|M.Sc Student||Qasem Abdallah Hiba|
|Subject||The Molecular Mechanism of Heme Transport by HmuU2V2-T|
from Yersinia Pestis
|Department||Department of Medicine||Supervisor||Professor Oded Lewinson|
|Full Thesis text|
ABC transporters are a large family of integral membrane complexes found in all kingdoms of life. They share a basic domain organization: 2 transmembrane domains (TMDs) that form the translocation pore, and 2 cytosolic nucleotide binding domains (NBDs) that hydrolyze ATP. ATP hydrolysis drives conformational changes, which enable the transport of solutes through the membrane, against a concentration gradient.
ABC transporters that function as importers require an additional component, a substrate binding protein (SBP) that binds the substrate molecule and brings it to its cognate transporter.
HmuU2V2 from Yersinia pestis is an ABC heme importer that accepts heme from its periplasmic SBP, HmuT. Iron acquisition is essential for bacterial virulence, and bacteria utilize iron compounds (e.g.,heme that is a critical component of the hemoglobin molecules) as an iron source. Yersinia pestis, can grow on iron deficient medium supplemented with heme as an iron source.
The atomic structure of HmuT(the SBP) has been solved. Unlike other SBPs HmuT has been shown to bind two heme molecules in its binding pocket, with a 100-fold difference in affinity between the first and the second binding events, suggesting that they may have different mechanistic roles.
The 3-D structure of HmuU2V2 (the core transporter subunit) was also solved in the absence of nucleotides and the transporter was found in an outward-facing conformation (TMD helices open toward the periplasm), similar to the structure of the nucleotide free bacterial vitamin B12 transporter, BtuC2D2. Studies of the mechanism of action of BtuC2D2 revealed it to function by a mechanism that is very different, if not opposite, from all ABC importers characterized to date. This finding raises the question whether the structural similarity between the BtuC2D2 and the HmuU2V2 also implies a mechanistic similarity?
A key step in the transport cycle of all ABC transporters is the formation of a complex between the core transporter unit and the cognate SBP. Unlike other ABC transporter, BtuC2D2 forms a high affinity stable complex with its Substrate Binding Protein in a substrate and nucleotide free conformation. In addition, ATP binding has an inhibitory effect on the complex formation of BtuC2D2. Moreover, high substrate levels have an inhibitory effect on the complex formation of BtuC2D2.
In my research I aimed to characterize the dynamics of interaction between the membrane embedded transporter HmuU2V2 and the heme binding protein HmuT by using Surface Plasmon Resonance (SPR) measurements and membrane sedimentation experiments. This will help determine whether HmuU2V2 and BtuC2D2 share a common mechanism of action, as well as help us understand the importance of the two heme binding events.
My results revealed significant mechanistic similarities between the well-characterized BtuC2D2 and my protein of interest HmuU2V2: both transporters form a high affinity stable complex with their SBP in the absence of substrate, in both cases the substrate has an inhibitory effect on complex formation, and ATP reduces the interaction between the transporters and their SBP.