Modelling the MHAS2168 in inward-open conformation

To build an inward open conformation of MHAS2168, MODELLER⁷⁹ was used. Initially, a web frontend (http://www.ebi.ac.uk/Tools/msa/clustalo) to CLUSTAL Omega⁹¹ was used to align the target protein sequence of MHAS2168 to the proton-dependent oligopeptide transporter PepTSo2 from Shewanella oneidensis⁹² (sequence identity is 23%). The obtained alignment was manually curated to avoid fragmentation mostly in the region of the linker helices. The PepTSo2 X-ray structure PDB 4LEP (resolution 3.20 Å) was used as template in combination with an initial mock-model of MHAS2168 in inward-open conformation. To obtain this mock-model, the N- (residues 1–183) and C-terminal regions (residues 263–495) of MHAS2168 were independently superposed to the homologous parts of PepTSo2. During the modeling procedure the linker helices were restrained to assume a canonical α-helix conformation, however, their overall position differed from the template 4LEP. Therefore, to correctly position the target linker-helices, the best structural model out of 100 generated ones was further used for a second round of MODELLER together with both linker-helices modeled in the initial step and in turn superposed to the 4LEP linker-helices.