[Structure of a membrane protein in eicosanoid and glutathione metabolism by electron crystallography].
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Amino Acid Sequence↗
Biomedical subjects
Kaoru Mitsuoka
Publications and source records attributed to Kaoru Mitsuoka.
Aquaporin-11 containing a divergent NPA motif has normal water channel activity.
Recently, two novel mammalian aquaporins (AQPs), AQPs 11 and 12, have been identified and classified as members of a new AQP subfamily, the "subcellular AQPs". In members of this subfamily one of the two asparagine-proline-alanine (NPA) motifs, which play a crucial role in selective water conduction, are not completely conserved. Mouse AQP11 (mAQP11) was expressed in Sf9 cells and purified using the detergent Fos-choline 10. The protein was reconstituted into liposomes, which were used for water conduction studies with a stopped-flow device. Single water permeability (pf) of AQP11 was measured to be 1.72+/-0.03x10(-13) cm(3)/s, suggesting that other members of the subfamily with incompletely conserved NPA motifs may also function as water channels.
Alanine↗
Structural basis for detoxification and oxidative stress protection in membranes.
Synthesis of mediators of fever, pain and inflammation as well as protection against reactive molecules and oxidative stress is a hallmark of the MAPEG superfamily (membrane associated proteins in eicosanoid and glutathione metabolism). The structure of a MAPEG member, rat microsomal glutathione transferase 1, at 3.2 A resolution, solved here in complex with glutathione by electron crystallography, defines the active site location and a cytosolic domain involved in enzyme activation. The glutathione binding site is found to be different from that of the canonical soluble glutathione transferases. The architecture of the homotrimer supports a catalytic mechanism involving subunit interactions and reveals both cytosolic and membraneous substrate entry sites, providing a rationale for the membrane location of the enzyme.
Amino Acid Sequence↗
Two-dimensional crystallization and analysis of projection images of intact Thermus thermophilus V-ATPase.
H(+)-ATPase/synthases are membrane-bound rotary nanomotors that are essential for energy conversion in nearly all life forms. A member of the family of the vacuolar-type ATPases (V-ATPases) from Thermus thermophilus, sometimes also termed A-type ATPase, was purified to homogeneity and subjected to two-dimensional (2D) crystallization trials. A novel approach to the 2D crystallization of unstable complexes yielded densely packed sheets of V-ATPase, exhibiting crystalline arrays. Aggregation of the V-ATPase under acidic conditions during reconstitution circumvented the continuous dissociation of the whole complex into the V(1) and V(o) domains. The resulting three-dimensional aggregates were converted into 2D sheets by the use of a basic buffer, and after a short annealing cycle, ordered arrays of up to 1.5 microm diameter appeared. Fourier transforms calculated from micrographs taken from the negatively stained sample showed diffraction spots to a resolution of 23A. The Fourier transforms of the untilted images revealed unit-cell dimensions of a=232A, b=132A, and gamma=90 degrees , and a projection map was calculated by merging 11 images. The most probable molecular packing suggests p22(1)2(1) symmetry of the crystals and dimer contacts between the V(1) domains.
Crystallization↗
Implications of the aquaporin-4 structure on array formation and cell adhesion.
Aquaporin-4 (AQP4) is the predominant water channel in the mammalian brain and an important drug target for treatment of cerebral edema, bipolar disorder and mesial temporal lobe epilepsy. We determined the AQP4 structure by electron crystallography of double-layered, two-dimensional (2D) crystals. The structure allows us to discuss how the expression ratio between the long and short AQP4 splicing variant can determine the size of in vivo orthogonal arrays. Furthermore, AQP4 contains a short 3(10) helix in an extracellular loop, which mediates weak but specific interactions between AQP4 molecules in adjoining membranes. This finding suggests a previously unexpected role for AQP4 in cell adhesion. This notion was corroborated by expression of AQP4 in L-cells, which resulted in clustering of the cells. Our AQP4 structure thus enables us to propose models for the size regulation of orthogonal arrays and channel-mediated cell adhesion.
Amino Acid Sequence↗
Electron tomography reveals diverse conformations of integrin alphaIIbbeta3 in the active state.
We used electron tomography to determine the three-dimensional (3D) structure of integrin alphaIIbbeta3 in the active state. We found that we obtained better density maps when we reconstructed a 3D volume for each individual particle in the tilt series rather than to extract the particle-containing subvolumes from a 3D reconstruction of the entire specimen area. The 3D tomographic reconstructions of 100 particles revealed that activated alphaIIbbeta3 adopts many different conformations. An average of all the individual 3D reconstructions nicely accommodated the crystal structure of the alphaVbeta3 headpiece, confirming the locations assigned to the alpha- and beta-subunit in the density map. The most striking finding of our study is the structural flexibility of the lower leg of the beta-subunit as opposed to the conformational stability of the leg of the alpha-subunit. The good fit of the atomic structure of the betaI domain and the hybrid domain in the active state showed that the hybrid domain swings out, and most particles used for tomography are in the active state. Multivariate statistical analysis and classification applied to the set of 3D reconstructions revealed that more than 90% reconstructions are grouped into the classes that show the active state. Our results demonstrate that electron tomography can be used to classify complexes with a flexible structure such as integrins.
Cell Line, Tumor↗
Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique.
Image shift due to beam-induced specimen charging has become the most severe problem in electron microscopy for imaging two-dimensional (2D) crystals of biological macromolecules, especially in the case of highly tilted specimens. Image shift causes diffraction spots perpendicular to the tilt axis to disappear even at medium or low resolution. The yield of good images from tilted specimens prepared on a single layer of continuous carbon support film is therefore very low. In this paper, we have used 2D crystals of aquaporin-4 to investigate the effect of a carbon sandwich preparation method on specimen charging. We find that a larger number of images show sharp diffraction spots perpendicular to the tilt axis if crystals are placed in between two sheets of carbon film as compared to images taken from specimens prepared by the conventional single carbon support film technique. Our results demonstrate that the reproducible carbon sandwich preparation technique overcomes the severe specimen charging problem and thus has the potential to significantly speed up structure analysis by electron crystallography.
Aquaporin 4↗
[Three-dimensional structural analysis of biological molecules by cryo-electron microscopy].
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Animals↗
Roles of Met-34, Cys-64, and Arg-75 in the assembly of human connexin 26. Implication for key amino acid residues for channel formation and function.
Connexins form a family of membrane proteins that assemble into communication channels and directly connect the cytoplasms of adjoining cells. Malfunctioning of connexin channels often cause disease, such as the mutations M34T and R75W in human connexin 26, which are associated with hereditary deafness. Another residue known to be essential for normal channel activity in the connexin is Cys-64. To obtain structural and functional insights of connexin 26, we studied the roles of these three residues by expressing mutant connexins in insect Sf9 and HeLa cells. The M34T and M34A mutants both formed gap junction plaques, but dye transfer assays showed that the M34A mutant had a significantly reduced permeability, suggesting that for proper channel function a side chain of adequate size is required at this position. We propose that Met-34 is located in the innermost helix of the channel, where it ensures a fully open channel structure via interactions with other transmembrane helices. Gap junction channels formed by the R75W and R75D mutants dissociated upon solubilization in dodecyl maltoside, whereas the R75A mutant remained hexameric. All gap junctions formed by Arg-75 mutants also showed only negligible activity in dye transfer experiments. These results suggest that residue Arg-75 plays a role in subunit interactions needed to retain a functional and stable connexin hexamer. The C64S mutant was suggested to be defective in oligomerization and/or protein folding even in the presence of wild-type connexin.
Animals↗
A new technique to co-localise membrane proteins with Homer/vesl.
The minimal requirements were defined as necessary for cluster formation of the group 1 metabotropic glutamate receptor (mGluR), which is regulated by the Homer/vesl family of scaffolding proteins [Curr. Opin. Neurobiol. 10 (2000) 370]. Cluster formation of G-protein-coupled receptors (GPCRs) plays a fundamental role in signal transduction, particularly at the neuronal synapse. To understand the interaction of mGluR with PSD-Zip45, a Homer/vesl family member, we designed a series of chimeric receptor proteins, consisting of C-terminal mGluR1alpha sequences that were fused to endothelin B receptors (ET(B)Rs). In vitro and in vivo studies revealed that an extended 20 amino acid long C-terminal mGluR1alpha peptide, including the proline-rich core motif PPXXF, is sufficient to induce clustering of chimeric ET(B)R/mGluR1alpha receptors by PSD-Zip45. This result is especially important because it constitutes the basis for a new approach to form two-dimensional crystals of membrane proteins in situ, which may render unstable membrane proteins amenable to electron crystallographic structure determination.
Animals↗
Crystal structure of the Homer 1 family conserved region reveals the interaction between the EVH1 domain and own proline-rich motif.
PSD-Zip45 (also named Homer 1c/Vesl-1L) is a synaptic scaffolding protein, which interacts with neurotransmitter receptors and other scaffolding proteins to target them into post-synaptic density (PSD), a specialized protein complex at the synaptic junction. Binding of the PSD-Zip45 to the receptors and scaffolding proteins results in colocalization and clustering of its binding partners in PSD. It has an Ena/VASP homology 1 (EVH1) domain in the N terminus for receptor binding, two leucine zipper motifs in the C terminus for clustering, and a linking region whose function is unclear despite the high level of conservation within the Homer 1 family. The X-ray crystallographic analysis of the largest fragment of residues 1-163, including an EVH1 domain reported here, demonstrates that the EVH1 domain contains an alpha-helix longer than that of the previous models, and that the linking part included in the conserved region of Homer 1 (CRH1) of the PSD-Zip45 interacts with the EVH1 domain of the neighbour CRH1 molecule in the crystal. The results suggest that the EVH1 domain recognizes the PPXXF motif found in the binding partners, and the SPLTP sequence (P-motif) in the linking region of the CRH1. The two types of binding are partly overlapped in the EVH1 domain, implying a mechanism to regulate multimerization of Homer 1 family proteins.
Amino Acid Motifs↗
Structure and function of water channels.
All aquaporins are efficient water transporters, while sustaining strict selectivity, even against protons, thereby maintaining the proton gradient across the cell membrane. Recently solved structures of these membrane channels have helped us to understand this remarkable property.
Aquaporins↗