Membrane Binding of Myristylated Peptides Corresponding to the NH2 Terminus of Src

Abstract

Membrane association is required for cell transformation by pp60v-src (v-Src), the product of the v-src oncogene of Rous sarcoma virus. Previous experiments have identified two NH2-terminal membrane-binding motifs: a myristate (14-carbon acyl chain) attached to the NH2-terminal glycine and three basic residues at positions 5, 7, and 9 of Src. We examined the membrane binding of each motif using myristylated (myr-src) and nonmyristylated (nonmyr-src) peptides corresponding to the NH2 terminus of Src. All myristylated peptides partitioned equally well onto electrically neutral phosphatidylcholine vesicles (K1 = 10(4) M-1). Identical binding has been observed for simple myristylated peptides (e.g., myr-Gly) and arises from the hydrophobic insertion of the myristate into the bilayer. A nonmyristylated peptide corresponding to residues 2-16 of Src [nonmyr-src(2-16), net charge = +5] bound to vesicles containing 33% monovalent acidic phospholipids with K1 = 10(3) M-1. Penta(lysine) (+5 net charge) exhibits the same binding behavior, which is due to the electrostatic interaction between basic residues and acidic lipids. The corresponding myristylated peptide, myr-src(2-16), binds 3 orders of magnitude more strongly to vesicles containing 33% acidic lipids than to neutral vesicles. The resulting apparent association constant, K1 = 10(7) M-1, is approximately equal to the product of the partition coefficients for the two individual interactions. This 10(7) M-1 binding is sufficiently strong to anchor the Src protein to biological membranes. We propose a simple model that explains the observed synergism between the two peptide-membrane interactions.

Total chemical synthesis of N-myristoylated HIV-1 matrix protein p17: Structural and mechanistic implications of p17 myristoylation

“…Myr(Ϫ)-p17 existed as a monomer under the experimental conditions, whereas myr(ϩ)-p17 was in a monomer-trimer equilibrium with a K eq value of 1.2 ϫ 10 8 M Ϫ2 . such interaction alone is insufficient to hold lipid-linked proteins at the membrane surface (36,37), additional stabilizing interactions between the protein and membrane are needed. Despite the multipoint anchoring, lipid proteins can still dissociate from the plasma membrane, an event coupled to functions important in HIV-1 pathogenesis in the case of p17.…”

Identification of the Endoplasmic Reticulum Targeting Signal in Vesicle-associated Membrane Proteins

“…Several myristoylated proteins such as Src and myristoylated alanine-rich protein kinase C substrate require both the hydrophobic acyl chain and a sequence of positively charged residues for membrane binding (31)(32)(33). In this case the cluster of basic residues is required for nonspecific interaction with acidic phospholipids, whereas the myristate is inserted into the lipid bilayer.…”

Interfacial Kinetic and Binding Properties of the Complete Set of Human and Mouse Groups I, II, V, X, and XII Secreted Phospholipases A2

“…However, not all of the human and mouse sPLA 2 s have tryptophans, and we have found that the energy transfer intensity varies dramatically with the phospholipid composition of the interface. 2 Thus, we decided to adopt the procedure of Buser et al (62) in which vesicles are loaded with sucrose so that they pellet in an ultracentrifuge. The amount of sPLA 2 added to DO et PC vesicles to determine the sPLA 2 binding requirement on anionic phospholipid.…”