Interactions Controlling the Membrane Binding of Basic Protein Domains:  Phenylalanine and the Attachment of the Myristoylated Alanine-Rich C-Kinase Substrate Protein to Interfaces

202

256

A peptide corresponding to the basic (؉13), unstructured effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) binds strongly to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Although aromatic residues contribute to the binding, three experiments suggest the binding is driven mainly by nonspecific local electrostatic interactions. First, peptides with 13 basic residues, Lys-13 and Arg-13, bind to PIP 2 -containing vesicles with the same high affinity as the effector domain peptide. Second, removing basic residues from the effector domain peptide reduces the binding energy by an amount that correlates with the number of charges removed. Third, peptides corresponding to a basic region in GAP43 and MARCKS effector domain-like regions in other proteins (e.g. MacMARCKS, adducin, Drosophila A kinase anchor protein 200, and N-methyl-D-aspartate receptor) also bind with an energy that correlates with the number of basic residues. Kinetic measurements suggest the effector domain binds to several PIP 2 . Theoretical calculations show the effector domain produces a local positive potential, even when bound to a bilayer with 33% monovalent acidic lipids, and should thus sequester PIP 2 laterally. This electrostatic sequestration was observed experimentally using a phospholipase C assay. Our results are consistent with the hypothesis that MARCKS could reversibly sequester much of the PIP 2 in the plasma membrane.Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 1 plays many important roles in cells (reviewed in Refs. 1-8). Not only is PIP 2 the source of 3 second messengers, its hydrolysis via phospholipase Cs (PLCs) (9, 10) produces inositol 1,4,5-trisphosphate and diacylglycerol (11,12), and its phosphorylation via phosphoinositide 3-kinases produces phosphatidylinositol 3,4,5-trisphosphate (6, 13-15), but it also can be a second messenger itself (4,8,16,17). Moreover, PIP 2 helps regulate cytoskeletal attachment (18 -20), exo-and endocytosis (1-3), enzyme activity (21), and ion channel function (17,22). Several groups (2,8,16,23) have suggested that these myriad functions can be explained if there are different pools of PIP 2 in the plasma membrane. One hypothesis is that proteins act as reversible buffers to bind much of the PIP 2 and then release it locally in response to specific signals (8,24,25). These proteins would have to be present at a concentration comparable with PIP 2 , be localized to the plasma membrane, bind PIP 2 with high affinity, and release it in response to physiological stimuli. Myristoylated alanine-rich C kinase substrate (MARCKS) satisfies these criteria.MARCKS (reviewed in Refs. 26 -30), a ubiquitous protein kinase C (PKC) (31) substrate, is present at high concentration in many cell types (e.g. ϳ10 M in brain tissue (27, 32)) and is localized to the plasma membrane in quiescent cells. The binding of MARCKS to plasma membranes requires both hydrophobic insertion of its N-terminal myristate into the bilayer and electrostatic interactions between its effecto...

Section: The Binding Of Truncated Marcks-(151-175) Peptides To Pc/pipsupporting

confidence: 90%

Lateral Sequestration of Phosphatidylinositol 4,5-Bisphosphate by the Basic Effector Domain of Myristoylated Alanine-rich C Kinase Substrate Is Due to Nonspecific Electrostatic Interactions

Wang

Gambhir

Hangyás-Mihályné

et al. 2002

202

256

“…residues within the effector domain and negatively charged phospholipids and via a penetration of the phenylalanine side chains within the effector domain into the lipid bilayer (33,34). Similar to MARCKS, it has been shown that PSA associates with liposomal membranes and penetrates lipid bilayers (35)(36)(37).…”

Section: Discussionmentioning

confidence: 99%

“…This result suggests that PSA at least partially triggers signal transduction via an interaction with MARCKS at the plasma membrane and that this interaction interferes with PKC-dependent signal transduction. (33,34). Exchanging the serine residues in the effector domain to alanine, asparagine, or aspartic acid or mutation of the myristoylation site prevents the membrane insertion of MARCKS (28).…”

Section: Psa Directly Binds To the Effector Domain Of Marcks-mentioning

confidence: 99%

Functional Role of the Interaction between Polysialic Acid and Myristoylated Alanine-rich C Kinase Substrate at the Plasma Membrane

Theis

Mishra

Ohe

et al. 2013

Background: Polysialic acid (PSA) plays important roles in the developing and adult nervous system. Results: The interaction of PSA with myristoylated alanine-rich C kinase substrate (MARCKS) at the plasma membrane regulates neurite outgrowth. Conclusion:The MARCKS/PSA interaction regulates PSA-triggered signal transduction. Significance: Study of the molecular mechanisms underlying PSA-induced cellular responses helps to understand the functions of PSA in the nervous system.

“…EPR spectroscopy (17) and circular dichroism (13) have shown that this peptide adopts an extended conformation when bound to membrane bilayers. Cafiso and co-workers (18,19) have undertaken an extensive series of EPR studies on MARCKS-(151-175) to probe its location relative to the membrane surface. They systematically substituted amino acids in the MARCKS-(151-175) sequence with cysteines derivatized with nitroxide spin labels and found that spin labels attached at the highly basic and hydrophilic N and C termini of the peptide reside on the aqueous side of the lipid phosphate head group, whereas spin labels attached to the central Phe-containing portions of the peptide are located several angstroms below the lipid head group (17).…”

mentioning

confidence: 99%

Binding of Peptides with Basic and Aromatic Residues to Bilayer Membranes

Zhang

Crocker

McLaughlin

et al. 2003

109

Many peripheral membrane proteins contain unstructured clusters of basic and aromatic residues that interact with lipid bilayers. In some cases, these clusters can bind phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) 1 (Fig. 1C), a key regulator of signal transduction in cell membranes (for reviews, see Refs. 1-8). For instance, binding of PI(4,5)P 2 to the basic-aromatic cluster on phospholipase D activates the enzyme (9 -11), whereas a similar cluster in the effector domain (residues 151-175) of the myristoylated alanine-rich C kinase substrate (MARCKS) protein may act as a reversible buffer for regulating free PI(4,5)P 2 levels in the plasma membrane (2). Peptides corresponding to basic-aromatic clusters of phospholipase D and MARCKS bind PI(4,5)P 2 with high affinity and specificity compared with monovalent acid lipids such as phosphatidylserine (11-13). To establish how these clusters can bind to the membrane, selectively sequester PI(4,5)P 2 , and possibly exert other effects on membrane structure, we need to establish their specific location within the bilayer. This study focuses on the effector domain of the MARCKS protein, one of the major substrates for protein kinase C (for reviews, see Refs. 14 -16). MARCKS associates with membranes through an N-terminal myristoyl group and a cluster of basic-aromatic amino acids in its effector domain (16). The five Phe residues in the basic effector domain may influence membrane binding or structure in several different ways, depending on the degree to which they penetrate the membrane bilayer. For example, as we discuss in more detail below, they could (i) contribute a hydrophobic term to the membrane-binding energy, (ii) enhance the electrostatic potential due to basic residues in the cluster, or (iii) induce membrane curvature. Determining the depth of the Phe residues within the membrane is the first step in understanding why they are often associated with basic residues in peripheral membrane proteins.An array of biophysical approaches has shed light on the conformation and location of the MARCKS-(151-175) peptide bound to lipid membranes. EPR spectroscopy (17) and circular dichroism (13) have shown that this peptide adopts an extended conformation when bound to membrane bilayers. Cafiso and co-workers (18, 19) have undertaken an extensive series of EPR studies on MARCKS-(151-175) to probe its location relative to the membrane surface. They systematically substituted amino acids in the MARCKS-(151-175) sequence with cysteines derivatized with nitroxide spin labels and found that spin labels attached at the highly basic and hydrophilic N and C termini of the peptide reside on the aqueous side of the lipid phosphate head group, whereas spin labels attached to the central Phe-containing portions of the peptide are located several angstroms below the lipid head group (17).In this study, we used a complementary technique, magic