Structural asymmetry in a trimeric Na+/betaine symporter, BetP, from Corynebacterium glutamicum

The Na⁺-coupled symporter BetP catalyzes the uptake of the compatible solute betaine in the soil bacterium Corynebacterium glutamicum. BetP also senses hyperosmotic stress and regulates its own activity in response to stress level. We determined a three-dimensional (3D) map (at 8 Å in-plane resolution) of a constitutively active mutant of BetP in a C. glutamicum membrane environment by electron cryomicroscopy of two-dimensional crystals. The map shows that the constitutively active mutant, which lacks the C-terminal domain involved in osmosensing, is trimeric like wild-type BetP. Recently, we reported the X-ray crystal structure of BetP at 3.35 Å, in which all three protomers displayed a substrate-occluded state. Rigid-body fitting of this trimeric structure to the 3D map identified the periplasmic and cytoplasmic sides of the membrane. Fitting of an X-ray monomer to the individual protomer maps allowed assignment of transmembrane helices and of the substrate pathway, and revealed differences in trimer architecture from the X-ray structure in the tilt angle of each protomer with respect to the membrane. The three protomer maps showed pronounced differences around the substrate pathway, suggesting three different conformations within the same trimer. Two of those protomer maps closely match those of the atomic structures of the outward-facing and inward-facing states of the hydantoin transporter Mhp1, suggesting that the BetP protomer conformations reflect key states of the transport cycle. Thus, the asymmetry in the two-dimensional maps may reflect cooperativity of conformational changes within the BetP trimer, which potentially increases the rate of glycine betaine uptake.     

Structural role of bacterioruberin in the trimeric structure of archaerhodopsin-2

Archaerhodopsin-2 (aR2), a retinal protein-carotenoid complex found in the claret membrane of Halorubrum sp. aus-2, functions as a light-driven proton pump. In this study, the membrane fusion method was utilized to prepare trigonal P321 crystals (a = b = 98.2 Å, c = 56.2 Å) and hexagonal P6₃ crystals (a = b = 108.8 Å, c = 220.7 Å). The trigonal crystal is made up of stacked membranes in which the aR2 trimers are arranged on a honeycomb lattice. Similar membranous structures are found in the hexagonal crystal, but four membrane layers with different orientations are contained in the unit cell. In these crystals, the carotenoid bacterioruberin [5,32-bis(2-hydroxypropan-2-yl)-2,8,12,16,21,25,29,35-octamethylhexatriaconta-6,8,10,12,14,16,18,20,22,24,26,28,30-tridecaene-2,35-diol] binds to crevices between the subunits of the trimer. Its polyene chain is inclined from the membrane normal by an angle of about 20° and, on the cytoplasmic side, it is surrounded by helices AB and DE of neighbouring subunits. This peculiar binding mode suggests that bacterioruberin plays a striking structural role for the trimerization of aR2. When compared with the aR2 structure in another crystal form containing no bacterioruberin, the proton release channel takes a more closed conformation in the P321 or P6₃ crystal; i.e., the native conformation of protein is stabilized in the trimeric protein-bacterioruberin complex. Interestingly, most residues participating in the trimerization are not conserved in bacteriorhodopsin, a homologous protein capable of forming a trimeric structure in the absence of bacterioruberin. Despite a large alteration in the amino acid sequence, the shape of the intratrimer hydrophobic space filled by lipids is highly conserved between aR2 and bacteriorhodopsin. Since a transmembrane helix facing this space undergoes a large conformational change during the proton pumping cycle, it is feasible that trimerization is an important strategy to capture special lipid components that are relevant to the protein activity.     

Conformational Changes and Ligand Recognition of Escherichia colid-Xylose Binding Protein Revealed

ATP binding cassette transport systems account for most import of necessary nutrients in bacteria. The periplasmic binding component (or an equivalent membrane-anchored protein) is critical to recognizing cognate ligand and directing it to the appropriate membrane permease. Here we report the X-ray structures of d-xylose binding protein from Escherichia coli in ligand-free open form, ligand-bound open form, and ligand-bound closed form at 2.15 Å, 2.2 Å, and 2.2 Å resolutions, respectively. The ligand-bound open form is the first such structure to be reported at high resolution; the combination of the three different forms from the same protein furthermore gives unprecedented details concerning the conformational changes involved in binding protein function. As is typical of the structural family, the protein has two similar globular domains, which are connected by a three-stranded hinge region. The open liganded structure shows that xylose binds first to the C-terminal domain, with only very small conformational changes resulting. After a 34° closing motion, additional interactions are formed with the N-terminal domain; changes in this domain are larger and serve to make the structure more ordered near the ligand. An analysis of the interactions suggests why xylose is the preferred ligand. Furthermore, a comparison with the most closely related proteins in the structural family shows that the conformational changes are distinct in each type of binding protein, which may have implications for how the individual proteins act in concert with their respective membrane permeases.     

Projection structure of channelrhodopsin-2 at 6 Å resolution by electron crystallography

Channelrhodopsin-2 (ChR2) is the prototype of a new class of light-gated ion channels that is finding widespread applications in optogenetics and biomedical research. We present a  6-Å projection map of ChR2, obtained by cryo-electron microscopy of two-dimensional crystals grown from pure, heterologously expressed protein. The map shows that ChR2 is the same dimer with non-crystallographic 2-fold symmetry in three different membrane crystals. This is consistent with biochemical analysis, which shows a stable dimer in detergent solution. Comparison to the projection map to bacteriorhodopsin indicates a similar structure of seven transmembrane alpha helices. Based on the projection map and sequence alignments, we built a homology model of ChR2 that potentially accounts for light-induced channel gating. Although a monomeric channel is not ruled out, comparison to other membrane channels and transporters suggests that the ChR2 channel is located at the dimer interface on the 2-fold axis, lined by transmembrane helices 3 and 4.     

Two structures of a lambda Cro variant highlight dimer flexibility but disfavor major dimer distortions upon specific binding of cognate DNA

Previously reported crystal structures of free and DNA-bound dimers of λ Cro differ strongly (about 4 Å backbone rmsd), suggesting both flexibility of the dimer interface and induced-fit protein structure changes caused by sequence-specific DNA binding. Here, we present two crystal structures, in space groups P3₂21 and C2 at 1.35 and 1.40 Å resolution, respectively, of a variant of λ Cro with three mutations in its recognition helix (Q27P/A29S/K32Q, or PSQ for short). One dimer structure (P3₂21; PSQ form 1) resembles the DNA-bound wild-type Cro dimer (1.0 Å backbone rmsd), while the other (C2; PSQ form 2) resembles neither unbound (3.6 Å) nor bound (2.4 Å) wild-type Cro. Both PSQ form 2 and unbound wild-type dimer crystals have a similar interdimer β-sheet interaction between the β1 strands at the edges of the dimer. In the former, an infinite, open β-structure along one crystal axis results, while in the latter, a closed tetrameric barrel is formed. Neither the DNA-bound wild-type structure nor PSQ form 1 contains these interdimer interactions. We propose that β-sheet superstructures resulting from crystal contact interactions distort Cro dimers from their preferred solution conformation, which actually resembles the DNA-bound structure. These results highlight the remarkable flexibility of λ Cro but also suggest that sequence-specific DNA binding may not induce large changes in the protein structure.     

Structure and stability of the neurotoxin PV2 from the eggs of the apple snail Pomacea canaliculata

There is little information on the egg proteins of gastropod mollusks. Here we focus on PV2, a novel neurotoxin from snail eggs, studying its size, shape, structure, and stability, using small angle X-ray scattering (SAXS), absorption and fluorescence spectroscopy, circular dichroism, electron microscopy and partial proteolysis. Results indicate that PV2 is a compact and well folded oligomer of 130 × 44 Å. It is an octamer of four 98 kDa heterodimers composed of 67 and 31 kDa subunits. Subunits are held together by disulfide bonds. Dimers are assembled into native PV2 by non-covalent forces. The larger subunit is more susceptible to proteolysis, indicating it is less compactly folded and/or more exposed. Quenching of tryptophan fluorescence showed a single class of tryptophyl side chains occluded in hydrophobic regions. Native structure shows loss of secondary structure (α+β) at 6 M urea or 60–70 °C; the effects on the quaternary structure suggest an unfolding without disassembling of the protein. The 3D model of PV2 presented here is the first for an egg proteinaceous neurotoxin in animals.     

Homologous sugar transport proteins in Escherichia coli and their relatives in both prokaryotes and eukaryotes

Separate proteins for proton-linked transport of D-xylose, L-arabinose, D-galactose, L-rhamnose and L-fucose into Escherichia coli are being studied. By cloning and sequencing the appropriate genes, the amino acid sequences of proteins for D-xylose/H+ symport (XylE), L-arabinose/H+ symport (AraE), and part of the protein for D-galactose/H+ symport (GalP) have been determined. These are homologous, with at least 28% identical amino acid residues conserved in the aligned sequences, although their primary sequences are not similar to those of other E. coli transport proteins for lactose, melibiose, or D-glucose. However, they are equally homologous to the passive D-glucose transport proteins from yeast, rat brain, rat adipocytes, human erythrocytes, human liver, and a human hepatoma cell line. The substrate specificity of GalP from E. coli is similar to that of the mammalian glucose transporters. Furthermore, the activities of GalP, AraE and the mammalian glucose transporters are all inhibited by cytochalasin B and N-ethylmaleimide. Conserved residues in the aligned sequences of the bacterial and mammalian transporters are identified, and the possible roles of some in sugar binding, cation binding, cytochalasin binding, and reaction with N-ethylmaleimide are discussed. Each protein is independently predicted to form 12 hydrophobic, membrane-spanning alpha-helices with a central hydrophilic segment, also comprised of alpha-helix. This unifying structural model of the sugar transporters shares features with other ion-linked transport proteins for citrate or tetracycline.     

Ligand binding mode of GABAA receptor-associated protein

The γ-aminobutyric acid type A (GABA_A) receptor-associated protein is a versatile adaptor protein playing an important role in intracellular vesicle trafficking, particularly in neuronal cells. We present the X-ray structure of the soluble form of human GABA_A receptor-associated protein complexed with a high-affinity synthetic peptide at 1.3 Å resolution. The data shed light on the probable binding modes of key interaction partners, including the GABA_A receptor and the cysteine protease Atg4. The resulting models provide a structural background for further investigation of the unique biological properties of this protein.     

The Three-dimensional Structure of a Mycobacterial DapD Provides Insights into DapD Diversity and Reveals Unexpected Particulars about the Enzymatic Mechanism

The enzyme tetrahydrodipicolinate N-succinyltransferase (DapD) is part of the L-lysine biosynthetic pathway. This pathway is crucial for the survival of the pathogen Mycobacterium tuberculosis (Mtb) and, consequently, the enzymes of the pathway are potential drug targets. We report here the crystal structures of Mtb-DapD and of Mtb-DapD in complex with the co-factor succinyl-CoA (SCoA) at 2.15 Å and 1.97 Å resolution, respectively. Each subunit of the trimeric enzyme consists of three domains, of which the second, a left-handed, parallel β-helix (LβH domain), is the common structural motif of enzymes belonging to the hexapeptide repeat superfamily. The trimeric quaternary structure is stabilized by Mg²⁺ and Na⁺ located on the 3-fold axis. The binary complex of Mtb-DapD and SCoA reveals the binding mode(s) of the co-factor and a possible covalent reaction intermediate. The N-terminal domain of Mtb-DapD exhibits a unique architecture, including an interior water-filled channel, which allows access to a magnesium ion located at the 3-fold symmetry axis.     

Cytochalasin B as a probe of protein structure and substrate recognition by the galactose/H+ transporter of Escherichia coli.

Cytochalasin B is a potent inhibitor of mammalian passive glucose transporters. The recent demonstration of sequence similarities between these proteins and several bacterial proton-linked sugar transporters suggested that cytochalasin B might be a useful tool for investigation of the galactose/H+ symport protein (GalP) of Escherichia coli. Equilibrium binding studies using membranes from a GalP-constitutive (GalPc) strain of E. coli revealed a single set of high affinity binding sites for cytochalasin B with a Kd of 0.8-2.2 microM. Binding was inhibited by D-glucose, but not by L-glucose. UV irradiation of the membranes in the presence of [4-3H]cytochalasin B photolabeled principally a protein of apparent Mr 38,000, corresponding to the GalP protein. Labeling was inhibited by greater than 80% in the presence of 500 mM D-glucose or D-galactose, the major substrates of the GalP system. The extent of inhibition of photolabeling by different sugars and sugar analogues showed that the substrate specificity of GalP closely resembles that of the mammalian passive glucose transporters. Structural similarity to the latter was revealed by tryptic digestion of [4-3H]cytochalasin B-photolabeled GalP, which yielded a radiolabeled fragment of apparent Mr 17,000-19,000, similar to that previously reported for the human erythrocyte glucose transporter.     

High-resolution crystal structures of trimeric and rod phycocyanin

The phycobilisome light-harvesting antenna in cyanobacteria and red algae is assembled from two substructures: a central core composed of allophycocyanin surrounded by rods that always contain phycocyanin (PC). Unpigmented proteins called linkers are also found within the rods and core. We present here two new structures of PC from the thermophilic cyanobacterium Thermosynechococcus vulcanus. We have determined the structure of trimeric PC to 1.35 Å, the highest resolution reported to date for this protein. We also present a structure of PC isolated in its intact and functional rod form at 1.5 Å. Analysis of rod crystals showed that in addition to the α and β PC subunit, there were three linker proteins: the capping rod linker (L_R^8.7), the rod linker (L_R), and only one of three rod-core linkers (L_RC, CpcG4) with a stoichiometry of 12:12:1:1:1. This ratio indicates that the crystals contained rods composed of two hexamers. The crystallographic parameters of the rod crystals are nearly identical with that of the trimeric form, indicating that the linkers do not affect crystal packing and are completely embedded within the rod cavities. Absorption and fluorescence emission spectra were red-shifted, as expected for assembled rods, and this could be shown for the rod in solution as well as in crystal using confocal fluorescence microscopy. The crystal packing imparts superimposition of the three rod linkers, canceling out their electron density. However, analysis of B-factors and the conformations of residues facing the rod channel indicate the presence of linkers. Based on the experimental evidence presented here and a homology-based model of the L_R protein, we suggest that the linkers do not in fact link between rod hexamers but stabilize the hexameric assembly and modify rod energy absorption and transfer capabilities.     

Structural polymorphism of oligomeric adiponectin visualized by electron microscopy

Adiponectin, a macromolecular complex similar to the members of the C1q and other collagenous homologues, elicits diverse biological functions, including anti-diabetes, anti-atherosclerosis, anti-inflammation and anti-tumor activities, which have been directly linked to the high molecular weight (HMW) oligomeric structures formed by multiples of adiponectin trimers. Here, we report the 3-D reconstructions of isolated full-length, recombinant murine C39A adiponectin trimer and hexamer of wild-type trimers (the major HMW form) determined by single-particle analysis of electron micrographs. The pleiomorphic ensemble of collagen-like stretches of the trimers leads to a dynamic structure of HMW that partition into two major classes, the fan-shaped (class I) and bouquet-shaped (class II). In both of these, while the N termini cluster into a compact ellipsoid-shaped (∼ 60 Å × 45 Å × 45 Å) volume, the collagenous domains assume a variety of arrangements. The domains are splayed by up to ∼ 90° in class I, can form a close-packed, up to ∼ 100 × 40 Å cylindrical assembly in class II, which can house about half of the 66 putative collagen-like sequence and the rest, tethered to the trimeric globular domains at the C terminus, are highly dynamic. As a result, the globular domains elaborate a variety of arrangements, covering an area of up to ∼ 4.9 × 10⁵ Ų and up to ∼ 320 Å apart, some of which were captured in reconstructions of class II. Our reconstructions suggest that the N-terminal structured domain, agreeing approximately with the expected volume for the octadecameric assembly of the terminal 27 amino acids, is crucial to the formation of the functionally active HMW. On the other hand, conformational flexibility of the trimers at the C terminus can allow the HMW to access and cluster disparate target ligands binding to the globular domains, which may be necessary to activate cellular signaling leading to the remarkable functional diversity of adiponectin.     

Crystal Structure of Mouse MD-1 with Endogenous Phospholipid Bound in Its Cavity

MD-1 is a glycoprotein that associates with a B-cell-specific RP105 protein and has a low sequence identity of 16% to MD-2 that associates with Toll-like receptor 4 and recognizes endotoxic lipopolysaccharide. MD-1 and RP105 are supposed to mediate lipopolysaccharide recognition; however, little is known about their structures and functions. Here, the crystal structure of mouse MD-1 is determined at 1.65 Å resolution. MD-1 has a hydrophobic cavity sandwiched by two β-sheets as is MD-2. The cavity is 25 Å long, 5 Å wide, and 10 Å deep: longer, narrower, and shallower than that of MD-2. No charged residues are located on the cavity entrance. MD-1 is primarily monomeric in solution but shows a dimeric assembly in the crystal lattices, with their cavity entrances facing each other. In the cavity, electron densities attributable to phosphatidylcholine are located. Together with the binding assay with tetra-acylated lipid IVa, MD-1 is shown to be a lipid-binding coreceptor.     

Crystal structure of a core domain of stomatin from Pyrococcus horikoshii Illustrates a novel trimeric and coiled-coil fold

Stomatin is a major integral membrane protein of human erythrocytes, the absence of which is associated with a form of hemolytic anemia known as hereditary stomatocytosis. However, the function of stomatin is not fully understood. An open reading frame, PH1511, from the hyperthermophilic archaeon Pyrococcus horikoshii encodes p-stomatin, a prokaryotic stomatin. Here, we report the first crystal structure of a stomatin ortholog, the core domain of the p-stomatin PH1511p (residues 56-234 of PH1511p, designated as PhSto^CD). PhSto^CD forms a novel homotrimeric structure. Three α/β domains form a triangle of about 50 Å on each side, and three α-helical segments of about 60 Å in length extend from the apexes of the triangle. The α/β domain of PhSto^CD is partly similar in structure to the band-7 domain of mouse flotillin-2. While the α/β domain is relatively rigid, the α-helical segment shows conformational flexibility, adapting to the neighboring environment. One α-helical segment forms an anti-parallel coiled coil with another α-helical segment from a symmetry-related molecule. The α-helical segment shows a heptad repeat pattern, and mainly hydrophobic residues form a coiled-coil interface. According to chemical cross-linking experiments, PhSto^CD would be able to assemble into an oligomeric form. The coiled-coil fold observed in the crystal probably contributes to self-association.     

Crystal Structures of Limulus SAP-Like Pentraxin Reveal Two Molecular Aggregations

The serum-amyloid-P-component-like pentraxin from Limulus polyphemus, a recently discovered pentraxin species and important effector protein of the hemolymph immune system, displays two distinct doubly stacked cyclic molecular aggregations, heptameric and octameric. The refined three-dimensional structures determined by X-ray crystallography, both based on the same cDNA sequence, show that each aggregate is constructed from a similar dimer of protomers, which is repeated to make up the ring structure. The native octameric form has been refined at a resolution of 3 Å, the native heptameric form at 2.3 Å, and the phosphoethanolamine (PE)-bound octameric form at 2.7 Å. The existence of the hitherto undescribed heptameric form was confirmed by single-particle analysis using cryo-electron microscopy. In the native structures, the calcium-binding site is similar to that in human pentraxins, with two calcium ions bound in each subunit. Upon binding PE, however, each subunit binds a third calcium ion, with all three calcium ions contributing to the binding and orientation of the bound phosphate group within the ligand-binding pocket. While the phosphate is well-defined in the electron density, the ethanolamine group is poorly defined, suggesting structural and binding variabilities of this group. Although sequence homology with human serum amyloid P component is relatively low, structural homology is high, with very similar overall folds and a common affinity for PE. This is due, in part, to a “topological” equivalence of side-chain position. Identical side chains that are important in both function and fold, from different regions of the sequence in human and Limulus structures, occupy similar space within the overall subunit fold. Sequence and structure alignment, based on the refined three-dimensional structures presented here and the known horseshoe crab pentraxin sequences, suggest that adaptation and refinement of C-reactive-protein-mediated immune responses in these ancient creatures lacking antibody-based immunity are based on adaptation by gene duplication.     

Structure of the Complex between HER2 and an Antibody Paratope Formed by Side Chains from Tryptophan and Serine

Engineered antibody paratopes with limited sequence diversity permit assessment of the roles played by different amino acid side chains in creating the high-affinity, high-specificity interactions characteristic of antibodies. We describe a paratope raised against the human ErbB family member HER2, using a binary diversity tryptophan/serine library displayed on phage. Fab37 binds to the extracellular domain of HER2 with sub-nanomolar affinity. An X-ray structure at 3.2 Å resolution reveals a contact paratope composed almost entirely of tryptophan and serine residues. Mutagenesis experiments reveal which of these side chains are more important for direct antigen interactions and which are more important for conformational flexibility. The crystal lattice contains an unprecedented trimeric arrangement of HER2 closely related to previously observed homodimers of the related epidermal growth factor receptor.     

The catalytic subunit of human protein kinase CK2 structurally deviates from its maize homologue in complex with the nucleotide competitive inhibitor emodin

The Ser/Thr kinase CK2 (former name: casein kinase 2) is a heterotetrameric enzyme composed of two catalytic chains (CK2α) attached to a dimer of noncatalytic subunits. Together with the cyclin-dependent kinases and the mitogen-activated protein kinases, CK2α belongs to the CMGC family of the eukaryotic protein kinases. CK2 is an important survival and stability factor in eukaryotic cells: its catalytic activity is elevated in a wide variety of tumors while its down-regulation can lead to apoptosis. Thus, CK2 is a valuable target for drug development and for chemical biology approaches of cell biological research, and small organic inhibitors addressing CK2 are of considerable interest. We describe here the complex structure between a C-terminal deletion mutant of human CK2α and the ATP-competitive inhibitor emodin (1,3,8-trihydroxy-6-methylanthraquinone, International Union of Pure and Applied Chemistry name: 1,3,8-trihydroxy-6-methylanthracene-9,10-dione) and compare it with a previously published complex structure of emodin and maize CK2α. With a resolution of 1.5 Å, the human CK2α/emodin structure has a much better resolution than its maize counterpart (2.6 Å). Even more important, in spite of a sequence identity of more than 77% between human and maize CK2α, the two structures deviate significantly in the orientation, in which emodin is trapped by the enzyme, and in the local conformations around the ligand binding site: maize CK2α shows its largest adaptations in the ATP-binding loop, whereas human CK2α shows its largest adaptations in the hinge region connecting the two main domains of the protein kinase core. These observations emphasize the importance of local plasticity for ligand binding and demonstrate that two orthologues of an enzyme can behave quite different in this respect.     

Crystal structure of the novel complex formed between zinc alpha2-glycoprotein (ZAG) and prolactin-inducible protein (PIP) from human seminal plasma

This is the first report on the formation of a complex between zinc α2-glycoprotein (ZAG) and prolactin-inducible protein (PIP). The complex was purified from human seminal plasma and crystallized using 20% polyethylene glycol 9000 and 5% hexaethylene glycol. The structure of the complex has been determined using X-ray crystallographic method and refined to an R_cryst of 0.199 (R_free = 0.239). The structure of ZAG is broadly similar to the structure of serum ZAG. The scaffolding of PIP consists of seven β-strands that are organized in the form of two antiparallel β-pleated sheets, resulting in the formation of a sandwiched β-sheet. The amino acid sequence of PIP contains one potential N-glycosylation site at Asn77, and the same is found glycosylated with four sugar residues. The structure of the complex shows that the β-structure of PIP is ideally aligned with the β-structure of domain α3 of ZAG to form a long interface between two proteins. The proximal β-strands at the long interface are arranged in an antiparallel manner. There are 12 hydrogen bonds and three salt bridges between ZAG and PIP. At the two ends of vertical interface, two salt bridges are formed between pairs of Lys41-Asp233 and Lys68-Glu229. On the perpendicular interface involving α1-α2 domains of ZAG and a loop of PIP, another salt bridge is formed. The internal space at the corner of the L-shaped structure is filled with solvent molecules including a carbonate ion. The overall buried area in the complex is approximately 914 Å², which is considerably higher than the 660 Å² reported for the class I major histocompatibility complex structures.     

Tetrameric structure of thermostable direct hemolysin from vibrio parahaemolyticus revealed by ultracentrifugation, small-angle X-ray scattering and electron microscopy

The thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus. We have characterized the conformational properties of TDH by small-angle X-ray scattering (SAXS), ultracentrifugation and transmission electron microscopy. Sedimentation equilibrium and velocity studies revealed that the protein is tetrameric in aqueous solvents. The Guinier plot derived from SAXS data provided a radius of gyration of 29.0 Å. The elongated pattern with a shoulder of a pair distance distribution function derived from SAXS data suggested the presence of molecules with an anisotropic shape having a maximum diameter of 98 Å. Electron microscopic image analysis of the negatively stained TDH oligomer showed the presence of C₄ symmetric particles with edge and diagonal lengths of 65 Å and 80 Å, respectively. Shape reconstruction was carried out by ab initio calculations using the SAXS data with a C₄ symmetric approximation. These results suggested that the tetrameric TDH assumes an oblate structure. The hydrodynamic parameters predicted from the ab initio model differed slightly from the experimental values, suggesting the presence of flexible segments.     

Influence of the Cytoplasmic Domains of Aquaporin-4 on Water Conduction and Array Formation

Phosphorylation of Ser180 in cytoplasmic loop D has been shown to reduce the water permeability of aquaporin (AQP) 4, the predominant water channel in the brain. However, when the structure of the S180D mutant (AQP4M23S180D), which was generated to mimic phosphorylated Ser180, was determined to 2.8 Å resolution using electron diffraction patterns, it showed no significant differences from the structure of the wild-type channel. High-resolution density maps usually do not resolve protein regions that are only partially ordered, but these can sometimes be seen in lower-resolution density maps calculated from electron micrographs. We therefore used images of two-dimensional crystals and determined the structure of AQP4M23S180D at 10 Å resolution. The features of the 10-Å density map are consistent with those of the previously determined atomic model; in particular, there were no indications of any obstruction near the cytoplasmic pore entrance. In addition, water conductance measurements, both in vitro and in vivo, show the same water permeability for wild-type and mutant AQP4M23, suggesting that the S180D mutation neither reduces water conduction through a conformational change nor reduces water conduction by interacting with a protein that would obstruct the cytoplasmic channel entrance. Finally, the 10-Å map shows a cytoplasmic density in between four adjacent tetramers that most likely represents the association of four N termini. This finding supports the critical role of the N terminus of AQP4 in the stabilization of orthogonal arrays, as well as their interference through lipid modification of cysteine residues in the longer N-terminal isoform.