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Structural Membrane Biochemistry

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    Structural Membrane Biochemistry / Structural Membrane Biochemistry
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    Structural Membrane Biochemistry
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    The dynamic regulation of water channels

    Water channels, or aquaporins, form specialized channels in membranes for water permeation. These are extremely efficient channels that allow millions of water molecules to permeate the pore per second. Because they are channels, the cell can regulate their activity dynamically to help maintain homeostasis. In the case of the eye lens water channel aquaporin-0 (AQP0), it can be regulated by at least 4 known mechanisms that we studied over the last decade. The first is irreve
    rsible and involves the cleavage of the C-terminal domain of AQP0. The cleavage results in complete pore closure and AQP0 ceases to act as a water channel. Instead it becomes an adhesive protein mediating cell-to-cell adhesive junctions.
     
    Full-length AQP0 is dynamically modulated by 3 mechanisms: pH, calcium/calmodulin (Ca2+/CaM) and protein phosphorylation. We recently showed that the binding of Ca2+/CaM to AQP0 results in partial pore closure. The net effect is that the permeability through AQP0 halves in the presence of Ca2+/CaM. Conversely, we showed that phosphorylation of AQP0 by anchored PKA (AKAP2/PKA complex) abolished CaM binding, keeping AQP0 in the open conformation and functioning at maximal activity.
     
    Our studies of channel phosphorylation led us to discover a new protein in the eye lens called AKAP2. Our biochemical and structural studies indicate that AKAPs anchor PKA onto substrate and provide the kinase a sphere of action in which the kinase could phosphorylate substrates in a cAMP independent way. This is fundamentally an exciting observation because it helps explain how fast phosphorylation can occur, as seen for example in heart cells. Moreover, we showed that inhibition of phosphorylation of AQP0 in the lens results in cataract formation. Essentially we recapitulated the lens disease ex vivo by inhibiting protein phosphorylation.

    Membrane protein complexes

    Our structure of the AQP0/CaM complex is the first for any full-length membrane channel in complex with this ubiquitous secondary messenger. Current efforts in the laboratory are to understand how Ca2+/CaM binds to and modulates the activity of other channels such as ion channels.
     
    We are also trying to understand more about the AQP-AKAP system, in particular we are trying to assemble the AQP2-AKAP18-PKA complex and AQP0-AKAP2-PKA complex for structural studies. Intrinsically disordered regions of proteins are widespread in nature yet the mechanistic roles they play in biology are underappreciated.  Such disordered segments can act simply to link functionally coupled structural domains or they can orchestrate enzymatic reactions through a variety of allosteric mechanisms.  The regulatory subunits of protein kinase A provide an example of this important phenomenon where functionally defined and structurally conserved domains are connected by intrinsically disordered regions of defined length with limited sequence identity.  Our studies show that this seemingly paradoxical amalgam of order and disorder permits fine-tuning of local protein phosphorylation events. The anchoring of PKA by AKAP affords the kinase a sphere of action in which multiple targets can get phosphorylated fast in a cAMP independent way.

     

    Membrane transporters involved in nutrient uptake

    Sugar uptake

    The major facilitator superfamily of membrane proteins is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been captured and structurally characterized in multiple transporting conformations including both the outward and inward facing states. We determined the crystal structure of XylE in a new inward-facing open conformation. Structural comparison of XylE in this conformation with its outward-facing partially occluded conformation reveals how this transporter functions through a non-symmetrical rocker switch movement of the N-domain as a rigid body and the C-domain as a flexible body. Molecular dynamics simulations were employed to help describe how XylE transitions in a lipid membrane to facilitate sugar transport.

     

    Nitrogen uptake

    Nitrate is the preferred nitrogen source for plants on which all higher forms of life ultimately depend. Plants and microorganisms evolved to efficiently assimilate nitrate. Despite decades of effort no structure was available for any nitrate transport protein and the mechanism by which nitrate is transported remained largely obscure until our study was published. We reported the structure of a bacterial nitrate/nitrite transport protein, NarK, from Escherichia coli, with and without substrate. The structures revealed a positively charged substrate-translocation pathway lacking protonatable residues, suggesting that NarK functions as a nitrate/nitrite exchanger and that H+s are unlikely to be co-transported. Conserved arginine residues form the substrate-binding pocket, which is formed by association of helices from the two halves of NarK. Key residues that are important for substrate recognition and transport were identified and related to extensive mutagenesis and functional studies. We proposed that NarK exchanges nitrate for nitrite by a rocker-switch mechanism facilitated by inter-domain H-bond networks.

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    Relevant papers:

    1. Gonen T., Sliz P., Cheng Y., Kistler J. and Walz T. (2004) Aquaporin-0 membrane junctions reveal the structure of a closed water pore. Nature. 429 : 193 – 197.

    2. Gonen T., Cheng Y., Kistler J. and Walz T. (2004) Aquaporin-0 membrane junctions form upon proteolytic cleavage. Journal of Molecular Biology 342 : 1337 - 1345.

    3. Gonen T., Cheng Y., Sliz P., Hiroaki Y., Fujiyoshi Y., Harrison SC., Walz T (2005) Lipid–protein interactions in double layered two dimensional crystals of AQP0. Nature 438: 633 - 638.

    4. Reichow L.S. and Gonen T*. (2008) Non-canonical binding of calmodulin to aquaporin-0: implications for channel regulation. Structure. 16: 1389 – 1398.

    5. Gold MG, Reichow SL., O’Neill SE, Weisbrod CR, Langeberg LK, Bruce JE, Gonen T* and Scott JD*. (2012) AKAP2 anchors PKA with aquaporin-0 to support ocular lens transparency. EMBO Molecular Medicine 4: 15-26.

    6. Reichow S.L, Clemens D.M., Freites J.A., Németh-Cahalan K.L., Heyden M., Tobias D.J., Hall J.E*. and Gonen T*. (2013) Allosteric mechanism of water channel gating by Ca2+–calmodulin. Nature  Structural and Molecular Biology – 20 (9): 1085 - 1092.

    7. Smith FD., Reichow LS., Esseltine JL., Shi D., Langeberg LK., Scott J* and Gonen T* (2013). Intrinsic disorder within an AKAP-Protein kinase A complex guides local substrate phosphorylation. eLife 2:e01319.

     

    Relevant Reviews:

    1. Gonen T*. and Walz T* (2006) The structure of Aquaporins. Quarterly Reviews in Biophysics 39 : 361 -396.

    2. Engel, A., Fujiyoshi, Y., Gonen, T. and Walz, T. (2008) Junction forming aquaporins. Current Opinion in Structural Biology. 18 : 229 - 235.

    3. Andrews, S.A., Reichow, L.S. and Gonen T*. (2008) Electron crystallography of aquaporins. IUBMB Life 60: 430 – 436.

    4. Reichow, S.L. and Gonen T*. (2009) Lipid-protein interactions probed by electron crystallography. Current Opinion in Structural Biology. 19: 560 - 565

    5. Gold M. Gonen T and Scott J. (2013) Local cAMP signaling in disease at a glance. Journal of Cell Science. 126: 4537 - 4543.

     

    Relevant papers

    1. Zheng H., Taraska J., Merz A. and Gonen T*. (2010) The prototypical H+/galactose symporter GalP assembles into functional trimers. Journal of Molecular Biology. 396 : 593 – 601.

    2. Wisedchaisri G., Dranow D.M., Lie T.J., Bonanno J.B., Patskovsky Y., Ozyurt S.A., Michael Sauder J.M., Almo S.C., Wasserman S.R., Burley S.K., Leigh J.A. and Gonen T*. (2010) Structural underpinnings of nitrogen regulation by the prototypical nitrogen-responsive transcriptional factor NrpR. Structure 18 1512 - 1521.

    3. Zheng H, Wisedchaisri W and Gonen T*. (2013) Crystal structure of a nitrate/nitrite exchanger. Nature – 497: 647-651.

    4. Wisedchaisri G., Park MS., Iadanza MG., Zheng H. and Gonen T*. (2014) Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE. Nature Communications – (5)4521: 1 - 11.