Bing K. Jap

Structural basis of water-specific transport through the AQP1 water channel.

Water channels facilitate the rapid transport of water across cell membranes in response to osmotic gradients. These channels are believed to be involved in many physiological processes that include renal water conservation, neuro-homeostasis, digestion, regulation of body temperature and reproduction. Members of the water channel superfamily have been found in a range of cell types from bacteria to human. In mammals, there are currently 10 families of water channels, referred to as aquaporins (AQP): AQP0–AQP9. Here we report the structure of the aquaporin 1 (AQP1) water channel to 2.2 Å resolution. The channel consists of three topological elements, an extracellular and a cytoplasmic vestibule connected by an extended narrow pore or selectivity filter. Within the selectivity filter, four bound waters are localized along three hydrophilic nodes, which punctuate an otherwise extremely hydrophobic pore segment. This unusual combination of a long hydrophobic pore and a minimal number of solute binding sites facilitates rapid water transport. Residues of the constriction region, in particular histidine 182, which is conserved among all known water-specific channels, are critical in establishing water specificity. Our analysis of the AQP1 pore also indicates that the transport of protons through this channel is highly energetically unfavourable.

Structural studies of the transmembrane C-terminal domain of the amyloid precursor protein (APP): does APP function as a cholesterol sensor?

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an alpha-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of alpha-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Abeta production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by beta- and gamma-secretase and resulting amyloid-beta production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.

Protein 4.1R core domain structure and insights into regulation of cytoskeletal organization.

The crystal structure of the core domain (N-terminal 30 kDa domain) of cytoskeletal protein 4.1R has been determined and shows a cloverleaf-like architecture. Each lobe of the cloverleaf contains a specific binding site for either band 3, glycophorin C/D or p55. At a central region of the molecule near where the three lobes are joined are two separate calmodulin (CaM) binding regions. One of these is composed primarily of an α-helix and is Ca 2+ insensitive; the other takes the form of an extended structure and its binding with CaM is dramatically enhanced by the presence of Ca 2+, resulting in the weakening of protein 4.1R binding to its target proteins. This novel architecture, in which the three lobes bind with three membrane associated proteins, and the location of calmodulin binding sites provide insight into how the protein 4.1R core domain interacts with membrane proteins and dynamically regulates cell shape in response to changes in intracellular Ca2+ levels.

Molecular design of PhoE porin and its functional consequences

The three-dimensional structure of PhoE porin from Escherichia coli, negatively stained with uranyl acetate, has been determined by electron crystallographic techniques to a resolution of about 18 A. The structure shows that PhoE porin consists of trimeric stain-filled channels as the basic unit. The trimeric channels converge as they transverse the membrane but they do not merge. Our three-dimensional structure of PhoE porin indicates that there is a short, narrower segment of channel, which extends beyond the visible strain-filled portion of the channel. The map of glucose-embedded PhoE porin in projection normal to the membrane has also been determined to a resolution of 6.5 A. The projected map shows trimeric ring-like structures, which are presumably cylindrical domains of beta-sheet. At the 3-fold symmetry axis of the trimer, there is a low density region, which is suggested to be a site of lipopolysaccharide that is required for channel and bacteriophage receptor activities. The structural model of the PhoE monomer consists of a flattened cylinder with a large water-filled vestibule about 35 A long with an elliptically shaped opening that is 27 A along the major axis and 18 A along the minor axis. The vestibule has a narrower extension about 10 A long with an average diameter of about 10 A. The vestibule wall is formed by beta-sheet, which may have a large fraction of the beta-strands oriented normal to membrane. Our structural model provides a clue as to how the surface charges on the outer membrane may regulate the permeation of ionic solutes through the channel.

Structural genomics of membrane proteins.

Improvements in the fields of membrane-protein molecular biology and biochemistry, technical advances in structural data collection and processing, and the availability of numerous sequenced genomes have paved the way for membrane-protein structural genomics efforts. There has been significant recent progress, but various issues essential for high-throughput membrane-protein structure determination remain to be resolved.

Three-dimensional electron diffraction of PhoE porin to 2·8resolution

A three-dimensional set of electron diffraction intensities of PhoE porin embedded in trehalose extending to 2.8 A resolution has been collected and analyzed. The strongest high-resolution intensities are distributed as a figure of revolution about the z*-axis and are located primarily in a resolution range of 4.5 A to 5.0 A. Within this region, centered near 4.8 A resolution the brightest intensities are clustered about inclination angles of 35 degrees and 0 degrees from the a*, b* plane. This distribution of intensities indicates that the beta-sheet in PhoE porin is arranged to form a cylinder-like structure that contains major populations of beta-sheet strands tilted an average of 35 degrees and 0 degrees with respect to the membrane plane normal. This cylindrical structure has been seen previously in the high-resolution projection map of PhoE as an elliptical ring of high density.

X-ray diffraction by crystals of beef heart ubiquinol: Cytochrome c oxidoreductase

Beef heart mitochondrial ubiquinol:cytochrome c oxidoreductase has been crystallized in the shape of hexagonal bipyramids. At present the crystals diffract X-rays to 4.7 A. From preliminary analysis the diffraction pattern appears to be consistent with space group P6(1)22 or P6(5)22 and with unit cell parameters a = b = 212 A and c = 352 A.

Hybrid molecular structure of the giant protease tripeptidyl peptidase II

Tripeptidyl peptidase II (TPP II) is the largest known eukaryotic protease (6 MDa). It is believed to act downstream of the 26S proteasome, cleaving tripeptides from the N termini of longer peptides, and it is implicated in numerous cellular processes. Here we report the structure of Drosophila TPP II determined by a hybrid approach. We solved the structure of the dimer by X-ray crystallography and docked it into the three-dimensional map of the holocomplex, which we obtained by single-particle cryo–electron microscopy. The resulting structure reveals the compartmentalization of the active sites inside a system of chambers and suggests the existence of a molecular ruler determining the size of the cleavage products. Furthermore, the structure suggests a model for activation of TPP II involving the relocation of a flexible loop and a repositioning of the active-site serine, coupling it to holocomplex assembly and active-site sequestration.

High-resolution electron diffraction of reconstituted PhoE porin.

PhoE porin has been reconstituted with phospholipid, forming large membrane patches. Electron diffraction shows that the reconstituted PhoE porin forms highly coherent crystalline arrays, giving structural information to a resolution of 3.4 A. The crystal form is of the orthorhombic space group P2(1)2(1)2, with unit cell dimensions a = 150 A and b = 129 A. Images of negatively stained PhoE crystalline patches show that there are four PhoE porin trimers in a unit cell.

Solution NMR Approaches for Establishing Specificity of Weak Heterodimerization of Membrane Proteins

Solution NMR provides a powerful approach for detecting complex formation involving weak to moderate intermolecular affinity. However, solution NMR has only rarely been used to detect complex formation between two membrane proteins in model membranes. The impact of specific binding on the NMR spectrum of a membrane protein can be difficult to distinguish from spectral changes that are induced by nonspecific binding and/or by changes that arise from forced cohabitation of the two proteins in a single model membrane assembly. This is particularly the case when solubility limits make it impossible to complete a titration to the point of near saturation of complex formation. In this work experiments are presented that provide the basis for establishing whether specific complex formation occurs between two membrane proteins under conditions where binding is not of high avidity. Application of these methods led to the conclusion that the membrane protein CD147 (also known as EMMPRIN or basigin) forms a specific heterodimeric complex in the membrane with the 99-residue transmembrane C-terminal fragment of the amyloid precursor protein (C99 or APP-βCTF), the latter being the immediate precursor of the amyloid-β polypeptides that are closely linked to the etiology of Alzheimers disease.