Conformational dynamics and pH-regulation in ApcT.

Abstract

ApcT is a LeuT-fold symporter of the amino acid/polyamine/organocation (APC) superfamily of transporters. On the basis of a crystal structure of ApcT captured in a putatively inward-facing conformation, a model of conformational dynamics triggered by the protonation of residue K158, has been proposed. To test this model and define the structural changes involved in transporter isomerization, we used EPR spectroscopy to measure distances between pairs of spin labels on both sides of the transporter. Moreover, we conducted mutational analyses which have identified novel ion interactions critical for pH regulation of conformational transitions. To complement the DEER distance data, a series of computational models have been generated, using an in-silico mutagenesis approach, yielding an ensemble of conformations in the inward- and outward-facing conformations. By integrating the computational models and experimental data, we propose a model of coupled transport by ApcT.