Anna Arbuzova

The Effector Domain of Myristoylated Alanine-rich C Kinase Substrate Binds Strongly to Phosphatidylinositol 4,5-Bisphosphate

Both the myristoylated alanine-rich protein kinase C substrate protein (MARCKS) and a peptide corresponding to its basic effector domain, MARCKS-(151−175), inhibit phosphoinositide-specific phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in vesicles (Glaser, M., Wanaski, S., Buser, C. A., Boguslavsky, V., Rashidzada, W., Morris, A., Rebecchi, M., Scarlata, S. F., Runnels, L. W., Prestwich, G. D., Chen, J., Aderem, A., Ahn, J., and McLaughlin, S. (1996) J. Biol. Chem. 271, 26187–26193). We report here that adding 10−100 nmMARCKS-(151−175) to a subphase containing either PLC-δ or -β inhibits hydrolysis of PIP2 in a monolayer and that this inhibition is due to the strong binding of the peptide to PIP2. Two direct binding measurements, based on centrifugation and fluorescence, show that ≈10 nmPIP2, in the form of vesicles containing 0.01%, 0.1%, or 1% PIP2, binds 50% of MARCKS-(151−175). Both electrophoretic mobility measurements and competition experiments suggest that MARCKS-(151−175) forms an electroneutral complex with ≈4 PIP2. MARCKS-(151−175) binds equally well to PI(4,5)P2 and PI(3,4)P2. Local electrostatic interactions of PIP2 with MARCKS-(151−175) contribute to the binding energy because increasing the salt concentration from 100 to 500 mm decreases the binding 100-fold. We hypothesize that the effector domain of MARCKS can bind a significant fraction of the PIP2 in the plasma membrane, and release the bound PIP2 upon interaction with Ca2+/calmodulin or phosphorylation by protein kinase C.

MARCKS, membranes, and calmodulin: kinetics of their interaction

It is well documented that membrane binding of MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate) requires both hydrophobic insertion of the N-terminal myristate into the bilayer and electrostatic interaction of the basic effector region with acidic lipids. The structure of a membrane-bound peptide corresponding to the effector region, residues 151-175 of bovine MARCKS, was recently determined using spin-labeled peptides and EPR. The kinetics of the peptide-membrane interaction were determined from stopped-flow fluorescence measurements; the adsorption of the peptide onto phospholipid vesicles is a diffusion-limited process. Five microM Ca2+-calmodulin decreases the lifetime of the peptide on a 100 nm diameter 10:1 PC/PS vesicle from 0.1 s to 0.01 s by rapidly pulling the peptide off the membrane. We propose a molecular mechanism, based on previous work by M. Eigen and colleagues, by which calmodulin may remove MARCKS(151-175) from the membrane at a diffusion-limited rate. Calmodulin may also use this mechanism to remove the pseudosubstrate region from the substrate binding site of enzymes such as calmodulin kinase II and myosin light chain kinase.

Kinetics of Interaction of the Myristoylated Alanine-rich C Kinase Substrate, Membranes, and Calmodulin

Membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) requires both its myristate chain and basic “effector” region. Previous studies with a peptide corresponding to the effector region, MARCKS-(151–175), showed that the 13 basic residues interact electrostatically with acidic lipids and that the 5 hydrophobic phenylalanine residues penetrate the polar head group region of the bilayer. Here we describe the kinetics of the membrane binding of fluorescent (acrylodan-labeled) peptides measured with a stopped-flow technique. Even though the peptide penetrates the polar head group region, the association of MARCKS-(151–175) with membranes is extremely rapid; association occurs with a diffusion-limited association rate constant. For example,k on = 1011 m −1 s−1 for the peptide binding to 100-nm diameter phospholipid vesicles. As expected theoretically,k on is independent of factors that affect the molar partition coefficient, such as the mole fraction of acidic lipid in the vesicle and the salt concentration. The dissociation rate constant (k off) is ∼10 s−1(lifetime = 0.1 s) for vesicles with 10% acidic lipid in 100 mm KCl. Ca2+-calmodulin (Ca2+·CaM) decreases markedly the lifetime of the peptide on vesicles, e.g. from 0.1 to 0.01 s in the presence of 5 μm Ca2+·CaM. Our results suggest that Ca2+·CaM collides with the membrane-bound MARCKS-(151–175) peptide and pulls the peptide off rapidly. We discuss the biological implications of this switch mechanism, speculating that an increase in the level of Ca2+-calmodulin could rapidly release phosphatidylinositol 4,5-bisphosphate that previous work has suggested is sequestered in lateral domains formed by MARCKS and MARCKS-(151–175).

The role of electrostatic and nonpolar interactions in the association of peripheral proteins with membranes

Abstract The membrane association of acylated and prenylated peripheral proteins, such as Src, the myristoylated alanine-rich C kinase substrate (MARCKS), and K-ras4B, plays an important role in cellular signal transduction. This chapter reviews experimental and computational studies of the membrane partitioning of peptides corresponding to the membrane-interacting regions of these proteins. The computational model partitions the membrane interaction free energies into three components: electrostatic attraction between basic groups on the protein and acidic phospholipids in the membrane, desolvation of the protein and membrane as they associate, and nonpolar burial of aromatic side chains into the membrane interface. The electrostatic components of the binding free energy are calculated by solving the Poisson-Boltzmann equation for protein/ membrane systems represented in atomic detail (finite-difference Poisson-Boltzmann [FDPB] method). The nonpolar component is calculated as the product of an interfacial hydrophobicity coefficient and the change in solvent-accessible surface area of aromatic side chains as they penetrate the interface. The model predicts how membrane association changes as a function of the electrostatic properties of the system and how different combinations of electrostatic and nonpolar forces dictate a wide range of membrane-binding properties. The success of the FDPB methodology in describing experimentally characterized biophysical systems establishes the applicability of classical electrostatics and the continuum approach to protein/ membrane systems and justifies its extension to predicting the structural origins of the interfacial association of proteins of known structure. The biological implications of recent experimental measurements of the partitioning of MARCKS onto membranes containing phosphatidylinositol 4,5-bisphosphate are discussed.

Fluorescently labeled neomycin as a probe of phosphatidylinositol-4,5-bisphosphate in membranes

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a minor component of the plasma membrane, is important in signal transduction, exocytosis, and ion channel activation. Thus fluorescent probes suitable for monitoring the PI(4,5)P(2) distribution in living cells are valuable tools for cell biologists. We report here three experiments that show neomycin labeled with either fluorescein or coumarin can be used to detect PI(4,5)P(2) in model phospholipid membranes. First, addition of physiological concentrations of PI(4,5)P(2) (2%) to lipid vesicles formed from mixtures of phosphatidylcholine (PC) and phosphatidylserine (PS) enhances the binding of labeled neomycin significantly (40-fold for 5:1 PC/PS vesicles). Second, physiological concentrations of inositol-1,4,5-trisphosphate (10 microM I(1,4,5)P(3)) cause little translocation of neomycin from PC/PS/PI(4,5)P(2) membranes to the aqueous phase, whereas the same concentrations of I(1,4,5)P(3) cause significant translocation of the green fluorescent protein/phospholipase C-delta pleckstrin homology (GFP-PH) constructs from membranes (Hirose et al., Science, 284 (1999) 1527). Third, fluorescence microscopy observations confirm that one can distinguish between PC/PS vesicles containing either 0 or 2% PI(4, 5)P(2) by exposing a mixture of the vesicles to labeled neomycin. Thus fluorescently labeled neomycin could complement GFP-PH constructs to investigate the location of PI(4,5)P(2) in cell membranes.

Pore-forming action of mastoparan peptides on liposomes: a quantitative analysis

We have investigated the wasp venom peptides mastoparan X and polistes mastoparan regarding their apparent potential to induce pore-like defects in phosphatidylcholine unilamellar vesicles. Based on a fundamental theoretical model, the pore activation and deactivation kinetics have been evaluated from the observed efflux of liposome entrapped carboxyfluorescein in relation to the bound peptide to lipid ratio. We can quantitatively describe our experimental data very well in terms of a specific reaction scheme resulting in only a few short-lived pores. They evidently emerge rapidly from a prepore nucleus being produced by two rate-limiting monomeric states of bound peptide. These peculiar states would be favorably populated in an early stage of bilayer perturbation, but tend to die out in the course of a peptide/lipid restabilization process.

Microelectrophoresis of a bilayer-coated silica bead in an optical trap: application to enzymology.

We describe an apparatus that combines microelectrophoresis and laser trap technologies to monitor the activity of phosphoinositide-specific phospholipase C-delta1 (PLC-delta) on a single bilayer-coated silica bead with a time resolution of approximately 1 s. A 1-microm-diameter bead was coated with a phospholipid bilayer composed of electrically neutral phosphatidylcholine (PC) and negatively charged phosphatidylinositol 4,5-bisphosphate (2% PIP2) and captured in a laser trap. When an AC field was applied (160 Hz, 20 V/cm), the electrophoretic force produced a displacement of the bead, Delta(x), from its equilibrium position in the trap; Delta(x), which was measured using a fast quadrant diode detector, is proportional to the zeta potential and thus to the number of PIP2 molecules on the outer leaflet (initially, approximately 10(5)). When a solution containing PLC-delta flows past the bead, the enzyme adsorbs to the surface and hydrolyzes PIP2 to form the neutral lipid diacylglycerol. We observed a nonexponential decay of PIP2 on the bead with time that is consistent with a model based on the known structural properties of PLC-delta.