Integrative dynamic structural biology unveils conformers essential for the oligomerization of a large GTPase
- Heinrich Heine University Düsseldorf, Germany
- Ruhr University Bochum, Germany
- Forschungszentrum Jülich, Germany
- Institut Laue-Langevin, France
- 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
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.
Reviewing Editor
- Hannes Neuweiler, University of Würzburg, Germany
Version history
- Preprint posted: April 8, 2020 (view preprint)
- Received: April 18, 2022
- Accepted: June 2, 2023
- Accepted Manuscript published: June 14, 2023 (version 1)
- Version of Record published: July 27, 2023 (version 2)
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.
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Integrative dynamic structural biology unveils conformers essential for the oligomerization of a large GTPase
eLife 12:e79565.
https://doi.org/10.7554/eLife.79565
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The Neuronal Calcium Sensor 1, an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Ga have revealed how Ric-8A phosphorylation promotes Ga recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Ga subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Ga. Our data show that the binding of NCS-1 and Ga to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to Casein Kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A GEF activity towards Ga when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.
