An Experimentally Based Computer Search Identifies Unstructured Membrane-binding Sites in Proteins: APPLICATION TO CLASS I MYOSINS, PAKS, AND CARMIL

Edward D Korn,Hanna Brzeska,Jake Guag,Susan Chacko,Kirsten Remmert

doi:10.1074/jbc.m109.066910

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 Lys-positive, rather than negative, values. Using this basic and hydrophobic scale with a standard search algorithm, we successfully identified previously determined unstructured membrane-binding 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.

Highlights

Programs exist for searching protein sequences for potential membrane-penetrating segments and for lipid-binding sites with highly defined tertiary structures, such as PH, FERM, C2, ENTH, and other domains
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 Acanthamoeba myosin IC (AMIC) heavy chain [3]
There was no significant increase in fluorescence when the peptide was exposed to vesicles composed of 100% PC (Fig. 1A) or when dansyl-L-glutamine was exposed to 50% PS or 100% PC vesicles. These results are consistent with the increase in fluorescence of the dansylAMIC(tail) peptide being due to binding of the peptide to the acidic phospholipid vesicles

Summary

EXPERIMENTAL PROCEDURES

Phospholipid Vesicles—PS and PC were purchased from Avanti Polar Lipids (Alabaster, AL). The expressed proteins were mixed with various concentrations of vesicles in 20 mM imidazole, pH 7.0, 100 mM NaCl, 1 mM EGTA, and 0.5 mg/ml bovine serum albumin and centrifuged at 200,000 ϫ g for 40 min at 20 °C. Binding of CARMIL Fragment to Phospholipid Vesicles—The CAH3 fragment of mouse CARMIL (Q6EDY6) corresponding to residues 961–1085 of the full-length protein, with N-terminal GFP and C-terminal His tags, was expressed in Escherichia coli and purified by absorption on nickel resin as described by Fujiwara et al [14]. The purified monomeric GFP-mouse CAH3 fragment (0.6 ␮M) was mixed with varied concentrations of 50% PS vesicles in the same buffer used for binding synthetic peptides and centrifuged as described above for the peptide binding assay. After BH search was developed, the EMBOSS program was modified to be more convenient for this application (http://helixweb.nih.gov/bhsearch)

RESULTS

Ն50 Ͼ125 Ն28

19 Binds calmodulin

DISCUSSION