Spin labeling analysis of structure and dynamics of the Na+/proline transporter of Escherichia coli

With respect to the functional importance attributed to the N-terminal part of the Na+/proline transporter of Escherichia coli (PutP), we report here on the structural arrangement and functional dynamics of transmembrane doma ins (TMs) II and III and the adjoining loop regions. Information on membran e topography was obtained by analyzing the residual mobility of site-specif ically-attached nitroxide spin label and by determination of collision freq uencies of the nitroxide with oxygen and a polar metal ion complex using el ectron paramagnetic resonance (EPR) spectroscopy. The studies suggest that amino acids Phe45, Ser50, Ser54, Trp59, and Met62 are part of TM II while G ly39 and Arg40 are located at a membrane-water interface probably forming t he cytoplasmic cap of the TM. Also Ala67 and Glu75 are at a membrane-water interface, suggesting a location close to the periplasmic ends of TMs II an d III, respectively. Ser71 between these residues is clearly in a water-exp osed loop (periplasmic loop 3). Spin labels attached to positions 80, 86, a nd 91 show EPR properties typical for a TM location (TM III). Leu97 may be part of a structured loop region while Ala107 is clearly located in a water -exposed loop (cytoplasmic loop 4). Finally, spin labels attached to the po sitions of Asp33 and Leu37 are clearly on the surface of the transporter an d are directed into an apolar environment. These findings strongly support the recently proposed 13-helix model of PutP [Jung, H., Rubenhagen, R., Teb be, S., Leifker, K,, Tholema, N., Quick, M., and Schmid, R. (1998) J. Biol. Chern. 273, 26400-26407] and suggest that TMs II and III of the transporte r are formed by amino acids Ser41 to Gly66 and Ser76 to Gly95, respectively . In addition to the topology analysis, it is shown that binding of Na+ and /or proline to the transporter alters the mobility of the nitroxide group a t the positions of Leu37 and Phe45. From these findings, it is concluded th at binding of the ligands induces conformational alterations of PutP that i nvolve at least parts of TM II and the preceding cytoplasmic loop.