Mapping the lipid-protein interface of the Torpedo californica and muscle-type acetylcholine receptors

Mapping the lipid-protein interface of the Torpedo californica and muscle-type acetylcholine receptors

Background

The nicotinic acetylcholine receptor (AChR) is a ligand-gated ion channel, integral membrane protein, which mediates fast chemical synaptic transmission at the neuromuscular junction and throughout the nervous system and is activated by binding of the chemical neurotransmitter acetylcholine (ACh). Topologically, AChR is a pentamerous receptor that consists of 20 putative transmembrane domains (4 transmembrane domains per AChR-subunit) of which 10 are lipid-exposed transmembrane domains (LETMDs) denoted as M3 and M4 domains. The studies that combined site-directed mutagenesis and electrophysiology (two-electrode voltage clamp and patch clamp) demonstrated that a single tryptophan substitution in the LETMDs could alter the kinetics rates, expression levels, and activatable fraction of the AChR. The extension of these studies along the LETMDs (tryptophan-scanning mutagenesis; TrpScanM) has revealed structure-function relationships, allosteric hydrophobic sites, helical structural models, transitional conformational changes, and functional and structural divergence between Torpedo and muscle-type AChRs and has provided the development of a hypothetical dynamic model (tilting-spring model). In addition, it develops a mathematical tool that combine the TrpScanM data with Fourier transform to reliably predict the helical structure of the LETMDs. In spite of valuable information about LETMDs provided by our laboratory, some LETMDs (ßM3, ßM4, ?M3, ?M4, dM3, dM4, eM3, and eM4 domains of the muscle-type AChR) have not been studied yet, keeping unrevealing structural and functional information of these LETMDs.

Long-term goal

Our long-term goal is to elucidate the role of lipid-protein interface of AChR in the cholinergic synapses of the nervous system.

Objective

The objective of this research project is to map the lipid-protein interface of the Torpedo and muscle-type AChRs. Hypothesis The central hypothesis of the research project is that the lipid-protein interface modulates AChR function and the fraction of receptors that could be activatable by ACh neurotransmitter.

Rationale

The rationale for the research project are to: (1) define the structure and spatial orientation of the LETMDs, (2) gain insight into the molecular basis for the lipid interaction and regulation of AChR function, (3) define the mechanistic link between LETMDs and gating machinery of the AChR, and (4) understand the structural basis for differential sensitivities of different AChRs species to the lipid environment.

Working objectives

The objective of this project is to complete the TrpScanM data for all the M3 and M4 lipid-exposed domains of the muscle-type AChRs to develop structural models that will be compared with the Torpedo AChR and with the most recent AChR structure (Unwin et al., 2005). The structural models generated from TrpScanM data will be used to estimate: (1) spatial orientation of the helix relative to the interior of the protein, (2) overall secondary structure, (3) membrane crossing angle predictions, (4) altered patterns of hydrogen bonds inside the helix, (5) periodicity of the helix at the center and at the edge with the membrane, (6) potential deviations from helical structure, (7) prediction on the degree of lipid contact and (8) overall movement on each of the lipid exposed domains upon agonist activation. These parameters will be estimated for the closed and open states of the Torpedo and muscle-type AChRs. These structural models will be use to generate a detailed structural model of the AChR lipid interface for Torpedo and muscle-type AChRs.

References

Caballero-Rivera D, Cruz-Nieves OA, Oyola-Cintrón J, del Hoyo-Rivera N, Otero-Cruz JD, Torres-Nuñez DA & Lasalde-Dominicci JA (2008) Role of the lipid-exposed dM4 transmembrane domain on the function and structure of the Torpedo californica nicotinic acetylcholine receptor. (Manuscript in preparation).

Díaz-De León R, Otero-Cruz JD, Torres-Nuñez DA, Casiano A & Lasalde-Dominicci JA (2008) Tryptophan scanning of the acetylcholine receptor’s bM4 transmembrane domain; decoding allosteric linkage at the lipid-protein interface with ion-channel gating. Channels (Manuscript in revision).

Otero-Cruz JD, Torres-Núñez DA, Báez-Pagán CA & Lasalde-Dominicci JA (2008) Fourier transform coupled to tryptophan-scanning mutagenesis: Lessons from its application to the prediction of secondary structure in the acetylcholine receptor lipid-exposed transmembrane domains. BBA – Proteins & Proteomics 1784(9):1200-7.

Otero-Cruz JD, Báez-Pagán CA, Caraballo-González IM & Lasalde-Dominicci JA (2007) Tryptophan-scanning mutagenesis in the aM3 transmembrane domain of the muscle-type acetylcholine receptor. A spring model revealed. J Biol Chem 282(12):9162-71.

Guzmán GR, Ortiz-Acevedo A, Ricardo A, Rojas LV & Lasalde-Dominicci JA (2006) The polarity of lipid-exposed residues contributes to the functional differences between Torpedo and muscle-type nicotinic receptors. J Membr Biol 214(3):131-8.

Ortiz-Acevedo A, Melendez M, Asseo AM, Biaggi N, Rojas LV & Lasalde-Dominicci JA (2004) Tryptophan scanning mutagenesis of the ?M4 transmembrane domain of the acetylcholine receptor from Torpedo californica. J Biol Chem 279(40):42250-7.

Santiago J, Guzmán GR, Torruellas K, Rojas LV & Lasalde-Dominicci JA (2004) Tryptophan scanning mutagenesis in the TM3 domain of the Torpedo californica acetylcholine receptor beta subunit reveals an a-helical structure. Biochemistry 43(31):10064-70.

Navedo M, Nieves M, Rojas L & Lasalde-Dominicci JA (2004) Tryptophan substitutions reveal the role of nicotinic acetylcholine receptor a-TM3 domain in channel gating: differences between Torpedo and muscle-type AChR. Biochemistry 43(1):78-84.

Guzmán GR, Santiago J, Ricardo A, Martí-Arbona R, Rojas LV & Lasalde-Dominicci JA (2003) Tryptophan scanning mutagenesis in the aM3 transmembrane domain of the Torpedo californica acetylcholine receptor: functional and structural implications. Biochemistry 42(42):12243-50.

Cruz-Martín A, Mercado JL, Rojas LV, McNamee MG & Lasalde-Dominicci JA (2001) Tryptophan substitutions at lipid-exposed positions of the ?M3 transmembrane domain increase the macroscopic ionic current response of the Torpedo californica nicotinic acetylcholine receptor. J Membr Biol 183(1):61-70.

Tamamizu S, Guzmán GR, Santiago J, Rojas LV, McNamee MG & Lasalde-Dominicci JA (2000) Functional effects of periodic tryptophan substitutions in the aM4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor. Biochemistry 39(16):4666-73.

Tamamizu S, Lee Y, Hung B, McNamee MG & Lasalde-Dominicci JA (1999) Alteration in ion channel function of mouse nicotinic acetylcholine receptor by mutations in the M4 transmembrane domain. J Membr Biol 170(2):157-64.

Ortiz-Miranda SI, Lasalde JA, Pappone PA & McNamee MG (1997) Mutations in the M4 domain of the Torpedo californica nicotinic acetylcholine receptor alter channel opening and closing. J Membr Biol 158(1):17-30.

Lasalde JA, Tamamizu S, Butler DH, Vibat CR, Hung B & McNamee MG (1996) Tryptophan substitutions at the lipid-exposed transmembrane segment M4 of Torpedo californica acetylcholine receptor govern channel gating. Biochemistry 35(45):14139-48.

Lee YH, Li L, Lasalde J, Rojas L, McNamee M, Ortiz-Miranda SI & Pappone P (1994) Mutations in the M4 domain of Torpedo californica acetylcholine receptor dramatically alter ion channel function. Biophys J 66(3 Pt 1):646-53.

Topological model of the four transmembrane domains (M1-M4) of the Torpedo californica gamma subunit using an α-helical segment. On the left a theoretical α-helical model of the γM3 shows the positions examined in this study. Tryptophan substitutions at positions F292, L296, and M299, which are presumably exposed to the lipid, produced significant increase in the macroscopic response of the AChR. J Membr Biol 183 (1):61-70

nAChR expression and functional data illustrated using the γ subunit model proposed by Miyazawa et al. 2003. TM domains 1–4 of the γ subunit are displayed as gray α-helices and marked as M1–M4 . Cys451 and Ser460 ( green ) have been proposed to be lipid exposed Blanton et al., 1994. Tryptophan replacement at residue Leu458 ( red ) resulted in a moderate increase in EC50. An increase in nAChR expression, decreased EC50, and increased macroscopic response were detected when tryptophan was introduced in residues Leu456, Phe459, and Gly462 ( blue ). Tryptophan replacement in residues Ile454, Ala455, Leu457, and Ile461 ( blue ) resulted in increased macroscopic and/or normalized macroscopic response. A , view from top of γTM4. B , side view of γTM4. J Biol Chem 279 (40):42250-7.

Molecular model of the a-TM3 domain of the AChR. The helical structure of muscle-type a-TM3 segment was modeled using Biomer V1.0 alpha (see http://www.scripps.edu/n_nwhite/B). An a-helical structure for the a-TM3 was minimized using the Fletcher-Reeves conjugated gradient algorithm (<2000 iterations), as described in Guzman et al., 2003. Minimized structures for wild type shows lipid exposed positions from a side and top views. Positions F284 and A287, which have been previously shown to be exposed to the membrane lipids, are oriented to the left side of the putative helix, while residues M282 and V285 are facing other transmembrane domains deep within the interior of the protein. This helical model predicts that position I290 is less embedded in the interior of the protein, possibly facing a neighboring transmembrane segment from the same subunit or of other subunit. Biochemistry 43(1):78-84.

Helical wheel diagram. (view from top) for an α-helical representation (3.6 amino acids per turn) of the β M3 showing the location of steric, constraint, inhibitory, and lipid exposed positions (Blanton et al., 1994) according to the present mutagenesis study. Positions at which tryptophan substitution results in relatively normal AChR expression levels compared to wild type, including M285, I289, and F293, are oriented toward the same face of the α-helix. The asterisk (*) indicates that the residue was presumably exposed to the lipid environment, as demonstrated by independent photolabeling studies (Blanton et al., 1994). Biochemistry 43 (31):10064-70.

Hypothetical topological model of the four transmembrane segments for the Torpedo californica AChR α-subunit. The structural models for the αM3 and αM4 domains were examined using Biomer V1.0 alpha (see http://www.scripps.edu/n_nwhite/B). The structures were minimized using the Fletcher-Reeves conjugated gradient algorithm (<1000 iterations). Inhibitory positions in the αM3 (M282, V285, and I289) are facing the same side of the helix, oriented toward the M2 domain, while lipid-exposed residues face the opposite side of the helix. Biochemistry 42 (42):12243-50.

αM3 structural models of the muscle-type AChR. A and B show the helical structure of the αM3 domain in the closed- ( A ) and open-channel ( B ) states using the data of the periodicity profiles of the closed and open-channel states, respectively (see Fig. 2, A and C ). Purple dashed dots represent hydrogen bonds. C shows the B-factor of the superposition between αM3 models. The B-factor is an atomic displacement parameter that reflects the degree of mobility of an atom in the protein structure (Smith et al., 2003). The colors and thickness emphasize the conformational change of the backbone atoms in the superposed helical structures of theαM3 models. The root mean square deviation of the fit was 3.307 Å in 72 atoms. The root mean square deviation parameter estimates the quality of the fit between two superposed protein structures. J Biol Chem 282 (12):9162-71.

Localization of the αM3-mutants of the muscle-type AChR in the open-channel state. A and C exhibit the helical net diagrams of the amount of amino acids per helical turn between the adjacent maximum ( A ) and minimum ( C ) oscillatory peaks of the periodicity profile of the muscle-type AChR αM3 domain in the open-channel state (see Fig. 2 C ). The gray box delimits the area of the αM3-mutants that produced nonfunctional ( blue circle ) and gain-of-function ( green circle ) AChRs using the data of the open-channel state profile (see Fig. 2 C ). B and D display theαM3 mutants that produced non-functional ( cyan stick with surface side chains ) and gain-of-function ( green stick with sphere side chains ) AChRs using the open-channel state model (see Fig. 3 B ). J Biol Chem 282 (12):9162-71.

Tryptophan-periodicity profiles, Fourier transform power spectra, and a-helical character curves generated with tryptophan-scanning mutagenesis data from AChR lipid-exposed transmembrane domains. (A–E) are TrpPPs. Values along the lines indicate the number of residues per helical turn between adjacent maximum and minimum peaks. The black dashed lines indicate the ACh EC50 value of the wild-type AChR. (F–J) are FT power spectra from entire sequences of the ACh EC50 values shown in the TrpPPs (A–E). The black headed arrows indicate the peaks that correspond to mean periodicities of the TrpPPs (A–E). Grey shadings demarcate the region 85°=?=115° that is used to calculate peak ratio values. (K–O) are a-helical character curves of different periodicity intervals as a function of the number of residues in the sliding window; red line (3.5–3.7) residues/turn; yellow line (3.4–3.8) residues/turn; green line (3.3–3.9) residues/turn; or blue line (3.2–4.0) residues/turn. BBA – Proteins & Proteomics 1784(9):1200-7.