‘alternating access’ ’ mechanism which proposes the conformational transitions of the substrate translocation pathway at transmembrane domain (TMD) between the outward-facing and inward-facing states. The conformational transition at TMD is assumed to couple with the movement of the cytoplasmic nucleotide-binding domains (NBDs) driven by ATP hydrolysis/binding. In this study, we performed targeted molecular dynamics (MD) simulations to explore the atomic details of the conformational transitions of BtuCD importer. The outward-facing to inward-facing (ORI) transition was found to be initiated by the conformational movement of NBDs. The subsequent reorientation of the substrate translocation pathway at TMD began with the closing of the periplasmic gate, followed by the opening of the cytoplamic gate in the last stage of the conformational transition due to the extensive hydrophobic interactions at this region, consistent with the functional requirement of unidirectional transport of the substrates. The reverse inward-facing to outward-facing (IRO) transition was found to exhibit intrinsic diversity of the conformational transition pathways and significant structural asymmetry, suggesting that the asymmetric crystal structure of BtuCD-F is a

Conformational changes at the periplasmic side of the translocation pathway.

<p>(a) Variation of the radius of the translocation pore during the O→I transition. The Z coordinate is along the membrane normal, and the layers denoted with residues are labeled. (b) Close-up top view from the periplasmic side of the translocation pore. Hydrophobic residues are shown with stick model. (c) Evolution of distances between Cα atoms on residues Y165 and M176 during the O→I transition. (d) Evolution of d_pairs of residues at the perplasmic gate along the simulation trajectory of O→I transition.</p

The core region in the middle of the translocation pathway.

<p>(a) The residues involved in core region are shown with stick model. TM5a helices are removed for a clear view. (b) Differences of d_pairs of residues between outBtuCD and inBtuCD states. The residues involved in the core region are colored cyan.</p

Conformational changes at the cytoplasmic side of the translocation pathway.

<p>(a) Close-up bottom view from the cytoplasmic side of the translocation pore. Hydrophobic residues are shown with stick model. (b) Evolution of d_pairs of residues at the cytoplasmic side along the simulation trajectory of O→I transition. (c) Cartoon representation of the hydrophobic network at the cytoplasmic gate in outBtuCD and inBtuCD. (d) Evolution of minimal distances between residues L90-V150 and L85-L146 during the O→I transition.</p

The conformational motions of NBD dimer.

<p>(a) The evolution of the spin angle between NBD and TMD during the O→I (light color) and I→O (deep color) transitions. (b) The evolution of the distance between the nucleotide-binding motifs (Walker A and LSGGQ) during O→I transition. (c) The evolution of the distances between the two NBDs and their sub-domains during the O→I transition.</p