Protein-Lipid Interactions: Correlation of a predictive algorithm for lipid-binding sites with three-dimensional structural data

Renal Unit, Leukocyte Biology & Inflammation Program, Structural Biology Program and the Massachusetts General Hospital/Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA.
Theoretical Biology and Medical Modelling (Impact Factor: 0.95). 02/2006; 3(1):17. DOI: 10.1186/1742-4682-3-17
Source: PubMed


Over the past decade our laboratory has focused on understanding how soluble cytoskeleton-associated proteins interact with membranes and other lipid aggregates. Many protein domains mediating specific cell membrane interactions appear by fluorescence microscopy and other precision techniques to be partially inserted into the lipid bilayer. It is unclear whether these protein-lipid-interactions are dependent on shared protein motifs or unique regional physiochemistry, or are due to more global characteristics of the protein.
We have developed a novel computational program that predicts a protein's lipid-binding site(s) from primary sequence data. Hydrophobic labeling, Fourier transform infrared spectroscopy (FTIR), film balance, T-jump, CD spectroscopy and calorimetry experiments confirm that the interfaces predicted for several key cytoskeletal proteins (alpha-actinin, Arp2, CapZ, talin and vinculin) partially insert into lipid aggregates. The validity of these predictions is supported by an analysis of the available three-dimensional structural data. The lipid interfaces predicted by our algorithm generally contain energetically favorable secondary structures (e.g., an amphipathic alpha-helix flanked by a flexible hinge or loop region), are solvent-exposed in the intact protein, and possess favorable local or global electrostatic properties.
At present, there are few reliable methods to determine the region of a protein that mediates biologically important interactions with lipids or lipid aggregates. Our matrix-based algorithm predicts lipid interaction sites that are consistent with the available biochemical and structural data. To determine whether these sites are indeed correctly identified, and whether use of the algorithm can be safely extended to other classes of proteins, will require further mapping of these sites, including genetic manipulation and/or targeted crystallography.

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    • "Most cell–matrix contacts are linked to the actin filaments via the heterodimeric transmembrane protein integrin and the focal adhesion complex (FAC) proteins. Within FAC, a variety of adaptor proteins have been implicated to associate with or in some cases insert into lipid membranes [1]. The focal adhesion protein vinculin shows such lipid binding ability. "
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    ABSTRACT: The focal adhesion protein vinculin has been implicated in associating with soluble and membranous phospholipids. Here, we investigated the intermolecular interactions of two vinculin tail domains with membrane phospholipids. Previous studies have shown that the tail's unstructured C-terminus affects the mechanical behavior of cells, but not the H3 region. The aim of this work was to establish whether the C-terminal or the H3 region either associate favorably with or anchor in lipid membranes. This work characterizes the energetics and dynamics of phospholipid interactions using differential scanning calorimetry (DSC) as well as circular dichroism (CD) spectroscopy. Biochemical data from tryptophan quenching and SDS-PAGE experiments support calorimetric and CD spectroscopic findings insofar that only vinculin's C-terminus inserts into lipid membranes. These in vitro results provide further insight into the mechanical behavior of vinculin tail regions in cells and contribute to the understanding of their structure and function.
    Preview · Article · Jul 2010 · Biochemical and Biophysical Research Communications
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    • "It is not yet clear whether these interactions in vivo are associated with membrane or soluble lipids in the cytosol. According to studies in my laboratory (Tempel et al., 1995; Scott et al., 2006), the entire 15 residues at the C-terminal region of the wild-type vinculin tail (1052–1066) can interact with lipids. These 15 residues are highly flexible in the crystal structure (Bakolitsa et al., 2004). "
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    ABSTRACT: Cell adhesion and cell-cell contacts are a pre-requisite for proper metabolism, protein synthesis and cell survival. Integrins are the transmembrane receptors that link the extracellular matrix via the FAC (focal adhesion complex) with the cytoskeleton. Vinculin is a pivotal FAC protein that has not only been implicated in regulating FAC formation and transmitting mechanical forces, but also in associating with membranous lipids in biological systems.
    Preview · Article · Apr 2010 · Cell Biology International
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    • "Some of these proteins are believed to interact transiently with the lipid membrane [4]. However, the nature and cellular function of these interactions are poorly understood [5] [6]. Vinculin is one of the FAC proteins that shows in vitro and in vivo lipid-binding capabilities [7] [8] [9] [10] [11] [12]. "
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    ABSTRACT: The focal adhesion protein vinculin (1066 residues) plays an important role in cell adhesion and migration. The interaction between vinculin and lipid membranes is necessary to ensure these processes. There are three putative lipid-membrane interaction sites located at the vinculin tail domain two that form amphipathic alpha-helices (residues 935-978 and 1020-1040) and one that remains unstructured (residues 1052-1066) during crystallization. In this work, the structural and biochemical properties of the last 21 residues of the vinculin tail domain were investigated. Differential scanning calorimetry was performed in the presence of lipid vesicles consisting of dimyristoyl-L-alpha-phosphatidylcholine and dimyristoyl-L-alpha-phosphatidylglycerol at various molar ratios. The results demonstrate that this peptide inserts into lipid vesicle membranes. Examining the secondary structure of this peptide by molecular dynamics simulations and circular dichroism spectroscopy, we show that it adopts an antiparallel beta sheet backbone geometry that could ensure the association with lipid vesicles.
    Full-text · Article · Sep 2008 · Biochemical and Biophysical Research Communications
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