Detecting molecular interactions that stabilize, activate and guide ligand-binding of the sodium/proton antiporter MjNhaP1 from Methanococcus jannaschii.

Integral membrane proteins are involved in virtually every cellular process. Precisely regulating these machineries would allow controlling many human and vertebrate diseases. Embedded into cellular membranes, membrane proteins establish molecular interactions that sensitively react to environmental changes and to molecular compounds, such as ligands or inhibitors. We applied atomic force microscopy (AFM) to image the Na(+)/H(+) antiporter MjNhaP1 from Methanococcus jannaschii, and single-molecule force spectroscopy (SMFS) to probe molecular interactions that drive the protein structure-function relationship. High-resolution AFM topographs showed the dimeric assembly of MjNhaP1 being reconstituted into a lipid bilayer. SMFS of MjNhaP1 unraveled molecular interactions stabilizing individual structural domains. Transmembrane domains exhibited certain probabilities to unfold individually or cooperatively with other domains resulting in different unfolding pathways. Helices VIII and X established pH sensitive interactions altering significantly upon MjNhaP1 activation, while removal of the ligand (Na(+)) destabilized the entire antiporter except helix VIII. It is assumed that Asp234/235 of helix VIII are involved in the ligand-binding site and that helix X plays a functional role in the activation of the transporter.