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.
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
-
- 913
- views
-
- 122
- 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)
Integrative dynamic structural biology unveils conformers essential for the oligomerization of a large GTPase
eLife 12:e79565.
https://doi.org/10.7554/eLife.79565
Further reading
-
- Structural Biology and Molecular Biophysics
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.
-
- Immunology and Inflammation
- Structural Biology and Molecular Biophysics
Increasing evidence suggests that mechanical load on the αβ T-cell receptor (TCR) is crucial for recognizing the antigenic peptide-bound major histocompatibility complex (pMHC) molecule. Our recent all-atom molecular dynamics (MD) simulations revealed that the inter-domain motion of the TCR is responsible for the load-induced catch bond behavior of the TCR-pMHC complex and peptide discrimination (Chang-Gonzalez et al., 2024). To further examine the generality of the mechanism, we perform all-atom MD simulations of the B7 TCR under different conditions for comparison with our previous simulations of the A6 TCR. The two TCRs recognize the same pMHC and have similar interfaces with pMHC in crystal structures. We find that the B7 TCR-pMHC interface stabilizes under ∼15 pN load using a conserved dynamic allostery mechanism that involves the asymmetric motion of the TCR chassis. However, despite forming comparable contacts with pMHC as A6 in the crystal structure, B7 has fewer high-occupancy contacts with pMHC and exhibits higher mechanical compliance during the simulation. These results indicate that the dynamic allostery common to the TCRαβ chassis can amplify slight differences in interfacial contacts into distinctive mechanical responses and nuanced biological outcomes.
