Aquaporin (AQP) 4 is the predominant water channel in the mammalian brain, abundantly expressed in the blood-brain and braincerebrospinal fluid interfaces of glial cells. Its function in cerebral water balance has implications in neuropathological disorders, including brain edema, stroke, and head injuries. The 1.8-Å crystal structure reveals the molecular basis for the water selectivity of the channel. Unlike the case in the structures of water-selective AQPs AqpZ and AQP1, the asparagines of the 2 Asn-Pro-Ala motifs do not hydrogen bond to the same water molecule; instead, they bond to 2 different water molecules in the center of the channel. Molecular dynamics simulations were performed to ask how this observation bears on the proposed mechanisms for how AQPs remain totally insulating to any proton conductance while maintaining a single file of hydrogen bonded water molecules throughout the channel.brain edema ͉ inhibitor discovery ͉ NPA motif

Many voltage-gated ion channel (VGIC) superfamily members contain six-transmembrane segments in which the first four form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltagegated sodium channel (Na V Sp1) PD forms a stand-alone, ion selective pore (Na V Sp1p) that is tetrameric, α-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the Na V Sp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel Na V Bh1 (NaChBac), creates a calcium-selective pore-only channel, Ca V Sp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile Na V s: one from the hydrocarbon degrader Alcanivorax borkumensis (Na V Ab1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (Na V Ae1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.V oltage-gated sodium channels (Na V s) are large polytopic membrane proteins involved in action potential generation in excitable cells and belong to an ion channel superfamily that includes voltage-gated calcium channels (Ca V s), voltage-gated potassium channels (K V s), and transient receptor potential (TRP) channels (1, 2). Within the voltage-gated ion channel (VGIC) superfamily, Na V s and Ca V s are close relatives (1-3) that share a topology of 24 transmembrane segments organized in four homologous six-transmembrane repeats. These two families are also thought to share some common structure in the ion selectivity filter despite having markedly different ion permeation properties (4). Both are central to human neuromuscular, cardiovascular, and neural physiology. Consequently, they are targets for a host of pharmaceuticals used to treat a diverse set of disorders and remain active targets for drug development (5-7). Recently, single subunit, six-transmembrane segment Na V s have been identified in a large number of bacteria from diverse environments (8, 9). These channels show clear similarities to eukaryotic Na V s and Ca V s (2, 9, 10), suggesting that the prokaryotic channels may have been ancestors of the more complex vertebrate channels.Despite the central importance of Na V s, nothing is known about the high-resolution structure o...

Resistance nodulation cell division (RND)-based efflux complexes mediate multidrug and heavy-metal resistance in many Gramnegative bacteria. Efflux of toxic compounds is driven by membrane proton/substrate antiporters (RND protein) in the plasma membrane, linked by a membrane fusion protein (MFP) to an outer-membrane protein. The three-component complex forms an efflux system that spans the entire cell envelope. The MFP is required for the assembly of this complex and is proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we chose ZneB, the MFP component of the ZneCAB heavy-metal efflux system from Cupriavidus metallidurans CH34. ZneB is shown to be highly specific for Zn 2+ alone. The crystal structure of ZneB to 2.8 Å resolution defines the basis for metal ion binding in the coordination site at a flexible interface between the β-barrel and membrane proximal domains. The conformational differences observed between the crystal structures of metal-bound and apo forms are monitored in solution by spectroscopy and chromatography. The structural rearrangements between the two states suggest an active role in substrate efflux through metal binding and release.Cupriavidus metallidurans CH34 | heavy-metal resistance | resistance nodulation cell division | periplasmic adaptor protein M icroorganisms depend on protective mechanisms to survive the effects of toxic compounds in the environment. In Gramnegative bacteria, resistance nodulation cell division (RND) -driven efflux systems confer resistance to many drugs and heavy metals (1, 2). The canonical RND-based system is formed by the association of three components: one integral to the plasma membrane, one integral to the outer membrane, and a periplasmic connector. The plasma membrane protein (RND) is a substrate/ proton antiporter. The outer-membrane factor (OMF) spans a large part of the periplasm and provides the exit portal. The periplasmic membrane fusion protein (MFP) links these two components together. Based on the nature of their substrate, tripartite RND-driven efflux systems are divided into two subclasses: the hydrophobe/amphiphile efflux (HAE) and the heavy-metal efflux (HME) subclasses. The selectivity and function of many HAE-RND systems such as the Acr and Mex families have been determined, alongside crystal structures of the RND, OMF, and MFP components of various systems (3-13). However, knowledge of HME-RND systems, including substrate specificity and the basis for selectivity, is much more limited.The integral plasma membrane components of the RND-based efflux systems are thought of as the pump; however, the discovery of increasing functions of MFPs shows that these proteins also play a major role in substrate transport. Several MFPs bind their respective substrates (14-16), facilitate substrate transport (17, 18), and are essential for transport in vitro (17). MFPs are also involved in OMF recruitment (19), and are also found in Grampositive bacteria, where no OMF is pr...

Efflux pumps belonging to the ubiquitous resistance-nodulationcell division (RND) superfamily transport substrates out of cells by coupling proton conduction across the membrane to a conformationally driven pumping cycle. The heavy metal-resistant bacteria Cupriavidus metallidurans CH34 relies notably on as many as 12 heavy metal efflux pumps of the RND superfamily. Here we show that C. metallidurans CH34 ZneA is a proton driven efflux pump specific for Zn(II), and that transport of substrates through the transmembrane domain may be electrogenic. We report two Xray crystal structures of ZneA in intermediate transport conformations, at 3.0 and 3.7 Å resolution. The trimeric ZneA structures capture protomer conformations that differ in the spatial arrangement and Zn(II) occupancies at a proximal and a distal substrate binding site. Structural comparison shows that transport of substrates through a tunnel that links the two binding sites, toward an exit portal, is mediated by the conformation of a short 14-aa loop. Taken together, the ZneA structures presented here provide mechanistic insights into the conformational changes required for substrate efflux by RND superfamily transporters.T he resistance-nodulation-cell division (RND) superfamily, named based on the original members' roles in metal resistance, root nodulation, and cell division (1), is found in all kingdoms of life and is comprised of nine phylogenetically distinct families (2-5). Functional characterization of RND proteins has shown that they are transmembrane efflux pumps that transport a variety of substrates out of cells, powered by an electrochemical proton gradient (6). In Gram-negative bacteria, RND pumps are specific for toxic substrates and largely belong to one of two families; the heavy metal efflux (HME) family and the multidrug hydrophobe/amphiphile efflux-1 (HAE1) family. For the HME-and HAE1-RND-driven efflux systems, a trimer of the RND pump in the plasma membrane is coupled by a hexamer of a periplasmic membrane fusion protein (MFP), also specific for the metal ion substrate in HME-RND-driven efflux systems, to a pore formed by a trimeric outer membrane factor (OMF), forming a continuous conduit that spans the inner and outer membranes (7-10). Each protomer of the RND trimer consists of a transmembrane domain of 12 transmembrane α-helices and two large hydrophilic loops that comprise the substratebinding porter (or pore) domain and the OMF-coupling docking domain (11).X-ray crystal structures of only four RND efflux pumps have been described, including just one of the HME family to date, shedding some light on the conformational changes that are necessary for transport (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In two of these structures, those of the HAE1 efflux pumps AcrB from Escherichia coli and of the closely related MexB from Pseudomonas aeruginosa, each protomer of the homotrimer is trapped in a unique conformation, with only a single protomer conducive for substrate binding, suggestive of a functionally rotating mechanism (12, ...