| الملخص: | <p>Integral membrane proteins are responsible for a wide range of cellular functions and are important targets for the development of novel therapeutics. Therefore, obtaining structural and mechanistic insights into their function is of central importance for rational drug design. In this thesis, I focused on characterising three human integral membrane proteins using X-ray crystallography, cryo-EM, mass spectrometry and other biochemical methods.</p>
<p>MFSD10 is a member of the drug:H+ antiporter family 1 (DHA1) of the Major Facilitator Superfamily of solute carriers and has been proposed to act as an efflux pump for organic anions, including several non-steroidal anti-inflammatory drugs (NSAIDs). Here, I present a crystal structure of human MFSD10 in an outward-facing partially occluded conformation, with a bound citrate ion from the crystallisation solution. The structure provides insights into the recognition of organic anions by MFSD10 and suggests a potential mechanism of proton coupling. An in vitro assay was employed to confirm the NSAID:H+ antiporter function of MFSD10 and will constitute the starting point for additional functional assays to dissect the mechanism of this transporter.</p>
<p>The second focus of this thesis falls on elucidating the mechanisms of two enzyme families responsible for the acyl chain specificity in fatty acid elongation and sphingolipid biosynthesis, respectively. ELOVL elongases catalyse the first rate-limiting condensation step of the fatty acid elongation cycle in the endoplasmic reticulum (ER). The recently solved crystal structure of human ELOVL7 provided the first structural insights into this enzyme family. Here, I followed up on the structural data by characterising the mechanism of this enzyme using mass spectrometry. I demonstrated that catalysis by human ELOVL7, and likely other ELOVLs, proceeds via a ping-pong type mechanism involving an unusual covalent acyl-imidazole intermediate. Lastly, I focused on obtaining structural insights into the function of human ceramide synthases, which catalyse the N-acylation of sphingoid bases in the ER and determine the acyl chain composition of sphingolipids. I conducted a systematic screening of expression and purification conditions for two members of this family, identified suitable truncations for structural studies, obtained and validated nanobody binders, and optimised cryo-EM conditions. Together, these provide an advanced position for future structural studies to obtain valuable three-dimensional information on these important drug targets.</p>
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