Zantza, Iliana Pyrris, Yiannis Raniolo, Stefano Papadaki, Georgia F. Lambrinidis, George Limongelli, Vittorio Diallinas, George Mikros, Emmanuel 2023 Transporters mediate the uptake of solutes, metabolites and drugs across the cell membrane. The eukaryotic FurE nucleobase/H+ symporter of Aspergillus nidulans has been used as a model protein to address structure–function relationships in the APC transporter superfamily, members of which are characterized by the LeuT-fold and seem to operate by the so-called ‘rocking-bundle’ mechanism. In this study, we reveal the binding mode, translocation and release pathway of uracil/H+ by FurE using path collective variable, funnel metadynamics and rational mutational analysis. Our study reveals a stepwise, induced-fit, mechanism of ordered sequential transport of proton and uracil, which in turn suggests that FurE, functions as a multi-step gated pore, rather than employing ‘rocking’ of compact domains, as often proposed for APC transporters. Finally, our work supports that specific residues of the cytoplasmic N-tail are involved in substrate translocation, in line with their essentiality for FurE function. application/pdf https://n2t.net/ark:/12658/srd1329319 https://susi.usi.ch/global/documents/329319 https://susi.usi.ch/documents/329319/files/Raniolo-Limongelli_2023_Elsevier_JMB.pdf eng info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jmb.2023.168226 info:eu-repo/semantics/altIdentifier/ark/12658/srd1329319 info:eu-repo/semantics/openAccess CC BY-NC-ND Journal of molecular biology. - 2023, vol. 435, no. 19, p. 168226 Aspergillus nidulans NCS1 transporters FurE nucleobase-proton symporter Funnel metadynamics Mechanism of function info:eu-repo/classification/udc/61 Uracil/H+ symport by FurE refines aspects of the rocking-bundle mechanism of APC-type transporters http://purl.org/coar/resource_type/c_6501