MECHANISMS OF GATING AND SELECTIVITY OF AQUAPORINS

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Aquaporins (AQPs) (URL: http://www.ks.uiuc.edu/Research/aquaporins/) are a family of membrane channels specializing in rapid water conduction across biological membranes [42, 43]. They are widely distributed in all forms of life. Through modulating water permeability of cellular membranes AQPs play an important role in water homeostasis of living cells. MD simulations of membrane-embedded, fully hydrated models of various AQPs with NAMD [44] have revealed novel mechansims for selective function of these channels. A proper description of these proteins in their natural environment of lipid and water requires system sizes of 100,000 atoms or more, and simulation times on the order of tens of nanoseconds. We have investigated the mechanisms that gate the water pores and the central pore of AQPs [45,46]. In close collaboration with A. Yool (U. Arizona) who was the first to propose cGMP-induced ion conductivity of AQPs [47,48], protein conformational changes induced by binding of cGMP were studied. Based on our simulations, a gating mechanism has been proposed: one of the cytoplasmic loops (loop D) plays a critical role in controlling the accessibility of the central pore to water and hydrated ions [46]. The conformation of this flexible loop is highly perturbed by freely diffusing cGMP during the simulations, mainly through strong interactions of the nucleotide with an arginine-rich region of the loop. Retraction of the D loops of the four monomers away from the central pore not only physically unblocks the entrance of the central pore, but also results in conformational changes of a ring of pore-lining, hydrophobic residues that form a gate and block the access of water. The helix bearing these hydrophobic residues is immediately connected to loop D, and it is very likely that the conformational coupling of loop D and this helix is the molecular mechanism of detecting cGMP binding inside the central pore [46]. The involvement of arginines in the gating mechanism was successfully verified by experimental measurements performed by our collaborator on a double mutant species in which two of the arginines had been knocked out [46]. The mechanism of phosphorylation mediated gating of water pores in a plant AQP was investigated through a close collaboration [45] with the group of R. Neutze (Chalmers U. Tech., Sweden). The x-ray structure of the channel in its closed form was used to study conformational changes triggered by phosphorylation. In the closed (unphosphorylated) form, loop D, which is 4-5 residues longer in plant AQPs vs. mammalian ones, is held in its closing position through hydrogen bonds with the N-terminus of the protein. Examination of simulation results using the visualization program VMD [49] shows that upon phosphorylation, the connection between the N-terminus and loop D breaks, and that the latter is free to undergo large conformational changes. These changes, in turn, result in the opening of the water pores through two complementary mechanisms: 1) displacement of loop D from the cytoplasmic mouth of the channel, and, 2) retraction of a hydrophobic residue (Leu197) from the pore [45]. Besides the investigation of gating mechanisms of AQPs, we also tackled the problem of selectivity in these water channels. A comparative study [50] was performed on two bacterial AQPs from the same species, i.e., E. coli GlpF and AqpZ, which are structurally highly homologous, but functionally different. While AqpZ is a pure water channel [51,52], GlpF also conducts glycerol. In an earlier study [53], we calculated the PMF associated with permeation of glycerol through GlpF. Calculation of accurate free energy profiles from MD simulations presents an important challenging problem. The Resource collaborates with several theoretical groups, such as Chris Chipot (France) and Ioan Kosztin (U. Missouri) to overcome technical difficulties associated with such calculations. Although AqpZ is not a glycerol channel, an artificially induced passage of glycerol through AqpZ was achieved in SMD simulations, from which we constructed the PMF of glycerol conduction along the pore,and identified the barriers that make AqpZ impermeable to glycerol under normal conditions [50]. The results indicate that a high barrier against glycerol permeation arises not only in the selectivity filter region of AqpZ, which is believed to account for the main structural difference between AqpZ and GlpF, but along the entire channel. Furthermore, the difference in substrate selectivities of AqpZ and GlpF may not be simply due to only those residues that directly line the channel [50]. To convert a water channel into a glycerol channel one may have to identify remote residues that do not directly interact with the permeant, but nevertheless control the channel diameter through shifting and tilting helices forming the pore [50].