Membrane Binding of Neuronal Calcium Sensor-1: Highly Specific Interaction with Phosphatidylinositol-3-Phosphate.

Viktoriia E Baksheeva,Evgeni Yu Zernii,Yuri N Antonenko,Alexander M Firsov,Dmitry V Zinchenko,Pavel P Philippov,Vasily I Vladimirov,Sergey E Permyakov,Arthur O Zalevsky,Andrey A Zamyatnin,Natalia K Tikhomirova,Ekaterina L Nemashkalova

doi:10.3390/biom10020164

Abstract

Neuronal calcium sensors are a family of N-terminally myristoylated membrane-binding proteins possessing a different intracellular localization and thereby targeting unique signaling partner(s). Apart from the myristoyl group, the membrane attachment of these proteins may be modulated by their N-terminal positively charged residues responsible for specific recognition of the membrane components. Here, we examined the interaction of neuronal calcium sensor-1 (NCS-1) with natural membranes of different lipid composition as well as individual phospholipids in form of multilamellar liposomes or immobilized monolayers and characterized the role of myristoyl group and N-terminal lysine residues in membrane binding and phospholipid preference of the protein. NCS-1 binds to photoreceptor and hippocampal membranes in a Ca2+-independent manner and the binding is attenuated in the absence of myristoyl group. Meanwhile, the interaction with photoreceptor membranes is less dependent on myristoylation and more sensitive to replacement of K3, K7, and/or K9 of NCS-1 by glutamic acid, reflecting affinity of the protein to negatively charged phospholipids. Consistently, among the major phospholipids, NCS-1 preferentially interacts with phosphatidylserine and phosphatidylinositol with micromolar affinity and the interaction with the former is inhibited upon mutating of N-terminal lysines of the protein. Remarkably, NCS-1 demonstrates pronounced specific binding to phosphoinositides with high preference for phosphatidylinositol-3-phosphate. The binding does not depend on myristoylation and, unexpectedly, is not sensitive to the charge inversion mutations. Instead, phosphatidylinositol-3-phosphate can be recognized by a specific site located in the N-terminal region of the protein. These data provide important novel insights into the general mechanism of membrane binding of NCS-1 and its targeting to specific phospholipids ensuring involvement of the protein in phosphoinositide-regulated signaling pathways.

Highlights

Many signaling pathways in neurons involve the recruitment of proteins to the membrane surface in response to stimuli
The N-terminus of neuronal calcium sensor-1 (NCS-1) ensures both general membrane anchoring of the protein by providing a myristoyl group and positively charged residues and its highly specific binding to PI3P by forming the respective site
The first mode represents myristoyl-dependent binding of neuronal calcium sensors (NCSs)-1 to cellular membranes, which involves N-terminal lysine residues K3, K7, and/or K9 ensuring preference of the protein to membranes enriched in acidic phospholipids

Summary

Introduction

Many signaling pathways in neurons involve the recruitment of proteins to the membrane surface in response to stimuli. Membrane targeting of proteins is achieved by a variety of specific lipid-binding domains as well as co-translational modifications, such as N-terminal myristoylation [1,2]. The latter strategy is used by neuronal calcium sensors (NCSs), a family of calcium-binding EF-hand proteins playing different signaling roles in the nervous system (for review, see [3]). In response to Ca2+ binding, the residues forming this pocket and the myristoyl group can be exposed to the surface of the protein molecule. Apart from the myristoyl group, each NCS might possess unique structural elements, which contain additional targeting information, selectively linking them to membranes based on their lipid composition

Methods

Findings

Discussion

Conclusion