Cholesterol-recognition motifs in the transmembrane domain of the tyrosine kinase receptor family: The case of TRKB.

Cecilia Cannarozzo,Ilpo Vattulainen,Plinio C Casarotto,Mykhailo Girych,Senem Merve Fred,Caroline Biojone,Eero Castrén,Tomasz Róg,Giray Enkavi

doi:10.1111/ejn.15218

Abstract

Cholesterol is an essential constituent of cell membranes. The discovery of cholesterol-recognition amino acid consensus (CRAC) motif in proteins indicated a putative direct, non-covalent interaction between cholesterol and proteins. In the present study, we evaluated the presence of a CRAC motif and its inverted version (CARC) in the transmembrane region (TMR) of the tyrosine kinase receptor family (RTK) in several species using in silico methods. CRAC motifs were found across all species analyzed, while CARC was found only in vertebrates. The tropomyosin-related kinase B (TRKB), a member of the RTK family, through interaction with its endogenous ligand brain-derived neurotrophic factor (BDNF) is a core participant in the neuronal plasticity process and exhibits a CARC motif in its TMR. Upon identifying the conserved CARC motif in the TRKB, we performed molecular dynamics simulations of the mouse TRKB.TMR. The simulations indicated that cholesterol interaction with the TRKB CARC motif occurs mainly at the central Y433 residue. Our binding assay suggested a bell-shaped effect of cholesterol on BDNF interaction with TRKB receptors, and our results suggest that CARC/CRAC motifs may play a role in the function of the RTK family TMR.

Highlights

The human brain contains 23% of the body's total cholesterol
We recently identified a CARC domain in tropomyosin- related kinase B (TRKB) and showed that its mutation interferes with plasticity-related brain-derived neurotrophic factor (BDNF) signaling (Casarotto et al, 2021)
We found that moderate concentrations of added cholesterol (20 μM) facilitate biotinylated BDNF (bBDNF) binding to TRKB, while higher amounts of cholesterol (50 or 100 μM) compromise bBDNF interaction with immobilized TRKB (Figure 3d)

Summary

| INTRODUCTION

The human brain contains 23% of the body's total cholesterol. Most of this cholesterol is found in the myelin sheath of oligodendrocytes (Dietschy & Turley, 2004; Martin et al, 2014). Cholesterol can interact with transmembrane domains to regulate protein function (Elkins et al, 2018; Fantini & Barrantes, 2013). In silico models suggest that two cholesterol molecules can interact in a tail-to-tail fashion as a transbilayer dimer (Harris et al, 1995; Rukmini et al, 2001) or back-to-back through their flat alpha faces, leaving the beta sides accessible for interactions with proteins (Hanson et al, 2008) On these target proteins, the following two consensus motifs with predictive value have been defined (Di Scala et al, 2017): the Cholesterol-Recognition Amino acid Consensus sequence (CRAC) and its “inverted” version (CARC) (Baier et al, 2011; Li & Papadopoulos, 1998). We expanded our previous findings about the mechanisms behind the cholesterol effect on BDNF-induced TRKB activity (Casarotto et al, 2021) by assaying the binding of biotinylated BDNF to immobilized TRKB

| METHODS

| RESULTS

Findings

| DISCUSSION