Kebbel, Fabian. Structural and functional characterization of the integral membrane proteins CitS and CCR5 by electron microscopy. 2013, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_10423
The main part of this PhD thesis is the structural and functional characterization of the secondary citrate/sodium symporter CitS from Klebsiella pneumonia, mainly by transmission electron microscopy (TEM). CitS is the best characterized member of the bacterial 2-hydroxycarboxylate transporter (2-HCT) family. It facilitates the secondary transport of bivalent citrate ions driven by a coupled flux of Na+ across the inner membrane of the host. Hydropathy profiling and extensive biochemical experimentation prior to this study predicted CitS to represent a new structural fold as paradigm for numerous related proteins, so that it constitutes a highly attractive target for structural studies.
As a first step, two-dimensional (2D) crystals of recombinant CitS were produced by dialysis assisted reconstitution of pure detergent solubilized protein into bilayer forming phospholipids. Extensive screening of crystallization conditions led to highly ordered tubular 2D crystals suitable for structure determination by cryo-electron crystallography. Therefore, numerous sample preparation methods were evaluated, while plunge-freezing provided significantly better results compared to commonly used sugar embedding methods. As described in chapter 2, image processing of electron micrographs from plunge-frozen 2D crystals provided the projection structure of CitS at 6 Å resolution. The transporter appears as oval shaped dimer measuring 5*9 nm in the membrane plane. The dimer reveals three distinct structural domains being formed by two dense clusters of α-helices at each molecule’s tip and a third, less dense domain in the center of the dimer. The domains are separated by solvent areas. Surprisingly, this architecture highly resembles that of the unrelated Na+/H+ antiporter NhaP1. In projection, each CitS monomer reveals eleven TMS that well match previous membrane topology predictions. Finally, we developed several models describing possible monomer-monomer interfaces and domain organizations.
In chapter 3, we describe the 3D structure of CitS at 6/15 Å resolution obtained by electron micrographs of tilted 2D crystal samples. Based on the 3D volume, we developed a molecular model that reveals eleven α-helices and two additional helical reentrant loops. The central dimerization domain is formed by seven partially tilted helices, while the distal cluster reveals 4 transmembrane segments surrounding the two reentrant loops. We also find internal structural symmetry for the strongly intertwined N- and C-terminal domains as prerequisite for substrate translocation by the ‘alternating access’ mechanism. Additional projection structures of CitS in various substrate environments (Na+, K+, acetate and citrate) allowed us to map the conformational space. The binding of citrate as main substrate induces a defined movement of α-helices spatially limited to the helix cluster in each monomer. This primarily occurs in the presence of Na+, and much less with K+ and highlights the high co-ion specificity. These findings also enable us to assign the dense helix cluster as substrate binding and translocation site.
In a second project, various biophysical techniques were used to characterize the recombinant G protein-coupled receptor (GPCR) CCR5. Besides its important role in immune responses, CCR5 also acts as co-receptor during HIV-1 target cell entry. In chapter 4, an innovative E. coli based expression platform is presented that enables the production of 10 mg purified protein from 1L cell culture. We could demonstrate ligand binding, structural integrity, homogeneity and stability of triply isotope labeled CCR5. This provides a promising starting point for ongoing structural studies, especially by nuclear magnetic resonance (NMR) spectroscopy.
|Committee Members:||Hiller, Sebastian|
|Faculties and Departments:||05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Stahlberg)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||101 S.|
|Last Modified:||30 Jun 2016 10:53|
|Deposited On:||17 Jul 2013 13:02|
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