Helix bundle structure of the sodium/proline symporter PutP obtained by DEER distance measurements in a native lipid environment.

The sodium/proline symporter PutP uses the free energy stored in an electrochemical sodium gradient to drive the selective transport of proline with a stoichiometry of 1:1. [1]. To understand the molecular mechanism underlying proline transport, knowledge of the structure is a basic prerequisite. However, due to their size, hydrophobicity, and conformational flexibility, the generation of high-resolution structural data of membrane proteins by X-ray crystallography and NMR is still a challenge. Here, we present a new modeling approach that allows determination of the helix bundle structure of polytopic membrane proteins in the native environment of the lipid bilayer. This approach was used to obtain the first structure of the helix bundle of PutP based on experimentally determined intramolecular distances and template restraints derived from the ten-helix core of the vSGLT crystal structure [2]. For this purpose, DEER distance measurements between spin labels attached to helix ends were conducted and mean interspin distances were determined. Fitting algorithm based on matrix geometry in combination with prediction of spin label conformations by a rotamer library approach [3] resulted in an ensemble of helix bundle structures. The central structure of the ensemble showed a core structure with a fold similar to that of the vSGLT template. However, the position and orientation of the three non-core helices of PutP was found to be different.