Mechanism and energetics of membrane transporters and channels

membrane transporters;membrane channels;major facilitator superfamily;antiporter;urea;aquaporin

Enkavi, Giray

关键词: membrane transporters;    membrane channels;    major facilitator superfamily;    antiporter;    urea;    aquaporin;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/46912/Giray_Enkavi.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Living cells have evolved specialized transport proteins called membrane transporters andchannels that catalyze exchange of materials across the cell membrane. Membrane trans-porters couple the active transport of their specific substrates against their electrochemicalgradient. On the other hand, membrane channels facilitate passive diffusion of polar orcharged molecules down their electrochemical gradient. We present here molecular dy-namics (MD) investigation of a membrane transporter, glycerol-3 phosphate transporter(GlpT) and two membrane channels, urea transporter (UT) and aquaporin (Aqp1). Eachsimulations presented here provided a dynamical and atomistic picture of the protein ofinterest in a collaborative effort with an experimental lab.Membrane transporters use various source of cellular energy, e.g., ATP binding andhydrolysis in primary active transporters, and pre-established electrochemical gradientof molecular species other than their substrate in the case of secondary active trans-porters. All membrane transporters use the widely-accepted "alternating-access mecha-nism", which ensures that the substrate is only accessible from one side of the membraneat a given time, and relies on complex protein conformational changes between outward-facing (OF) and inward-facing (IF) states, going through several intermediate states.The first system that we investigated is the glycerol-3-phosphate transporter (GlpT),an antiporter member of the MFS. GlpT transports glycerol-3-phosphate (G3P) into thecell in exchange for inorganic phosphate (Pi ). Major facilitator superfamily (MFS) isthe largest superfamily of secondary active transporters and catalyze the transport ofan enormous variety of small solute molecules across biological membranes. IndividualMFS members, despite their architectural similarities, exhibit strict specificity towardthe substrates that they transport. The structural basis of this specificity, however, ispoorly understood. Our collaborators, Da-Neng Wang Lab (New York University, NY)performed mutagenesis studies and transport assays, while we performed equilibrium sim-ulations of wild-type GlpT and several of its mutant forms in membrane in the presenceof all physiologically relevant substrates (Pi­ , Pi2 ­ , G3P ­ , and G3P 2 ­ ) to characterizethe determinants of substrate selectivity and conformational response of the protein tosubstrate binding. The positive electrostatic potential of the lumen of GlpT recruitssubstrate and drives binding. Only a few amino acid residues that line the transporterlumen act as specificity determinants. The phosphate moiety of Pi and G3P bind to acommon binding site and residues involved solely in recognition of the glycerol moiety ofG3P confers it a higher binding affinity. Furthermore, the simulations characterized theprocess and mechanism of substrate binding, and the protein's initial conformational re-sponse. All substrate-bound systems resulted in partial closing of the cytoplasmic half ofGlpT. Extended simulations of substrate-bound systems also captured a water-conducting"channel-like" state. These states were also observed in several other transporters, sug-gesting that alternating-access mechanism tolerates transient states that are partiallyopen to both sides of the membrane. We, later, obtained a model of the outward-facing(OF) state of GlpT using nonequilibrium molecular dynamics and calculated free energiesto investigate the energetics associated with the transport cycle of GlpT.The second system we report here is a membrane channel that facilitates passive diffu-sion of urea across the membrane, namely the urea transporter (UT). Urea is ubiquitouslyused as a nitrogen source by bacteria and a safe end product of protein catabolism. Dueto its highly polar nature, urea relies on the UTs to permeate through the cell membrane.UTs are most frequently found in kidneys of mammals and allow rapid equilibrationof urea between the urinary space and the hyperosmotic tissue fluid to prevent osmoticdiuresis. Our collaborators, Ming Zhou lab (Columbia University, NY), crystallized struc-ture of a mammalian UT (UT-B). UT-B is a homotrimer and each monomer contains aurea conduction pore with a narrow selectivity filter. We performed an extensive set ofmolecular dynamics simulations combined with free energy calculations to elucidate thestructural determinants of the selectivity in UT-B and the associated energetics. Theorientation of the urea as its goes through the channel, as well as specific water-urea andprotein-urea interactions determine the specificity of the channel. The free energy barrierat the selectivity filter appears to be approximately 5.0 kcal/mol. We, then, investigatedthe gas permeability of UT in collaboration with Walter F. Boron lab (Case Western Uni-versity, Cleveland). Our free energy calculations along with the physiological experimentsindicate that UTs can function as gas channels and identified the monomeric pores as themain conduction pathway for both water and NH3 . Our work characterized UTs as thethird family of gas channels along with aquaporins (water channels), and Rh-associatedglycoprotein (RhAG) (ammonia channels).The other membrane channel system that we investigated is an aquaporin. Aqua-porins are ubiquitous integral membrane channels that maintain water homeostasis ofthe cell by facilitating selective diffusion of water across the membrane while preventingproton diffusion. Two conserved regions located along the pore are responsible for theselectivity: the dual asparagine, proline, alanine (NPA) aquaporin signature motif, andthe aromatic/arginine selectivity filter (SF). Recently, our collaborators, Richard Neutzelab (University of Gotenburg, Sweden), have crystallized a yeast aquaporin at 0.88 ° res-Aolution, the highest resolution achieved to date for a membrane protein. The structurereveals a great deal of novel information on the structure of hydrogen-bonded network ofwater and protein side chains. To determine the dynamics and energetics of water diffu-sion along the channel, we performed molecular dynamics simulations of this impressivelyhigh quality crystal structure. The results show disruption of the water chain in both NPAand SF regions in this aquaporin, due to characteristic hydrogen-bonding patterns thatdictate specific orientations to water molecules. The motion of water molecules is highlycorrelated on either side of the NPA region. The correlation, however, is lower at theNPA region, attesting yet another possible mechanism for this region to contribute to abarrier against proton transport. Besides, the NPA region appears as a barrier region withlow occupancy for water, a feature not seen in other aquaporins. The correlated motionof adjacent water molecules along with their binary co-occupancies in the SF show thatwater molecules move in pairs in this region. Specific hydrogen-bonding patterns in theSF region may also play a role in exclusion of hydronium (H3 O+ ) and/or hydroxide ions(OH ­ ). These simulations have helped elucidate the dynamical basis of many intricatefeatures revealed by this new structure.