Interaction between Polysialic acid and MARCKS-ED peptide at the molecular level.

Polysialic acid (polySia) is a highly negatively-charged linear homopolymer comprising α-2,8-linked sialic acids. It is abundant in the embryonic brain and modulates various functions such as differentiation and synaptic plasticity in the adult central nervous system by direct binding to its protein partners. One such example is the binding of polySia to myristoylated-alanine rich C-kinase substrate (MARCKS) to modulate neuritogenesis. To understand their interaction mechanism at the molecular level, we performed binding assay which showed a direct binding of MARCKS-ED peptide (KKKKKRFSFKKSFKLSGFSFKKNKK) with polySia in a concentration-dependent manner. Molecular dynamics simula-tions revealed that this binding is not exclusively dominated by electrostatics, but can in part be attributed to the presence of near-regularly spaced Phe residues, that confer a compact 3D conformation based on pseudo-glycine loop structures sup-ported by Phe-Phe interactions. Our simulations, which are confirmed by circular dichroism measurements, also indicate that the peptide-polySia binding induces large-scale conformational rearrangement of polySia into coils at the binding site, whereas the peptide conformation is relatively unperturbed. As a consequence we predict that each peptide can bind to a domain extending ~14 polySia repeat units. Using fluorescently tagged MARCKS-ED peptide on rat brainstem tissue sections, we demonstrate the ability of the peptide to detect polySia, similarly to polySia-specific antibody mAb735, especially in the spinal trigeminal nucleus and the dorsal vagal complex. This study provides information about the interaction be-tween polySia and its CNS protein binding partner, MARCKS, and provides a fundamental platform for further studies to explore the prospect of the MARCKS-ED as an effective polySia-binding peptide for bioimaging and drug delivery applications.