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The CcdAB system expressed in the E.coli cells is a prototypical example of the bacterial toxin-antitoxin (TA) systems that ensure the survival of the bacterial population under adverse environmental conditions. The solution and crystal structures of CcdA, CcdB and of CcdB in complex with the toxin-binding C-terminal domain of CcdA have been reported. Our interest lies in the dynamics of CcdB-CcdA complex formation. Solution NMR studies have shown that CcdB_G100T, in presence of saturating concentrations of CcdA-c, a truncated C-terminal fragment of CcdA exists in equilibrium between two major populations. Sequence specific backbone resonance assignments of both equilibrium forms of the ~ 27 kDa complex, have been obtained from a suite of triple resonance NMR experiments acquired on 2H, 13C, 15N enriched samples of CcdB_G100T. Analysis of 1H, 13Cα, 13Cβ secondary chemical shifts, shows that both equilibrium forms of CcdB_G100T have five beta-strands and one alpha-helix as the major secondary structural elements in the tertiary structure. The results of these studies are presented below. [ABSTRACT FROM AUTHOR]

Conformational heterogeneity of biological macromolecules is a challenge in single-particle averaging (SPA). Current standard practice is to employ classification and filtering methods that may allow a discrete number of conformational states to be reconstructed. However, the conformation space accessible to these molecules is continuous and, therefore, explored incompletely by a small number of discrete classes. Recently developed heterogeneous reconstruction algorithms (HRAs) to analyse continuous heterogeneity rely on machine-learning methods that employ low-dimensional latent space representations. The non-linear nature of many of these methods poses a challenge to their validation and interpretation and to identifying functionally relevant conformational trajectories. These methods would benefit from in-depth benchmarking using high-quality synthetic data and concomitant ground truth information. We present a framework for the simulation and subsequent analysis with respect to the ground truth of cryo-EM micrographs containing particles whose conformational heterogeneity is sourced from molecular dynamics simulations. These synthetic data can be processed as if they were experimental data, allowing aspects of standard SPA workflows as well as heterogeneous reconstruction methods to be compared with known ground truth using available utilities. The simulation and analysis of several such datasets are demonstrated and an initial investigation into HRAs is presented.

In their folded state, biomolecules exchange between multiple conformational states that are crucial for their function. Traditional structural biology methods, such as X-ray crystallography and cryogenic electron microscopy (cryo-EM), produce density maps that are ensemble averages, reflecting molecules in various conformations. Yet, most models derived from these maps explicitly represent only a single conformation, overlooking the complexity of biomolecular structures. To accurately reflect the diversity of biomolecular forms, there is a pressing need to shift toward modeling structural ensembles that mirror the experimental data. However, the challenge of distinguishing signal from noise complicates manual efforts to create these models. In response, we introduce the latest enhancements to qFit, an automated computational strategy designed to incorporate protein conformational heterogeneity into models built into density maps. These algorithmic improvements in qFit are substantiated by superior Rfree and geometry metrics across a wide range of proteins. Importantly, unlike more complex multicopy ensemble models, the multiconformer models produced by qFit can be manually modified in most major model building software (e.g., Coot) and fit can be further improved by refinement using standard pipelines (e.g., Phenix, Refmac, Buster). By reducing the barrier of creating multiconformer models, qFit can foster the development of new hypotheses about the relationship between macromolecular conformational dynamics and function.

Intrinsically disordered regions (IDRs) are crucial to homeostatic and organellar remodeling pathways. In reticulophagy/ER-phagy, long cytosolic IDR-containing receptors (e.g. RETREG1/FAM134B) house the LC3-interacting region (LIR) motif to recruit the phagophore. The precise functions of the IDR beyond engaging the autophagic machinery are unclear. Here, we comment on the role of the RETREG1-IDR based on our recent computer modeling and molecular dynamics (MD) simulations. Extensive analysis of the RETREG1-IDR indicates a continuum of conformations between expanded and compact structures, displaying a Janus-like feature. Using an adapted MARTINI model, we find that the IDR ensemble properties vary widely depending on the membrane anchor. IDRs alone are sufficient to promote and sense membrane curvature and can act as entropic tethers. When anchored to the Reticulon homology domain (RHD), they adopt compact collapsed conformations, acting as effector scaffolds that amplify RHD membrane remodeling properties, enhancing receptor-clustering and accelerating spontaneous budding. These findings expand the operational scope of IDRs within reticulophagy, offering fresh insights into a mechanistic understanding of membrane remodeling. [ABSTRACT FROM AUTHOR]

Trans-activation response (TAR) RNA-binding protein (TRBP) has emerged as a key player in the RNA interference pathway, wherein it binds to different pre-microRNAs (miRNAs) and small interfering RNAs (siRNAs), each varying in sequence and/or structure. We hypothesize that TRBP displays dynamic adaptability to accommodate heterogeneity in target RNA structures. Thus, it is crucial to ascertain the role of intrinsic and RNA-induced protein dynamics in RNA recognition and binding. We have previously elucidated the role of intrinsic and RNA-induced conformational exchange in the double-stranded RNA-binding domain 1 (dsRBD1) of TRBP in shape-dependent RNA recognition. The current study delves into the intrinsic and RNA-induced conformational dynamics of the TRBP-dsRBD2 and then compares it with the dsRBD1 study carried out previously. Remarkably, the two domains exhibit differential binding affinity to a 12-bp dsRNA owing to the presence of critical residues and structural plasticity. Furthermore, we report that dsRBD2 depicts constrained conformational plasticity when compared to dsRBD1. Although, in the presence of RNA, dsRBD2 undergoes induced conformational exchange within the designated RNA-binding regions and other residues, the amplitude of the motions remains modest when compared to those observed in dsRBD1. We propose a dynamics-driven model of the two tandem domains of TRBP, substantiating their contributions to the versatility of dsRNA recognition and binding.

In their recent commentary in Protein Science, Jaskolski et al. analyzed three randomly picked diffraction data sets from fragment-screening group depositions from the PDB and, based on that, they claimed that such data are principally problematic. We demonstrate here that if such data are treated properly, none of the proclaimed criticisms persist.

Proteins are well known 'shapeshifters' which change conformation to function. In crystallography, multiple conformational states are often present within the crystal and the resulting electron‐density map. Yet, explicitly incorporating alternative states into models to disentangle multi‐conformer ensembles is challenging. We previously reported the tool FLEXR, which, within a few minutes, automatically separates conformational signal from noise and builds the corresponding, often missing, structural features into a multi‐conformer model. To make the method widely accessible for routine multi‐conformer building as part of the computational toolkit for macromolecular crystallography, we present a graphical user interface (GUI) for FLEXR, designed as a plugin for Coot 1. The GUI implementation seamlessly connects FLEXR models with the existing suite of validation and modeling tools available in Coot. We envision that FLEXR will aid crystallographers by increasing access to a multi‐conformer modeling method that will ultimately lead to a better representation of protein conformational heterogeneity in the Protein Data Bank. In turn, deeper insights into the protein conformational landscape may inform biology or provide new opportunities for ligand design. The code is open source and freely available on GitHub at https://github.com/TheFischerLab/FLEXR‐GUI. [ABSTRACT FROM AUTHOR]

In their folded state, biomolecules exchange between multiple conformational states that are crucial for their function. Traditional structural biology methods, such as X-ray crystallography and cryogenic electron microscopy (cryo-EM), produce density maps that are ensemble averages, reflecting molecules in various conformations. Yet, most models derived from these maps explicitly represent only a single conformation, overlooking the complexity of biomolecular structures. To accurately reflect the diversity of biomolecular forms, there is a pressing need to shift toward modeling structural ensembles that mirror the experimental data. However, the challenge of distinguishing signal from noise complicates manual efforts to create these models. In response, we introduce the latest enhancements to qFit, an automated computational strategy designed to incorporate protein conformational heterogeneity into models built into density maps. These algorithmic improvements in qFit are substantiated by superior Rfree and geometry metrics across a wide range of proteins. Importantly, unlike more complex multicopy ensemble models, the multiconformer models produced by qFit can be manually modified in most major model building software (e.g., Coot) and fit can be further improved by refinement using standard pipelines (e.g., Phenix, Refmac, Buster). By reducing the barrier of creating multiconformer models, qFit can foster the development of new hypotheses about the relationship between macromolecular conformational dynamics and function.

Cryo electron microscopy (cryo-EM) instrumentation allows obtaining 3D reconstruction of the structure of biomolecular complexes in vitro (purified complexes studied by single particle analysis) and in situ (complexes studied in cells by cryo electron tomography). Standard cryo-EM approaches allow high-resolution reconstruction of only a few conformational states of a molecular complex, as they rely on data classification into a given number of classes to increase the resolution of the reconstruction from the most populated classes while discarding all other classes. Such discrete classification approaches result in a partial picture of the full conformational variability of the complex, due to continuous conformational transitions with many, uncountable intermediate states. In this article, we present the software with a user-friendly graphical interface for running two recently introduced methods, namely, MDSPACE and MDTOMO, to obtain continuous conformational landscapes of biomolecules by analyzing in vitro and in situ cryo-EM data (single particle images and subtomograms) based on molecular dynamics simulations of an available atomic model of one of the conformations. The MDSPACE and MDTOMO software is part of the open-source ContinuousFlex software package (starting from version 3.4.2 of ContinuousFlex), which can be run as a plugin of the Scipion software package (version 3.1 and later), broadly used in the cryo-EM field. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphatase 1B (PTP1B) plays important roles in cellular homeostasis and is a highly validated therapeutic target for multiple human ailments, including diabetes, obesity and breast cancer. However, much remains to be learned about how conformational changes may convey information through the structure of PTP1B to enable allosteric regulation by ligands or functional responses to mutations. High‐resolution X‐ray crystallography can offer unique windows into protein conformational ensembles, but comparison of even high‐resolution structures is often complicated by differences between data sets, including non‐isomorphism. Here, the highest resolution crystal structure of apo wild‐type (WT) PTP1B to date is presented out of a total of ∼350 PTP1B structures in the PDB. This structure is in a crystal form that is rare for PTP1B, with two unique copies of the protein that exhibit distinct patterns of conformational heterogeneity, allowing a controlled comparison of local disorder across the two chains within the same asymmetric unit. The conformational differences between these chains are interrogated in the apo structure and between several recently reported high‐resolution ligand‐bound structures. Electron‐density maps in a high‐resolution structure of a recently reported activating double mutant are also examined, and unmodeled alternate conformations in the mutant structure are discovered that coincide with regions of enhanced conformational heterogeneity in the new WT structure. These results validate the notion that these mutations operate by enhancing local dynamics, and suggest a latent susceptibility to such changes in the WT enzyme. Together, these new data and analysis provide a detailed view of the conformational ensemble of PTP1B and highlight the utility of high‐resolution crystallography for elucidating conformational heterogeneity with potential relevance for function. [ABSTRACT FROM AUTHOR]

Rate dispersion is commonly observed when measuring relaxation in complex media such as solution of a protein. Its origin is often associated to molecular heterogeneity in the system. However, interpretation of rate dispersion is not straightforward, and it can be exhibited by both heterogeneous (all molecules are different) and homogeneous (all molecules are identical) systems. Here, we report two-dimensional (2D) correlation analysis of intensity fluctuations originating from translational diffusion-lead fluctuations in number of particles simulated in an observation volume resembling fluorescence correlation spectroscopy experiment. Translational diffusion of particles representing a protein having two different conformations are simulated. Homogeneous and heterogeneous systems are simulated to have similar one-dimensional (1D) autocorrelation curves with comparable extent of rate dispersion, α ∼ 0.8. Time slices of 2D autocorrelation curves and rate-rate spectra obtained from them clearly demonstrate that similar extent of rate dispersion observed in 1D autocorrelation can originate from both heterogeneous and homogeneous systems. We establish that one-dimensional analysis only reports on the extent of rate dispersion. Two-dimensional analysis reports on the origin of rate dispersion and distinguishes heterogeneous from homogeneous.

Movement of the Rieske domain of the iron–sulfur protein is essential for intramolecular electron transfer within complex III2 (CIII2) of the respiratory chain as it bridges a gap in the cofactor chain towards the electron acceptor cytochrome c. We present cryo-EM structures of CIII2 from Yarrowia lipolytica at resolutions up to 2.0 Å under different conditions, with different redox states of the cofactors of the high-potential chain. All possible permutations of three primary positions were observed, indicating that the two halves of the dimeric complex act independently. Addition of the substrate analogue decylubiquinone to CIII2 with a reduced high-potential chain increased the occupancy of the Qo site. The extent of Rieske domain interactions through hydrogen bonds to the cytochrome b and cytochrome c1 subunits varied depending on the redox state and substrate. In the absence of quinols, the reduced Rieske domain interacted more closely with cytochrome b and cytochrome c1 than in the oxidized state. Upon addition of the inhibitor antimycin A, the heterogeneity of the cd1-helix and ef-loop increased, which may be indicative of a long-range effect on the Rieske domain.

Epigenetic modifications of chromatin play a critical role in regulating the fidelity of the genetic code and in controlling the translation of genetic information into the protein components of the cell. One key posttranslational modification is acetylation of histone lysine residues. Molecular dynamics simulations, and to a smaller extent experiment, have established that lysine acetylation increases the dynamics of histone tails. However, a systematic, atomic resolution experimental investigation of how this epigenetic mark, focusing on one histone at a time, influences the structural dynamics of the nucleosome beyond the tails, and how this translates into accessibility of protein factors such as ligases and nucleases, has yet to be performed. Herein, using NMR spectroscopy of nucleosome core particles (NCPs), we evaluate the effects of acetylation of each histone on tail and core dynamics. We show that for histones H2B, H3, and H4, the histone core particle dynamics are little changed, even though the tails have increased amplitude motions. In contrast, significant increases to H2A dynamics are observed upon acetylation of this histone, with the docking domain and L1 loop particularly affected, correlating with increased susceptibility of NCPs to nuclease digestion and more robust ligation of nicked DNA. Dynamic light scattering experiments establish that acetylation decreases inter-NCP interactions in a histone-dependent manner and facilitates the development of a thermodynamic model for NCP stacking. Our data show that different acetylation patterns result in nuanced changes to NCP dynamics, modulating interactions with other protein factors, and ultimately controlling biological output. [ABSTRACT FROM AUTHOR]

[Display omitted] • New triphenylpyrimidines with different donors synthesized. • Experimental signs of SSLE, TADF, RTP and TTA detected for one compound. • Mechanochromism predetermined by conformational heterogeneity. • Efficient OLED and oxygen sensor application operating utilizing triplet states. Three derivatives of fluorinated triphenylpyrimidine with the attached carbazole, phenothiazine, or acridan donor moieties are synthesized by Buchwald-Hartwig reactions. The impact of the donor units on emissive and other properties of the compounds is reported. The compounds exhibit excellent thermal stability, competitive photophysical phenomena such as room temperature phosphorescence (RTP) appearing when compounds are molecularly dispersed in the rigid polymer matrix and thermally activated delayed fluorescence (TADF). The compounds with carbazole and phenothiazine donor moieties show the manifestation of triplet–triplet annihilation in the electroluminescence when used as emitters in organic light-emitting diodes (OLEDs). The phenothiazine-containing compound exhibit dual photoluminescence with the blue-shifted peak corresponding to the quasi-axial conformer and a red-shifted peak to the quasi-equatorial conformer. This derivative shows reversible shifts of emission spectra exceeding 100 nm due to the stable (at least 4 cycles) mechanochromic luminescence under the application of external stimuli. After grinding the emission intensity maximum is observed at 555 nm, after fuming at. ca 448 nm and after melting at 555 nm. The photoluminescence shifts and ON/OFF delayed fluorescence of the phenothiazine-based emitter occur due to the alteration between the crystalline and amorphous states. Optimization of the device structure allows to control the charge balance resulting in external quantum efficiency of up to 5.7 % observed for the OLED based on the phenothiazine-based emitter. This compound also shows the biggest response to the presence of oxygen acting as the quencher of triplet excited energy. The film of the compound doped in rigid Zeonex shows an 8.4-fold increase in emission intensity after evacuation. The optical sensor fabricated using the derivative of fluorinated triphenylpyrimidine and phenothiazine is characterized by the Stern-Volmer constant 1.37 × 10-4 ppm−1. [ABSTRACT FROM AUTHOR]

For roughly two decades, cryocrystallography has been the overwhelmingly dominant method for determining high‐resolution biomolecular structures. Competition from single‐particle cryo‐electron microscopy and micro‐electron diffraction, increased interest in functionally relevant information that may be missing or corrupted in structures determined at cryogenic temperature, and interest in time‐resolved studies of the biomolecular response to chemical and optical stimuli have driven renewed interest in data collection at room temperature and, more generally, at temperatures from the protein–solvent glass transition near 200 K to ∼350 K. Fischer has recently reviewed practical methods for room‐temperature data collection and analysis [Fischer (2021), Q. Rev. Biophys.54, e1]. Here, the key advantages and physical principles of, and methods for, crystallographic data collection at noncryogenic temperatures and some factors relevant to interpreting the resulting data are discussed. For room‐temperature data collection to realize its potential within the structural biology toolkit, streamlined and standardized methods for delivering crystals prepared in the home laboratory to the synchrotron and for automated handling and data collection, similar to those for cryocrystallography, should be implemented. [ABSTRACT FROM AUTHOR]

Biomolecular condensates are membraneless organelles that concentrate proteins and nucleic acids. One of the primary components of condensates is multidomain proteins, whose domains can be broadly classified as structured and disordered. While structured protein domains are ubiquitous within biomolecular condensates, the physical ramifications of their unique properties have been relatively underexplored. Therefore, this review synthesizes current literature pertaining to structured protein domains within the context of condensates. We examine how the propensity of structured domains for high interaction specificity and low conformational heterogeneity contributes to the formation, material properties, and functions of biomolecular condensates. Finally, we propose unanswered questions on the behavior of structured protein domains within condensates, the answers of which will contribute to a more complete understanding of condensate biophysics., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Visualizing a protein's molecular motions has been a long standing topic of research in the biophysics community. Largely this has been done by exploiting nuclear magnetic resonance spectroscopy (NMR), and arguably no protein's molecular motions have been better characterized by NMR than that of ubiquitin (Ub), a 76 amino acid polypeptide essential in ubiquitination-a key regulatory system within cells. Herein, we discuss ubiquitin's conformational plasticity as visualized, at atomic resolution, by more than 35 years of NMR work. In our discussions we point out the differences between data acquired in vitro, ex vivo, as well as in vivo and stress the need to investigate Ub's conformational plasticity in more biologically representative backgrounds., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Cryogenic electron microscopy (cryo-EM) has become widely used for the past few years in structural biology, to collect single images of macromolecules "frozen in time". As this technique facilitates the identification of multiple conformational states adopted by the same molecule, a direct product of it is a set of 3D volumes, also called EM maps. To gain more insights on the possible mechanisms that govern transitions between different states, and hence the mode of action of a molecule, we recently introduced a bioinformatic tool that interpolates and generates morphing trajectories joining two given EM maps. This tool is based on recent advances made in optimal transport, that allow efficient evaluation of Wasserstein barycenters of 3D shapes. As the overall performance of the method depends on various key parameters, including the sensitivity of the regularization parameter, we performed various numerical experiments to demonstrate how MorphOT can be applied in different contexts and settings. Finally, we discuss current limitations and further potential connections between other optimal transport theories and the conformational heterogeneity problem inherent with cryo-EM data.

The three‐dimensional structure of the enzymes provides very relevant information on the arrangement of the catalytic machinery and structural elements gating the active site pocket. The recent success of the neural network Alphafold2 in predicting the folded structure of proteins from the primary sequence with high levels of accuracy has revolutionized the protein design field. However, the application of Alphafold2 for understanding and engineering function directly from the obtained single static picture is not straightforward. Indeed, understanding enzymatic function requires the exploration of the ensemble of thermally accessible conformations that enzymes adopt in solution. In the present study, we evaluate the potential of Alphafold2 in assessing the effect of the mutations on the conformational landscape of the beta subunit of tryptophan synthase (TrpB). Specifically, we develop a template‐based Alphafold2 approach for estimating the conformational heterogeneity of several TrpB enzymes, which is needed for enhanced stand‐alone activity. Our results show the potential of Alphafold2, especially if combined with molecular dynamics simulations, for elucidating the changes induced by mutation in the conformational landscapes at a rather reduced computational cost, thus revealing its plausible application in computational enzyme design. [ABSTRACT FROM AUTHOR]

Continuous developments in cryogenic X‐ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room‐temperature X‐ray crystallographic method using temperature as a trigger to record movie‐like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15 Da fragment, undergoes binding‐mode changes in endothiapepsin. A surprising fragment‐binding discrepancy was observed between the cryo‐cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure‐based drug discovery. [ABSTRACT FROM AUTHOR]

Small-angle X-ray scattering (SAXS) is an increasingly common and useful technique for structural characterization of molecules in solution. A SAXS experiment determines the scattering intensity of a molecule as a function of spatial frequency, termed SAXS profile. SAXS profiles can be utilized in a variety of molecular modeling applications, such as comparing solution and crystal structures, structural characterization of flexible proteins, assembly of multi-protein complexes, and modeling of missing regions in the high-resolution structure. Here, we describe protocols for modeling atomic structures based on SAXS profiles. The first protocol is for comparing solution and crystal structures including modeling of missing regions and determination of the oligomeric state. The second protocol performs multi-state modeling by finding a set of conformations and their weights that fit the SAXS profile starting from a single-input structure. The third protocol is for protein-protein docking based on the SAXS profile of the complex. We describe the underlying software, followed by demonstrating their application on interleukin 33 (IL33) with its primary receptor ST2 and DNA ligase IV-XRCC4 complex.

Conformational equilibria of G-protein-coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron-electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ∼16 min at 20 °C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners.

TFIID is a large protein complex required for the recognition and binding of eukaryotic gene core promoter sequences and for the recruitment of the rest of the general transcription factors involved in initiation of eukaryotic protein gene transcription. Cryo-electron microscopy studies have demonstrated the conformational complexity of human TFIID, where one-third of the mass of the complex can shift its position by well over 100 Å. This conformational plasticity appears to be linked to the capacity of TFIID to bind DNA, and suggests how it would allow both the recognition of different core promoter elements and the tuning of its binding affinity by regulatory factors.

TDP-43 proteinopathies is a disease hallmark that characterizes amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The N-terminal domain of TDP-43 (NTD) is important to both TDP-43 physiology and TDP-43 proteinopathy. However, its folding and dimerization process is still poorly characterized. In the present study, we have investigated the folding/unfolding of NTD employing all-atom molecular dynamics (MD) simulations in 8 M dimethylsulfoxide (DMSO) at high temperatures. The MD results showed that the unfolding of the NTD at high temperature evolves through the formation of a number of conformational states differing in their stability and free energy. The presence of structurally heterogeneous population of intermediate ensembles was further characterized by the different extents of solvent exposure of Trp80 during unfolding. We suggest that these non-natives unfolded intermediate ensembles may facilitate NTD oligomerization and subsequently TDP-43 oligomerization, which might lead to the formation of irreversible pathological aggregates, characteristics of disease pathogenesis. [ABSTRACT FROM AUTHOR]

TDP-43 proteinopathies is a disease hallmark that characterizes amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The N-terminal domain of TDP-43 (NTD) is important to both TDP-43 physiology and TDP-43 proteinopathy. However, its folding and dimerization process is still poorly characterized. In the present study, we have investigated the folding/unfolding of NTD employing all-atom molecular dynamics (MD) simulations in 8 M dimethylsulfoxide (DMSO) at high temperatures. The MD results showed that the unfolding of the NTD at high temperature evolves through the formation of a number of conformational states differing in their stability and free energy. The presence of structurally heterogeneous population of intermediate ensembles was further characterized by the different extents of solvent exposure of Trp80 during unfolding. We suggest that these non-natives unfolded intermediate ensembles may facilitate NTD oligomerization and subsequently TDP-43 oligomerization, which might lead to the formation of irreversible pathological aggregates, characteristics of disease pathogenesis.

NOSs are homodimeric multidomain enzymes responsible for producing NO. In mammals, NO acts as an intercellular messenger in a variety of signaling reactions, as well as a cytotoxin in the innate immune response. Mammals possess three NOS isoforms--inducible, endothelial, and neuronal NOS--that are composed of an N-terminal oxidase domain and a C-terminal reductase domain. Calmodulin (CaM) activates NO synthesis by binding to the helical region connecting these two domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length NOS. We used high-throughput single-particle EM to obtain the structures and higher-order domain organization of all three NOS holoenzymes. The structures of inducible, endothelial, and neuronal NOS with and without CaM bound are similar, consisting of a dimerized oxidase domain flanked by two separated reductase domains. NOS isoforms adopt many conformations enabled by three flexible linkers. These conformations represent snapshots of the continuous electron transfer pathway from the reductase domain to the oxidase domain, which reveal that only a single reductase domain participates in electron transfer at a time, and that CaM activates NOS by constraining rotational motions and by directly binding to the oxidase domain. Direct visualization of these large conformational changes induced during electron transfer provides significant insight into the molecular underpinnings governing NO formation.

Studies of large proteins, protein complexes, and membrane protein complexes pose new challenges, most notably the need for increased ion mobility (IM) and mass spectrometry (MS) resolution. This review covers evolutionary developments in IM‐MS in the authors' and key collaborators' laboratories with specific focus on developments that enhance the utility of IM‐MS for structural analysis. IM‐MS measurements are performed on gas phase ions, thus "structural IM‐MS" appears paradoxical—do gas phase ions retain their solution phase structure? There is growing evidence to support the notion that solution phase structure(s) can be retained by the gas phase ions. It should not go unnoticed that we use "structures" in this statement because an important feature of IM‐MS is the ability to deal with conformationally heterogeneous systems, thus providing a direct measure of conformational entropy. The extension of this work to large proteins and protein complexes has motivated our development of Fourier‐transform IM‐MS instruments, a strategy first described by Hill and coworkers in 1985 (Anal Chem, 1985, 57, pp. 402–406) that has proved to be a game‐changer in our quest to merge drift tube (DT) and ion mobility and the high mass resolution orbitrap MS instruments. DT‐IMS is the only method that allows first‐principles determinations of rotationally averaged collision cross sections (CSS), which is essential for studies of biomolecules where the conformational diversities of the molecule precludes the use of CCS calibration approaches. The Fourier transform‐IM‐orbitrap instrument described here also incorporates the full suite of native MS/IM‐MS capabilities that are currently employed in the most advanced native MS/IM‐MS instruments. © 2020 John Wiley & Sons Ltd. Mass Spec Rev [ABSTRACT FROM AUTHOR]

Myosin motor domains perform an extraordinary diversity of biological functions despite sharing a common mechanochemical cycle. Motors are adapted to their function, in part, by tuning the thermodynamics and kinetics of steps in this cycle. However, it remains unclear how sequence encodes these differences, since biochemically distinct motors often have nearly indistinguishable crystal structures. We hypothesized that sequences produce distinct biochemical phenotypes by modulating the relative probabilities of an ensemble of conformations primed for different functional roles. To test this hypothesis, we modeled the distribution of conformations for 12 myosin motor domains by building Markov state models (MSMs) from an unprecedented two milliseconds of all-atom, explicit-solvent molecular dynamics simulations. Comparing motors reveals shifts in the balance between nucleotide-favorable and nucleotide-unfavorable P-loop conformations that predict experimentally measured duty ratios and ADP release rates better than sequence or individual structures. This result demonstrates the power of an ensemble perspective for interrogating sequence-function relationships.

Although structure-based virtual drug discovery is revolutionizing the conventional high-throughput cell-based screening system, its limitation is obvious, together with other critical challenges. In particular, the resolved static snapshots fail to represent a full free-energy landscape due to homogenization in structural determination processing. The loss of conformational heterogeneity and related functional diversity emphasize the necessity of developing an approach that can fill this space. In this regard, NMR holds undeniable potential. However, outstanding questions regarding the NMR application remain. This review summarizes the limitations of current drug discovery and explores the potential of 19F NMR in establishing a conformation-guided drug screening system, advancing the cell- and structure-based discovery strategy for G protein-coupled receptor (GPCR) biased drug screening. Studies indicate that G protein-coupled receptor (GPCR) activation is a multistate transition process instead of a simple switch, suggesting the possibility of modulating the GPCR function through tuning of these individual states. A biased ligand was proposed to specifically target one signaling pathway over others in a multiple-signaling coexistence system. However, despite the progress in structural biology, a detailed mechanism of how this is achieved remains elusive. Research has indicated that different GPCR conformations interact with different downstream partners, dictating various pharmacological outputs. 19F NMR has been demonstrated to be extremely promising in delineating GPCR conformations, attributable to its quantitative properties and high sensitivity toward electrostatic changes of the probe microenvironment. Thus, there is tremendous potential for biased drug discovery by establishing a rigorous correlation between individual receptor conformations and the pharmacological consequence upon ligand binding. [ABSTRACT FROM AUTHOR]

Arylamines are known to form covalent-DNA adducts upon metabolic activation. These covalent adducts adopt different conformational attributes, viz., major groove (B), stacked (S), and minor groove (W), and lead to different types of mutations. The conformation depends on the flanking and next flanking bases at the 3′ position of the adduct. Early detection of these conformations by simple probes is an ideal and challenging task. Here, we have reported two Ir(III)-based cyclometalated complexes, viz., [Ir(ppy)2(imiphen)]+ (1) (ppy: 2-phenylpyridine; imiphen: 2-(1H-imidazol-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and [Ir(ppy)2(furphen)]+ (2) (furphen: 2-(furan-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its interaction with N-acetyl-2-aminofluorene-dG (AAF-dG). The sequences used in this work are NarI sequence (–CG1G2CG3CX–) in which Gs are modified with AAF and X is either C or T. Luminescence studies reveal that the Ir(III) complexes bind to AAF-dG adduct with high specificity toward G1 and G3 compared to G2 and unmodified control. The selectivity also depends on the next flanking base as cytosine favors G3AAF, while thymine favors G1AAF in complex 1 and vice versa for complex 2. The quenching studies confirm that Ir(III) complexes bind with AAF-dG sequences through the minor groove. The outcome of this work reveals that the switch-on effect by the complexes can be utilized for determining the conformational heterogeneity of the adduct and also for similar covalent-DNA adducts. [ABSTRACT FROM AUTHOR]

Relating the native fold of a protein to its amino acid sequence remains a fundamental problem in biology. While computer algorithms have demonstrated recently their prowess in predicting what structure a particular amino acid sequence will fold to, an understanding of how and why a specific protein fold is achieved remains elusive. A major challenge is to define the role of conformational heterogeneity during protein folding. Recent experimental studies, utilizing time-resolved FRET, hydrogen-exchange coupled to mass spectrometry, and single-molecule force spectroscopy, often in conjunction with simulation, have begun to reveal how conformational heterogeneity evolves during folding, and whether an intermediate ensemble of defined free energy consists of different sub-populations of molecules that may differ significantly in conformation, energy and entropy. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins involved in signal transduction and are characterized by seven transmembrane domain architecture interconnected by extra- and intracellular loops. These loops, along with the N- and C-terminal domains, constitute the extramembranous regions in GPCRs. These regions, accounting for ~ 40% or more amino acid residues across different GPCR classes, are distinct from the conserved transmembrane domains in terms of nonconservation of sequence, diversity in length, and conformational heterogeneity. Due to technical challenges in exploring the molecular basis underlying the relation between structure, dynamics, and function in these regions, their contribution to GPCR organization and signaling remain underappreciated. Despite existing literature on the involvement of GPCR loops in numerous aspects of GPCR biology, the functional relevance of GPCR loops in the context of their inherent conformational heterogeneity and probable membrane interaction are not well understood. This review focuses on highlighting these aspects of GPCR extramembranous regions in the overall context of GPCR organization, dynamics, and biology. We envision that a judicious combination of insights obtained from structured transmembrane domains and disordered extramembranous regions in GPCRs would be crucial in arriving at a comprehensive understanding of GPCR structure, function, and dynamics, thereby leading to efficient drug discovery. [ABSTRACT FROM AUTHOR]

Proteins inherently fluctuate between conformations to perform functions in the cell. For example, they sample product‐binding, transition‐state‐stabilizing and product‐release states during catalysis, and they integrate signals from remote regions of the structure for allosteric regulation. However, there is a lack of understanding of how these dynamic processes occur at the basic atomic level. This gap can be at least partially addressed by combining variable‐temperature (instead of traditional cryogenic temperature) X‐ray crystallography with algorithms for modeling alternative conformations based on electron‐density maps, in an approach called multitemperature multiconformer X‐ray crystallography (MMX). Here, the use of MMX to reveal alternative conformations at different sites in a protein structure and to estimate the degree of energetic coupling between them is discussed. These insights can suggest testable hypotheses about allosteric mechanisms. Temperature is an easily manipulated experimental parameter, so the MMX approach is widely applicable to any protein that yields well diffracting crystals. Moreover, the general principles of MMX are extensible to other perturbations such as pH, pressure, ligand concentration etc. Future work will explore strategies for leveraging X‐ray data across such perturbation series to more quantitatively measure how different parts of a protein structure are coupled to each other, and the consequences thereof for allostery and other aspects of protein function. Crystallography at multiple temperatures can reveal the collective shifts of alternative conformations that underlie allosteric communication through protein structures. [ABSTRACT FROM AUTHOR]

The extent of ribosomal heterogeneity has caught increasing interest over the past few years, as recent studies have highlighted the presence of structural variations of the ribosome. More precisely, the heterogeneity of the ribosome covers multiple scales, including the dynamical aspects of ribosomal motion at the single particle level, specialization at the cellular and subcellular scale, or evolutionary differences across species. Upon solving the ribosome atomic structure at medium to high resolution, cryogenic electron microscopy (cryo-EM) has enabled investigating all these forms of heterogeneity. In this review, we present some recent advances in quantifying ribosome heterogeneity, with a focus on the conformational and evolutionary variations of the ribosome and their functional implications. These efforts highlight the need for new computational methods and comparative tools, to comprehensively model the continuous conformational transition pathways of the ribosome, as well as its evolution. While developing these methods presents some important challenges, it also provides an opportunity to extend our interpretation and usage of cryo-EM data, which would more generally benefit the study of molecular dynamics and evolution of proteins and other complexes.

The complement component fragment 5a (C5a) binds and activates two complement receptors like C5aR1 and C5aR2, which play a significant role in orchestrating the proinflammatory function of C5a in tissues through the recruitment of heterotrimeric G-proteins and β-arrestins. Dysregulation of the complement induces excessive production of C5a, which triggers aberrant activation of the C5a-C5aR1-G-protein and C5a-C5aR2-β-arrestin signalling axes in tissues, contributing to the pathology of numerous immune-inflammatory diseases. Thus, understanding the interaction of C5a with C5aR1 and C5aR2, as well as the interaction of G-protein and β-arrestins, respectively, with C5a-C5aR1 and C5a-C5aR2, holds tremendous therapeutic value. In the absence of structural data, we have previously elaborated the binary complexes of C5a-C5aR1 and C5a-C5aR2, as well as the ternary complex of C5a-C5aR2-β-arrestin1, in highly refined model structures. While our ternary model complex of C5a-C5aR1-G-protein was in progress, two cryo-electron microscopy-based ternary structural complexes of C5aR1 were made available by others. However, it is observed that the interaction of the crucial NT-peptide of C5aR1 with C5a, including the portion of the G⍺ i -subunit that harbors the switch-I region, is not fully resolved in both complexes. The current study addresses the issues and provides two highly refined alternative model ternary complexes of C5a-C5aR1-G-protein. The study highlights the conformational heterogeneity in C5aR1 by comparing the two conformational variants of the model ternary complex in the context of C5a-C5aR2-β-arrestin1 for further devising methods and molecules targeting both surface and intracellular C5aR1/C5aR2 for effectively mitigating the proinflammatory role of C5a in various disease settings.Communicated by Ramaswamy H. Sarma.

Intrinsically disordered proteins (IDPs) are a novel class of proteins that have established a significant importance and attention within a very short period of time. These proteins are essentially characterized by their inherent structural disorder, encoded mainly by their amino acid sequences. The profound abundance of IDPs and intrinsically disordered regions (IDRs) in the biological world delineates their deep-rooted functionality. IDPs and IDRs convey such extensive functionality through their unique dynamic nature, which enables them to carry out huge number of multifaceted biomolecular interactions and make them "interaction hub" of the cellular systems. Additionally, with such widespread functions, their misfunctioning is also intimately associated with multiple diseases. Thus, understanding the dynamic heterogeneity of various IDPs along with their interactions with respective binding partners is an important field with immense potentials in biomolecular research. In this context, molecular docking-based computational approaches have proven to be remarkable in case of ordered proteins. Molecular docking methods essentially model the biomolecular interactions in both structural and energetic terms and use this information to characterize the putative interactions between the two participant molecules. However, direct applications of the conventional docking methods to study IDPs are largely limited by their structural heterogeneity and demands for unique IDP-centric strategies. Thus, in this chapter, we have presented an overview of current methodologies for successful docking operations involving IDPs and IDRs. These specialized methods majorly include the ensemble-based and fragment-based approaches with their own benefits and limitations. More recently, artificial intelligence and machine learning-assisted approaches are also used to significantly reduce the complexity and computational burden associated with various docking applications. Thus, this chapter aims to provide a comprehensive summary of major challenges and recent advancements of molecular docking approaches in the IDP field for their better utilization and greater applicability.Asp (D)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

ABSTRACT Single particle analysis, which can be regarded as an average of signals from thousands or even millions of particle projections, is an efficient method to study the three-dimensional structures of biological macromolecules. An intrinsic assumption in single particle analysis is that all the analyzed particles must have identical composition and conformation. Thus specimen heterogeneity in either composition or conformation has raised great challenges for high-resolution analysis. For particles with multiple conformations, inaccurate alignments and orientation parameters will yield an averaged map with diminished resolution and smeared density. Besides extensive classification approaches, here based on the assumption that the macromolecular complex is made up of multiple rigid modules whose relative orientations and positions are in slight fluctuation around equilibriums, we propose a new method called as local optimization refinement to address this conformational heterogeneity for an improved resolution. The key idea is to optimize the orientation and shift parameters of each rigid module and then reconstruct their three-dimensional structures individually. Using simulated data of 80S/70S ribosomes with relative fluctuations between the large (60S/50S) and the small (40S/30S) subunits, we tested this algorithm and found that the resolutions of both subunits are significantly improved. Our method provides a proof-of-principle solution for high-resolution single particle analysis of macromolecular complexes with dynamic conformations.

The proposal that coupled folding to binding is not an obligatory mechanism for intrinsically disordered (ID) proteins was put forward 10 years ago. The notion of fuzziness implies that conformational heterogeneity can be maintained upon interactions of ID proteins, which has a functional impact either on regulated assembly or activity of the corresponding complexes. Here I review how the concept has evolved in the past decade, via increasing experimental data providing insights into the mechanisms, pathways and regulatory modes. The effects of structural diversity and transient contacts on protein assemblies have been collected and systematically analyzed (Fuzzy Complexes Database, http://protdyn-database.org ). Fuzziness has also been exploited as a framework to decipher molecular organization of higher-order protein structures. Quantification of conformational heterogeneity opens exciting future perspectives for drug discovery from small molecule–ID protein interactions to supramolecular assemblies. [ABSTRACT FROM AUTHOR]

The modulation of main‐chain and side‐chain conformational heterogeneity and solvent structure in monoclinic lysozyme crystals by dehydration (related to water activity) and temperature is examined. Decreasing the relative humidity (from 99 to 11%) and decreasing the temperature both lead to contraction of the unit cell, to an increased area of crystal contacts and to remodeling of primarily contact and solvent‐exposed residues. Both lead to the depopulation of some minor side‐chain conformers and to the generation of new conformations. Side‐chain modifications and main‐chain r.m.s.d.s associated with cooling from 298 to 100 K depend on relative humidity and are minimized at 85% relative humidity (r.h.). Dehydration from 99 to 93% r.h. and cooling from 298 to 100 K result in a comparable number of remodeled residues, with dehydration‐induced remodeling somewhat more likely to arise from contact interactions. When scaled to equivalent temperatures based on unit‐cell contraction, the evolution of side‐chain order parameters with dehydration shows generally similar features to those observed on cooling to T = 100 K. These results illuminate the qualitative and quantitative similarities between structural perturbations induced by modest dehydration, which routinely occurs in samples prepared for 298 and 100 K data collection, and cryocooling. Differences between these perturbations in terms of energy landscapes and occupancies, and implications for variable‐temperature crystallography between 180 and 298 K, are discussed. It is also noted that remodeling of a key lysozyme active‐site residue by dehydration, which is associated with a radical decrease in the enzymatic activity of lysozyme powder, arises due to a steric clash with the residue of a symmetry mate. [ABSTRACT FROM AUTHOR]

The conformational landscape of multi-domain proteins is inherently linked to their specific functions. This also holds for polyubiquitin chains that are assembled by two or more ubiquitin domains connected by a flexible linker thus showing a large interdomain mobility. However, molecular recognition and signal transduction are associated with particular conformational substates that are populated in solution. Here, we apply high-resolution NMR spectroscopy in combination with dual-scale MD simulations to explore the conformational space of K6-, K29-, and K33-linked diubiquitin molecules. The conformational ensembles are evaluated utilizing a paramagnetic cosolute reporting on solvent exposure plus a set of complementary NMR parameters. This approach unravels a conformational heterogeneity of diubiquitins and explains the diversity of structural models that have been determined for K6-, K29-, and K33-linked diubiquitins in free and ligand-bound states so far. We propose a general application of the approach developed here to demystify multi-domain proteins occurring in nature. [Display omitted] • General workflow for studying multi-domain proteins at atomic resolution • Heterogeneous conformational character of K6-, K29-, and K33-linked ubiquitin dimers • Integrative approach combines chemical biology, NMR spectroscopy, and MD simulations • Structural basis for the observed diversity of ubiquitin dimer crystal structures Schneider et al. investigated the conformational landscape of various ubiquitin dimers by applying an integrative approach. The ubiquitin dimers probed in their study show a pronounced conformational heterogeneity. Besides ubiquitin dimers, the here designed workflow will make it possible to study the conformational space of multi-domain proteins in general. [ABSTRACT FROM AUTHOR]

The Hsp70 chaperone system is integrated into a myriad of biochemical processes that are critical for cellular proteostasis. Although detailed pictures of Hsp70 bound with peptides have emerged, correspondingly detailed structural information on complexes with folding-competent substrates remains lacking. Here we report a methyl-TROSY based solution NMR study showing that the Escherichia coli version of Hsp70, DnaK, binds to as many as four distinct sites on a small 53-residue client protein, hTRF1. A fraction of hTRF1 chains are also bound to two DnaK molecules simultaneously, resulting in a mixture of DnaK-substrate sub-ensembles that are structurally heterogeneous. The interactions of Hsp70 with a client protein at different sites results in a fuzzy chaperone-substrate ensemble and suggests a mechanism for Hsp70 function whereby the structural heterogeneity of released substrate molecules enables them to circumvent kinetic traps in their conformational free energy landscape and fold efficiently to the native state.

Bulky organic carcinogens are activated in vivo and subsequently react with nucleobases of cellular DNA to produce adducts. Some of these DNA adducts exist in multiple conformations that are slowly interconverted to one another. Different conformations have been implicated in different mutagenic and repair outcomes. However, studies on the conformation-specific inhibition of replication, which is more relevant to cell survival, are scarce, presumably due to the structural dynamics of DNA lesions at the replication fork. It is difficult to capture the exact nature of replication inhibition by existing end-point assays, which usually detect either the ensemble of consequences of all the conformers or the culmination of all cellular behaviors, such as mutagenicity or survival rate. We previously reported very unusual sequence-dependent conformational heterogeneities involving FABP-modified DNA under different sequence contexts (TG1*G2T [67%B:33%S] and TG1G2*T [100%B], G*, N-(2′-deoxyguanosin-8-yl)-4′-fluoro-4-aminobiphenyl) (Cai et al. Nucleic Acids Research, 46, 6356–6370 (2018)). In the present study, we attempted to correlate the in vitro inhibition of polymerase activity to different conformations from a single FABP-modified DNA lesion. We utilized a combination of surface plasmon resonance (SPR) and HPLC-based steady-state kinetics to reveal the differences in terms of binding affinity and inhibition with polymerase between these two conformers (67%B:33%S and 100%B).

Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain. [ABSTRACT FROM AUTHOR]