Probing binding and occlusion of substrate in the human creatine transporter‐1 by computation and mutagenesis

Abstract

AbstractIn chordates, energy buffering is achieved in part through phosphocreatine, which requires cellular uptake of creatine by the membrane‐embedded creatine transporter (CRT1/SLC6A8). Mutations in human slc6a8 lead to creatine transporter deficiency syndrome, for which there is only limited treatment. Here, we used a combined homology modeling, molecular dynamics, and experimental approach to generate a structural model of CRT1. Our observations support the following conclusions: contrary to previous proposals, C144, a key residue in the substrate binding site, is not present in a charged state. Similarly, the side chain D458 must be present in a protonated form to maintain the structural integrity of CRT1. Finally, we identified that the interaction chain Y148‐creatine‐Na+ is essential to the process of occlusion, which occurs via a “hold‐and‐pull” mechanism. The model should be useful to study the impact of disease‐associated point mutations on the folding of CRT1 and identify approaches which correct folding‐deficient mutants.