Membrane lipids have been implicated to influence the activity of G protein-coupled receptors (GPCRs). Almost all of our knowledge on the role of lipids on GPCR and G protein function comes from work on the visual pigment rhodopsin and its G protein transducin, which reside in a highly specialized membrane environment. Thus insight gained from rhodopsin signaling may not be simply translated to other non-visual GPCRs. Here, we investigated the effect of lipid head group charges on the signal transduction properties of the class A GPCR neurotensin receptor 1 (NTS1) under defined experimental conditions, using self-assembled phospholipid nanodiscs prepared with the zwitter-ionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), the negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), or a POPC/POPG mixture. A combination of dynamic light scattering and sedimentation velocity showed that NTS1 was monomeric in POPC-, POPC/POPG- and POPG-nanodiscs. Binding of the agonist neurotensin to NTS1 occurred with similar affinities and was essentially unaffected by the phospholipid composition. In contrast, Gq protein coupling to NTS1 in various lipid nanodiscs was significantly different and the apparent affinity of Gαq and Gβ1γ1 to activated NTS1 increased with increasing POPG content. NTS1-catalyzed GDP/GTPγS nucleotide exchange at Gαq in the presence of Gβ1γ1 and neurotensin was crucially affected by the lipid type, with exchange rates higher by one or two orders of magnitude in POPC/POPG- and POPG-nanodiscs, respectively, compared to POPC-nanodiscs. Our data demonstrate that negatively charged lipids in the immediate vicinity of a non-visual GPCR modulate the G protein-coupling step.

Nanodiscs are self-assembled discoidal phospholipid bilayers surrounded and stabilized by membrane scaffold proteins (MSP), that have become a powerful and promising tool for the study of membrane proteins. Even though their reconstitution is highly regulated by the type of MSP and phospholipid input, a biophysical characterization leading to the determination of the stoichiometry of MSP, lipid and membrane protein is essential. This is important for biological studies, as the oligomeric state of membrane proteins often correlates with their functional activity. Typically combinations of several methods are applied using, for example, modified samples that incorporate fluorescent labels, along with procedures that result in nanodisc disassembly and lipid dissolution. To obtain a comprehensive understanding of the native properties of nanodiscs, modification-free analysis methods are required. In this work we provide a strategy, using a combination of dynamic light scattering and analytical ultracentrifugation, for the biophysical characterization of unmodified nanodiscs. In this manner we characterize the nanodisc preparation in terms of its overall polydispersity and characterize the hydrodynamically resolved nanodisc of interest in terms of its sedimentation coefficient, Stokes’ radius and overall protein and lipid stoichiometry. Functional and biological applications are also discussed for the study of the membrane protein embedded in nanodiscs under defined experimental conditions.

Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends.