Diana Murray

Electrostatic interaction of myristoylated proteins with membranes: simple physics, complicated biology

Cell membrane association by several important peripheral proteins, such as Src, MARCKS, HIV-1 Gag, and K-Ras, requires nonspecific electrostatic interactions between a cluster of basic residues on the protein and acidic phospholipids in the plasma membrane. A simple theoretical model based on the nonlinear Poisson-Boltzmann equation describes well the experimentally measured electrostatic association between such proteins and the cell membrane.

Electrostatic Properties of Membranes Containing Acidic Lipids and Adsorbed Basic Peptides: Theory and Experiment

The interaction of heptalysine with vesicles formed from mixtures of the acidic lipid phosphatidylserine (PS) and the zwitterionic lipid phosphatidylcholine (PC) was examined experimentally and theoretically. Three types of experiments showed that smeared charge theories (e.g., Gouy-Chapman-Stern) underestimate the membrane association when the peptide concentration is high. First, the zeta potential of PC/PS vesicles in 100 mM KCl solution increased more rapidly with heptalysine concentration (14.5 mV per decade) than predicted by a smeared charge theory (6.0 mV per decade). Second, changing the net surface charge density of vesicles by the same amount in two distinct ways produced dramatically different effects: the molar partition coefficient decreased 1000-fold when the mole percentage of PS was decreased from 17% to 4%, but decreased only 10-fold when the peptide concentration was increased to 1 microM. Third, high concentrations of basic peptides reversed the charge on PS and PC/PS vesicles. Calculations based on finite difference solutions to the Poisson-Boltzmann equation applied to atomic models of heptalysine and PC/PS membranes provide a molecular explanation for the observations: a peptide adsorbing to the membrane in the presence of other surface-adsorbed peptides senses a local potential more negative than the average potential. The biological implications of these discreteness-of-charge effects are discussed.

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.

Outcome of a Workshop on Applications of Protein Models in Biomedical Research

We describe the proceedings and conclusions from the Workshop on Applications of Protein Models in Biomedical Research (the Workshop) that was held at the University of California, San Francisco on 11 and 12 July, 2008. At the Workshop, international scientists involved with structure modeling explored (i) how models are currently used in biomedical research, (ii) the requirements and challenges for different applications, and (iii) how the interaction between the computational and experimental research communities could be strengthened to advance the field.

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.

The “Electrostatic-Switch” Mechanism: Monte Carlo Study of MARCKS-Membrane Interaction

The binding of the myristoylated alanine-rich C kinase substrate (MARCKS) to mixed, fluid, phospholipid membranes is modeled with a recently developed Monte Carlo simulation scheme. The central domain of MARCKS is both basic (zeta = +13) and hydrophobic (five Phe residues), and is flanked with two long chains, one ending with the myristoylated N-terminus. This natively unfolded protein is modeled as a flexible chain of beads representing the amino acid residues. The membranes contain neutral (zeta = 0), monovalent (zeta = -1), and tetravalent (zeta = -4) lipids, all of which are laterally mobile. MARCKS-membrane interaction is modeled by Debye-Hückel electrostatic potentials and semiempirical hydrophobic energies. In agreement with experiment, we find that membrane binding is mediated by electrostatic attraction of the basic domain to acidic lipids and membrane penetration of its hydrophobic moieties. The binding is opposed by configurational entropy losses and electrostatic membrane repulsion of the two long chains, and by lipid demixing upon adsorption. The simulations provide a physical model for how membrane-adsorbed MARCKS attracts several PIP(2) lipids (zeta = -4) to its vicinity, and how phosphorylation of the central domain (zeta = +13 to zeta = +7) triggers an electrostatic switch, which weakens both the membrane interaction and PIP(2) sequestration. This scheme captures the essence of discreteness of charge at membrane surfaces and can examine the formation of membrane-mediated multicomponent macromolecular complexes that function in many cellular processes.

A computational interactome and functional annotation for the human proteome.

We present a database, PrePPI (Predicting Protein-Protein Interactions), of more than 1.35 million predicted protein-protein interactions (PPIs). Of these at least 127,000 are expected to constitute direct physical interactions although the actual number may be much larger (~500,000). The current PrePPI, which contains predicted interactions for about 85% of the human proteome, is related to an earlier version but is based on additional sources of interaction evidence and is far larger in scope. The use of structural relationships allows PrePPI to infer numerous previously unreported interactions. PrePPI has been subjected to a series of validation tests including reproducing known interactions, recapitulating multi-protein complexes, analysis of disease associated SNPs, and identifying functional relationships between interacting proteins. We show, using Gene Set Enrichment Analysis (GSEA), that predicted interaction partners can be used to annotate a protein’s function. We provide annotations for most human proteins, including many annotated as having unknown function. DOI: http://dx.doi.org/10.7554/eLife.18715.001

Protein kinase Cθ C2 domain is a phosphotyrosine binding module that plays a key role in its activation

Background: PKCθ plays a key role in T lymphocyte activation, but its regulatory mechanism is not understood. Results: Phosphotyrosine binds the PKCθ C2 domain and activates PKCθ. Conclusion: The PKCθ C2 domain-phosphotyrosine binding is important for PKCθ activation in T cells. Significance: This study provides new mechanistic insight into the regulation of PKCθ in T cells. Protein kinase Cθ (PKCθ) is a novel PKC that plays a key role in T lymphocyte activation. To understand how PKCθ is regulated in T cells, we investigated the properties of its N-terminal C2 domain that functions as an autoinhibitory domain. Our measurements show that a Tyr(P)-containing peptide derived from CDCP1 binds the C2 domain of PKCθ with high affinity and activates the enzyme activity of the intact protein. The Tyr(P) peptide also binds the C2 domain of PKCδ tightly, but no enzyme activation was observed with PKCδ. Mutations of PKCθ-C2 residues involved in Tyr(P) binding abrogated the enzyme activation and association of PKCθ with Tyr-phosphorylated full-length CDCP1 and severely inhibited the T cell receptor/CD28-mediated activation of a PKCθ-dependent reporter gene in T cells. Collectively, these studies establish the C2 domain of PKCθ as a Tyr(P)-binding domain and suggest that the domain may play a major role in PKCθ activation via its Tyr(P) binding.

Genome-wide Structural Analysis Reveals Novel Membrane Binding Properties of AP180 N-terminal Homology (ANTH) Domains

Background: There is a great need for a high throughput computational tool for predicting the function of lipid binding domains on a genomic scale. Results: A newly developed computation protocol allows for genome-wide prediction of membrane binding properties of ANTH domains. Conclusion: Membrane binding properties of proteins can be systematically and reliably predicted by our combinatorial approach. Significance: A novel functionality of lipid binding domains can be computationally predicted. An increasing number of cytosolic proteins are shown to interact with membrane lipids during diverse cellular processes, but computational prediction of these proteins and their membrane binding behaviors remains challenging. Here, we introduce a new combinatorial computation protocol for systematic and robust functional prediction of membrane-binding proteins through high throughput homology modeling and in-depth calculation of biophysical properties. The approach was applied to the genomic scale identification of the AP180 N-terminal homology (ANTH) domain, one of the modular lipid binding domains, and prediction of their membrane binding properties. Our analysis yielded comprehensive coverage of the ANTH domain family and allowed classification and functional annotation of proteins based on the differences in local structural and biophysical features. Our analysis also identified a group of plant ANTH domains with unique structural features that may confer novel functionalities. Experimental characterization of a representative member of this subfamily confirmed its unique membrane binding mechanism and unprecedented membrane deforming activity. Collectively, these studies suggest that our new computational approach can be applied to genome-wide functional prediction of other lipid binding domains.