Programs exist for searching protein sequences for potential membrane-penetrating segments (hydrophobic regions) and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains. However, a rapidly growing number of membrane-associated proteins (including cytoskeletal proteins, kinases, GTP-binding proteins, and their effectors) bind lipids through less structured regions. Here, we describe the development and testing of a simple computer search program that identifies unstructured potential membrane-binding sites. Initially, we found that both basic and hydrophobic amino acids, irrespective of sequence, contribute to the binding to acidic phospholipid vesicles of synthetic peptides that correspond to the putative membrane-binding domains of Acanthamoeba class I myosins. Based on these results, we modified a hydrophobicity scale giving Arg-and Lyspositive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membranebinding sites in all 16 proteins tested. Importantly, basic and hydrophobic searches identified previously unknown potential membrane-binding sites in class I myosins, PAKs and CARMIL (capping protein, Arp2/3, myosin I linker; a membrane-associated cytoskeletal scaffold protein), and synthetic peptides and protein domains containing these newly identified sites bound to acidic phospholipids in vitro.Recently, there has been considerable interest in characterizing protein domains, such as PH, FERM, C2, and ENTH, that are responsible for specific binding to membrane lipids (for review, see Ref. 1). These domains have highly defined tertiary structures comprising ␣-helices, -sheets and loops, and there are multiple programs for recognizing them in protein sequences. However, a growing number of membrane-binding proteins, including cytoskeletal proteins (2), GTP-binding proteins (e.g. Rit), and GTP-binding protein effectors (e.g. some PAKs) (see references below), do not fit within these categories and bind membrane lipids through much less structured regions. Here, we describe the development and testing of a simple computer search program that identifies such potential membrane-binding sites. This novel search program evolved from our previous effort (3) to identify the membrane-binding site in the heavy chain of Acanthamoeba myosin IC (AMIC) 2 (4), which was known to bind to acidic phospholipids (3, 5, 6) and cell membranes (7-10) through its 220-residue basic region (5).We had found (3) that AMIC binds nonspecifically to acidic phospholipid vesicles in proportion to their negative charge. Prompted by the report (11) that several proteins bind to acidic phospholipids through a basic-hydrophobic-basic (BHB) region consisting of two small clusters of basic amino acids separated by hydrophobic residues, we identified, by visual inspection, a 13-residue BHB sequence, KVKPFLYVLKRR, within the basic region of the AMIC heavy chain (3). A synthetic ...
Background: Class I myosins contribute to membrane-associated events. Results: A short segment of basic/hydrophobic amino acids in the tail which binds acidic phospholipids and the actin binding site in the head is required for relocalization of Dictyostelium myosin IB. Conclusion: Dynamic relocalization results from competition between membrane acidic phospholipids and cytoplasmic F-actin. Significance: The molecular basis of myosin I relocation is fundamental to understanding cell motility.
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