How Do Branched Detergents Stabilize GPCRs in Micelles?

Abstract

The structural and functional properties of G protein-coupled receptors (GPCRs) are often studied in a detergent micellar environment, but many GPCRs tend to denature or aggregate in short alkyl chain detergents. In our previous work [Lee, S., et al. (2016) J. Am. Chem. Soc. 138, 15425–15433], we showed that GPCRs in alkyl glucosides were highly dynamic, resulting in the penetration of detergent molecules between transmembrane α-helices, which is the initial step in receptor denaturation. Although this was not observed for GPCRs in dodecyl maltoside (DDM, also known as lauryl maltoside), even this detergent is not mild enough to preserve the integrity of many GPCRs during purification. Lauryl maltose neopentylglycol (LMNG) detergents have been found to have significant advantages for purifying GPCRs in a native state as they impart more stability to the receptor than DDM. To gain insights into how they stabilize GPCRs, we used atomistic molecular dynamics simulations of wild type adenosine A2A receptor (WT-A2AR), thermostabilized A2AR (tA2AR), and wild type β2-adrenoceptor (β2AR) in a variety of detergents (LMNG, DMNG, OGNG, and DDM). Analysis of molecular dynamics simulations of tA2AR in LMNG, DMNG, and OGNG showed that this series of detergents exhibited behavior very similar to that of an analogous series of detergents DDM, DM, and OG in our previous study. However, there was a striking difference upon comparison of the behavior of LMNG to that of DDM. LMNG showed considerably less motion than DDM, which resulted in the enhanced density of the aliphatic chains around the hydrophobic regions of the receptor and considerably more hydrogen bond formation between the head groups. This contributed to enhanced interaction energies between both detergent molecules and between the receptor and detergent, explaining the enhanced stability of GPCRs purified in this detergent. Branched detergents occlude between transmembrane helices and reduce their flexibility. Our results provide a rational foundation to develop detergent variants for stabilizing membrane proteins.

Allosteric modulators enhance agonist efficacy by increasing the residence time of a GPCR in the active state

“…The combination of MNG and CHS was shown to be required for a proper activation of purified mGlu5 to the Gq protein(Nasrallah et al 2018). Improved GPCR functionality by the branched nonionic detergent LMNG has been described to result from an enhanced stabilization of the 7TM due to a lower detergent exchange rate as compared to its single aliphatic counterpart DDM, thereby confining receptor motions in a way similar to lipids(S. Lee et al 2020). In addition, enhanced polar interactions of the maltosid head of LMNG with loops and 7TM ends may play an important role in maintaining the functional link between the VFT and the 7TM through the CRD, e.g.…”

“…It is not surprising that a functional reconstitution of the multidomain, multimeric mGlu receptor requires adapted characteristics to account for proper folding, ligand binding and activity. Improved functionality by the branched nonionic detergent LMNG through enhanced stabilization of the 7TM (Lee et al 2020) and beneficial polar interactions of the maltoside head with loops and 7TM ends may play an important role in maintaining the functional link between the ECD and the 7TM (Koehl et al 2019). Further stabilization and functionality are provided by the two sterol-containing compounds CHS and GDN.…”

Structure and Dynamics of GPCRs in Lipid Membranes: Physical Principles and Experimental Approaches

“…MD simulations comparing LMNG to DDM (a non-branched alkyl glucoside) showed LMNG displayed increased density of alkyl chains covering the hydrophobic region of the receptor and higher levels of hydrogen bonding between detergent head groups. These interactions led to reduced mobility of the receptor [ 183 ]. Reduced mobility may, however, be deleterious for studying conformational changes in receptors; investigations by NMR of the β 2 AR in either DDM or LMNG showed the latter to slow ligand induced conformational exchange, attributed to the lower off-rates of this detergent [ 184 ].…”

Activation Microswitches in Adenosine Receptor A2A Function as Rheostats in the Cell Membrane

“…1B) using procedures described in the Methods section. For comparisons, we have also used analysis of our previous MD simulations using branched detergent Lauryl Maltose Neopentylglycol, LMNG (30).…”

“…We performed 5 production runs with different starting velocities each 400ns long. The trajectories of the all-atom MD simulations in the LMNG series of detergents were taken from our previous work (30) and used here for comparison. We used the TIP3 force field for describing the waters.…”