Human red cell AQP1 is the first functionally defined member of the aquaporin family of membrane water channels. Here we describe an atomic model of AQP1 at 3.8A resolution from electron crystallographic data. Multiple highly conserved amino-acid residues stabilize the novel fold of AQP1. The aqueous pathway is lined with conserved hydrophobic residues that permit rapid water transport, whereas the water selectivity is due to a constriction of the pore diameter to about 3 A over a span of one residue. The atomic model provides a possible molecular explanation to a longstanding puzzle in physiology-how membranes can be freely permeable to water but impermeable to protons.

Bacteriorhodopsin is a transmembrane protein that uses light energy, absorbed by its chromophore retinal, to pump protons from the cytoplasm of bacteria such as Halobacterium salinarium into the extracellular space. It is made up of seven alpha-helices, and in the bacterium forms natural, two-dimensional crystals called purple membranes. We have analysed these crystals by electron cryo-microscopy to obtain images of bacteriorhodopsin at 3.0 A resolution. The structure covers nearly all 248 amino acids, including loops outside the membrane, and reveals the distribution of charged residues on both sides of the membrane surface. In addition, analysis of the electron-potential map produced by this method allows the determination of the charge status of these residues. On the extracellular side, four glutamate residues surround the entrance to the proton channel, whereas on the cytoplasmic side, four aspartic acids occur in a plane at the boundary of the hydrophobic-hydrophilic interface. The negative charges produced by these aspartate residues is encircled by areas of positive charge that may facilitate accumulation and lateral movement of protons on this surface.

The entry and exit of water from cells is a fundamental process of life. Recognition of the high water permeability of red blood cells led to the proposal that specialized water pores exist in the plasma membrane. Expression in Xenopus oocytes and functional studies of an erythrocyte integral membrane protein of relative molecular mass 28,000, identified it as the mercury-sensitive water channel, aquaporin-1 (AQP1). Many related proteins, all belonging to the major intrinsic protein (MIP) family, are found throughout nature. AQP1 is a homotetramer containing four independent aqueous channels. When reconstituted into lipid bilayers, the protein forms two-dimensional lattices with a unit cell containing two tetramers in opposite orientation. Here we present the three-dimensional structure of AQP1 determined at 6A resolution by cryo-electron microscopy. Each AQP1 monomer has six tilted, bilayer-spanning alpha-helices which form a right-handed bundle surrounding a central density. These results, together with functional studies, provide a model that identifies the aqueous pore in the AQP1 molecule and indicates the organization of the tetrameric complex in the membrane.

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The major facilitator superfamily (MFS) represents one of the largest classes of evolutionarily related membrane transporter proteins. Here we present the three-dimensional structure at 6.5 A resolution of a bacterial member of this superfamily, OxlT. The structure, derived from an electron crystallographic analysis of two-dimensional crystals, reveals that the 12 helices in the OxlT molecule are arranged around a central cavity, which is widest at the center of the membrane. The helices divide naturally into three groups: a peripheral set comprising helices 3, 6, 9 and 12; a second set comprising helices 2, 5, 8 and 11 that faces the central substrate transport pathway across most of the length of the membrane; and a third set comprising helices 1, 4, 7 and 10 that participate in the pathway either on the cytoplasmic side (4 and 10) or on the periplasmic side (1 and 7). Overall, the architecture of the protein is remarkably symmetric, providing a compelling molecular explanation for the ability of such transporters to carry out bi-directional substrate transport.

The serine receptor (Tsr) from Escherichia coli is representative of a large family of transmembrane receptor proteins that mediate bacterial chemotaxis by influencing cell motility through signal transduction pathways. Tsr and other chemotaxis receptors form patches in the inner membrane that are often localized at the poles of the bacteria. In an effort to understand the structural constraints that dictate the packing of receptors in the plane of the membrane, we have used electron microscopy to examine ordered assemblies of Tsr in membrane extracts isolated from cells engineered to overproduce the receptor. Three types of assemblies were observed: ring-like "micelles" with a radial arrangement of receptor subunits, two-dimensional crystalline arrays with approximate hexagonal symmetry, and "zippers," which are receptor bilayers that result from the antiparallel interdigitation of cytoplasmic domains. The registration among Tsr molecules in the micelle and zipper assemblies was sufficient for identification of the receptor domains and for determination of their contributions to the total receptor length. The overall result of this analysis is compatible with an atomic model of the receptor dimer that was constructed primarily from the X-ray crystal structures of the periplasmic and cytoplasmic domains. Significantly, the micelle and zipper structures were also observed in fixed, cryosectioned cells expressing the Tsr receptor at high abundance, suggesting that the modes of Tsr assembly found in vitro are relevant to the situation in the cell.The serine receptor (Tsr), one of four methyl-accepting chemotaxis proteins (MCPs) that span the inner membrane of Escherichia coli, initiates responses and governs adaptation to changes in the serine concentration. MCPs belong to a large class of transducers (21, 46), which sense a variety of environmental cues and are the inputs to sensory pathways that bias cell movement toward favorable environments (12). The chemotaxis pathways belong to the two-component superfamily of signal transduction pathways (17, 42), which are chiefly found in prokaryotes. A two-component pathway consists of a sensor, which is frequently an integral membrane protein possessing kinase activity, and one or more cytoplasmic phosphate-accepting response regulator proteins. The transmembrane sensor-kinases of the chemotaxis pathways are often noncovalent complexes between MCPs (which have no enzyme activity) and two soluble cytoplasmic proteins, namely, an adaptor protein (CheW) and a kinase (CheA) (15, 39).Elucidation of the structure and distribution of receptors in the membrane of the cell is integral to understanding the molecular basis of signaling by the transmembrane sensor (MCP-CheW-CheA) complexes. X-ray structure determination of the soluble domains has clearly defined the dimeric organization of the 60-kDa receptor subunits (19,31,45), and functional studies have helped to elucidate the role of dimer organization in the mechanism of transmembrane signaling (references 32 and 12 and refer...

The major facilitator superfamily includes a large collection of evolutionarily related proteins that have been implicated in the transport of a variety of solutes and metabolites across the membranes of organisms ranging from bacteria to humans. We have recently reported the three-dimensional structure, at 6.5 Å resolution, of the oxalate transporter, OxlT, a representative member of this superfamily. In the oxalate-bound state, 12 helices surround a central cavity to form a remarkably symmetrical structure that displays a well-defined pseudo twofold axis perpendicular to the plane of the membrane as well as two less pronounced, mutually perpendicular pseudo twofold axes in the plane of the membrane. Here, we combined this structural information with sequence information from other members of this protein family to arrive at models for the arrangement of helices in this superfamily of transport proteins. Our analysis narrows down the number of helix arrangements from about a billion starting possibilities to a single probable model for the relative spatial arrangement for the 12 helices, consistent both with our structural findings and with the majority of previous biochemical studies on members of this superfamily.The movement of substrates across biological membranes is mediated by a large variety of membrane proteins that function as transporters. The major facilitator superfamily (MFS) (7, 19) is one of the largest classes of evolutionarily related transporters found in nature, representing a wide range of membrane proteins whose functions range from accumulating nutrients in bacteria to the cycling of neurotransmitters across human synaptic membranes. Sequence and biochemical analyses of several MFS proteins have suggested that most of these secondary active transporters are likely to contain 12 membrane-spanning segments (18). We have confirmed this prediction for the case of the oxalate transporter, OxlT, for which we recently reported a three-dimensional structure at a resolution of 6.5 Å (9).At the present (6.5 Å ) resolution, it is not possible to directly assign the 12 helices in the sequence of OxlT to the 12 transmembrane densities observed in the three-dimensional map. However, the unexpectedly high symmetry in the organization of the 12 helices leads to a significant reduction in the number of ways in which the transmembrane helices are likely to be arranged. By combining this structural information with selected biochemical and sequence information on other MFS proteins, we show that the number of helix assignments can be further reduced, leading to the deduction of a single, highly probable model for the spatial connectivity of the 12 helices. We propose that this arrangement is likely to reflect helix packing in the large variety of prokaryotic and eukaryotic transporters in the MFS. Figure 1 shows a representation of the density map derived from our electron crystallographic studies together with an idealized representation of the 12 helical transmembrane segments fitted into the density map....

Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable. Here we used electron tomographic imaging to determine the molecular architecture of Escherichia coli cells engineered to overproduce the bacterial chemotaxis receptor Tsr. Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures. We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters. Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr. We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type E. coli and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis.Over the last three decades, methods for three-dimensional reconstruction of objects (5) imaged with an electron microscope have been used to determine the structures of a variety of biological assemblies by two types of approaches. One approach, which has been used extensively in analyses of large macromolecular assemblies, involves three-dimensional reconstruction of a structure by averaging images recorded from several identical copies oriented randomly relative to the electron beam (11, 31). The other approach, which has been used for reconstruction of objects that cannot be easily averaged, such as whole cells, involves tomographic reconstruction by combining projection images of an object recorded with an electron microscope over a range of tilt angles (4). Electron tomography is therefore a potentially powerful tool for threedimensional imaging of the spatial arrangement of proteins that make up complex and dynamic assemblies, such as those involved in bacterial chemotaxis.At least 12 proteins act in concert to convert the signal of ligand binding at the periplasmic end of a chemotaxis receptor into rotation of the flagellar motor (6, 27). The principal protein components at the input end include one of the chemotaxis receptors (Tsr, Tar, Trg, Tap, or Aer), and the cytoplasmic signaling proteins CheA and CheW, which are thought to form a noncovalent complex with the chemotaxis receptors. Knowledge of the structure and spatial arrangement of the chemotaxis receptors is therefore fundamental to understanding the structural biology of signaling. X-ray crystallographic studies of the periplasmic fragments of the aspartate receptor fragments have revealed the dimeric organization of the ligand binding domain, in which the ligand binding pocket is located at...