Role of Membrane Lipids in Peptide Hormone Function

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Role of Membrane Lipids in Peptide Hormone Function Proc. Nati. Acad. Sci. USA Vol. 81, pp. 61-65, January 1984 Biochemistry Role of membrane lipids in peptide hormone function: Binding of enkephalins to micelles (NMR/peptide-lipid interactions/opioid peptides/attraction-interaction model) CHARLES M. DEBER*t AND BASIL A. BEHNAM* *Research Institute, The Hospital for Sick Children, Toronto, ON, M5G 1X8, Canada; and tDepartment of Biochemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada Communicated by Elkan R. Blout, September 9, 1983 ABSTRACT In the course of their biological function, Physicochemical studies of the association of materials peptide hormones must be transferred from an aqueous phase such as insulin (2), glucagon (3), and the apolipoproteins (4) to the lipid-rich environment of their membrane-bound recep- with membrane preparations in vitro have been performed to tor proteins. We have investigated the possible influence of determine the specific manner through which phospholipids phospholipids in this process, using 360-MHz 'H and 90-MHz contribute to their functioning in vivo. Enkephalin itself has 13C NMR spectroscopy to examine the association of the opioid been shown to associate through ionic attraction to negative peptides [Met]- and [Leu]enkephalins (Tyr-Gly-Gly-Phe-Met/ lipids such as phosphatidylserine (5). Using high-resolution Leu) with phospholipid micelles. Binding of peptides to lipid 1H and 13C NMR techniques, we have now obtained evi- was monitored in NMR spectra by selective chemical shift dence for hydrophobic interaction of two endogenous 1 2 3 movements (e.g., the Phe aromatic ring protons) and residue- opioid peptides, [Met]- and [Leu]enkephalin (Tyr-Gly-Gly- specific line broadening (e.g., of Met/Leu carbonyl- and a- 4 5 carbon resonances). Results established that the zwitterionic Phe-Met/Leu), with a neutral phospholipid, lysophosphati- hormones associate hydrophobically both with a neutral lipid dylcholine (lysoPtdCho). The peptides also interact through (lysophosphatidylcholine) and (also electrostatically) with a a combination of electrostatic and hydrophobic interactions negative lipid (lysophosphatidylglycerol). An association coh- with the anionic lipid lysophosphatidylglycerol (lysoPtd- stant of Ka = 3.7 x 101 M-1 was calculated for the hydropho- Gro). In addition, we have been able to infer specific sites on bic binding of enkephalin to lysophosphatidylcholine. NMR the peptide of major interaction with lipid and to obtain a data suggested that enkephalin binds to the lipid with Met/ quantitative measure of the hydrophobic component of pep- Led, Phe, and likely Tyr side-chain substituents associated tide-lipid interaction. The possible role(s) of endogenous with nonpolar interior regions of the micelle, whereas the phospholipids is evaluated on the basis of these data. COOH-terminal carboxylate moiety of the peptide is located in the surface of the lipid particle. An "attraction-interaction" MATERIALS AND METHODS model is proposed for hormone-lipid association wherein neg- [Met]Enkephalin (Tyr-Gly-Gly-Phe-Met; Bachem Fine ative lipids attract the hormone electrostatically, while site- Chemicals, Torrance, CA), [Leulenkephalin (Tyr-Gly-Gly- specific hydrophobic contacts facilitate its entry, concentra- Phe-Leu; Fluka, Hauppauge, NY), egg L-a-lysoPtdCho (Sig- tion, and orientation into the lipid phase. ma), L-a-lysoPtd-DL-Gro (Sigma), and praseodymium nitrate pentahydrate (99.9%; Alfa-Ventron, Danvers, MA) were Aqueous-soluble proteins and peptide hormones are often used without further purification. bound to or transferred into membranes in conjunction with NMR samples were prepared with a peptide concentration their biological functions. Because a hormone as well as its of 8.72 mM for 1H NMR and from 27.3 to 28.2 mM for 13C membrane-embedded receptor protein contain potential NMR studies. Lipid concentrations, given in figure legends, sites of association with lipids, one may hypothesize that were generally above critical micelle concentration levels surrounding endogenous phospholipids could potentially (6). The quoted pH values are pH meter readings in 2H20 play any of several roles in mediating the transfer. These (Merck Sharp and Dohme, Montreal; 99.8%) that were un- could include: facilitating the capture, entry, and concentra- corrected for the deuterium isotope effect and were mea- tion of the aqueous-soluble hormone or neurotransmitter sured directly in the NMR tubes at room temperature. into the microenvironment of the receptor; orienting the pep- 1H NMR spectra were determined at 360 MHz with the tide in the membrane vis-d-vis the receptor by restricting mo- Nicolet NIC spectrometer operating in the Fourier transform lecular motions; and/or, in a more specific function, con- mode with 16,000 data points and typically 250 accumula- verting the hormone into a conformation required for elicit- tions for each spectrum. A 5-sec frequency pulse was used to ing biological activity. suppress the residual 1H2HO resonance. Temperature was These circumstances are relevant to the enkephalins-the 23 ± 10C. Chemical shifts are given in ppm after standardiza- peptide neurotransmitters that compete with morphine and tion of the spectrometer to external tetramethylsilane. Lipid- its derivatives for opiate binding sites in the brain (1). To induced shifts were measured after introducing successive, examine the above possibilities, we have initiated a study of weighed amounts of the lipid into the 2H20 solution of pep- some enkephalin-lipid complexes to determine (i) whether tide. hydrophobic interactions as well as electrostatic attractions 'H-decoupled 13C NMR spectra were determined at 90 contribute significantly to their stabilization, particularly in MHz with the Nicolet NIC spectrometer operating in the complexes with zwitterionic (net neutral) lipids, and (ii) the Fourier transform mode with 16,000 data points and typical- extent to which these interactions influence hormone mo- ly 12,000 accumulations for each spectrum. Chemical shifts tional and conformational parameters. are reported in ppm downfield from internal [2-'3C]acetoni- trile reference standard. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: PtdCho, phosphatidylcholine; lysoPtdCho, lyso- in accordance with 18 U.S.C. §1734 solely to indicate this fact. phosphatidylcholine; lysoPtdGro, lysophosphatidylglycerol. 61 Downloaded by guest on September 26, 2021 62 Biochemistry: Deber and Behnam Proc. NatL Acad ScL USA 81 (1984) For the measurements of Pr(III)-induced shifts, the pep- tides were dissolved in 2H20 at 28.2 mM in the case of [Leu]enkephalin and 15.3 mM in the [Met]enkephalin experi- ment. Successive weighed amounts of Pr(NO3)3 5H2O were introduced into a solution of the peptide in 2H20. RESULTS 13C NMR Spectra of Enkephalins and Phospholipids. 13C NMR spectra (90 MHz) of [Met]enkephalin carbonyl car- bons in the presence of increasing concentrations of the neu- tral (zwitterionic) lipid lysoPtdCho are shown in Fig. LA Be- cause of the clarity of spectra obtainable, this lipid and oth- ers that form micellar particles are emerging as valuable tools for investigation of protein-membrane interactions (9- 12). Significant upfield chemical shift changes (A8) were ob- B. +35 mg served for three of the five enkephalin carbonyl carbons lysoPtdCho (Gly-2, Phe, and Met), while the NH2-terminal Tyr and Gly-3 carbonyl-carbon resonances displayed little or no change up Wet l'dE < to approximately 5-fold molar excess of lipid (Fig. 1, spec- trum D).t Binding of [Leu]enkephalin to lysoPtdCho micelles pro- A duced similar results (Fig. 2B), whereas the chemical shift Phe changes observed for the titration of [Leu]enkephalin with micelles prepared from the negatively charged lipid lysoPtdGro are shown in Fig. 2A. In the latter experiment, the chemical shift of the NH2-terminal Tyr carbonyl-carbon resonance shifted downfield from 169.1 to 169.9 ppm after addition of the equivalent amount of lysoPtdGro. This be- havior should be contrasted with the negligible chemical 177 175 173 171 169 shift change for Tyr carbonyl-carbon resonances of both PPm [Met]- and [Leu]enkephalin over the range of 0 to 6-fold mo- lar excess of lysoPtdCho (Fig. 1, spectrum D and Fig. 2B). It FIG. 1. Regions of carbonyl-carbon resonances in 13C NMR is emphasized that throughout all additions of lipids, the pH spectra (90 MHz) of [Met]enkephalin (23.5 mg in 1.5 ml of 2H20, pH remained constant ±0.1 unit. The Gly-3 carbonyl-carbon - 6) (spectrum A) to which increasing portions of lysoPtdCho have resonance also reflected the "titration" behavior between 0 been added (spectra B-D). Mole ratios of lipid/peptide range from ca. 2 in spectrum B to ca. 6 in spectrum D. Resonances offree pep- and 1:1 molar ratio of lysoPtdGro/peptide (Fig. 2A) vs. its tide (spectrum A) were assigned in accordance with Khaled et al. relative insensitivity to added lysoPtdCho (Fig. 2B). An (ref. 7; see also footnote t). Numbers in spectrum D indicate the overall comparison of the trends above 1:1 lysoPtdGro/ total chemical shift changes (Hz) from spectrum A to spectrum D; peptide in Fig. 2A with Fig. 2B suggests that the upfield positive shifts are upfield. _L, ester carbonyl-carbon resonance of movements of Leu, Phe, and Gly-2 carbonyl-carbon reso- lysoPtdCho. nances are comparable, indicating that the enkephalin-lipid interactions manifested by these shifts are taking place in a and lysoPtdGro. Similar phenomena are observed in the a- corresponding manner with both lipids, being masked, in ef- carbon region of micelle-bound peptides (Fig. 3); for exam- fect, by the initial primary attraction apparent in the lysoPtd- ple, the [Leu]enkephalin a-carbon resonance is selectively Gro system. broadened in the presence of lysoPtdGro [linewidth = 3.5 The chemical shift changes upon binding lysoPtdCho mi- Hz for free [Leu]enkephalin (Fig. 3, spectrum A) and 10.5 celles are accompanied by general line broadening (Fig. 1, Hz at 1:1 lysoPtdGro/peptide (Fig. 3, spectrum B)]. spectra A-D), but additional selective line broadening clear- 'H NMR Spectra.
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