P-glycoprotein is an ATP-binding cassette transporter that is associated with multidrug resistance and the failure of chemotherapy in human patients. We have previously shown, based on two-dimensional projection maps, that P-glycoprotein undergoes conformational changes upon binding of nucleotide to the intracellular nucleotide binding domains. Here we present the threedimensional structures of P-glycoprotein in the presence and absence of nucleotide, at a resolution limit of ϳ2 nm, determined by electron crystallography of negatively stained crystals. The data reveal a major reorganization of the transmembrane domains throughout the entire depth of the membrane upon binding of nucleotide. In the absence of nucleotide, the two transmembrane domains form a single barrel 5-6 nm in diameter and about 5 nm deep with a central pore that is open to the extracellular surface and spans much of the membrane depth. Upon binding nucleotide, the transmembrane domains reorganize into three compact domains that are each 2-3 nm in diameter and 5-6 nm deep. This reorganization opens the central pore along its length in a manner that could allow access of hydrophobic drugs (transport substrates) directly from the lipid bilayer to the central pore of the transporter. ATP Binding Cassette (ABC)1 transporters are an extended family of membrane proteins defined by a highly conserved domain, the ATP binding cassette (1); they mediate the ATP-dependent transport of a wide variety of compounds across cellular membranes (2, 3). The core ABC transporter consists of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). The NBDs are peripherally located at the cytoplasmic face of the membrane, bind ATP, and couple ATP hydrolysis to the transport process. All NBDs whose structures have been determined have very similar tertiary folds (4 -8). The TMDs bind the transported substrate and form the pathway through which it crosses the membrane. In contrast to the NBDs, the TMDs of different ABC transporters share little primary sequence similarity, except between closely related members of a subfamily; this may be because of the variety of substrates transported by different ABC proteins. Little is known about the structures of the TMDs of ABC transporters or how the binding/ hydrolysis of ATP by the NBDs is coupled to transmembrane transport of solute. Hydrophobicity plots typically predict six transmembrane ␣-helices per TMD, but there are notable exceptions with additional predicted transmembrane ␣-helices (9, 10). The structures of two complete bacterial ABC transporters (9, 11) have confirmed that the membrane-spanning segments are indeed ␣-helical, although the packing of these ␣-helices within the membrane differs markedly between the two structures.P-glycoprotein (P-gp) is a mammalian ABC transporter that pumps hydrophobic drugs across the cell membrane and can confer multidrug resistance on cells and tumors. P-gp is probably the best characterized ABC transporter, and much is known about the ATP hydrolytic cycle (1...

The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that is mutated in patients suffering from cystic fibrosis. Here we report the purification and first crystallization of wild-type human CFTR. Functional characterization of the material showed it to be highly active. Electron crystallography of negatively stained two-dimensional crystals of CFTR has revealed the overall architecture of this channel for two different conformational states. These show a strong structural homology to two conformational states of another eukaryotic ATP-binding cassette transporter, P-glycoprotein. In contrast to P-glycoprotein, however, both conformational states can be observed in the presence of a nucleotide, which may be related to the role of CFTR as an ion channel rather than a transporter. The hypothesis that the two conformations could represent the "open" and "closed" states of the channel is considered.

Wza is a highly conserved multimeric outer membrane protein complex required for the surface expression of the serotype K30 group 1 capsular polysaccharide in Escherichia coli. Here we present the first three-dimensional structure of this type of polysaccharide exporter at a 15.5-Å resolution obtained using single particle averaging on a dataset of cryo-negatively stained protein.Previous structural studies on purified Wza have revealed a homo-oligomeric ring structure that is most probably composed of eight subunits. Symmetry analysis of the three-dimensional structure combined with biochemical two-and three-dimensional crystallographic data strongly suggest that Wza is an octameric complex with a C4 quasi-rotational symmetry and is organized as a tetramer of dimeric subunits. Wza is best described as a stack of two 4-Å high rings with differing diameters providing a mushroom-like aspect from the side. The larger ring has a distinctive square shape with a diameter of 115 Å, whereas the smaller is almost circular with a diameter of 90 Å. In the center of the complex and enclosed by the four symmetrical arms is a small elliptical cagelike cavity of ϳ40 Å in diameter. The central cavity is effectively sealed at the top and bottom of the complex but has small inter-arm holes when viewed from the side. We discuss the structure of this complex and implications in the surface translocation of cell-surface polysaccharide.

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Cystic fibrosis, one of the major human inherited diseases, is caused by defects in the CFTR (cystic fibrosis transmembrane conductance regulator), a cell-membrane protein. CFTR acts as a chloride channel which can be opened by ATP. Low-resolution structural studies of purified recombinant human CFTR are described in the present paper. Localization of the C-terminal decahistidine tag in CFTR was achieved by Ni 2+ -nitriloacetate nanogold labelling, followed by electron microscopy and single-particle analysis. The presence of the gold label appears to improve the single-particle-alignment procedure. Projection structures of CFTR from twodimensional crystals analysed by electron crystallography displayed two alternative conformational states in the presence of nucleotide and nanogold, but only one form of the protein was observed in the quiescent (nucleotide-free) state.The ABC (ATP-binding cassette) proteins are a superfamily of active transporter membrane proteins and are composed of approx. 50 functionally diverse classes of prokaryotic and eukaryotic transmembrane proteins [1]. Despite the differences in the type of substances that are transported by each protein, most members of the ABC proteins have a similar pattern of two NBDs (nucleotide-binding domains) and two TMDs (transmembrane domains) [2]. The CFTR (cystic fibrosis transmembrane conductance regulator), also termed ABCC7, is a unique member of the ABC superfamily of transporter proteins, which works as an ion channel and has an extra domain called the regulatory domain (R) located between NBD1 and TMD2 [3]. Channel activity is affected by phosphorylation (by protein kinases A and C) and by ATP. CFTR regulates secretion and reabsorption of ions at epithelial surfaces [4]. Defects in the channel function and/or folding and processing lead to cystic fibrosis [5].Structural data for many ABC transporter proteins suspected to be responsible for human diseases are mostly lacking. Structural studies of ABC proteins were successful in determining the high-resolution structures of MsbA (bacterial lipid transporter) [6,7] and BtuCD (bacterial vitamin B 12 transporter) [8] and the low-resolution structures