Trans reentrant loop structures in secondary transporters

PhD ceremony: Mr. A.J. Dobrowolski, 12.45 uur, Academiegebouw, Broerstraat 5, Groningen

Dissertation: Trans reentrant loop structures in secondary transporters

Promotor(s): prof. A.J.M. Driessen

Faculty: Mathematics and Natural Sciences

Biological membranes have a very important role in protecting the cell against the stressful environment and, at the same time, they have to enable uptake of different substrates, like nutrition factors, and extrusion of waste products. Essential transport functions are facilitated by transport proteins, which catalyze the physical process of movement of substrates from one side of the membrane to the other. The work described in this thesis focuses on the investigation of structural similarity between families of transporter proteins, and, in particular, the glutamate symporter GltS of Escherichia colii and the citrate symporter CitS of Klebsiella pneumoniae were studied. These two transporter proteins, and other members of their families, are not related in amino acid sequence but share similar hydropathy profiles, which suggests a similar folding of the proteins in the membrane and a similar mechanism of transport. Starting from the bioinformatics data obtained from the MemGen classification system and a previous topology study of CitS we were able to predict and confirm experimentally the membrane topology of GltS and in this way show that analysis by hydropathy profiles of membrane proteins is a powerful tool to study structures of membrane proteins in the absence of X-ray structural data. The obtained data suggest that both proteins consist of two homologous domains containing trans reentrant loops, and that are connected by a long cytoplasmic loop. The reentrant loops are important for the activity of the transporters, interact at the interface of the two domains and form a translocation pathway for substrates and co-ions.