Myristoylation, lipid anchors

Proteins can be attached to the membrane in a variety of ways. When a protein completely spans the membrane, it is often in the form of an a-helix or jS-sheet. These structures minimize contact of the polar parts of the peptide backbone with the nonpolar lipids in the interior of the bilayer (Figure 8.17). Proteins can also be anchored to the lipids via covalent bonds from cysteines or free amino groups on the protein to one of several lipid anchors. Myristoyl and palmitoyl groups are common anchors (Figure 8.17). 

Four different types of lipid-anchoring motifs have been found to date. These are amide-linked myristoyl anchors, thioester-linked fatty acyl anchors, thioether-linked prenyl anchors, and amide-linked glycosyl phosphatidylinosi-tol anchors. Each of these anchoring motifs is used by a variety of membrane proteins, but each nonetheless exhibits a characteristic pattern of structural requirements. 

Fig. 3.13. Lipid anchors and basic regions as elements of the membrane association of proteins. Examples for proteins which exhibit basic residues near a lipid anchor, a) Src kinase (see ch. 8) possess a myristoyl anchor at the N-terminus as well as a stretch of basic residues, b) In Ki-Ras proteins (see ch. 9) there is a farnesyl residue at the C-terminus that serves as a lipid anchor, as weU as a stretch of Lys residues. Negatively charged head groups of phospholipids are shown as filled circles. X any amino acid.

Palmitoylation is, after myristoylation, the most common modification of the a-sub-rmit of the heterotrimeric G-proteins (see chapter 5). The a-subunit of G-proteins can be lipidated in a two-fold marmer, with a myristoic acid and a pahnitoic acid anchor at the N-terminus. It appears in this case that two lipid anchors are necessary to mediate a stable association of the protein with the membrane. The lipidation of cytoplasmic protein tyrosine kinase also includes both myristoylation and palmitoylation. H-Ras protein also requires, apart from C-terminal farnesylation (see below), a pahnitoyl modification in order to bind to the plasma membrane. In all mentioned examples the fatty acid anchors play an essential role in the signal transduction. 

Recoverin is a Ca receptor with four EF structures and two Ca binding sites it can exist in the cytosol or associated with the membrane and has an N-terminal myristoyl residue as a lipid anchor. The distribution between free and membrane-associated forms is regulated by Ca. Binding of Ca to recoverin leads to its translocation from the cytosol to the membrane of the rod cells. Structural determination of recoverin in the Ca bound and Ca free forms (Ames et al., 1997) indicates that membrane association of recoverin is regulated by a Ca -myristoyl switch. The myristoyl residue can adopt two alternative positions in recoverin. In the absence of Ca, recoverin exists in a conformation in which the myristoyl residue is hidden in the iimer of the protein and is not available for membrane association. On Ca binding, a conformation change of recoverin takes place the myristoyl residue moves to the outside and can now associate with the membrane. 

The number of myristoylated proteins identified in viruses is constantly increasing. Nevertheless, substantial additional work will be required to shed light on the multiple functions that could be executed by the respective proteins as well as their involvement in the viral life cycle and elucidation of the role of the lipid anchor. Inhibition of NMT or replacing myristic acid by analogs in infected cells is known to affect numerous viruses of medical importance, for example, HIV (Bryant et al. 1989), hepatitis B virus (Parang et al. 1997), herpesviruses (Harper et al. 1993), and... 

Most of the biological functions of the members of the Ras superfamily are linked to the cytoplasmic side of the cell membrane, where specific signals are received and transmitted further. Accordingly, Ras superfamily members contain structural features that mediate membrane association and serve as lipid anchors (see Section 3.7). Prenylation, palmitoylation and myristoylation are post-translational modifications frequently found on Ras superfamily members. 

Lipidation -acylation TV -myristoylation Myristoy-lation S-prenylation Prenylation Palmitoylation Isoprenylation GPI anchors Glypiation... 

G-protein a-subunits also possess specific residues that can be covalently modified by bacterial toxins. Cholera toxin catalyzes the transfer of ADP-ribose moiety of NAD to a specific arginine residue in certain a-subunits, whereas pertussis toxin ADP-ribosylates those a-subunits that contain a specific cysteine residue near the carboxy-terminus. Modification of the a-subunit by cholera toxin persistently activates these protein by inhibiting their GTPase activity, whereas pertussis toxin inactives Gia protein and thereby results in the uncoupling of receptor from the effector. G-protein a-subunits are regulated by covalent modifications by fatty acids myristate and palmate. These lipid modifications serve to anchor the subunits to the membrane and increase the interaction with other protein and also increase the affinity of the a-subunit for 3y. In this regard, the myristoylation of Gia is required for adenylyl cyclase inhibition in cell-free assay (Taussig et al. 1993). 

In addition to creating recognition motifs to recruit proteins, a few PTM can also increase interaction with other species, such as the lipid bilayer of different cellular membranes. These modifications include the formation of GPI-anchored proteins (20), protein myristoylation on the a-amino group of the N-terminal... 

Fig. 1. Structures of lipids covalently attached to proteins. Panel A shows proteins that are lipidated on cytoplasmi-cally exposed amino acids, whereas panel B shows lipidated proteins in the extracellular leaflet. (A) iV-myristoyl glycine, palmitate thioester-linked to cysteine, farnesyl, or geranylgeranyl (prenyl) thioether-linked to cysteine. (B) A/-palmitoyl cysteine, cholesterol ester-linked to glycine, and a minimal GPI anchor linked to the to amino acid in a GPI-anchored protein. The GPI structure is shown with a diacylglycerol moiety containing two ester-linked fatty acids. Other GPI anchors are based on ceramide, while yet others have monoacylglycerol, a fatty acid ether-linked to glycerol, and/or a fatty acid ester-linked to inositol.

Lipopeptides, peptides modified with lipid residues which are preferentially bound at the thiol function of cysteine or at the a-amino group of N-terminal amino acids. The a-subunits of heteromeric G proteins and non-tyrosine receptor kinases contain N-myristoylated N-terminal glycine residues together with S-palmitoylation of a neighboring cysteine residue. Lipid moieties are necessary to recruit and anchor peptides and proteins to the membrane. Furthermore, it has been postulated that lipidation of proteins represents an event in signal transduction. The synthesis of lipid-modified peptides is not easy to perform as all coupling and deprotection reactions must be carried out under very mild conditions [S. Moffet et al., EMBO J. 1993, 12, 349 D. Kadereit et al., Chem. Fur./. 2001, 7,1184]. 

The four major types of protein lipidation are A-myristoylation, palmitoylation, prenylation and glycosylphosphatidylinositol-anchor (GPI-anchor) addition (Table 1). 

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