Integrative dynamic structural biology unveils conformers essential for the oligomerization of a large GTPase

  1. Thomas O Peulen
  2. Carola S Hengstenberg
  3. Ralf Biehl
  4. Mykola Dimura
  5. Charlotte Lorenz
  6. Alessandro Valeri
  7. Julian Folz
  8. Christian A Hanke
  9. Semra Ince
  10. Tobias Vöpel
  11. Bela Farago
  12. Holger Gohlke
  13. Johann P Klare  Is a corresponding author
  14. Andreas M Stadler  Is a corresponding author
  15. Claus AM Seidel  Is a corresponding author
  16. Christian Herrmann  Is a corresponding author
  1. Heinrich Heine University Düsseldorf, Germany
  2. Ruhr University Bochum, Germany
  3. Forschungszentrum Jülich, Germany
  4. Institut Laue-Langevin, France
  5. University of Osnabrück, Germany

Abstract

Guanylate binding proteins (GBPs) are soluble dynamin-like proteins. They undergo a conformational transition for GTP-controlled oligomerization and disrupt membranes of intra-cellular parasites to exert their function as part of the innate immune system of mammalian cells. We apply neutron spin echo, X-ray scattering, fluorescence, and EPR spectroscopy as techniques for integrative dynamic structural biology to study the structural basis and mechanism conformational transitions in the human GBP1 (hGBP1). We mapped hGBP1’s essential dynamics from nanoseconds to milliseconds by motional spectra of sub-domains. We find a GTP-independent flexibility of the C-terminal effector domain in the μs-regime and resolve structures of two distinct conformers essential for an opening of hGBP1 like a pocketknife and oligomerization. Our results show that an intrinsic flexibility, a GTP-triggered association of the GTPase-domains, and the assembly-dependent GTP-hydrolysis are functional design principles that control hGBP1’s reversible oligomerization.

Data availability

Data availabilityThe following material is available at Zenodo in two locations: Experimental data (doi 10.5281/zenodo.6534557): (i) fluorescence decays recorded by eTCSPC used to compute the distance restraints in Supp. Tab. 3A, (ii) single-molecule multiparameter fluorescence data: all raw data, burst selection and calibration measurements, fFCS (filters and generated correlation curves) (iii) double electron-electron resonance (DEER) EPR data used for structural modeling, (iv) neutron spin-echo data and SAXS structure factor of hGBP1. Scripts for structural modeling of conformational ensembles through integrative/hybrid methods using FRET, DEER and SAXS together with the initial and selected structural ensembles (10.5281/zenodo.6565895). The experimental SAXS data and the ab initio analysis thereof are available in the SASBDB (ID SASDDD6). Structure models of hGBP1 based on experimental restraints were deposited to PDB-Dev (PDB-Dev ID: PDBDEV_00000088) using the FLR-dictionary extension (developed by PDB and the Seidel group) available on the IHM working group GitHub site (https://github.com/ihmwg/FLR-dictionary). Further data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Code availabilityMost general custom-made software is directly available from http://www.mpc.hhu.de/en/software. General algorithms and source code are published under https://github.com/Fluorescence-Tools. Additional computer code custom-made for this publication is available upon request from the corresponding authors.

Article and author information

Author details

  1. Thomas O Peulen

    Chair for Molecular Physical Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Carola S Hengstenberg

    Physical Chemistry I, Ruhr University Bochum, Bochum, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Ralf Biehl

    Jülich Centre for Neutron Science, Forschungszentrum Jülich, Jülich, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Mykola Dimura

    Chair for Molecular Physical Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9462-0264
  5. Charlotte Lorenz

    Jülich Centre for Neutron Science, Forschungszentrum Jülich, Jülich, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3614-341X
  6. Alessandro Valeri

    Chair for Molecular Physical Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Julian Folz

    Chair for Molecular Physical Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Christian A Hanke

    Chair for Molecular Physical Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4826-4908
  9. Semra Ince

    Physical Chemistry I, Ruhr University Bochum, Bochum, Germany
    Competing interests
    The authors declare that no competing interests exist.
  10. Tobias Vöpel

    Physical Chemistry I, Ruhr University Bochum, Bochum, Germany
    Competing interests
    The authors declare that no competing interests exist.
  11. Bela Farago

    Institut Laue-Langevin, Grenoble, France
    Competing interests
    The authors declare that no competing interests exist.
  12. Holger Gohlke

    Institut für Pharmazeutische und Medizinische Chemie, Heinrich Heine University Düsseldorf, Dusseldorf, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8613-1447
  13. Johann P Klare

    Department of Physics, University of Osnabrück, Osnabrück, Germany
    For correspondence
    jklare@uos.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5761-5968
  14. Andreas M Stadler

    Jülich Centre for Neutron Science, Forschungszentrum Jülich, Jülich, Germany
    For correspondence
    a.stadler@fz-juelich.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2272-5232
  15. Claus AM Seidel

    Institute for Molecular Physical Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany
    For correspondence
    cseidel@hhu.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5171-149X
  16. Christian Herrmann

    Physical Chemistry I, Ruhr University Bochum, Bochum, Germany
    For correspondence
    Chr.Herrmann@rub.de
    Competing interests
    The authors declare that no competing interests exist.

Funding

Deutsche Forschungsgemeinschaft (EXC 2033 - 390677874 - RESOLV)

  • Christian Herrmann

Deutsche Forschungsgemeinschaft (HE 2679/6-1)

  • Christian Herrmann

Deutsche Forschungsgemeinschaft (SE 1195/17-1)

  • Claus AM Seidel

Deutsche Forschungsgemeinschaft (STA 1325/2-1)

  • Andreas M Stadler

Deutsche Forschungsgemeinschaft (KL2077/1-2)

  • Johann P Klare

European Research Council (Advanced Grant 2014 hybridFRET (671208))

  • Claus AM Seidel

Deutsche Forschungsgemeinschaft (project no. 267205415 / CRC 1208,subproject A03)

  • Holger Gohlke

Heinrich-Heine-Universität Düsseldorf (Zentrum für Informations- und Medientechnologie (ZIM)")

  • Holger Gohlke

Jülich Supercomputing Centre, Forschungszentrum Jülich (user ID: HKF7,VSK33)

  • Holger Gohlke

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2023, Peulen 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

  • 914
    views
  • 123
    downloads
  • 10
    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. Thomas O Peulen
  2. Carola S Hengstenberg
  3. Ralf Biehl
  4. Mykola Dimura
  5. Charlotte Lorenz
  6. Alessandro Valeri
  7. Julian Folz
  8. Christian A Hanke
  9. Semra Ince
  10. Tobias Vöpel
  11. Bela Farago
  12. Holger Gohlke
  13. Johann P Klare
  14. Andreas M Stadler
  15. Claus AM Seidel
  16. Christian Herrmann
(2023)
Integrative dynamic structural biology unveils conformers essential for the oligomerization of a large GTPase
eLife 12:e79565.
https://doi.org/10.7554/eLife.79565

Share this article

https://doi.org/10.7554/eLife.79565

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Yamato Niitani, Kohei Matsuzaki ... Michio Tomishige
    Research Article

    The two identical motor domains (heads) of dimeric kinesin-1 move in a hand-over-hand process along a microtubule, coordinating their ATPase cycles such that each ATP hydrolysis is tightly coupled to a step and enabling the motor to take many steps without dissociating. The neck linker, a structural element that connects the two heads, has been shown to be essential for head–head coordination; however, which kinetic step(s) in the chemomechanical cycle is ‘gated’ by the neck linker remains unresolved. Here, we employed pre-steady-state kinetics and single-molecule assays to investigate how the neck-linker conformation affects kinesin’s motility cycle. We show that the backward-pointing configuration of the neck linker in the front kinesin head confers higher affinity for microtubule, but does not change ATP binding and dissociation rates. In contrast, the forward-pointing configuration of the neck linker in the rear kinesin head decreases the ATP dissociation rate but has little effect on microtubule dissociation. In combination, these conformation-specific effects of the neck linker favor ATP hydrolysis and dissociation of the rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.

    1. Structural Biology and Molecular Biophysics
    Christopher T Schafer, Raymond F Pauszek III ... David P Millar
    Research Article

    The canonical chemokine receptor CXCR4 and atypical receptor ACKR3 both respond to CXCL12 but induce different effector responses to regulate cell migration. While CXCR4 couples to G proteins and directly promotes cell migration, ACKR3 is G-protein-independent and scavenges CXCL12 to regulate extracellular chemokine levels and maintain CXCR4 responsiveness, thereby indirectly influencing migration. The receptors also have distinct activation requirements. CXCR4 only responds to wild-type CXCL12 and is sensitive to mutation of the chemokine. By contrast, ACKR3 recruits GPCR kinases (GRKs) and β-arrestins and promiscuously responds to CXCL12, CXCL12 variants, other peptides and proteins, and is relatively insensitive to mutation. To investigate the role of conformational dynamics in the distinct pharmacological behaviors of CXCR4 and ACKR3, we employed single-molecule FRET to track discrete conformational states of the receptors in real-time. The data revealed that apo-CXCR4 preferentially populates a high-FRET inactive state, while apo-ACKR3 shows little conformational preference and high transition probabilities among multiple inactive, intermediate and active conformations, consistent with its propensity for activation. Multiple active-like ACKR3 conformations are populated in response to agonists, compared to the single CXCR4 active-state. This and the markedly different conformational landscapes of the receptors suggest that activation of ACKR3 may be achieved by a broader distribution of conformational states than CXCR4. Much of the conformational heterogeneity of ACKR3 is linked to a single residue that differs between ACKR3 and CXCR4. The dynamic properties of ACKR3 may underly its inability to form productive interactions with G proteins that would drive canonical GPCR signaling.