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

Guanylate binding proteins (GBPs) are soluble dynamin-like proteins that undergo a conformational transition for GTP-controlled oligomerization and disrupt membranes of intracellular 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 of 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 pocket knife and for oligomerization. Our results on hGBP1's conformational heterogeneity and dynamics (intrinsic flexibility) deepen our molecular understanding relevant for its reversible oligomerization, GTP-triggered association of the GTPase-domains and assembly-dependent GTP-hydrolysis.

Resolution of Maximum Entropy Method-Derived Posterior Conformational Ensembles of a Flexible System Probed by FRET and Molecular Dynamics Simulations.

Maximum entropy methods (MEMs) determine posterior distributions by combining experimental data with prior information. MEMs are frequently used to reconstruct conformational ensembles of molecular systems for experimental information and initial molecular ensembles. We performed time-resolved Förster resonance energy transfer (FRET) experiments to probe the interdye distance distributions of the lipase-specific foldase Lif in the apo state, which likely has highly flexible, disordered, and/or ordered structural elements. Distance distributions estimated from ensembles of molecular dynamics (MD) simulations serve as prior information, and FRET experiments, analyzed within a Bayesian framework to recover distance distributions, are used for optimization. We tested priors obtained by MD with different force fields (FFs) tailored to ordered (FF99SB, FF14SB, and FF19SB) and disordered proteins (IDPSFF and FF99SBdisp). We obtained five substantially different posterior ensembles. As in our FRET experiments the noise is characterized by photon counting statistics, for a validated dye model, MEM can quantify consistencies between experiment and prior or posterior ensembles. However, posterior populations of conformations are uncorrelated to structural similarities for individual structures selected from different prior ensembles. Therefore, we assessed MEM simulating varying priors in synthetic experiments with known target ensembles. We found that (i) the prior and experimental information must be carefully balanced for optimal posterior ensembles to minimize perturbations of populations by overfitting and (ii) only ensemble-integrated quantities like inter-residue distance distributions or density maps can be reliably obtained but not ensembles of atomistic structures. This is because MEM optimizes ensembles but not individual structures. This result for a highly flexible system suggests that structurally varying priors calculated from varying prior ensembles, e.g., generated with different FFs, may serve as an ad hoc estimate for MEM reconstruction robustness.

Automated and optimally FRET-assisted structural modeling.

FRET experiments can provide state-specific structural information of complex dynamic biomolecular assemblies. However, to overcome the sparsity of FRET experiments, they need to be combined with computer simulations. We introduce a program suite with (i) an automated design tool for FRET experiments, which determines how many and which FRET pairs should be used to minimize the uncertainty and maximize the accuracy of an integrative structure, (ii) an efficient approach for FRET-assisted coarse-grained structural modeling, and all-atom molecular dynamics simulations-based refinement, and (iii) a quantitative quality estimate for judging the accuracy of FRET-derived structures as opposed to precision. We benchmark our tools against simulated and experimental data of proteins with multiple conformational states and demonstrate an accuracy of ~3 Å RMSDCα against X-ray structures for sets of 15 to 23 FRET pairs. Free and open-source software for the introduced workflow is available at https://github.com/Fluorescence-Tools . A web server for FRET-assisted structural modeling of proteins is available at http://nmsim.de .

Resolving dynamics and function of transient states in single enzyme molecules.

We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, and other biochemical and biophysical tools, we characterize three short-lived conformational states over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen available T4L structures, reveal that T4L in solution mainly adopts the known open and closed states in exchange at 4 µs. A newly found minor state, undisclosed by, at present, more than 500 crystal structures of T4L and sampled at 230 µs, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit will likely accelerate the development of dynamic structural biology by identifying transient conformational states that are highly abundant in biology and critical in enzymatic reactions.

Dynamics of the nucleosomal histone H3 N-terminal tail revealed by high precision single-molecule FRET.

Chromatin compaction and gene accessibility are orchestrated by assembly and disassembly of nucleosomes. Although the disassembly process was widely studied, little is known about the structure and dynamics of the disordered histone tails, which play a pivotal role for nucleosome integrity. This is a gap filling experimental FRET study from the perspective of the histone H3 N-terminal tail (H3NtT) of reconstituted mononucleosomes. By systematic variation of the labeling positions we monitored the motions of the H3NtT relative to the dyad axis and linker DNA. Single-molecule FRET unveiled that H3NtTs do not diffuse freely but follow the DNA motions with multiple interaction modes with certain permitted dynamic transitions in the µs to ms time range. We also demonstrate that the H3NtT can allosterically sense charge-modifying mutations within the histone core (helix α3 of histone H2A (R81E/R88E)) resulting in increased dynamic transitions and lower rate constants. Those results complement our earlier model on the NaCl induced nucleosome disassembly as changes in H3NtT configurations coincide with two major steps: unwrapping of one linker DNA and weakening of the internal DNA - histone interactions on the other side. This emphasizes the contribution of the H3NtT to the fine-tuned equilibrium between overall nucleosome stability and DNA accessibility.

Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α.

The dynamic architecture of chromatin fibers, a key determinant of genome regulation, is poorly understood. Here, we employ multimodal single-molecule Förster resonance energy transfer studies to reveal structural states and their interconversion kinetics in chromatin fibers. We show that nucleosomes engage in short-lived (micro- to milliseconds) stacking interactions with one of their neighbors. This results in discrete tetranucleosome units with distinct interaction registers that interconvert within hundreds of milliseconds. Additionally, we find that dynamic chromatin architecture is modulated by the multivalent architectural protein heterochromatin protein 1α (HP1α), which engages methylated histone tails and thereby transiently stabilizes stacked nucleosomes. This compacted state nevertheless remains dynamic, exhibiting fluctuations on the timescale of HP1α residence times. Overall, this study reveals that exposure of internal DNA sites and nucleosome surfaces in chromatin fibers is governed by an intrinsic dynamic hierarchy from micro- to milliseconds, allowing the gene regulation machinery to access compact chromatin.

Quantitative FRET studies and integrative modeling unravel the structure and dynamics of biomolecular systems.

Förster Resonance Energy Transfer (FRET) combined with single-molecule spectroscopy probes macromolecular structure and dynamics and identifies coexisting conformational states. We review recent methodological developments in integrative structural modeling by satisfying spatial restraints on networks of FRET pairs (hybrid-FRET). We discuss procedures to incorporate prior structural knowledge and to obtain optimal distance networks. Finally, a workflow for hybrid-FRET is presented that automates integrative structural modeling and experiment planning to put hybrid-FRET on rails. To test this workflow, we simulate realistic single-molecule experiments and resolve three protein conformers, exchanging at 30µs and 10ms, with accuracies of 1-3Å RMSD versus the target structure. Incorporation of data from other spectroscopies and imaging is also discussed.

Structural assemblies of the di- and oligomeric G-protein coupled receptor TGR5 in live cells: an MFIS-FRET and integrative modelling study.

TGR5 is the first identified bile acid-sensing G-protein coupled receptor, which has emerged as a potential therapeutic target for metabolic disorders. So far, structural and multimerization properties are largely unknown for TGR5. We used a combined strategy applying cellular biology, Multiparameter Image Fluorescence Spectroscopy (MFIS) for quantitative FRET analysis, and integrative modelling to obtain structural information about dimerization and higher-order oligomerization assemblies of TGR5 wildtype (wt) and Y111 variants fused to fluorescent proteins. Residue 111 is located in transmembrane helix 3 within the highly conserved ERY motif. Co-immunoprecipitation and MFIS-FRET measurements with gradually increasing acceptor to donor concentrations showed that TGR5 wt forms higher-order oligomers, a process disrupted in TGR5 Y111A variants. From the concentration dependence of the MFIS-FRET data we conclude that higher-order oligomers - likely with a tetramer organization - are formed from dimers, the smallest unit suggested for TGR5 Y111A variants. Higher-order oligomers likely have a linear arrangement with interaction sites involving transmembrane helix 1 and helix 8 as well as transmembrane helix 5. The latter interaction is suggested to be disrupted by the Y111A mutation. The proposed model of TGR5 oligomer assembly broadens our view of possible oligomer patterns and affinities of class A GPCRs.