An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins

Abstract

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 ...

Cell Migration and Invadopodia Formation Require a Membrane-binding Domain of CARMIL2

“…To investigate the molecular mechanism for membrane targeting, we started with a computer-based algorithm that identifies clusters of basic and hydrophobic residues, as a tool to find potential intrinsically disordered membrane-binding domains (23). An 11-aa region (residues 1060 -1070) was identified (Fig.…”

“…Membrane-binding Domain Search-The CARMIL2 membrane-binding domain was initially identified with a computer program using an algorithm that detects basic and hydrophobic residues (helixweb.nih.gov/bhsearch) (23 PM Index Calculation-We quantitated targeting of GFP fusions to the plasma membrane from fluorescence images by calculating a plasma-membrane index as described (24). To account for volume effects, td-Tomato was co-expressed from a second plasmid (pBJ 2090, Table 1).…”

“…One is the PH domain (20); the other is a basic and hydrophobic domain in a position analogous to the one described here for CARMIL2. The basic and hydrophobic domain was predicted from sequence analysis, and a relatively large fragment containing that region was found to bind liposomes (23).…”

Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI

“…Furthermore, our sequence analysis predicted six lipid-binding domains along the myosin-XXI C terminus following the converter, with basic residues flanking a central hydrophobic patch, as described for nonspecific charged interactions with phospholipids for Acanthamoeba, Dictyostelium, and human myosin class I (25)(26)(27). Two of those regions scored significantly on the basic-hydrophobic scale, as described by Brzeska et al (27), to identify putative unstructured lipid-binding sites in myosin tail sequences. The consensus sequence for a phospholipid-binding PX domain that overlaps with the converter region of myosin XXI seemed to be unique among myosins.…”

“…Two of those domains overlapped with potentially unstructured lipid-binding domains (ULBDs in Fig. 5A) rich in basic and hydrophobic residues, which were identified by calculating a basic-hydrophobic score over a running window of 20-amino-acid residues along the sequence, as described by Brzeska et al (27) (Fig. 5D).…”