Relative Affinities of Protein-Cholesterol Interactions from Equilibrium Molecular Dynamics Simulations

Abstract

Specific interactions of lipids with membrane proteins contribute to protein stability and function. Multiple lipid interactions surrounding a membrane protein are often identified in molecular dynamics (MD) simulations and are, increasingly, resolved in cryo-EM densities. Determining the relative importance of specific interaction sites is aided by determination of lipid binding affinities by experimental or simulation methods. Here, we develop a method for determining protein-lipid binding affinities from equilibrium coarse-grained MD simulations using binding saturation curves, designed to mimic experimental protocols. We apply this method to directly obtain affinities for cholesterol binding to multiple sites on a range of membrane proteins and compare our results with free energies obtained from density-based equilibrium methods and with potential of mean force calculations, getting good agreement with respect to the ranking of affinities for different sites. Thus, our binding saturation method provides a robust, high-throughput alternative for determining the relative consequence of individual sites seen in e.g. cryo-EM derived membrane protein structures surrounded by a plethora of ancillary lipid densities.

Hepatocyte cholesterol content modulates glucagon receptor signalling

“…The site-4 K d app was comparable to ‘strong’ cholesterol binding sites observed on various other membrane proteins e.g. on the TRP channel Polycystin-2 (11 ± 1%), on Patched1 (6.8 ± 0.3%) and on the GPCR 5-HT 1A (4–9%) where cholesterol densities have been observed in structures and cholesterol is implicated in biological function [ 31 ]. While interpreting the relative specificity of cholesterol sites from kinetics alone can be challenging, saturable binding curves were observed which were distinct from background non-specific interactions ( Figure 3 E, black line).…”

“…To further evaluate the relevance of identified sites we performed simulations using membrane bilayers with varying cholesterol content [ 31 ] which allowed us to estimate apparent dissociation constants for cholesterol at each site ( Figure 3 E): K d app at site-1: 10.7 ± 0.3%; site-2: 18.3 ± 0.4%; site-3: 15.0 ± 0.8%; and site-4: 6.6 ± 0.1%. The site-4 K d app was comparable to ‘strong’ cholesterol binding sites observed on various other membrane proteins e.g.…”

LipIDens: Simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins

“…In particular, if there is already data suggesting a biological role for a specific lipid, it would of course be wise to include this in the bilayer component of the simulation. In addition we note there is likely to be some bias in the initial density map towards lipids with strong interactions which are able to survive membrane protein purification, as has been suggested by previous affinity calculations 46 .…”

Specific interactions of peripheral membrane proteins with lipids: what can molecular simulations show us?

“…As MSM provides information about transitions, it constitutes a promising tool for precise descriptions of complex binding events involving intermediate states. Another intriguing new method demonstrates that it may be possible to directly extract affinities in the form of apparent dissociation constants (K d ) from the simulation of binding saturation curves (174).…”