Hossain, KR & Clarke, RJ 2019, 'General and specific interactions of the phospholipid bilayer with P-type ATPases.', Biophysical reviews, vol. 11, no. 3, pp. 353-364.View/Download from: Publisher's site
Protein structure and function are modulated via interactions with their environment, representing both the surrounding aqueous media and lipid membranes that have an active role in shaping the structural topology of membrane proteins. Compared to a decade ago, there is now an abundance of crystal structural data on membrane proteins, which together with their functional studies have enhanced our understanding of the salient features of lipid-protein interactions. It is now important to recognize that membrane proteins are regulated by both (1) general lipid-protein interactions, where the general physicochemical properties of the lipid environment affect the conformational flexibility of a membrane protein, and (2) by specific lipid-protein interactions, where lipid molecules directly interact via chemical interactions with specific lipid-binding sites located on the protein. However, due to local differences in membrane composition, thickness, and lipid packing, local membrane physical properties and hence the associated lipid-protein interactions also differ due to membrane location, even for the same protein. Such a phenomenon has been shown to be true for one family of integral membrane ion pumps, the P2-type adenosine triphosphatases (ATPases). Despite being highly homologous, individual members of this family have distinct structural and functional activity and are an excellent candidate to highlight how the local membrane physical properties and specific lipid-protein interactions play a vital role in facilitating the structural rearrangements of these proteins necessary for their activity. Hence in this review, we focus on both the general and specific lipid-protein interactions and will mostly discuss the structure-function relationships of the following P2-type ATPases, Na+,K+-ATPase (NKA), gastric H+,K+-ATPase (HKA), and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), in concurrence with their lipid environment.
Garcia, A, Lev, B, Hossain, KR, Gorman, A, Diaz, D, Pham, THN, Cornelius, F, Allen, TW & Clarke, RJ 2019, 'Cholesterol depletion inhibits Na+,K+-ATPase activity in a near-native membrane environment.', The Journal of biological chemistry, vol. 294, no. 15, pp. 5956-5969.View/Download from: Publisher's site
Cholesterol's effects on Na+,K+-ATPase reconstituted in phospholipid vesicles have been extensively studied. However, previous studies have reported both cholesterol-mediated stimulation and inhibition of Na+,K+-ATPase activity. Here, using partial reaction kinetics determined via stopped-flow experiments, we studied cholesterol's effect on Na+,K+-ATPase in a near-native environment in which purified membrane fragments were depleted of cholesterol with methyl-β-cyclodextrin (mβCD). The mβCD-treated Na+,K+-ATPase had significantly reduced overall activity and exhibited decreased observed rate constants for ATP phosphorylation (ENa3 + → E2P, i.e. phosphorylation by ATP and Na+ occlusion from the cytoplasm) and K+ deocclusion with subsequent intracellular Na+ binding (E2K2 + → E1Na3 +). However, cholesterol depletion did not affect the observed rate constant for K+ occlusion by phosphorylated Na+,K+-ATPase on the extracellular face and subsequent dephosphorylation (E2P → E2K2 +). Thus, partial reactions involving cation binding and release at the protein's intracellular side were most dependent on cholesterol. Fluorescence measurements with the probe eosin indicated that cholesterol depletion stabilizes the unphosphorylated E2 state relative to E1, and the cholesterol depletion-induced slowing of ATP phosphorylation kinetics was consistent with partial conversion of Na+,K+-ATPase into the E2 state, requiring a slow E2 → E1 transition before the phosphorylation. Molecular dynamics simulations of Na+,K+-ATPase in membranes with 40 mol % cholesterol revealed cholesterol interaction sites that differ markedly among protein conformations. They further indicated state-dependent effects on membrane shape, with the E2 state being likely disfavored in cholesterol-rich bilayers relative to the E1P state because of a greater hydrophobic mismatch. In summary, cholesterol extraction from membranes significantly decreases Na+,K+-ATPase steady-state activity.
Garcia, A, Zou, H, Hossain, KR, Xu, QH, Buda, A & Clarke, RJ 2019, 'Polar Interactions Play an Important Role in the Energetics of the Main Phase Transition of Phosphatidylcholine Membranes', ACS Omega, vol. 4, no. 1, pp. 518-527.View/Download from: Publisher's site
© 2019 American Chemical Society. Conformational changes of membrane proteins are accompanied by deformation in the surrounding lipid bilayer. To gain insight into the energetics of membrane deformation, the phase behavior of dimyristoylphosphatidylcholine (DMPC) membranes in the presence of the dipole potential, d, modifiers was investigated by differential scanning calorimetry. 7-Ketocholesterol, which weakens d and reduces membrane-perpendicular dipole-dipole repulsion, causes a discrete second peak on the high-temperature side of the main transition, whereas 6-ketocholestanol, which strengthens d and increases membrane-perpendicular dipole-dipole repulsion, merely produces a shoulder. Measurements on pure DMPC vesicles showed that the observed temperature profile could not be explained by a single endothermic process, that is, breaking of van der Waals forces between hydrocarbon chains alone. Removal of NaCl from the buffer caused an increase in the main transition temperature and the appearance of an obvious shoulder, implicating polar interactions. Consideration of the phosphatidylcholine (PC) head group dipole moment indicates direct interactions between PC dipoles that are unlikely to account for the additional process. It seems more likely that the breaking of an in-plane hydrogen-bonded network consisting of hydrating water dipoles together with zwitterionic lipid head groups is responsible. The evidence presented supports the idea that the breaking of van der Waals forces between lipid tails required for the main phase transition of PC membranes is coupled to partial breaking of a hydrogen-bonded network at the membrane surface.
Hossain, KR, Turkewitz, DR, Holt, SA, Herson, L, Brown, LJ, Cornell, BA, Curmi, PMG & Valenzuela, SM 2019, 'A conserved GXXXG motif in the transmembrane domain of CLIC proteins is essential for their cholesterol-dependant membrane interaction.', Biochimica et biophysica acta. General subjects, vol. 1863, no. 8, pp. 1243-1253.View/Download from: Publisher's site
BACKGROUND:Sterols have been reported to modulate conformation and hence the function of several membrane proteins. One such group is the Chloride Intracellular Ion Channel (CLIC) family of proteins. The CLIC protein family consists of six evolutionarily conserved protein members in vertebrates. These proteins exist as both monomeric soluble proteins and as membrane bound proteins. To date, the structure of their membrane-bound form remains unknown. In addition to several studies indicating cellular redox environment and pH as facilitators of CLIC1 insertion into membranes, we have also demonstrated that the spontaneous membrane insertion of CLIC1 is regulated by membrane cholesterol. METHOD:We have performed Langmuir-film, Impedance Spectroscopy and Molecular Docking Simulations to study the role of this GXXXG motif in CLIC1 interaction with cholesterol. RESULTS:Unlike CLIC1-wild-type protein, the G18A and G22A mutants, that form part of the GXXXG motif, showed much slower initial kinetics and lower ion channel activity compared to the native protein. This difference can be attributed to the significantly reduced membrane interaction and insertion rate of the mutant proteins and/or slower formation of the final membrane configuration of the mutant proteins once in the membrane. CONCLUSION:In this study, our findings uncover the identification of a GXXXG motif in CLIC1, which likely serves as the cholesterol-binding domain, that facilitates the protein's membrane interaction and insertion. Furthermore, we were able to postulate a model by which CLIC1 can autonomously insert into membranes to form functional ion channels. GENERAL SIGNIFICANCE:Members of the CLIC family of proteins demonstrate unusual structural and dual functional properties - as ion channels and enzymes. Elucidating how the CLIC proteins' interact with membranes, thus allowing them to switch between their soluble and membrane form, will provide key information as to a mechanism of moonlighting ac...
Bray, K, Cheung, L, Hossain, KR, Aharonovich, I, Valenzuela, SM & Shimoni, O 2018, 'Versatile multicolor nanodiamond probes for intracellular imaging and targeted labeling', JOURNAL OF MATERIALS CHEMISTRY B, vol. 6, no. 19, pp. 3078-3084.View/Download from: Publisher's site
Hossain, KR, Holt, SA, Le Brun, AP, Al Khamici, H & Valenzuela, SM 2017, 'X-ray and Neutron Reflectivity Study Shows That CLIC1 Undergoes Cholesterol-Dependent Structural Reorganization in Lipid Monolayers.', Langmuir, vol. 33, no. 43, pp. 12497-12509.View/Download from: Publisher's site
CLIC1 belongs to the ubiquitous family of chloride intracellular ion channel proteins that are evolutionarily conserved across species. The CLICs are unusual in that they exist mainly as soluble proteins but possess the intriguing property of spontaneous conversion from the soluble to an integral membrane-bound form. This conversion is regulated by the membrane lipid composition, especially by cholesterol, together with external factors such as oxidation and pH. However, the precise physiological mechanism regulating CLIC1 membrane insertion is currently unknown. In this study, X-ray and neutron reflectivity experiments were performed to study the interaction of CLIC1 with different phospholipid monolayers prepared using POPC, POPE, or POPS with and without cholesterol in order to better understand the regulatory role of cholesterol in CLIC1 membrane insertion. Our findings demonstrate for the first time two different structural orientations of CLIC1 within phospholipid monolayers, dependent upon the absence or presence of cholesterol. In phospholipid monolayers devoid of cholesterol, CLIC1 was unable to insert into the lipid acyl chain region. However, in the presence of cholesterol, CLIC1 showed significant insertion within the phospholipid acyl chains occupying an area per protein molecule of 6-7 nm(2) with a total CLIC1 thickness ranging from ∼50 to 56 Å across the entire monolayer. Our data strongly suggests that cholesterol not only facilitates the initial docking or binding of CLIC1 to the membrane but also promotes deeper penetration of CLIC1 into the hydrophobic tails of the lipid monolayer.
Al Khamici, H, Hossain, KR, Cornell, BA & Valenzuela, SM 2016, 'Investigating Sterol and Redox Regulation of the Ion Channel Activity of CLIC1 Using Tethered Bilayer Membranes.', Membranes, vol. 6, no. 4, pp. 1-13.View/Download from: Publisher's site
The Chloride Intracellular Ion Channel (CLIC) family consists of six conserved proteins in humans. These are a group of enigmatic proteins, which adopt both a soluble and membrane bound form. CLIC1 was found to be a metamorphic protein, where under specific environmental triggers it adopts more than one stable reversible soluble structural conformation. CLIC1 was found to spontaneously insert into cell membranes and form chloride ion channels. However, factors that control the structural transition of CLIC1 from being an aqueous soluble protein into a membrane bound protein have yet to be adequately described. Using tethered bilayer lipid membranes and electrical impedance spectroscopy system, herein we demonstrate that CLIC1 ion channel activity is dependent on the type and concentration of sterols in bilayer membranes. These findings suggest that membrane sterols play an essential role in CLIC1's acrobatic switching from a globular soluble form to an integral membrane form, promoting greater ion channel conductance in membranes. What remains unclear is the precise nature of this regulation involving membrane sterols and ultimately determining CLIC1's membrane structure and function as an ion channel. Furthermore, our impedance spectroscopy results obtained using CLIC1 mutants, suggest that the residue Cys24 is not essential for CLIC1's ion channel function. However Cys24 does appear important for optimal ion channel activity. We also observe differences in conductance between CLIC1 reduced and oxidized forms when added to our tethered membranes. Therefore, we conclude that both membrane sterols and redox play a role in the ion channel activity of CLIC1.
Cranfield, CG, Berry, T, Holt, SA, Hossain, KR, Le Brun, AP, Carne, S, Al Khamici, H, Coster, H, Valenzuela, SM & Cornell, B 2016, 'Evidence of the Key Role of H3O+ in Phospholipid Membrane Morphology', LANGMUIR, vol. 32, no. 41, pp. 10725-10734.View/Download from: Publisher's site
Hossain, KR, Al Khamici, H, Holt, SA & Valenzuela, SM 2016, 'Cholesterol Promotes Interaction of the Protein CLIC1 with Phospholipid Monolayers at the Air-Water Interface.', Membranes, vol. 6, no. 1, pp. 1-13.View/Download from: Publisher's site
CLIC1 is a Chloride Intracellular Ion Channel protein that exists either in a soluble state in the cytoplasm or as a membrane bound protein. Members of the CLIC family are largely soluble proteins that possess the intriguing property of spontaneous insertion into phospholipid bilayers to form integral membrane ion channels. The regulatory role of cholesterol in the ion-channel activity of CLIC1 in tethered lipid bilayers was previously assessed using impedance spectroscopy. Here we extend this investigation by evaluating the influence of cholesterol on the spontaneous membrane insertion of CLIC1 into Langmuir film monolayers prepared using 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine alone or in combination with cholesterol. The spontaneous membrane insertion of CLIC1 was shown to be dependent on the presence of cholesterol in the membrane. Furthermore, pre-incubation of CLIC1 with cholesterol prior to its addition to the Langmuir film, showed no membrane insertion even in monolayers containing cholesterol, suggesting the formation of a CLIC1-cholesterol pre-complex. Our results therefore suggest that CLIC1 membrane interaction involves CLIC1 binding to cholesterol located in the membrane for its initial docking followed by insertion. Subsequent structural rearrangements of the protein would likely also be required along with oligomerisation to form functional ion channels.
Al Khamici, H, Brown, LJ, Hossain, KR, Hudson, AL, Sinclair-Burton, AA, Jane, PMN, Daniel, EL, Hare, JE, Cornell, BA, Curmi, PMG, Davey, MW & Valenzuela, SM 2015, 'Members of the Chloride Intracellular Ion Channel Protein Family Demonstrate Glutaredoxin-Like Enzymatic Activity', PLOS ONE, vol. 10, no. 1.View/Download from: Publisher's site
Yepuri, NR, Holt, SA, Moraes, G, Holden, PJ, Hossain, KR, Valenzuela, SM, James, M & Darwish, TA 2014, 'Stereoselective synthesis of perdeuterated phytanic acid, its phospholipid derivatives and their formation into lipid model membranes for neutron reflectivity studies', CHEMISTRY AND PHYSICS OF LIPIDS, vol. 183, pp. 22-33.View/Download from: Publisher's site
Hossain, KR, Al Khamici, H, Holt, SA & Valenzuela, SM 2016, 'Elucidating the Mechanism for Sterol Regulation of Chloride Intracellular Ion Channel Protein Interactions with Lipid Membranes', BIOPHYSICAL JOURNAL, 60th Annual Meeting of the Biophysical-Society, CELL PRESS, Los Angeles, CA, pp. 204A-204A.View/Download from: Publisher's site