Extracellular Loop 4 of the Proline Transporter PutP Controls the Periplasmic Entrance to Ligand Binding Sites

•External loop 4 of PutP has two helical segments forming a hairpin-like structure•Phe314 in eL4b of PutP anchors the loop in the core of the protein•A pulling force on Phe314 causes movement of eL4 and closes the extracellular gate

SummaryThe Na+/proline symporter (PutP), like several other Na+-coupled symporters, belongs to the so-called LeuT-fold structural family, which features ten core transmembrane domains (cTMs) connected by extra- and intracellular loops. The role of these loops has been discussed in context with the gating function in the alternating access model of secondary active transport processes. Here we report the complete spin-labeling site scan of extracellular loop 4 (eL4) in PutP that reveals the presence of two α-helical segments, eL4a and eL4b. Among the eL4 residues that are directly implicated in the functional dynamics of the transporter, Phe314 in eL4b anchors the loop by means of hydrophobic contacts to cTM1 close to the ligand binding sites. We propose that ligand-induced conformational changes at the binding sites are transmitted via the anchoring residue to eL4 and through eL4 further to adjacent cTMs, leading to closure of the extracellular gate.

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