Biophysical and biochemical characterization of fumarases fromLeishmania major

Abstract

Corticosteroid-binding globulin (CBG) is a blood plasma protein that transports the weakly water-soluble hormone, cortisol, throughout the circulation. It is a member of the serine protease inhibitor (SERPIN) structural family; upon cleavage by human neutrophil elastase, it undergoes the canonical S-to-R transition, which results in a change in its binding affinity for cortisol, allowing it to release the hormone at sites of inflammation. We plan to redesign CBG to transport compounds other than its physiological ligand and, by harnessing the S-to-R transition, release them at specific sites in the body. In preliminary experiments, we succeeded in altering the proteinase specificity of CBG, making it susceptible to cleavage by human αthrombin. We plan to build on this and prove the principle that by altering the amino acid sequence of the reactive centre loop, CBG can be made to release its ligand in response to specific cleavage by various proteinases. We are especially interested in making it susceptible to proteinases that are tissue-specific so that the engineered protein would be able to deliver its cargo at very specific sites in the body. We are also looking at redesigning the steroid-binding pocket of CBG so that it is able to bind other compounds with high affinities. Apart from that, it is also necessary to make the change in affinity between the S-state and the R-state much larger should the engineered protein ever be considered for use as a drug-delivery system. In order to study the binding site, and to understand in depth how the change in binding affinity is achieved by the S-to-R transition, it is necessary to obtain crystal structures of CBG in both forms from the same organism as the structures currently available for the native and cleaved forms of CBG are from different organisms [1], [2]. This makes it difficult to tell if the differences in the positions of various residues in the binding pocket are due to conformational changes or a lack of sequence identity. These structural studies will be complemented with molecular modeling and biochemical experiments to determine how the residues lining the binding pocket contribute to ligand binding and to the mechanism that results in the change in ligand-binding affinity following proteolytic cleavage of the reactive centre loop. Whilst studying other factors that can affect ligand binding, we also discovered that binding affinity decreases when temperature increases, even within the small range allowed physiologically. This observation was corroborated by a recent study by Cameron et al [3]. We think this is another property we can exploit because the local temperature in different parts of the body can vary with tissue type and disease state, and consequently may add another level of specificity for the release of potentially therapeutic compounds from the engineered protein.