A facile approach for the in vitro assembly of multimeric membrane transport proteins
Abstract
Membrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a homomeric protein can be independently modified for functional or spectroscopic studies. Here, we describe a general approach for in vitro assembly that can be used for the generation of heteromeric variants of homomeric membrane proteins. We establish the approach using GltPh, a glutamate transporter homolog that is trimeric in the native state. We use heteromeric GltPh transporters to directly demonstrate the lack of coupling in substrate binding and demonstrate how heteromeric transporters considerably simplify the application of DEER spectroscopy. Further, we demonstrate the general applicability of this approach by carrying out the in vitro assembly of VcINDY, a Na+-coupled succinate transporter and CLC-ec1, a Cl-/H+ antiporter.
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All data generated or analyzed during this study are included in the manuscript and supporting files.
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Funding
National Institute of General Medical Sciences (R01 GM087546)
- Francis I Valiyaveetil
Howard Hughes Medical Institute
- Olga Boudker
National Institute of Neurological Disorders and Stroke (R37 NS085318)
- Scott C Blanchard
- Olga Boudker
- Francis I Valiyaveetil
National Institute of General Medical Sciences (P41GM103521)
- Jack H Freed
American Heart Association (12POST1910068)
- Paul J Focke
National Institute of General Medical Sciences (R01 GM123779)
- Elka R Georgieva
- Jack H Freed
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2018, Riederer et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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- Structural Biology and Molecular Biophysics
In eukaryotes, RNAs transcribed by RNA Pol II are modified at the 5′ end with a 7-methylguanosine (m7G) cap, which is recognized by the nuclear cap binding complex (CBC). The CBC plays multiple important roles in mRNA metabolism, including transcription, splicing, polyadenylation, and export. It promotes mRNA export through direct interaction with a key mRNA export factor, ALYREF, which in turn links the TRanscription and EXport (TREX) complex to the 5′ end of mRNA. However, the molecular mechanism for CBC-mediated recruitment of the mRNA export machinery is not well understood. Here, we present the first structure of the CBC in complex with an mRNA export factor, ALYREF. The cryo-EM structure of CBC-ALYREF reveals that the RRM domain of ALYREF makes direct contact with both the NCBP1 and NCBP2 subunits of the CBC. Comparing CBC-ALYREF with other cellular complexes containing CBC and/or ALYREF components provides insights into the coordinated events during mRNA transcription, splicing, and export.
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Under physiological conditions, proteins continuously undergo structural fluctuations on different timescales. Some conformations are only sparsely populated, but still play a key role in protein function. Thus, meaningful structure–function frameworks must include structural ensembles rather than only the most populated protein conformations. To detail protein plasticity, modern structural biology combines complementary experimental and computational approaches. In this review, we survey available computational approaches that integrate sparse experimental data from electron paramagnetic resonance spectroscopy with molecular modeling techniques to derive all-atom structural models of rare protein conformations. We also propose strategies to increase the reliability and improve efficiency using deep learning approaches, thus advancing the field of integrative structural biology.