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Aktivitätsbasierte Sonden sind wertvolle Werkzeuge in der chemischen Biologie. Nach wie vor ist es jedoch eine Herausforderung, molekulare Sonden zu entwickeln, die spezifisch an das aktive Zentrum eines bestimmten Enzyms binden. Wir stellen hier eine Strategie zur Ligandenselektion vor, die es ermöglicht, rasch elektrophile Sonden auf ausgewählte Enzyme zuzuschneiden, und zeigen in einer Machbarkeitsstudie ihre Anwendung für die beiden Cysteinproteasen von SARS‐CoV‐2. Die resultierenden Sonden markieren spezifisch die aktiven Zentren von 3CLpro und PLpro mit hinreichender Selektivität sowohl in einem lebenden Zellmodell als auch vor dem Hintergrund eines nativen menschlichen Proteoms. Durch die Nutzung der Sonden als Werkzeuge für das kompetitive Screening einer Bibliothek von Naturstoffen wurden Salvianolsäurederivate als vielversprechende 3CLpro‐Inhibitoren identifiziert. Unsere Strategie zur Ligandenselektion wird für die schnelle Entwicklung von maßgeschneiderten Sonden von großem Nutzen sein und die Entdeckung von Inhibitoren für eine Vielzahl von Zielproteinen ermöglichen, die auch über Coronavirus‐Proteasen hinausgehen. published

Regulation of gene expression via riboswitches is a widespread mechanism in bacteria. Here, we investigate ligand binding of a member of the guanidine sensing riboswitch family, the guanidine-II riboswitch (Gd-II). It consists of two stem–loops forming a dimer upon ligand binding. Using extensive molecular dynamics simulations we have identified conformational states corresponding to ligand-bound and unbound states in a monomeric stem–loop of Gd-II and studied the selectivity of this binding. To characterize these states and ligand-dependent conformational changes we applied a combination of dimensionality reduction, clustering, and feature selection methods. In absence of a ligand, the shape of the binding pocket alternates between the conformation observed in presence of guanidinium and a collapsed conformation, which is associated with a deformation of the dimerization interface. Furthermore, the structural features responsible for the ability to discriminate against closely related analogs of guanidine are resolved. Based on these insights, we propose a mechanism that couples ligand binding to aptamer dimerization in the Gd-II system, demonstrating the value of computational methods in the field of nucleic acids research.

Striated muscle undergoes remodelling in response to mechanical and physiological stress, but little is known about the integration of such varied signals in the myofibril. The interaction of the elastic kinase region from sarcomeric titin (A168‐M1) with the autophagy receptors Nbr1/p62 and MuRF E3 ubiquitin ligases is well suited to link mechanosensing with the trophic response of the myofibril. To investigate the mechanisms of signal cross‐talk at this titin node, we elucidated its 3D structure, analysed its response to stretch using steered molecular dynamics simulations and explored its functional relation to MuRF1 and Nbr1/p62 using cellular assays. We found that MuRF1‐mediated ubiquitination of titin kinase promotes its scaffolding of Nbr1/p62 and that the process can be dynamically down‐regulated by the mechanical unfolding of a linker sequence joining titin kinase with the MuRF1 receptor site in titin. We propose that titin ubiquitination is sensitive to the mechanical state of the sarcomere, the regulation of sarcomere targeting by Nbr1/p62 being a functional outcome. We conclude that MuRF1/Titin Kinase/Nbr1/p62 constitutes a distinct assembly that predictably promotes sarcomere breakdown in inactive muscle., The pseudokinase domain of the titin myofilament is a node for the cross‐talk of mechanical signals and turn‐over pathways in the sarcomere, contributing to a coordinated response to cellular stress.

Activity‐based probes are valuable tools for chemical biology. However, finding probes that specifically target the active site of an enzyme remains a challenging task. Herein, we present a ligand selection strategy that allows to rapidly tailor electrophilic probes to a target of choice and showcase its application for the two cysteine proteases of SARS‐CoV‐2 as proof of concept. The resulting probes were specific for the active site labeling of 3CLpro and PLpro with sufficient selectivity in a live cell model as well as in the background of a native human proteome. Exploiting the probes as tools for competitive profiling of a natural product library identified salvianolic acid derivatives as promising 3CLpro inhibitors. We anticipate that our ligand selection strategy will be useful to rapidly develop customized probes and discover inhibitors for a wide range of target proteins also beyond corona virus proteases., Tuning the ligand space allows to rapidly customize chemical probes for efficacy and active site specificity for a protease of interest. The application of this ligand selection approach is exemplified by generating probes for the two non‐homologous yet essential proteases of the new corona virus SARS‐CoV‐2 providing complementary chemical tools for proteomic labeling and inhibitor discovery.

Understanding the conformational characteristics of protein complexes in solution is crucial for a deeper insight in their biological function. Molecular dynamics simulations performed on high performance computing plants and with modern simulation techniques can be used to obtain large data sets that contain conformational and thermodynamic information about biomolecular systems. While this can in principle give a detailed picture of protein-protein interactions in solution and therefore complement experimental data, it also raises the challenge of processing exceedingly large high-dimensional data sets with several million samples. Here we present a novel method for the characterization of protein-protein interactions, which combines a neural network based dimensionality reduction technique to obtain a two-dimensional representation of the conformational space with a density based clustering algorithm for state detection and a metric which assesses the (dis)similarity between different conformational spaces. This method is highly scalable and therefore makes the analysis of massive data sets computationally tractable. We demonstrate the power of this approach to large scale data analysis by characterizing highly dynamic conformational phase spaces of differently linked ubiquitin (Ub) oligomers from coarse-grained simulations. We are able to extract a protein-protein interaction model for two unlinked Ub proteins which is then used to determine how the Ub-Ub interaction pattern is altered in Ub oligomers by the introduction of a covalent linkage. We find that the Ub chain conformational ensemble depends highly on the linkage type and for some cases also on the Ub chain length. By this, we obtain insight into the conformational characteristics of different Ub chains and how this may contribute to linkage type and chain length specific recognition.

Significance Despite being evolved to process the four canonical nucleotides, DNA polymerases are known to incorporate and extend from modified nucleotides, which is the key to numerous core biotechnology applications. The structural basis for postincorporation elongation remained elusive. We successfully crystallized KlenTaq DNA polymerase in six complexes, providing high-resolution snapshots of the modification “moving” from the 3′ terminus upstream to the sixth nucleotide in the primer strand. Combining these data with quantum mechanics/molecular mechanics calculations and biochemical studies elucidates how the enzyme and the modified substrate mutually modulate their conformations without compromising the enzyme’s activity. This highlights the unexpected plasticity of the system as origin of the broad substrate properties of the DNA polymerase and guide for the design of improved systems., DNA polymerases have evolved to process the four canonical nucleotides accurately. Nevertheless, these enzymes are also known to process modified nucleotides, which is the key to numerous core biotechnology applications. Processing of modified nucleotides includes incorporation of the modified nucleotide and postincorporation elongation to proceed with the synthesis of the nascent DNA strand. The structural basis for postincorporation elongation is currently unknown. We addressed this issue and successfully crystallized KlenTaq DNA polymerase in six closed ternary complexes containing the enzyme, the modified DNA substrate, and the incoming nucleotide. Each structure shows a high-resolution snapshot of the elongation of a modified primer, where the modification “moves” from the 3′-primer terminus upstream to the sixth nucleotide in the primer strand. Combining these data with quantum mechanics/molecular mechanics calculations and biochemical studies elucidates how the enzyme and the modified substrate mutually modulate their conformations without compromising the enzyme’s activity significantly. The study highlights the plasticity of the system as origin of the broad substrate properties of DNA polymerases and facilitates the design of improved systems.

RNase H is a prototypical example for two metal ion catalysis in enzymes. An RNase H activity is present in the HIV-1 reverse transcriptase but also in many other nucleases such as Homo sapiens (Hs) or Escherichia coli (Ec) RNase H. The mechanism of the reaction has already been extensively studied based on the Bacillus halodurans (Bh) RNase H crystal structures, most recently using time-resolved X-Ray crystallography. However, kinetic and mutation experiments with HIV-1, Hs and Ec RNase H implicate a catalytic histidine in the reaction that is not present in Bh RNase H, and the protonation of the leaving group also remains poorly understood. We use quantum mechanics/molecular mechanics (QM/MM) calculations combining Hamiltonian replica exchange with a finite-temperature string method to study the cleavage of the ribonucleic acid (RNA) backbone of a DNA/RNA hybrid catalyzed by the HIV-1 RNase H with a focus on the proton transfer pathway and the role of the histidine. The reported pathway is consistent with kinetic data obtained with mutant HIV-1, Hs and Ec RNase H, the calculated pKa values of the DEDD residues and crystallographic studies. The overall reaction barrier of ∼18 kcal mol-1, encountered in the first step, matches the slow experimental rate of ∼1-100 min-1. Using Molecular dynamics (MD) calculations we are able to sample the recently identified binding site for a third transient divalent metal ion in the vicinity of the scissile phosphate in the product complex. Our results account for the experimental observation of a third metal ion facilitating product release in an Aquifex aeolicus RNase III crystal structure and the Bh RNase H in crystallo reaction. Based on our data we are able to show that the third ion and the histidine are key to product release as had been hypothesized.

Hsp70 chaperones assist de novo folding of newly synthesized proteins in all cells. In yeast, the specialized Hsp70 Ssb directly binds to ribosomes. The structural basis and functional mode of recruitment of Ssb to ribosomes is not understood. Here, we present the molecular details underlying ribosome binding of Ssb in Saccharomyces cerevisiae. This interaction is multifaceted, involving the co-chaperone RAC and two specific regions within Ssb characterized by positive charges. The C-terminus of Ssb mediates the key contact and a second attachment point is provided by a KRR-motif in the substrate binding domain. Strikingly, ribosome binding of Ssb is not essential. Autonomous ribosome attachment becomes necessary if RAC is absent, suggesting a dual mode of Ssb recruitment to nascent chains. We propose, that the multilayered ribosomal interaction allows positioning of Ssb in an optimal orientation to the tunnel exit guaranteeing an efficient nascent polypeptide interaction., The correct folding of proteins often requires the intervention molecular chaperones, which can occur co-translationally. Here the authors identify elements of yeast Ssb (Hsp70) that mediate ribosomal binding, and suggest a mechanism that directs efficient interaction of Ssb with the nascent chain.

We use coarse-grained (CG) simulations to study the deformation of empty Cowpea Chlorotic Mottle Virus (CCMV) capsids under uniaxial compression, from the initial elastic response up to capsid breakage. Our CG model is based on the MARTINI force field and has been amended by a stabilizing elastic network, acting only within individual proteins, that was tuned to capture the fluctuation spectrum of capsid protein dimers, obtained from all atom simulations. We have previously shown that this model predicts force-compression curves that match AFM indentation experiments on empty CCMV capsids. Here we investigate details of the actual breaking events when the CCMV capsid finally fails. We present a symmetry classification of all relevant protein contacts and show that they differ significantly in terms of stability. Specifically, we show that interfaces which break readily are precisely those which are believed to form last during assembly, even though some of them might share the same contacts as other non-breaking interfaces. In particular, the interfaces that form pentamers of dimers never break, while the virtually identical interfaces within hexamers of dimers readily do. Since these units differ in the large-scale geometry and, most noticeably, the cone-angle at the center of the 5- or 6-fold vertex, we propose that the hexameric unit fails because it is pre-stressed. This not only suggests that hexamers of dimers form less frequently during the early stages of assembly; it also offers a natural explanation for the well-known β-barrel motif at the hexameric center as a post-aggregation stabilization mechanism. Finally, we identify those amino acid contacts within all key protein interfaces that are most persistent during compressive deformation of the capsid, thereby providing potential targets for mutation studies aiming to elucidate the key contacts upon which overall stability rests. published

FAT10 is a ubiquitin-like modifier that directly targets proteins for proteasomal degradation. Here, we report the high-resolution structures of the two individual ubiquitin-like domains (UBD) of FAT10 that are joined by a flexible linker. While the UBDs of FAT10 show the typical ubiquitin-fold, their surfaces are entirely different from each other and from ubiquitin explaining their unique binding specificities. Deletion of the linker abrogates FAT10-conjugation while its mutation blocks auto-FAT10ylation of the FAT10-conjugating enzyme USE1 but not bulk conjugate formation. FAT10- but not ubiquitin-mediated degradation is independent of the segregase VCP/p97 in the presence but not the absence of FAT10’s unstructured N-terminal heptapeptide. Stabilization of the FAT10 UBDs strongly decelerates degradation suggesting that the intrinsic instability of FAT10 together with its disordered N-terminus enables the rapid, joint degradation of FAT10 and its substrates without the need for FAT10 de-conjugation and partial substrate unfolding., The ubiquitin-like modifier FAT10 is composed of two ubiquitin-like domains (UBDs). Here the authors present the FAT10 UBD structures and show that the unstructured FAT10 N-terminal heptapeptide together with the poor stability of FAT10 facilitate the rapid proteasomal targeting of FAT10 along with its substrates.

Covalent modification of proteins by ubiquitin or ubiquitin chains is one of the most prevalent post-translational modifications in eukaryotes. Different types of ubiquitin chains are assumed to selectively signal respectively modified proteins for different fates. In support of this hypothesis, structural studies have shown that the eight possible ubiquitin dimers adopt different conformations. However, at least in some cases, these structures cannot sufficiently explain the molecular basis of the selective signaling mechanisms. This indicates that the available structures represent only a few distinct conformations within the entire conformational space adopted by a ubiquitin dimer. Here, molecular simulations on different levels of resolution can complement the structural information. We have combined exhaustive coarse grained and atomistic simulations of all eight possible ubiquitin dimers with a suitable dimensionality reduction technique and a new method to characterize protein-protein interfaces and the conformational landscape of protein conjugates. We found that ubiquitin dimers exhibit characteristic linkage type-dependent properties in solution, such as interface stability and the character of contacts between the subunits, which can be directly correlated with experimentally observed linkage-specific properties., Author summary Post-translational modification of proteins by covalent attachment of ubiquitin is a key cellular process, regulating for example the fate and recycling of proteins. We present a new method to combine multiscale simulation with advanced analysis methods to characterize the states of ubiquitin-ubiquitin conjugates. We found that the linkage position affects the conformational space of ubiquitin dimers, determining the number and stability of relevant states, the character of subunit contacts and the nature of the surface exposed to possible binding partners.

FAT10 is a ubiquitin-like modifier that directly targets proteins for proteasomal degradation. Here, we report the high-resolution structures of the two individual ubiquitin-like domains (UBD) of FAT10 that are joined by a flexible linker. While the UBDs of FAT10 show the typical ubiquitin-fold, their surfaces are entirely different from each other and from ubiquitin explaining their unique binding specificities. Deletion of the linker abrogates FAT10-conjugation while its mutation blocks auto-FAT10ylation of the FAT10-conjugating enzyme USE1 but not bulk conjugate formation. FAT10- but not ubiquitin-mediated degradation is independent of the segregase VCP/p97 in the presence but not the absence of FAT10’s unstructured N-terminal heptapeptide. Stabilization of the FAT10 UBDs strongly decelerates degradation suggesting that the intrinsic instability of FAT10 together with its disordered N-terminus enables the rapid, joint degradation of FAT10 and its substrates without the need for FAT10 de-conjugation and partial substrate unfolding.

A combination of coarse-grained (CG) and atomistic simulations provides a suitable computational framework to study unstructured regions of proteins, for which experimental data are often lacking or limited. In this work, we combine CG and atomistic simulations with clustering algorithms and free energy reweighting methods to explore the conformational equilibrium of certain regions of the salt-stable cowpea chlorotic mottle virus (SS-CCMV). In particular, we focus on the geometry of converging strands (residues 26-49) from contacting subunits at the 3-fold (hexamer) and 5-fold (pentamer) symmetry points of the capsid. We show the following: (i) The simulations reproduce the experimentally observed β-barrel for the hexamer. (ii) The pentamer geometry is unable to stabilize a β-barrel conformation; it assumes various states instead, again in accordance with the experimental results which do not indicate a well-defined structure for the pentameric interface. (iii) Atomistic simulations of the backmapped CG structures remain relatively stable, indicative of plausible CG conformations and slow kinetics on the atomistic level.

The molecular basis of microtubule nucleation is still not known in higher plant cells. This process is better understood in yeast and animals cells. In the yeast spindle pole body and the centrosome in animal cells, gamma-tubulin small complexes and gamma-tubulin ring complexes, respectively, nucleate all microtubules. In addition to gamma-tubulin, Spc98p or its homologues plays an essential role. We report here the characterization of rice and Arabidopsis homologues of SPC98. Spc98p colocalizes with gamma-tubulin at the nuclear surface where microtubules are nucleated on isolated tobacco nuclei and in living cells. AtSpc98p-GFP also localizes at the cell cortex. Spc98p is not associated with gamma-tubulin along microtubules. These data suggest that multiple microtubule-nucleating sites are active in plant cells. Microtubule nucleation involving Spc98p-containing gamma-tubulin complexes could then be conserved among all eukaryotes, despite differences in structure and spatial distribution of microtubule organizing centers.

γ-Tubulin is a protein found in all eukaryotic cells, where it plays a key role in the nucleation of microtubules. In higher plant cells, γ-tubulin is localized at the nuclear surface, a known microtubule-organizing centre, and is codistributed with all microtubule arrays. Functions of plant γ-tubulin remain to be determined. This study describes some properties of higher plant γ-tubulin. The overall level of γ-tubulin was constant during the cell cycle in synchronized tobacco BY-2 cells. Biochemical analysis of the subcellular distribution of γ-tubulin in maize cells revealed that, in contrast with animal γ-tubulin, plant γ-tubulin is mainly associated with endomembranes. We showed for the first time that the pool of soluble cytosolic γ-tubulin contained two main γ-tubulin complexes. γ-tubulin, Hsp70 and TCP1-related proteins might interact in a small complex of 750 kDa. A second γ-tubulin complex, larger than 1500 kDa was purified. The protein profile of this large complex was very similar to animal γ-tubulin complexes. The putative functions of these two complexes in plant microtubule nucleation are discussed.

The structure and the molecular composition of the microtubule-organizing centers in acentriolar higher plant cells remain unknown. We developed an in vitro complementation assay where tobacco BY-2 extracts can restore the microtubule-nucleating activity of urea-inactivated mammalian centrosomes. Our results provide first evidence that soluble microtubule-nucleating factors are present in the plant cytosolic fraction. The implication for microtubule nucleation in higher plants is discussed.

In higher-plant cells, the different cell-cycle-dependent microtubule arrays are involved in a wide range of activities including chromosome segregation, cell-plate formation and cellulose microfibril distribution and orientation. A wealth of data, obtained using animal cells, has indicated that the differential stability and function of microtubules during cell-cycle and/or differentiation could be primarily regulated by selective microtubule-associated proteins (MAP). Compared to animal MAP, our knowledge of plant MAP is so far very limited. In this study, we have identified a maize heat-stable protein with apparent molecular mass 100 kDa (P-100) which binds to taxol-stabilized neurotubules and copolymerizes in vitro with purified neural tubulin. Moreover, P-100 cross-reacts with affinity-purified tau antibodies like a maize 83-kDa putative MAP described previously [Vantard, M., Schellenbaum, P., Fellous, A. & Lambert, A. M. (1991) Biochemistry 30, 9334-9340]. Polyclonal antibodies directed against P-100 were obtained and indicated that this protein is found in diverse higher-plant cultured cells suggesting the ubiquitous nature of this protein. P-100 can be phosphorylated in vitro by protein kinases present in a maize cytosol extract. Together, our data suggest that P-100 could be a higher plant MAP.

We use a systematically coarse-grained model for the protein capsid of Cowpea Chlorotic Mottle Virus (CCMV) to study its deformation under uniaxial compression, all the way from its initial elastic response to the capsid's ultimate structural failure. Our model amends the MARTINI force field with an iteratively refined elastic network, and we have previously shown that it reproduces the fluctuations of small fragments as well as the large-scale stress-strain response.We developed an automated identification method that classifies the contacting protein interfaces in the CCMV capsid into symmetry-classes and characterizes their structural changes upon deformation in residue-level detail. We observed that the symmetry-classes differ markedly in their stability, in a way that appears to backtrack the putative assembly pathway: interfaces that are believed to form last are most likely to break first. For instance, neither protein dimers (the first assembly step) nor pentamers of dimers (the second step) were ever seen to fail, while the hexameric association site (presumably the last to form) ruptures most readily. Interestingly, the wild type capsid fortifies this location with a cooperatively formed 6-stranded beta-barrel motif, which is missing in the mutant we employed in our studies. We hypothesize that interfacial binding strengths regulate the assembly order, but that later (and hence weaker) contacts may be reinforced by cooperative motifs that form post-assembly.

The major protective coat of most viruses is a highly symmetric protein capsid that forms spontaneously from many copies of identical proteins. Structural and mechanical properties of such capsids, as well as their self-assembly process, have been studied experimentally and theoretically, including modeling efforts by computer simulations on various scales. Atomistic models include specific details of local protein binding but are limited in system size and accessible time, while coarse grained (CG) models do get access to longer time and length scales but often lack the specific local interactions. Multi-scale models aim at bridging this gap by systematically connecting different levels of resolution. A CG model for CCMV (Cowpea Chlorotic Mottle Virus), a virus with an icosahedral shell of 180 identical proteins, is developed, where parameters are derived from atomistic simulations of capsid protein dimers in aqueous solution. In particular, a new method is introduced to combine the MARTINI CG model with a supportive elastic network based on structural fluctuations of individual proteins. In the parametrization process, both network connectivity and strength are optimized. This elastic-network optimized CG model, which solely relies on atomistic data of small units (dimers), is able to correctly predict inter- protein conformational flexibility and properties of larger aggregates. Here, aggregates of 20 and more capsid proteins are chosen that are possible intermediates in the assembly process or otherwise relevant for the mechanical stability of the CCMV virus shell. Furthermore, it is shown that this CG model reproduces experimental (Atomic Force Microscopy) indentation measurements of the entire viral capsid. Thus it is shown that one obvious goal for hierarchical modeling, namely predicting elastic aspects of larger protein complexes from models that are carefully parametrized on smaller units, is achievable.

RNase H is a prototypical example for two-metal-ion catalysis in enzymes. An RNase H activity cleaving the ribonucleic acid (RNA) backbone of a DNA/RNA hybrid is present not only in important drug targets, such as the HIV-1 reverse transcriptase, but also in many other nucleases, such as Homo sapiens (Hs) and Escherichia coli (Ec) RNase H or, notably, in enzymes that are part of the CRISPR gene editing molecular machinery. Despite its importance, the reaction mechanism uncovering the proton-transfer events is not yet understood. In particular, it is not known, which group is the proton donor for the leaving group. Moreover, several different proton acceptors were proposed, and the exact identity of the proton acceptor is also elusive. Here, we revisit the mechanism for RNAse H, whereby we find that the highly conserved Glu residue of the DDE motif acts as a proton donor via a mechanism further stabilized by the 2′O atom of the sugar. Additionally, we also describe an alternative proton-transfer mechanism via a conserved catalytic His residue to deprotonate the attacking water molecule. Furthermore, our quantum mechanics/molecular mechanics (QM/MM) calculations combining Hamiltonian replica exchange with a finite-temperature string method provide an accurate free-energy profile for the reaction catalyzed by the HIV-1 RNase H. Our reported pathway is consistent with kinetic data obtained for mutant HIV-1, Hs, and Ec RNase H, with the calculated pKa values of the DEDD residues and with crystallographic studies. The overall reaction barrier of ∼19 kcal mol–1, encountered in the phosphate-cleavage step, matches the slow experimental rate of ∼1–100 min–1. Additionally, using molecular dynamics (MD) calculations, we sample the recently identified binding site for a third transient divalent metal ion in the vicinity of the scissile phosphate in the product complex. Our results account for the experimental observation of a third metal ion facilitating product release in an Aquifex aeolicus RNase III crystal structure and the Bh RNase H in crystallo reaction. Taken together, we provide a molecular mechanism of the nuclease catalytic reaction that is likely common for the broad family of two-metal-ion catalytic phosphate-cleaving enzymes with a DDE motif. published