Presentation Information
[4GteX-07]Ancestral reconstruction and in vitro transport assays for the small multidrug resistance protein EmrE
Yuta Ogawa1, Eishin Yamazaki1, ○Hironori Sugiyama1, Liam Longo1, Tomoaki Matsuura1 (1. Institute of Science Tokyo (Japan))
Keywords:
Transporter,EmrE,Small multidrug resistance protein,liposome
One key challenge in transporter engineering is to make substrate promiscuity tunable. On-demand, rational control of affinity and selectivity would broaden the utility of transporters and unlock new opportunities in biomanufacturing. However, practical design principles that link sequence to multisubstrate transport behavior remain poorly defined. The small multidrug resistance (SMR) family provides an experimentally tractable chassis for developing such principles, with EmrE serving as a well-studied model. Ancestral sequence reconstruction (ASR) offers an evolution-guided strategy to efficiently sample informative variants, and promiscuity has been suggested to vary with phylogenetic depth within a lineage. Thus, sampling reconstructed ancestral nodes may provide an interpretable route to connect sequence and function beyond what ad hoc mutagenesis typically affords. Here, we are establishing an integrated pipeline that links ASR to in vitro functional assays of EmrE, enabling systematic comparisons between extant proteins and ancestrally inferred variants.
The SMR superfamily is broadly divided into Qac-type and Gdx-type transporters: Qac proteins typically export hydrophobic cations, including quaternary ammonium compounds, whereas Gdx proteins export guanidinium ions. We curated about 10,000 SMR homologs spanning both groups and constructed a phylogenetic tree to guide node selection for reconstruction. From this tree, we prioritized five internal nodes predicted to differ in substrate preference. Notably, Node 1 corresponds to the deepest split separating the Qac and Gdx clades, providing a key reference point for probing how substrate recognition diverged early in SMR evolution.
We assessed transport activity of reconstructed variants using solid-supported membrane electrophysiology (SSME) with three substrates: tetrapropylammonium (TPA) and tetramethylammonium (TMA) as representative Qac-type substrates, and guanidinium (Gdm) as a Gdx-type substrate. Wild-type EmrE transported TPA and TMA, whereas no transport signal was detected with Gdm; the signal was consistent with antiport activity as reported previously [1,2]. Although still preliminary, ancestral variants exhibited distinct behaviors. For example, the Node 1 variant showed a weak signal with TPA and Gdm but not with TMA, and the current signatures were inverted relative to wild-type. One possible interpretation is that Node 1 operates as a symporter rather than an antiporter. Detailed validation is ongoing. In the presentation, we will also report recent progress in improving the efficiency and reproducibility of in vitro reconstitution, including potential contributions of the SRP/SP pathway and membrane glycolipids in addition to SecYEG and YidC [3].
Overall, this platform enables efficient sampling of evolutionarily informed sequences that shift substrate preference and transport coupling, providing a path toward rational engineering of controllable efflux for biomanufacturing.
References[1] Kermani, et al. Elife 11, (2022).[2] Kermani, et al. Nat. Commun. 11, 6064 (2020).[3] Nishiyama, et. al., Nat. Commun., 3 (2012)
The SMR superfamily is broadly divided into Qac-type and Gdx-type transporters: Qac proteins typically export hydrophobic cations, including quaternary ammonium compounds, whereas Gdx proteins export guanidinium ions. We curated about 10,000 SMR homologs spanning both groups and constructed a phylogenetic tree to guide node selection for reconstruction. From this tree, we prioritized five internal nodes predicted to differ in substrate preference. Notably, Node 1 corresponds to the deepest split separating the Qac and Gdx clades, providing a key reference point for probing how substrate recognition diverged early in SMR evolution.
We assessed transport activity of reconstructed variants using solid-supported membrane electrophysiology (SSME) with three substrates: tetrapropylammonium (TPA) and tetramethylammonium (TMA) as representative Qac-type substrates, and guanidinium (Gdm) as a Gdx-type substrate. Wild-type EmrE transported TPA and TMA, whereas no transport signal was detected with Gdm; the signal was consistent with antiport activity as reported previously [1,2]. Although still preliminary, ancestral variants exhibited distinct behaviors. For example, the Node 1 variant showed a weak signal with TPA and Gdm but not with TMA, and the current signatures were inverted relative to wild-type. One possible interpretation is that Node 1 operates as a symporter rather than an antiporter. Detailed validation is ongoing. In the presentation, we will also report recent progress in improving the efficiency and reproducibility of in vitro reconstitution, including potential contributions of the SRP/SP pathway and membrane glycolipids in addition to SecYEG and YidC [3].
Overall, this platform enables efficient sampling of evolutionarily informed sequences that shift substrate preference and transport coupling, providing a path toward rational engineering of controllable efflux for biomanufacturing.
References[1] Kermani, et al. Elife 11, (2022).[2] Kermani, et al. Nat. Commun. 11, 6064 (2020).[3] Nishiyama, et. al., Nat. Commun., 3 (2012)
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