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

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.

Disulfide Dimerization of Neuronal Calcium Sensor-1: Implications for Zinc and Redox Signaling

“…To address alterations in these features of the protein, associated with its disulfide dimerization, we used functional assays based on components of the visual cascade, namely photoreceptor membranes/rhodopsin and rhodopsin kinase (GRK1). Indeed, NCS-1 displays a relatively high affinity towards photoreceptor membranes (comparable to those of other neuronal membranes), whereas GRK1 represents one of the structurally confirmed targets of the protein [10,54]. We found that disulfide dimerization produced a moderate negative effect on the membrane association of NCS-1 (Figure 6A).…”

“…Urea-washed photoreceptor (rod outer segment) membranes were prepared from frozen retinae, as previously described [110]. The binding of NCS-1 and dNCS-1 to the membranes was analyzed by equilibrium centrifugation assay, as described in [10,111], under non-reducing conditions. Membrane-bound NCS-1 was visualized by Western blotting (see below) and weight fractions of the bound proteins were quantified by densitometric analysis of the bands using GelAnalyzer.2010a software (Istvan Lazar Jr., University of Budapest, Budapest, Hungary).…”

“…Neuronal calcium sensor-1 (NCS-1) is the ancestral member of the NCS family. It possesses relatively high Ca 2+ -binding affinity (K D ~0.3 µM) and is constitutively associated with plasma or Golgi membranes, preferring sites enriched in negatively charged phospholipids, such as phosphatidylserine or signaling phosphatidylinositol phosphates [10][11][12]. NCS-1 has three functional EF-hand motifs, which can coordinate magnesium and display different affinity and filling orders for calcium: EF2 and EF3 are occupied first, followed by the occupation of EF4 [13].…”

Zinc Modulation of Neuronal Calcium Sensor Proteins: Three Modes of Interaction with Different Structural Outcomes

“…All these structural features govern the Ca 2+ -affinity of each NCS protein, which, taken together, covers the entire range of Ca 2+ -signals in neurons (K D values for calcium vary from 60–200 nM for GCAPs and 90 nM for NCS1 to 1.2 µM for VILIP1 and 17 µM for recoverin [ 26 , 27 , 31 , 32 , 33 ]). Besides the number of functional EF-hands and the nature of the residues in the Ca 2+ -binding loop, the affinity to calcium is determined by the energy cost of conformational changes associated with its coordination, and is affected by the presence of cellular membranes, individual phospholipids, and/or target proteins [ 22 , 34 ].…”

A Novel Approach to Bacterial Expression and Purification of Myristoylated Forms of Neuronal Calcium Sensor Proteins

“…In recoverin, the functioning of the Ca 2+ -myristoyl switch and subsequent membrane binding are affected by a number of different factors, such as proper structure and conformation of its unique N-terminal and C-terminal segments, electrostatic contacts between the protein and the membrane, phosphatidylserine content and potential of the membrane as well as the presence of zinc and redox state of the medium [ 12 , 31 , 36 , 37 , 38 , 39 ]. NCALD [ 40 ], VILIP1 [ 41 ] and VILIP3 [ 42 ] possess a similar Ca 2+ -myristoyl switch mechanism, whereas NCS-1 has its myristoyl group permanently exposed in the presence of membranes [ 28 , 43 , 44 , 45 ] and its membrane localization is predominantly governed by the interaction of the protein with specific phospholipids in the bilayer [ 46 ]. In contrast to the other NCSs, in GCAP1 the myristoyl group is not involved in membrane binding.…”