Apelin conformational and binding equilibria upon micelle interaction primarily depend on membrane-mimetic headgroup

Abstract

Apelin is one of two peptide hormones that activate the apelin receptor (AR or APJ) to regulate the cardiovascular system, central nervous system, and adipoinsular axis. Here, we apply circular dichroism (CD) spectropolarimetry and nuclear magnetic resonance (NMR) spectroscopy to characterize the potential membrane binding by the two longest bioactive apelin isoforms, apelin-55 and -36, using membrane-mimetic dodecylphosphocholine (DPC), sodium dodecyl sulfate (SDS), and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) micelles. Pulsed field gradient diffusion NMR experiments demonstrated preferential interaction of both apelin-55 and -36 with anionic SDS and LPPG micelles over zwitterionic DPC micelles. Chemical shift perturbations and changes in ps-ns scale dynamics of apelin-55 in all micelles were similarly localized along the polypeptide backbone, demonstrating clear dependence upon detergent headgroup, while comparison of chemical shifts between apelin-55 and apelin-36 showed negligible differences indicative of highly similar modes of micelle interaction. Notably, the observed behaviour was consistent with an ensemble averaged pair of free and bound states in fast exchange on the NMR timescale proportional to the fraction of micelle-bound protein, implying a similar conformational equilibrium regardless of headgroup and tailgroup. Membrane catalysis of apelin-AR binding would thus give rise to analogous behaviour in the essential C-terminal region common to all apelin isoforms.

Simultaneous Ligand and Receptor Tracking through NMR Spectroscopy Enabled by Distinct 19F Labels

“…It should be noted that the observed relatively modest increase in 19 F resonance linewidth for the 2F substituent (Figure 3) is consistent with our prior observation of fast exchange for various apelin [12,13] and apela [10] species interacting with micelles, or even with slower-tumbling bicelles [14]. For future application, one may envision the 2F substituent—in particular—as a highly valuable probe to distinguish between a transient surface interaction vs. burial of the ligand C-terminus into a binding pocket, given the likelihood that the latter scenario would perturb the chemical shift at this site.…”

“…From a biophysical standpoint, the “RPRL” motif in apelin (found at the N-terminal of the apelin-13 isoform) is relatively conformationally restrained in buffer regardless of isoform [9,11]. This motif shows both involvement in and increased structuring upon binding to membrane-mimetic micelles [12,13]. Although this phenomenon has not been probed at the atomic-level in a bilayer setting, bicelle binding by apelin has been demonstrated from the perspective of the phospholipid headgroup [14], with likely extension to cell membrane interactive behaviour.…”

“…Although this phenomenon has not been probed at the atomic-level in a bilayer setting, bicelle binding by apelin has been demonstrated from the perspective of the phospholipid headgroup [14], with likely extension to cell membrane interactive behaviour. The apelin C-terminus (of sequence GPMPF), conversely, becomes structurally converged both when bound to micelles [12,13] and at low temperature [9,11].…”

Nuclear Magnetic Resonance Spectroscopy: A Multifaceted Toolbox to Probe Structure, Dynamics, Interactions, and Real-Time In Situ Release Kinetics in Peptide-Liposome Formulations

“…When incorporated in liposomes, pegylated apelin-13 could be efficiently delivered to endothelial cells in tumors, showing the potential of liposomes as a DDS to prolong apelin-like activity . The interaction mode of apelin-derived peptides with liposomes is still largely unknown, although NMR has been used previously to get detailed insight into the structure of apelin peptides, their molecular interactions with membrane models, and the self-aggregation properties of their various derivatives. − …”

The Influence of the Mixed DPC:SDS Micelle on the Structure and Oligomerization Process of the Human Cystatin C

“…Therefore, we decided to focus on the studies on the influence of one of the proposed earlier modulators of amyloidogenic proteins’ oligomerization, i.e., the biological membranes, on the dimerization and oligomerization of the h CC, using the micellar membrane mimetics. The most commonly used micellar systems for membrane mimicking involve dodecylphosphocholine (DPC) and sodium dodecyl sulfate (SDS) surfactants [ 22 , 23 , 24 , 25 , 26 ]. The use of micellar membrane mimetics is especially common in NMR experiments due to significant methodological issues, causing the use of natural membranes to be tricky or even impossible [ 27 ].…”