Structure and function of amino acid and peptide transport proteins

Casagrande, Fabio. Structure and function of amino acid and peptide transport proteins. 2008, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_8365

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All living cells are enclosed by biological membranes that separate the interior cytoplasm from the outer environment. Composed of a phospholipid bilayer with embedded proteins biological membranes act as insulators and filters. The transfer of selected substances and information across the membrane is controlled and mediated through membrane proteins. As a result, membrane proteins are central to almost all cellular processes. They play key roles in signalling between cells, in transport across cell membranes and in energy transduction processes. To accomplish these versatile cell functions, membrane proteins are available in an abundant diversity. Accordingly, it’s not astounding that about 30% of all genes encoded in the human genome are membrane proteins. However, only 158 (Status: May 2008) unique membrane protein structures, mainly from bacterial membrane proteins, have been deposited thus far in the Protein Data Bank (PDB), compared to the more than 10’000 soluble protein structures. These numbers highlight the enormous structural work that remains to be done in the field of membrane proteins. From a biomedical perspective, membrane proteins constitute about 50% of possible targets for novel drugs. The threedimensional (3D) structure of a protein is an essential starting point for the investigation of its molecular mechanisms of action, the basis for drug design. Therefore structural information of membrane proteins is of fundamental importance for human health. High-resolution structure determination of membrane proteins is currently one of the greatest challenges in cell biology. Membrane proteins possess a hydrophobic belt that is required for their incorporation into lipid membranes. For the extraction and purification of membrane proteins detergents are used to keep the proteins in a solubilized state. The lack of structural information on membrane proteins is mainly related to their low expression levels, the instability in the detergent solution and their resistance to crystallization. The latter is a considerable limitation because X-ray crystallography, which at the present time is the most powerful technique for determining protein structures, requires highly ordered 3D protein crystals. Besides structure determination by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, the method of electron crystallography on 2D crystals has become increasingly important in membrane protein research. The striking advantage of 2D crystals is that the membrane protein is analyzed in its native environment, the lipid bilayer. However, as with 3D crystals, the production of highly ordered 2D crystals is a major barrier. Electron crystallography was first applied in 1975 by Henderson and Unwin to study the structure of bacteriorhodopsin in purple membranes (1, 2). Since then, substantial progress has been made in further development of electron crystallography, especially in sample preparation, cryo-transmission electron microscopy (cryoTEM) imaging and data processing. Thus, improved cryo-TEM in combination with electron diffraction of 2D crystals is used to establish the 3D protein structure (3). The recent structure determination of the mammalian aquaporin-0 (AQP-0) at the remarkable resolution of 1.9 Å demonstrated impressively the potential of the cryo-TEM approach. So far seven atomic models (<4 Å) of membrane proteins have been determined by high-resolution electron crystallography (plant light harvesting complex 2 (4), AQP-1 (5, 6), nicotinic acetylcholine receptor (7, 8), AQP-0 (9, 10), AQP-4 (11), glutathione transferase 1 (2)). Thereby one has to notice that less than two dozen groups are pursuing electron crystallography, compared to the hundreds of groups in X-ray crystallography. Even more mportant, 2D crystallization combined with TEM is not an all-or-nothing approach, meaning that also from poorly ordered crystals low resolution structures are obtainable. In conclusion, electron crystallography presents a highly favourable and successful method to explore the structures of membrane proteins (13). The atomic force microscope (AFM) is a powerful tool to investigate the surface topography of membrane proteins embedded in lipid bilayers under near-physiological conditions, i.e., in buffer solution, at room temperature and under normal pressure. The high lateral (≥5 Å) and vertical resolution (~1 Å), and the high signal-to-noise ratio of the topographs acquired by AFM make this instrument unique to study surface structure and dynamics of single functional membrane proteins. Besides high-resolution surface imaging, structural and mechanical properties of single membrane proteins can be studied by single molecule force spectroscopy (SMFS), an AFMrelated technique. In a typical SMFS experiment the cantilever tip approaches the membrane protein, pushes on it and then retracts. During this approach-retraction cycle the force acting on the molecule is measured and plotted as a function of the tip-surface distance: the so-called ‘force curve’ is thus obtained. Such force curves reveal details about inter- and intra-molecular interactions, unfolding barriers and energy landscapes in membrane proteins. Because these measurements take place in solution at physiological conditions, the binding of ligands and the subsequent alteration within the protein may be detected and visualized. This offers the unique possibility to directly monitor structural changes related to biological processes.
eugindat/). In Eugindat biological and medical scientists with various backgrounds were united in one consortium to concentrate the research on amino acid and peptide transporters. The transport of amino acids into cells is a crucial process for all living species from bacteria to humans. Defects on proteins involved in this transport lead to strong disturbances in the amino acid metabolism of the organism. Humans strongly rely on amino acids in their diet, since nine essential amino acids cannot be synthesized from other precursors. Consequently, it’s all the more important that systems for the uptake, distribution and reabsorption of amino acids work properly. Thereby, the proximal tubule plays a central role by reabsorbing over 95% of the filtered amino acid load. In the case where elevated levels (>5%) are detected in urine, the term aminoaciduria is applied. Primary Inherited Aminoacidurias (PIA) is a group of rare diseases arisen from genetic defects in amino acid transporters expressed in the plasma membrane of renal epithelial cells. PIA members are classified by the target amino acid or acids involved. The group includes Cystinuria, Lysinuric Protein Intolerance (LPI), Dicarboxylic Aminoaciduria (DA), Hartnup Disorder (HDis), Iminoglycinuria (IG) and unlabeled aminoacidurias. Cystinuria and LPI are the best studied PIAs. It was demonstrated that members of the heteromeric amino acid transporter family (HAT family) are the molecular base of cystinuria and LPI. HATs are composed of a heavy subunit (HSHAT) and the corresponding light subunit (LSHAT) that are liked together by a disulfide bridge. HSHATs are N-glycosylated type II membrane glycoproteins, whereas LSHATs are nonglycosylated polytopic membrane proteins with twelve putative transmembrane segments (TMS) (14-16). Two genes rBAT (HSHAT) and b0,+AT (LSHAT) could be identified responsible for cystinuria (17, 18) while mutations in the system 4F2hc (HSHAT) and y+LAT1 (LSHAT) lead to LPI (19, 20).
The structural studies of amino acid and peptide transporters by TEM and AFM reported in this thesis became possible thank to an European initiative called Eugindat (European genomics initiative on disorders of plasma membrane To acquire a thorough knowledge of the amino acid transporters, http://www.ub.es/ structure of relevant transporters for PIA and renal reabsorption of amino acids, 2D and 3D crystallization of membrane and soluble proteins for structure determination was addressed within Eugindat. Recently, Eugindat members reported the X-ray structure of 4F2hc protein at 2.1 Å (monoclinic) and 2.8 Å (orthorhombic) resolutions (21). In contrast, there are no structures from eukaryotic or human amino acid transporters available. So far, only two structures of bacterial transport proteins with homology to their eukaryotic counterparts were successfully solved: the atomic structures of a bacterial leucine transporter (22) and of a bacterial glutamate transporter (23). Despite the awarded effort, these two transporters do not correspond to the class of LSHAT proteins. However, these examples indicate that important information to understand structure-function relationships of amino acid transporters is gained from prokaryotic homologues. In pursuit of our aim to reveal first structural information of PIA related amino acid transporters, we searched and studied prokaryotic homologues of LSHAT.
Within Eugindat, structural and functional studies were extended to cover other important transporters involved in human health, i.e. peptide transporters. Members of this second class of transport proteins were extensively studied in the past and belong to the peptide transporter (PTR) family (26, 27). Peptide transporters are integral membrane proteins that mediate the cellular uptake of di- and tripeptides. Similar to the amino acid transport, peptide transport is of fundamental importance in all species. In human peptide transport at the brush border membranes of small intestine, kidney and lung is handled by two members from the PTR family designated as PEPT1 and PEPT2. PEPT1 is considered as the major route by which protein digestion products enter the body. Previous studies demonstrated that peptide transporters have broad substrate specificity transporting essentially all 400 possible dipetides, 8000 possible tripeptides and a large spectrum of peptidomimetics into the cell (i.e. pharmacologically active compounds) (2831). Peptide transporters are therefore potent drug delivery systems. Substrate translocation The LSHAT membrane proteins b0,+AT and is coupled to the proton movement down y+LAT1 are members of the L-type amino acid an electrochemical proton gradient with the transporter family (LAT) that is a subfamily of membrane potential as the main driving force. the amino acid/polyamine/organocation (APC) transporter superfamily. The APC superfamily In contrast to the wealth of functional counts nearby 250 members in prokaryotes information, no structural information on peptide and eukaryotes that function as solute cation transporters is available. For our structural and symporters and solute cation antiporters (17). functional studies within Eugindat, we selected Most APC members are predicted to possess from several possible candidates the bacterial twelve α-helical transmembrane segments PTR family members YbgH, YdgR (TppB, (TMS) with cytosolic located N- and C-termini DtpA) and YhiP (DtpB) from Escherichia coli. (24, 25). According to the high sequence identity The structural work on these bacterial peptide and homology to eukaryotic and human APC transporters presented in this thesis represents transporters, we selected after an exhaustive search the first published structural information of these for structural (TEM/AFM) and functional studies important class of transport proteins. the two prokaryotic amino acid transporters AdiC and SteT. As shown in the presented thesis, the L-arginine/agmatine antiporter AdiC and the threonine/serine exchange transporter SteT represent excellent proteins to elucidate the molecular architecture of transporters from the APC superfamily.
Advisors:Engel, Andreas
Committee Members:Palacin, Manuel and Fotiadis, Dimitrios
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Structural Biology (Engel)
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:8365
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:125
Identification Number:
Last Modified:23 Feb 2018 11:44
Deposited On:08 Jan 2010 11:44

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