A non-helical region in transmembrane helix 6 of hydrophobic amino acid transporter MhsT mediates substrate recognition

Dorota Focht; Caroline Neumann; Joseph Lyons; Ander Eguskiza Bilbao; Rickard Blunck; Lina Malinauskaite; Ilona O. Schwarz; Jonathan A. Javitch; Matthias Quick; Poul Nissen

doi:10.15252/embj.2020105164

A non-helical region in transmembrane helix 6 of hydrophobic amino acid transporter MhsT mediates substrate recognition

Dorota Focht, Caroline Neumann, Joseph Lyons, Ander Eguskiza Bilbao, Rickard Blunck, Lina Malinauskaite, Ilona O. Schwarz, Jonathan A. Javitch, Matthias Quick^*, Poul Nissen^*

^*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

18 Citations (Scopus)

Abstract

MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na⁺-dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate-bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly-Met-Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.

Original language	English
Article number	e105164
Journal	EMBO Journal
Volume	40
Issue	1
Number of pages	13
ISSN	0261-4189
DOIs	https://doi.org/10.15252/embj.2020105164
Publication status	Published - Jan 2021

Keywords

amino acids uptake
MhsT
neurotransmitter
sodium symporters
substrate recognition
X-ray crystallography

Access to Document

10.15252/embj.2020105164

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780149/pdf/EMBJ-40-e105164.pdf

Cite this

@article{fdc59653af4d4ad6931092884fa9080c,

title = "A non-helical region in transmembrane helix 6 of hydrophobic amino acid transporter MhsT mediates substrate recognition",

abstract = "MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na+-dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate-bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly-Met-Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.",

keywords = "amino acids uptake, MhsT, neurotransmitter, sodium symporters, substrate recognition, X-ray crystallography",

author = "Dorota Focht and Caroline Neumann and Joseph Lyons and {Eguskiza Bilbao}, Ander and Rickard Blunck and Lina Malinauskaite and Schwarz, {Ilona O.} and Javitch, {Jonathan A.} and Matthias Quick and Poul Nissen",

note = "Funding Information: The authors are grateful to technical assistance by Tetyana Klymchuk, Lotte T. Pedersen, Anna Marie Nielsen, and Audrey Warren and support for computing by Jesper L. Karlsen. We thank Steffen Sinning and Birgit Schi{\o}tt for fruitful discussions. Work on the project was supported by a Short Term EMBO Fellowship [ASTF 80‐2016] and Boehringer Ingelheim Fonds travel grant (2015) to DF, a PhD fellowship from the Lundbeck Foundation to CN (2015‐3225), a PhD fellowship from the Boehringer Ingelheim Fonds to LM (2010), and a post‐doctoral fellowship from the Lundbeck Foundation to JAL (2015‐2704). The research was supported by NIH grants U54GM087519 and R01DA04510 to JAJ, R01GM119396 to MQ, and by the Lundbeck Foundation grants 2011‐3868 and 2016‐2518 to PN. Publisher Copyright: {\textcopyright} 2020 The Authors Copyright: Copyright 2021 Elsevier B.V., All rights reserved.",

year = "2021",

month = jan,

doi = "10.15252/embj.2020105164",

language = "English",

volume = "40",

journal = "EMBO Journal",

issn = "0261-4189",

publisher = "EMBO Press",

number = "1",

}

A non-helical region in transmembrane helix 6 of hydrophobic amino acid transporter MhsT mediates substrate recognition. / Focht, Dorota; Neumann, Caroline; Lyons, Joseph et al.
In: EMBO Journal, Vol. 40, No. 1, e105164, 01.2021.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - A non-helical region in transmembrane helix 6 of hydrophobic amino acid transporter MhsT mediates substrate recognition

AU - Focht, Dorota

AU - Neumann, Caroline

AU - Lyons, Joseph

AU - Eguskiza Bilbao, Ander

AU - Blunck, Rickard

AU - Malinauskaite, Lina

AU - Schwarz, Ilona O.

AU - Javitch, Jonathan A.

AU - Quick, Matthias

AU - Nissen, Poul

N1 - Funding Information: The authors are grateful to technical assistance by Tetyana Klymchuk, Lotte T. Pedersen, Anna Marie Nielsen, and Audrey Warren and support for computing by Jesper L. Karlsen. We thank Steffen Sinning and Birgit Schiøtt for fruitful discussions. Work on the project was supported by a Short Term EMBO Fellowship [ASTF 80‐2016] and Boehringer Ingelheim Fonds travel grant (2015) to DF, a PhD fellowship from the Lundbeck Foundation to CN (2015‐3225), a PhD fellowship from the Boehringer Ingelheim Fonds to LM (2010), and a post‐doctoral fellowship from the Lundbeck Foundation to JAL (2015‐2704). The research was supported by NIH grants U54GM087519 and R01DA04510 to JAJ, R01GM119396 to MQ, and by the Lundbeck Foundation grants 2011‐3868 and 2016‐2518 to PN. Publisher Copyright: © 2020 The Authors Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

PY - 2021/1

Y1 - 2021/1

N2 - MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na+-dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate-bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly-Met-Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.

AB - MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na+-dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate-bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly-Met-Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.

KW - amino acids uptake

KW - MhsT

KW - neurotransmitter

KW - sodium symporters

KW - substrate recognition

KW - X-ray crystallography

UR - http://www.scopus.com/inward/record.url?scp=85096754415&partnerID=8YFLogxK

U2 - 10.15252/embj.2020105164

DO - 10.15252/embj.2020105164

M3 - Journal article

C2 - 33155685

AN - SCOPUS:85096754415

SN - 0261-4189

VL - 40

JO - EMBO Journal

JF - EMBO Journal

IS - 1

M1 - e105164

ER -

A non-helical region in transmembrane helix 6 of hydrophobic amino acid transporter MhsT mediates substrate recognition

Abstract

Keywords

Access to Document

Other files and links

Fingerprint

Cite this