Structural studies of the transport cycle of the ABC transporter ABCB1
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- Tez No: 402989
- Danışmanlar: DR. THOMAS STOCKNER, PROF. DR. PETER CHIBA
- Tez Türü: Doktora
- Konular: Biyokimya, Genetik, Tıbbi Biyoloji, Biochemistry, Genetics, Medical Biology
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2015
- Dil: İngilizce
- Üniversite: Medical University of Vienna
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 132
Özet
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Özet (Çeviri)
The multispecific drug efflux transporter P-glycoprotein (P-gp, ABCB1) is a member of the ATP binding cassette (ABC) transporter superfamily. P-gp plays an important role in drug disposition and resistance, therefore a deeper understanding of its structure and function is essential for development of novel therapeutics. Its two transmembrane domains form composite solute translocation paths, which allow for movement of structurally diverse compounds across the plasma membrane. The rotational pseudo-symmetry of the transporter implies the existence of two binding modes. Solutes typically prefer one of them. We identified two conserved tyrosine residues Y950 and Y953 in the preferred translocation path for propafenone type ligands and studied their contribution to ligand interaction. Tyrosine residues are known to be essential for molecular recognition in many biological systems. The first aim of this thesis was to assess the role of tyrosyl hydroxyl groups to hydrogen bond formation with rhodamine 123 (rh123) and propafenones. We demonstrated the active site tyrosines Y950 and Y953 as direct hydrogen bonding partners of both rh123 and propafenone type ligands. Moreover, we confirmed the previously reported dual substrate binding mode of P-gp. The second aim addressed the importance of conformational changes of the protein for achieving an outward facing substrate releasing state. For this aim the Sav1866 crystal structure, which shows wing-like extensions on the extracellular side, was an ideal starting structure. Wing formation corresponds to a possible movement of helices within the membrane spanning helical bundles, the extent of which was explored in a physiological membrane environment by introduction of cross-linkable cysteine residues in the extracellular portion of P-gp. We demonstrated spontaneous formation of cross-links at zero length and further confirmed that TM helices 6 and 12 can come in close proximity at their extracellular ends using MTS cross-linkers of different lengths. Nucleotide binding did not prevent formation of zero-length cross-links. We also demonstrated a requirement of residues I328 and F971 for protein mechanics. The third and final aim was to provide insights into the catalytic cycle of ABC transporters with one non-canonical nucleotide binding site (NBS). Both NBSs are active in P-gp, while the bile salt export pump (BSEP) has four non-canonical substitutions in NBS1. One of these is the catalytic glutamate residue which is replaced by methionine. When introduced to P-gp, methionine at this position yielded a non-functional transporter that was also found to show weak UIC2 probed conformational changes in the presence of substrates. Simultaneous mutation of the additional three residues that differ at the NBD/NBD interface to those found in BSEP did not rescue P-gp transport function. However, we found recurrence of conformational changes in the presence of the well-known modulator tariquidar. We posit that these additional mutations lower the energy barrier of NBS1 and consequently lead to partial regain in function. P-gp and BSEP may differ in the mechanism by which ATP binding and hydrolysis are coupled to drug transport; the NBD:NBD interface, however, may be expected to have a strong influence on substrate dependent conformational changes. Neverthless, it is not the sole determinant for function. Overall, these data contribute to further define P-gp solute interactions in the drug binding cavity, as well as conformational changes, which are associated with the P-gp transport cycle. Data thus resulted in an improved characterization of model ABC transporters of the ABCB subfamily at a molecular level and results are expected to aid in drug development process.
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