1. Structural Biology and Molecular Biophysics

A facile approach for the in vitro assembly of multimeric membrane transport proteins

  1. Erika A Riederer
  2. Paul J Focke
  3. Elka R Georgieva
  4. Nurunisa Akyuz
  5. Kimberly Matulef
  6. Peter P Borbat
  7. Jack H Freed
  8. Scott C Blanchard
  9. Olga Boudker
  10. Francis I Valiyaveetil  Is a corresponding author
  1. Oregon Health and Science University, United States
  2. Cornell University, United States
  3. Weill Cornell Medicine, United States
https://doi.org/10.7554/eLife.36478
Share this article
Cite this article
  1. Erika A Riederer
  2. Paul J Focke
  3. Elka R Georgieva
  4. Nurunisa Akyuz
  5. Kimberly Matulef
  6. Peter P Borbat
  7. Jack H Freed
  8. Scott C Blanchard
  9. Olga Boudker
  10. Francis I Valiyaveetil
(2018)
A facile approach for the in vitro assembly of multimeric membrane transport proteins
eLife 7:e36478.
https://doi.org/10.7554/eLife.36478
  2. Download BibTeX
  3. Download .RIS

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.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Erika A Riederer

    Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1011-6536

  2. Paul J Focke

    Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, United States
    Competing interests
    No competing interests declared.

  3. Elka R Georgieva

    Department of Chemistry and Chemical Biology, Cornell University, Ithaca, United States
    Competing interests
    No competing interests declared.

  4. Nurunisa Akyuz

    Weill Cornell Medicine, New York, United States
    Competing interests
    No competing interests declared.

  5. Kimberly Matulef

    Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5011-9064

  6. Peter P Borbat

    Department of Chemistry and Chemical Biology, Cornell University, Ithaca, United States
    Competing interests
    No competing interests declared.

  7. Jack H Freed

    Department of Chemistry and Chemical Biology, Cornell University, Ithaca, United States
    Competing interests
    No competing interests declared.

  8. Scott C Blanchard

    Weill Cornell Medicine, New York, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2717-9365

  9. Olga Boudker

    Weill Cornell Medicine, New York, United States
    Competing interests
    Olga Boudker, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6965-0851

  10. Francis I Valiyaveetil

    Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, United States
    For correspondence
    valiyave@ohsu.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3387-8018

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.

Metrics

  • 2,553
    views
  • 419
    downloads
  • 17
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Erika A Riederer
  2. Paul J Focke
  3. Elka R Georgieva
  4. Nurunisa Akyuz
  5. Kimberly Matulef
  6. Peter P Borbat
  7. Jack H Freed
  8. Scott C Blanchard
  9. Olga Boudker
  10. Francis I Valiyaveetil
(2018)
A facile approach for the in vitro assembly of multimeric membrane transport proteins
eLife 7:e36478.
https://doi.org/10.7554/eLife.36478

Share this article

https://doi.org/10.7554/eLife.36478

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    A conformational fingerprint for amyloidogenic light chains

    Cristina Paissoni, Sarita Puri ... Carlo Camilloni
    Research Article

    Both immunoglobulin light-chain (LC) amyloidosis (AL) and multiple myeloma (MM) share the overproduction of a clonal LC. However, while LCs in MM remain soluble in circulation, AL LCs misfold into toxic-soluble species and amyloid fibrils that accumulate in organs, leading to distinct clinical manifestations. The significant sequence variability of LCs has hindered the understanding of the mechanisms driving LC aggregation. Nevertheless, emerging biochemical properties, including dimer stability, conformational dynamics, and proteolysis susceptibility, distinguish AL LCs from those in MM under native conditions. This study aimed to identify a2 conformational fingerprint distinguishing AL from MM LCs. Using small-angle X-ray scattering (SAXS) under native conditions, we analyzed four AL and two MM LCs. We observed that AL LCs exhibited a slightly larger radius of gyration and greater deviations from X-ray crystallography-determined or predicted structures, reflecting enhanced conformational dynamics. SAXS data, integrated with molecular dynamics simulations, revealed a conformational ensemble where LCs adopt multiple states, with variable and constant domains either bent or straight. AL LCs displayed a distinct, low-populated, straight conformation (termed H state), which maximized solvent accessibility at the interface between constant and variable domains. Hydrogen-deuterium exchange mass spectrometry experimentally validated this H state. These findings reconcile diverse experimental observations and provide a precise structural target for future drug design efforts.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Electrostatics of salt-dependent reentrant phase behaviors highlights diverse roles of ATP in biomolecular condensates

    Yi-Hsuan Lin, Tae Hun Kim ... Hue Sun Chan
    Research Article

    Liquid-liquid phase separation (LLPS) involving intrinsically disordered protein regions (IDRs) is a major physical mechanism for biological membraneless compartmentalization. The multifaceted electrostatic effects in these biomolecular condensates are exemplified here by experimental and theoretical investigations of the different salt- and ATP-dependent LLPSs of an IDR of messenger RNA-regulating protein Caprin1 and its phosphorylated variant pY-Caprin1, exhibiting, for example, reentrant behaviors in some instances but not others. Experimental data are rationalized by physical modeling using analytical theory, molecular dynamics, and polymer field-theoretic simulations, indicating that interchain ion bridges enhance LLPS of polyelectrolytes such as Caprin1 and the high valency of ATP-magnesium is a significant factor for its colocalization with the condensed phases, as similar trends are observed for other IDRs. The electrostatic nature of these features complements ATP’s involvement in π-related interactions and as an amphiphilic hydrotrope, underscoring a general role of biomolecular condensates in modulating ion concentrations and its functional ramifications.

    1. Structural Biology and Molecular Biophysics

    PROTAC-induced protein structural dynamics in targeted protein degradation

    Kingsley Y Wu, Ta I Hung, Chia-en A Chang
    Research Article

    PROteolysis TArgeting Chimeras (PROTACs) are small molecules that induce target protein degradation via the ubiquitin-proteasome system. PROTACs recruit the target protein and E3 ligase; a critical first step is forming a ternary complex. However, while the formation of a ternary complex is crucial, it may not always guarantee successful protein degradation. The dynamics of the PROTAC-induced degradation complex play a key role in ubiquitination and subsequent degradation. In this study, we computationally modelled protein complex structures and dynamics associated with a series of PROTACs featuring different linkers to investigate why these PROTACs, all of which formed ternary complexes with Cereblon (CRBN) E3 ligase and the target protein bromodomain-containing protein 4 (BRD4BD1), exhibited varying degrees of degradation potency. We constructed the degradation machinery complexes with Culling-Ring Ligase 4A (CRL4A) E3 ligase scaffolds. Through atomistic molecular dynamics simulations, we illustrated how PROTAC-dependent protein dynamics facilitating the arrangement of surface lysine residues of BRD4BD1 into the catalytic pocket of E2/ubiquitin cascade for ubiquitination. Despite featuring identical warheads in this PROTAC series, the linkers were found to affect the residue-interaction networks, and thus governing the essential motions of the entire degradation machine for ubiquitination. These findings offer a structural dynamic perspective on ligand-induced protein degradation, providing insights to guide future PROTAC design endeavors.