@article {6252, title = {Allosteric mechanism of water-channel gating by Ca(2+)-calmodulin.}, journal = {Nature Structural \& Molecular Biology}, volume = {20}, year = {2013}, month = {2013 Jul 28}, pages = {1085-92}, abstract = {

Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudoatomic structure of full-length mammalian aquaporin-0 (AQP0, Bos taurus) in complex with CaM, using EM to elucidate how this signaling protein modulates water-channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits.

}, issn = {1545-9985}, doi = {10.1038/nsmb.2630}, author = {Reichow, Steve L and Clemens, Daniel M and Freites, J Alfredo and N{\'e}meth-Cahalan, Karin L and Heyden, Matthias and Tobias, Douglas J and Hall, James E and Gonen, Tamir} } @article {6578, title = {Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation.}, journal = {eLife}, volume = {2}, year = {2013}, month = {2013 Nov 5}, pages = {e01319}, abstract = {

Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an \~{}16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.01319.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.01319}, author = {Smith, F Donelson and Reichow, Steve L and Esseltine, Jessica L and Shi, Dan and Langeberg, Lorene K and Scott, John D and Gonen, Tamir} } @article {4922, title = {AKAP2 anchors PKA with aquaporin-0 to support ocular lens transparency.}, journal = {EMBO Molecular Medicine}, volume = {4}, year = {2012}, month = {2012 Jan}, pages = {15-26}, abstract = {

A decline in ocular lens transparency known as cataract afflicts 90\% of individuals by the age 70. Chronic deterioration of lens tissue occurs as a pathophysiological consequence of defective water and nutrient circulation through channel and transporter proteins. A key component is the aquaporin-0 (AQP0) water channel whose permeability is tightly regulated in healthy lenses. Using a variety of cellular and biochemical approaches we have discovered that products of the A-kinase anchoring protein 2 gene (AKAP2/AKAP-KL) form a stable complex with AQP0 to sequester protein kinase A (PKA) with the channel. This permits PKA phosphorylation of serine 235 within a calmodulin (CaM)-binding domain of AQP0. The additional negative charge introduced by phosphoserine 235 perturbs electrostatic interactions between AQP0 and CaM to favour water influx through the channel. In isolated mouse lenses, displacement of PKA from the AKAP2-AQP0 channel complex promotes cortical cataracts as characterized by severe opacities and cellular damage. Thus, anchored PKA modulation of AQP0 is a homeostatic mechanism that must be physically intact to preserve lens transparency.

}, keywords = {A Kinase Anchor Proteins, Animals, Aquaporins, Calmodulin, Cataract, Crystalline, Cyclic AMP-Dependent Protein Kinases, Eye Proteins, Inbred C57BL, Lens, Membrane Proteins, Mice, Phosphopeptides, Phosphorylation, Protein Binding, Sheep, Static Electricity, Water}, issn = {1757-4684}, doi = {10.1002/emmm.201100184}, author = {Gold, Matthew G and Reichow, Steve L and O{\textquoteright}Neill, Susan E and Weisbrod, Chad R and Langeberg, Lorene K and Bruce, James E and Gonen, Tamir and Scott, John D} } @article {4935, title = {Advances in structural and functional analysis of membrane proteins by electron crystallography.}, journal = {Structure}, volume = {19}, year = {2011}, month = {2011 Oct 12}, pages = {1381-93}, abstract = {

Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography.

}, keywords = {Aquaporin 1, Archaea, Automatic Data Processing, Bacteriorhodopsins, Cell Membrane, Computer-Assisted, Cryoelectron Microscopy, Crystallography, Detergents, Dialysis, Humans, Image Processing, Lipid Bilayers, Membrane Proteins, Membrane Transport Proteins, Protein Conformation, Static Electricity}, issn = {1878-4186}, doi = {10.1016/j.str.2011.09.001}, author = {Wisedchaisri, Goragot and Reichow, Steve L and Gonen, Tamir} } @article {4928, title = {The binding of cholera toxin to the periplasmic vestibule of the type II secretion channel.}, journal = {Channels}, volume = {5}, year = {2011}, month = {2011 May-Jun}, pages = {215-8}, abstract = {

The type II secretion system (T2SS) is a large macromolecular complex spanning the inner and outer membranes of many gram-negative bacteria. The T2SS is responsible for the secretion of virulence factors such as cholera toxin (CT) and heat-labile enterotoxin (LT) from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. CT and LT are closely related AB5 heterohexamers, composed of one A subunit and a B-pentamer. Both CT and LT are translocated, as folded protein complexes, from the periplasm across the outer membrane through the type II secretion channel, the secretin GspD. We recently published the 19 {\r A} structure of the V. cholerae secretin (VcGspD) in its closed state and showed by SPR measurements that the periplasmic domain of GspD interacts with the B-pentamer complex. Here we extend these studies by characterizing the binding of the cholera toxin B-pentamer to VcGspD using electron microscopy of negatively stained preparations. Our studies indicate that the pentamer is captured within the large periplasmic vestibule of VcGspD. These new results agree well with our previously published studies and are in accord with a piston-driven type II secretion mechanism.

}, keywords = {Bacterial Proteins, Bacterial Secretion Systems, Cholera Toxin, Periplasm, Porins, Protein Folding, Protein Structure, Protein Subunits, Protein Transport, Quaternary, Tertiary, Vibrio cholerae}, issn = {1933-6969}, author = {Reichow, Steve L and Korotkov, Konstantin V and Gonen, Melissa and Sun, Ji and Delarosa, Jaclyn R and Hol, Wim G J and Gonen, Tamir} } @article {3632, title = {Structure of the cholera toxin secretion channel in its closed state.}, journal = {Nature Structural \& Molecular Biology}, volume = {17}, year = {2010}, month = {2010 Oct}, pages = {1226-32}, abstract = {

The type II secretion system (T2SS) is a macromolecular complex spanning the inner and outer membranes of Gram-negative bacteria. Remarkably, the T2SS secretes folded proteins, including multimeric assemblies such as cholera toxin and heat-labile enterotoxin from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. The major outer membrane T2SS protein is the {\textquoteright}secretin{\textquoteright} GspD. Cryo-EM reconstruction of the V. cholerae secretin at 19-{\r A} resolution reveals a dodecameric structure reminiscent of a barrel, with a large channel at its center that contains a closed periplasmic gate. The GspD periplasmic domain forms a vestibule with a conserved constriction, and it binds to a pentameric exoprotein and to the trimeric tip of the T2SS pseudopilus. By combining our results with structures of the cholera toxin and T2SS pseudopilus tip, we provide a structural basis for a possible secretion mechanism of the T2SS.

}, keywords = {Amino Acid, Amino Acid Sequence, Bacterial, Bacterial Outer Membrane Proteins, Cholera Toxin, Computer-Assisted, Conserved Sequence, Cryoelectron Microscopy, Fimbriae, Image Processing, Models, Molecular, Molecular Sequence Data, Periplasm, Protein Conformation, Protein Structure, Recombinant Fusion Proteins, Sequence Alignment, Sequence Homology, Surface Plasmon Resonance, Tertiary, Vibrio cholerae}, issn = {1545-9985}, doi = {10.1038/nsmb.1910}, author = {Reichow, Steve L and Korotkov, Konstantin V and Hol, Wim G J and Gonen, Tamir} } @article {3631, title = {Tension directly stabilizes reconstituted kinetochore-microtubule attachments.}, journal = {Nature}, volume = {468}, year = {2010}, month = {2010 Nov 25}, pages = {576-9}, abstract = {

Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct {\textquoteright}bi-oriented{\textquoteright} kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for \>30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

}, keywords = {Chromosomes, Fungal Proteins, Kinetochores, Microtubules, Saccharomyces cerevisiae}, issn = {1476-4687}, doi = {10.1038/nature09594}, author = {Akiyoshi, Bungo and Sarangapani, Krishna K and Powers, Andrew F and Nelson, Christian R and Reichow, Steve L and Arellano-Santoyo, Hugo and Gonen, Tamir and Ranish, Jeffrey A and Asbury, Charles L and Biggins, Sue} } @article {3638, title = {Lipid-protein interactions probed by electron crystallography.}, journal = {Current Opinion in Structural Biology}, volume = {19}, year = {2009}, month = {2009 Oct}, pages = {560-5}, abstract = {

Electron crystallography is arguably the only electron cryomicroscopy (cryoEM) technique able to deliver an atomic-resolution structure of membrane proteins embedded in the lipid bilayer. In the electron crystallographic structures of the light driven ion pump, bacteriorhodopsin, and the water channel, aquaporin-0, sufficiently high resolution was obtained and both lipid and protein were visualized, modeled, and described in detail. An extensive network of lipid-protein interactions mimicking native membranes is established and maintained in two-dimensional (2D) crystalline vesicles used for structural analysis by electron crystallography. Lipids are tightly integrated into the protein{\textquoteright}s architecture where they can affect the function, structure, quaternary assembly, and the stability of the membrane protein.

}, keywords = {Crystallography, Electron, Lipids, Microscopy, Models, Molecular, Proteins, X-Ray}, issn = {1879-033X}, doi = {10.1016/j.sbi.2009.07.012}, author = {Reichow, Steve L and Gonen, Tamir} } @article {45966, title = {Electron crystallography of aquaporins.}, journal = {IUBMB Life}, volume = {60}, year = {2008}, month = {2008 Jul}, pages = {430-6}, abstract = {

Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions.

}, keywords = {Animals, Aquaporins, Cell Membrane, Crystallization, Crystallography, Crystallography, X-Ray, Electrons, Humans, Lipids, Models, Molecular, Protein Conformation, Protein Folding, Proteins, Water}, issn = {1521-6551}, doi = {10.1002/iub.53}, author = {Andrews, Simeon and Reichow, Steve L and Gonen, Tamir} } @article {45967, title = {Noncanonical binding of calmodulin to aquaporin-0: implications for channel regulation.}, journal = {Structure}, volume = {16}, year = {2008}, month = {2008 Sep 10}, pages = {1389-98}, abstract = {

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homotetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens, where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show that CaM binds the AQP0 C-terminal domain in a calcium-dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a noncanonical fashion, suggesting a form of cooperativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0{\textquoteright}s CaM binding domain inhibits the AQP0/CaM interaction, suggesting a temporal regulatory mechanism for complex formation.

}, keywords = {Amino Acid Sequence, Animals, Aquaporins, Calcium, Calmodulin, Crystallins, Eye Proteins, Ion Channels, Models, Molecular, Molecular Sequence Data, Phosphorylation, Protein Binding, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Sequence Homology, Amino Acid, Sheep}, issn = {0969-2126}, doi = {10.1016/j.str.2008.06.011}, author = {Reichow, Steve L and Gonen, Tamir} }