Membrane topology screen of secondary transport proteins in structural class ST[3] of the MemGen classification. Confirmation and structural diversity.

The MemGen structural classification of membrane proteins groups families of proteins by hydropathy profile alignment. Class ST[3] of the MemGen classification contains 32 families of transporter proteins including the IT superfamily. Transporters from 19 different families in class ST[3] were evaluated by the TopScreen experimental topology screening method to verify the structural classification by MemGen. TopScreen involves the determination of the cellular disposition of three sites in the polypeptide chain of the proteins which allows for discrimination between different topology models. For nearly all transporters at least one of the predicted localizations is different in the models produced by MemGen and predictor TMHMM. Comparison to the experimental data showed that in all cases the prediction by MemGen was correct. It is concluded that the structural model available for transporters of the [st324]ESS and [st326]2HCT families is also valid for the other families in class ST[3]. The core structure of the model consists of two homologous domains, each containing 5 transmembrane segments, which have an opposite orientation in the membrane. A reentrant loop is present in between the 4th and 5th segments in each domain. Nearly all of the identified and experimentally confirmed structural variations involve additions of transmembrane segments at the boundaries of the core model, at the N- and C-termini or in between the two domains. Most remarkable is a domain swap in two subfamilies of the [st312]NHAC family that results in an inverted orientation of the proteins in the membrane.