On-cell nuclear magnetic resonance spectroscopy to probe cell surface interactions.

Nuclear magnetic resonance (NMR) spectroscopy allows the determination of atomic-level information on intermolecular interactions, molecular structure, and molecular dynamics in the cellular environment. This may be broadly divided into studies focused on obtaining detailed molecular information in the intracellular context ("in-cell") or those focused on characterizing molecules or events at the cell surface ("on-cell"). In this review, we outline some key NMR techniques applied for on-cell NMR studies through both solution- and solid-state NMR and survey studies that have used these techniques to uncover key information. In particular, we focus on the application of on-cell NMR spectroscopy to characterize ligand interactions with cell surface membrane proteins such as G-protein coupled receptors (GPCRs) and receptor tyrosine kinases. These techniques allow for quantification of binding affinities, competitive binding assays, delineation of ligands involved in binding, ligand bound-state conformational determination, evaluation of receptor structuring and dynamics, and inference of distance constraints characteristic of the ligand-receptor bound state. Interestingly, it is possible to avoid the barriers of production and purification of membrane proteins while obtaining directly physiologically relevant information through on-cell NMR. We also provide a brief survey of the applicability of on-cell NMR approaches to other classes of cell surface molecules.

Localized apelin-17 analogue-bicelle interactions as a facilitator of membrane-catalyzed receptor recognition and binding.

The apelinergic system encompasses two peptide ligand families, apelin and apela, along with the apelin receptor (AR or APJ), a class A G-protein-coupled receptor. This system has diverse physiological effects, including modulating heart contraction, vasodilation/constriction, glucose regulation, and vascular development, with involvement in a variety of pathological conditions. Apelin peptides have been previously shown to interact with and become structured upon binding to anionic micelles, consistent with a membrane-catalyzed mechanism of ligand-receptor binding. To overcome the challenges of observing nuclear magnetic resonance (NMR) spectroscopy signals of a dilute peptide in biological environments, 19F NMR spectroscopy, including diffusion ordered spectroscopy (DOSY) and saturation transfer difference (STD) experiments, was used herein to explore the membrane-interactive behaviour of apelin. NMR-optimized apelin-17 analogues with 4-trifluoromethyl-phenylalanine at various positions were designed and tested for bioactivity through ERK activation in stably-AR transfected HEK 293 T cells. Far-UV circular dichroism (CD) spectropolarimetry and 19F NMR spectroscopy were used to compare the membrane interactions of these analogues with unlabelled apelin-17 in both zwitterionic/neutral and net-negative bicelle conditions. Each analogue binds to bicelles with relatively weak affinity (i.e., in fast exchange on the NMR timescale), with preferential interactions observed at the cationic residue-rich N-terminal and mid-length regions of the peptide leaving the C-terminal end unencumbered for receptor recognition, enabling a membrane-anchored fly-casting mechanism of peptide search for the receptor. In all, this study provides further insight into the membrane-interactive behaviour of an important bioactive peptide, demonstrating interactions and biophysical behaviour that cannot be neglected in therapeutic design.

Structure, amphipathy, and topology of the membrane-proximal helix 8 influence apelin receptor plasma membrane localization.

G-protein coupled receptors (GPCRs) typically have an amphipathic helix ("helix 8") immediately C-terminal to the transmembrane helical bundle. To date, a number of functional roles have been associated with GPCR helix 8 segments, but structure-function analysis for this region remains limited. Here, we examine helix 8 of the apelin receptor (AR or APJ), a class A GPCR with wide physiological and pathophysiological relevance. The 71 residue C-terminal tail of the AR is primarily intrinsically disordered, with a detergent micelle-induced increase in helical character. This helicity was localized to the helix 8 region, in good agreement with the recent AR crystal structure. A series of helix 8 mutants were made to reduce helicity, remove amphipathy, or flip the hydrophobic and hydrophilic faces. Each mutant AR was tested both biophysically, in the isolated C-terminal tail, and functionally in HEK 293 T cells, for full-length AR. In all instances, micelle interactions were maintained, and steady-state AR expression was efficient. However, removal of amphipathy or helical character led to a significant decrease in cell surface localization. Flipping of helix 8 amphipathic topology restored cell surface localization to some degree, but still was significantly reduced relative to wild-type. Structural integrity, amphipathy to drive membrane association, and correct topology of helix 8 membrane association all thus appear important for cell surface localization of the AR. This behavior correlates well to GPCR C-terminal tail sequence motifs, implying that these serve to specify key topological features of helix 8 and its proximity to the transmembrane domain.