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Title: Crystallographic and ligand binding studies on human secretory Rab27a
Author: Jamshidiha, Mostafa
ISNI:       0000 0004 7969 8180
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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Rab27a is a member of Rab GTPase family of proteins, which plays a key role in cellular transport and docking of secretory vesicles at the plasma membrane. Rab27a is involved in transport/secretion of melanosomes and vesicles containing insulin, histamine, amylase, chemokines, metaloproteases and exosomes. Rab27a has been shown to promote the growth and invasion of multiple cancer types such as breast, lung, pancreatic and hepatocellular carcinoma. Evidence suggesting the significant role of Rab27a in multiple cancer types suggests Rab27a inhibition could potentially impact invasive primary tumors and metastases. The reliance of drug discovery on high-resolution structures prompted us to design suitable Rab27a constructs that crystallize but leave biologically relevant pockets of interest unoccupied. We used Rab27a-Slp2a complex and Rab27a homologue crystal structures to modify Rab27a into a suitable construct. In the first approach, I fused the Rab-binding domain (RBD) of an effector to Rab27a to promote crystallization. Crystals from this construct diffracted to a resolution better than 2 Å. The main advantage for this approach is leaving the SF4 pocket, which is an important interface for Rab27a Slp2a interaction, empty. This pocket is not accessible in crystals obtained from Rab27a-Slp2a complex. In the second approach, I analyzed the crystal contact residues of Rab3a, a homologue of Rab27a. This information was used to make site-specific mutations based on residues important for crystallization of Rab3a, to assist the crystallization of Rab27a. This approach produced a construct that crystallizes consistently in short period of time, while leaving large surface of the interaction interface empty. Apart from crystallization, we carried out screening campaigns using differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR) against Rab27a to identify ligands that bind to Rab27a-effector interaction interface. In the first approach, a fragment library using differential scanning fluorimetry (DSF) was screened against Rab27a. In the second method, a chemical library enriched with molecules complementary to the SF4 pocket was screened against Rab27a using surface plasmon resonance (SPR). At least one chemical series targeting the SF4 pocket was obtained from screening. Altogether, in this work, I have obtained crystals of Rab27a suitable for ligand interaction studies and ligands targeting Rab27a-effector interaction interface.
Supervisor: Cota, Ernesto ; Tate, Edward Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral