Structural basis for oligomerization of the prokaryotic peptide transporter PepT_So2

Abstract

Proton-dependent oligopeptide transporters (POTs) belong to the major facilitator superfamily (MFS) and transport dipeptides and tripeptides from the extracellular environment into the target cell. The human POTs PepT1 and PepT2 are also involved in the absorption of various orally ingested drugs. Previously reported structures revealed that the bacterial POTs possess 14 helices, of which H1–H6 and H7–H12 constitute the typical MFS fold and the residual two helices are involved in the cytoplasmic linker. PepTSo2 from Shewanella oneidensis is a unique POT which reportedly assembles as a 200 kDa tetramer. Although the previously reported structures suggested the importance of H12 for tetramer formation, the structural basis for the PepTSo2-specific oligomerization remains unclear owing to the lack of a high-resolution tetrameric structure. In this study, the expression and purification conditions for tetrameric PepTSo2 were optimized. A single-particle cryo-EM analysis revealed the tetrameric structure of PepTSo2 incorporated into Salipro nanoparticles at 4.1 Å resolution. Furthermore, a combination of lipidic cubic phase (LCP) crystallization and an automated data-processing system for multiple microcrystals enabled crystal structures of PepTSo2 to be determined at resolutions of 3.5 and 3.9 Å. The present structures in a lipid bilayer revealed the detailed mechanism for the tetrameric assembly of PepTSo2, in which a characteristic extracellular loop (ECL) interacts with two asparagine residues on H12 which were reported to be important for tetramerization and plays an essential role in oligomeric assembly. This study provides valuable insights into the oligomerization mechanism of this MFS-type transporter, which will further pave the way for understanding other oligomeric membrane proteins.

Structures and General Transport Mechanisms by the Major Facilitator Superfamily (MFS)

“… 357 Tetrameric organization of PepT So2 is mediated through a small extracellular loop domain in the C-terminal bundle of one protomer and two asparagine residues in TM12 of the C-terminal bundle of another; 356 the loop domain sequence is not found in PepT So . It has been put forward that such an assembly might be regulated by lipids, 356 yet the rationale and physiological significance of a tetramer remains unclear. Furthermore, because intracellular gating helices in PepT So and PepT So2 both involve the local movement of TM10 and TM11 in the C-terminal bundle in the transition from inward-occluded to inward-open states, 107 , 355 it further makes such oligomeric assembles difficult to rationalize as this could potentially restrict local gating.…”

DirectMX – One-Step Reconstitution of Membrane Proteins From Crude Cell Membranes Into Salipro Nanoparticles

“…The flexibility of the Salipro technology has been demonstrated by the reconstitution of a multitude of IMP sizes and shapes, ranging from small monomeric proteins, such as the 16.5-kDa bacterial outer membrane protein OmpX (Chien et al, 2017), to large protein oligomers, illustrated by the tetrameric peptide transporter PepT So2 with a total of 56 transmembrane helices (Frauenfeld et al, 2016). The Salipro nanoparticles have been shown to be compatible with a wide range of high-resolution structural, biophysical and biochemical methods (Frauenfeld et al, 2016;Chien et al, 2017;Lyons et al, 2017;Flayhan et al, 2018;Kintzer et al, 2018;Nguyen et al, 2018;Kanonenberg et al, 2019;Kehlenbeck et al, 2019;Nagamura et al, 2019). Moreover, Salipro nanoparticles can be functionalized by a diverse range of tags on the SapA scaffold protein, useful for downstream applications relevant to pharmaceutical research.…”

“…In both cases, the reconstituted protein is shown to form pure, stable and homogenous Salipro nanoparticles. Previously, detergent-purified IMPs reconstituted into Salipro nanoparticles were reported to remain structurally intact and functionally active (Chien et al, 2017;Flayhan et al, 2018;Kanonenberg et al, 2019;Kehlenbeck et al, 2019;Nagamura et al, 2019). We envision that the DirectMX methodology will enable studies on IMPs that remained so far inaccessible to other solubilization or reconstitution methods, in particular IMPs representing unstable therapeutic targets from the families of GPCRs, ion channels and transporters.…”

“…Salipro nanoparticles are highly homogeneous, displaying both increased thermostability and half-life. In addition, Salipro-reconstituted IMPs have been shown to be functional and structurally intact by a wide range of biochemical and biophysical methods such as cryogenic EM, nuclear magnetic resonance spectroscopy or enzymatic assays (Frauenfeld et al, 2016;Chien et al, 2017;Flayhan et al, 2018;Kintzer et al, 2018;Nguyen et al, 2018;Kanonenberg et al, 2019;Kehlenbeck et al, 2019;Nagamura et al, 2019).…”

Unified Nanotechnology Format: One Way to Store Them All

“… Smiley face DNA origami structure [ 44 ] combined with a protein (PDB: 6JI1 [ 45 ]), visualized in the UNF Viewer from the contents of a single UNF file. …”