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Title: Structural and functional studies of the Apical Sodium Dependent Bile Acid Transporter
Author: Sekiguchi, Yusuke
ISNI:       0000 0004 5922 7330
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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The apical sodium dependent transporter (ASBT) is responsible for the uptake of bile acids from the apical membrane of the ileum cell. Loss of function mutations of ASBT relate to several diseases, such as Crohn's disease and Primary Bile Acid Malabsorption (PBAM). It is also a target for drugs aimed at lowering cholesterol. The X-ray crystal structure of a bacterial homologue of ASBT, ASBTNM, was previously solved at 2.2Å. In order to understand mechanism of ASBT transport in more detail, further structural and functional information are required. In this thesis, a proteoliposome transport assay was performed for various mutations of residues in the sodium and substrate binding sites. ASBTNM has two sodium binding sites and the residues involved in the sites are functionally important. Both E260A and Q77A showed significantly lower transport activities compared to wild type at 40% and 60% respectively. E260 binds to one sodium ion and Q77 the other. The X-ray crystal structures of these two ASBTNM mutants were solved at 2.9 Å and 3.2 Å respectively. These mutant structures revealed that no large scale conformational changes from the wild type were observed though the mutations clearly affected sodium ion binding. These structural and functional data suggest that both two sodium ions are important for the transport activity of ASBTNM. However even without sodium ions in the binding sites, the protein can take the same conformation as the wild type structure. The data presented in this thesis, together with a comparison with the results from another bile acid transporter that was recently solved, and a homology model of human ASBT, provide insight into the transport mechanism of ASBT.
Supervisor: Beis, Konstantinos ; Drew, David Sponsor: Japan Student Services Organisation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral