Juxtamembrane Protein Segments that Contribute to Recruitment of Cholesterol into Domains

Abstract

We investigated the properties of several peptides with sequences related to LWYIK, a segment found in the gp41 protein of HIV and believed to play a role in sequestering this protein to a cholesterol-rich domain in the membrane. This segment fulfills the requirements to be classified as a CRAC motif that has been suggested to predict those proteins that will partition into cholesterolrich regions of the membrane. All of the peptides were studied with the terminal amino and carboxyl groups blocked, i.e. as N-acetyl-peptide-amides. Effects of cholesterol on the intensity of W emission generally parallel DSC evidence of sequestration of cholesterol. Modeling studies indicate that all of these peptides tend to partition with their mass center at the membrane interface at the level of the hydroxyl of cholesterol. Interaction with cholesterol is dual: van der Waals interactions between mainly hydrophobic surfaces and, electrostatic stabilization of the cholesterol OH group. Thus, both experiments and modeling studies indicate that preference of CRAC motifs for cholesterol-rich domains might be related to a membrane interfacial preference of the motif, to a capacity to wrap and block the cholesterol polar OH by H-bond interactions and to a capacity for peptide aromatic side chains to stack with cholesterol. These results were supported by studies of single mutations in the gp41 protein of HIV-1, in which L 679 is replaced with I. Despite the similarity of the properties of these amino acid residues, this single substitution resulted in a marked decrease in the capability of JC53-BL HeLa-based HIV-1 indicator cells to form syncytia.It has been demonstrated that peptides and proteins can cause the redistribution of cholesterol in membranes (1). Triggering domain formation may play an important role in signal transduction (2). One class of domains thought to be present in biological membranes has been termed "rafts". Although there is controversy about the nature and prevalence of such domains, it is well established that caveolae are distinct domains in the membrane with many chemical and physical properties similar to those proposed for "rafts".

Only two amino acids are essential for cytolytic toxin recognition of cholesterol at the membrane surface

“…28). Significant structural and operational differences, however, exist between the CDC cholesterol recognition motif and the CRAC motif.…”

Identification of a Binding Motif in the S5 Helix That Confers Cholesterol Sensitivity to the TRPV1 Ion Channel

“…One established sequence selective for binding cholesterol is the putative CRAC motif, first identified in the benzodiazepine receptor (43) and later identified in many other proteins and peptides (40,44,45). This motif is defined as a sequence pattern of -(Leu/Val-(X) 1-5 -Tyr-(X) 1-5 -Arg/Lys)-, in which (X) [1][2][3][4][5] represents between one and five residues of any amino acid (40,45). Cholesterol interacts with the CRAC motif with both attractive van der Waals interactions between hydrophobic surfaces and electrostatic interactions between the positively charged Arg or Lys residue and the cholesterol -OH group (40,45).…”

“…This motif is defined as a sequence pattern of -(Leu/Val-(X) 1-5 -Tyr-(X) 1-5 -Arg/Lys)-, in which (X) [1][2][3][4][5] represents between one and five residues of any amino acid (40,45). Cholesterol interacts with the CRAC motif with both attractive van der Waals interactions between hydrophobic surfaces and electrostatic interactions between the positively charged Arg or Lys residue and the cholesterol -OH group (40,45). In a study of a peptide from the fusogenic gp41 protein of HIV-1, Epand et al (40) found that the single Leu to Ile substitution in the CRAC motif (from LWYIK to IWYIK) resulted in no preferential interaction with cholesterol (33).…”

“…These correspond to a larger cluster (36 of 200 results with root mean square deviations below 2 Å) interacting at the site identified by mutagenesis, calculated with the Autodock suite. In this model of cholesterol binding, the OH group makes a possible electrostatic interaction with Arg 579 and the flat ␣-face of cholesterol is stacked against Phe 582 , which is an interaction seen very often in crystal structures of proteins bound to cholesterol and other sterols (20,40). The important interaction with residue Leu 585 seems to be mediated by the aliphatic tail of cholesterol, which enters an incipient cavity formed by Leu 585 and other hydrophobic residues.…”

Membrane-Transferring Regions of gp41 as Targets for HIV-1 Fusion Inhibition and Viral Neutralization

“…MPER carboxy-terminal LWYIK sequence has been identified as a potential "cholesterol recognition/interaction amino acid consensus" (CRAC) domain functional in fusion [83][84][85][86][87]. Recently reported experimental evidence and modeling studies provide the physicochemical grounds for a direct interaction of MPER-CRAC with Chol [84,85,88,89]. MPER-Chol interaction would be based on two capacities of CRAC sequence, namely, wrapping and blocking of the interfacial cholesterol OH group by H-bond interactions, and stacking of aromatic side chains with A ring of cholesterol.…”