Kaoru Mitsuoka

Structural determinants of water permeation through aquaporin-1.

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 3.8u2009Å 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 3u2009Å 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.

The three-dimensional structure of aquaporin-1

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 6Å resolution by cryo-electron microscopy. Each AQP1 monomer has six tilted, bilayer-spanning α-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.

Surface of bacteriorhodopsin revealed by high-resolution electron crystallography

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 α-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 3.0u2009å 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.

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.

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.

Two-dimensional crystals: a powerful approach to assess structure, function and dynamics of membrane proteins

Electron crystallography and atomic force microscopy allow the study of two‐dimensional membrane protein crystals. While electron crystallography provides atomic scale three‐dimensional density maps, atomic force microscopy gives insight into the surface structure and dynamics at sub‐nanometer resolution. Importantly, the membrane protein studied is in its native environment and its function can be assessed directly. The approach allows both the atomic structure of the membrane protein and the dynamics of its surface to be analyzed. In this way, the function‐related conformational changes can be assessed, thus providing a detailed insight on the molecular mechanisms of essential biological processes.

The three-dimensional map of microsomal glutathione transferase 1 at 6 A resolution.

Microsomal glutathione transferase 1 (MGST1) is representative of a superfamily of membrane proteins where different members display distinct or overlapping physiological functions, including detoxication of reactive electrophiles (glutathione transferase), reduction of lipid hydroperoxides (glutathione peroxidase), and production of leukotrienes and prostaglandin E. It follows that members of this superfamily constitute important drug targets regarding asthma, inflammation and the febrile response. Here we propose that this superfamily consists of a new class of membrane proteins built on a common left‐handed four‐helix bundle motif within the membrane, as determined by electron crystallography of MGST1 at 6 Å resolution. Based on the 3D map and biochemical data we discuss a model for the membrane topology. The 3D structure differs significantly from that of soluble glutathione transferases, which display overlapping substrate specificity with MGST1.

Projection map of the reaction center-light harvesting 1 complex from Rhodopseudomonas viridis at 10 Å resolution

The photosynthetic reaction center‐light harvesting 1 complex from Rhodopseudomonas viridis was purified and reconstituted into two‐dimensional crystals. The single‐layered crystalline sheets with lattice parameters a=b=133.3 Å and γ=120° were investigated by electron cryo‐microscopy and the projection map at 10 Å resolution was calculated. The opening diameter of the light‐harvesting ring of 72 Å is sufficient to allow slight movement of the reaction center within the ring. Based on characteristic features observed in the projection map, the mechanism of energy transfer from the light‐harvesting 1 complex to the reaction center was discussed.

Electron and atomic force microscopy of membrane proteins

Electron crystallography is becoming a powerful tool for the resolution of membrane protein structures. The past year has seen the production of a bacteriorhodopsin model at 3.5 A and the structure of aquaporin 1 approaching atomic resolution. Determination of surface topographies of 2D crystals using the atomic force microscope is similarly advancing to a level that reveals submolecular details. As the latter is operated in solution, membrane proteins can be observed at work.

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.