A de novo dominant-negative variant is associated with OTULIN-related autoinflammatory syndrome.

OTULIN-related autoinflammatory syndrome (ORAS), a severe autoinflammatory disease, is caused by biallelic pathogenic variants of OTULIN, a linear ubiquitin-specific deubiquitinating enzyme. Loss of OTULIN attenuates linear ubiquitination by inhibiting the linear ubiquitin chain assembly complex (LUBAC). Here, we report a patient who harbors two rare heterozygous variants of OTULIN (p.P152L and p.R306Q). We demonstrated accumulation of linear ubiquitin chains upon TNF stimulation and augmented TNF-induced cell death in mesenchymal stem cells differentiated from patient-derived iPS cells, which confirms that the patient has ORAS. However, although the de novo p.R306Q variant exhibits attenuated deubiquitination activity without reducing the amount of OTULIN, the deubiquitination activity of the p.P152L variant inherited from the mother was equivalent to that of the wild-type. Patient-derived MSCs in which the p.P152L variant was replaced with wild-type also exhibited augmented TNF-induced cell death and accumulation of linear chains. The finding that ORAS can be caused by a dominant-negative p.R306Q variant of OTULIN furthers our understanding of disease pathogenesis.

Conformational Fluctuations and Induced Orientation of a Protein, Its Solvation Shell, and Bulk Water in Weak Non-Unfolding External Electric Fields.

Extreme external electric fields have been reported to disrupt the tertiary structure of stably folded proteins; however, the effects of weaker electric fields on many biomolecules, especially net-uncharged proteins, and on the surrounding aqueous environment have been rarely discussed. To explore these effects at the atomic level, here, we have used molecular dynamics simulations to estimate rotational motion and induced structural fluctuations in the model protein ubiquitin and its hydration layer due to applied non-unfolding electrostatic fields. When exposed to weak electric fields of up to 0.2 V nm-1, ubiquitin displayed competition between internal structure-maintaining molecular interactions and the external orienting force, which disrupted the local structure in certain regions of the protein. Moreover, relative to hydration water, bulk water showed a greater tendency to align with the electric field, indicating that the presence of protein caused hydration water to acquire rotational mobility different from that in a pure-water system. The differential influence of the applied electric field on the hydration and bulk water surrounding ubiquitin will be common to almost all (nonmembrane) biomacromolecules. Our findings highlight the importance of local dipoles and their electric polarizability even in net-uncharged biomolecules.

Solution structure of the HOIL-1L NZF domain reveals a conformational switch regulating linear ubiquitin affinity.

Attachment of polyubiquitin (poly-Ub) chains to proteins is a major posttranslational modification in eukaryotes. Linear ubiquitin chain assembly complex, consisting of HOIP (HOIL-1-interacting protein), HOIL-1L (heme-oxidized IRP2 Ub ligase 1), and SHARPIN (Shank-associated RH domain-interacting protein), specifically synthesizes "head-to-tail" poly-Ub chains, which are linked via the N-terminal methionine α-amino and C-terminal carboxylate of adjacent Ub units and are thus commonly called "linear" poly-Ub chains. Linear ubiquitin chain assembly complex-assembled linear poly-Ub chains play key roles in immune signaling and suppression of cell death and have been associated with immune diseases and cancer; HOIL-1L is one of the proteins known to selectively bind linear poly-Ub via its Npl4 zinc finger (NZF) domain. Although the structure of the bound form of the HOIL-1L NZF domain with linear di-Ub is known, several aspects of the recognition specificity remain unexplained. Here, we show using NMR and orthogonal biophysical methods, how the NZF domain evolves from a free to the specific linear di-Ub-bound state while rejecting other potential Ub species after weak initial binding. The solution structure of the free NZF domain revealed changes in conformational stability upon linear Ub binding, and interactions between the NZF core and tail revealed conserved electrostatic contacts, which were sensitive to charge modulation at a reported phosphorylation site: threonine-207. Phosphomimetic mutations reduced linear Ub affinity by weakening the integrity of the linear di-Ub-bound conformation. The described molecular determinants of linear di-Ub binding provide insight into the dynamic aspects of the Ub code and the NZF domain's role in full-length HOIL-1L.

An integrated approach of NMR experiments and MD simulations visualizes structural dynamics of a cyclic multi-domain protein.

Cyclization can stabilize the structure of proteins, as previously demonstrated in single-domain proteins. Although Lys48-linked polyubiquitin, a multi-domain protein, is also known to be cyclized in human cells, the structural effects of cyclization remain unclear. Here, we examined the impact of cyclization on the structural stability and dynamics of cyclic Lys48-linked diubiquitin (Ub2 ). As expected, cyclization increased the thermal stability of Ub2 and its resistance to proteolytic digestion, indicating that cyclization stabilized the structure of Ub2 . Furthermore, cyclization repressed the interdomain motion in Ub2 , but cyclic Ub2 still exhibited microsecond conformational exchange in NMR relaxation dispersion experiments. A series of long coarse-grained (CG) MD simulations visualized how cyclization slowed down the intrinsic nanosecond open-closed domain motion of Ub2 to microseconds. Thus, CG-MD analysis helped to explain the unexpected NMR relaxation results, thereby facilitating characterization of the structural stabilization of cyclic Ub2 .

Rheo-NMR Spectroscopy for Cryogenic-Probe-Equipped NMR Instruments to Monitor Protein Aggregation.

Cryogenic-probe-based Rheo-NMR spectroscopy is a recently developed methodology to obtain solution NMR spectra of protein samples in situ under external shear. It is applicable to atomic-resolution monitoring of protein aggregation in situ, thereby aiding understanding of the transient structural changes and state conversion of amyloidogenic proteins, which are strongly associated with the both the onset and the progression of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Here, we present detailed experimental procedures for the instrumental setup and practical tips for preparation of NMR measurement to analyze protein aggregation by this technique. This protocol will thus aid future Rheo-NMR spectroscopic studies not only of protein aggregation but also of other phenomena related to shear stress, such as shear-induced viscosity increase and shear-enhanced crystallization. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Setup of a Rheo-NMR Instrument Basic Protocol 2: Adjustment of the Vertical and Horizontal Positions of the Glass Stick Basic Protocol 3: Monitoring Protein Aggregation by Rheo-NMR Spectroscopy.

Structural Insights into Methylated DNA Recognition by the Methyl-CpG Binding Domain of MBD6 from Arabidopsis thaliana.

Cytosine methylation is an epigenetic modification essential for formation of mature heterochromatin, gene silencing, and genomic stability. In plants, methylation occurs not only at cytosine bases in CpG but also in CpHpG and CpHpH contexts, where H denotes A, T, or C. Methyl-CpG binding domain (MBD) proteins, which recognize symmetrical methyl-CpG dinucleotides and act as gene repressors in mammalian cells, are also present in plant cells, although their structural and functional properties still remain poorly understood. To fill this gap, in this study, we determined the solution structure of the MBD domain of the MBD6 protein from Arabidopsis thaliana and investigated its binding properties to methylated DNA by binding assays and an in-depth NMR spectroscopic analysis. The AtMBD6 MBD domain folds into a canonical MBD structure in line with its binding specificity toward methyl-CpG and possesses a DNA binding interface similar to mammalian MBD domains. Intriguingly, however, the binding affinity of the AtMBD6 MBD domain toward methyl-CpG-containing DNA was found to be much lower than that of known mammalian MBD domains. The main difference arises from the absence of positively charged residues in AtMBD6 that supposedly interact with the DNA backbone as seen in mammalian MBD/methyl-CpG-containing DNA complexes. Taken together, we have established a structural basis for methyl-CpG recognition by AtMBD6 to develop a deeper understanding how MBD proteins work as mediators of epigenetic signals in plant cells.

Rigorous analysis of the interaction between proteins and low water-solubility drugs by qNMR-aided NMR titration experiments.

Drugs are designed and validated based on physicochemical data on their interactions with target proteins. For low water-solubility drugs, however, quantitative analysis is practically impossible without accurate estimation of precipitation. Here we combined quantitative NMR with NMR titration experiments to rigorously quantify the interaction of the low water-solubility drug pimecrolimus with its target protein FKBP12. Notably, the dissociation constants estimated with and without consideration of precipitation differed by more than tenfold. Moreover, the method enabled us to quantitate the FKBP12-pimecrolimus interaction even under a crowded condition established using the protein crowder BSA. Notably, the FKBP12-pimecrolimus interaction was slightly hampered under the crowded environment, which is explained by transient association of BSA with the drug molecules. Collectively, the described method will contribute to both quantifying the binding properties of low water-solubility drugs and to elucidating the drug behavior in complex crowded solutions including living cells.

Effects of Weak Nonspecific Interactions with ATP on Proteins.

Adenosine triphosphate (ATP) is an immensely well-studied metabolite serving multiple key biochemical roles as the major chemical energy currency in living systems, a building block of ribonucleic acids, and a phosphoryl group donor in kinase-mediated signaling. Intriguingly, ATP has been recently proposed to act as a hydrotrope that inhibits aggregation of amyloidogenic proteins; however, the underlying mechanism and the general physicochemical effect that coexistence with ATP exerts on proteins remain unclear. By combining NMR spectroscopy and MD simulations, here we observed weak but unambiguously measurable and concentration-dependent noncovalent interactions between ATP and various proteins. The interactions were most pronounced for an intrinsically disordered protein (α-synuclein) and for residues in flexible regions (e.g., loops or termini) of two representative folded proteins (ubiquitin and the dimeric ubiquitin-binding domain of p62). As shown by solution NMR, a consequence of the ATP-protein interaction was altered hydration of solvent-exposed residues in the protein. The observation that ATP interacted with all three proteins suggests that ATP is a general nonspecific binder of proteins. Several complementary biophysical methods further confirmed that, at physiological concentrations of ∼5-10 mM, ATP starts to form oligomeric states via magnesium-chelating and chelation-independent mechanisms, in agreement with previous studies. Although the observed ATP-protein interaction was relatively weak overall, the high ratio of ATP (monomeric free ATP, mono- and divalent ion-bound ATP, oligomeric and chelated ATP) to proteins in cells suggests that most proteins are likely to encounter transient interactions with ATP (and chemically similar metabolites) that confer metabolite-mediated protein surface protection.

Expression, solubility monitoring, and purification of the co-folded LUBAC LTM domain by structure-guided tandem folding in autoinducing cultures.

The linear ubiquitin chain assembly complex tethering motif (LUBAC-LTM) domain is composed of two different accessory LUBAC components (HOIL-1L and SHARPIN) but folds as a single globular domain. Targeted disruption of the intricate LTM-LTM interaction destabilizes LUBAC in lymphoma cells, thereby attenuating LUBAC stability, which highlights that targeting the interaction between the two LTM motifs is a promising strategy for the development of new agents against cancers that depend on LUBAC activity for their survival. To further screen for small-molecule inhibitors that can selectively disrupt the LTM-LTM interaction, it is necessary to obtain high-purity samples of the LTM domain. Ideally, such a sample would not contain any components other than the LTM itself, so that false positives (molecules binding to other parts of LUBAC) could be eliminated from the screening process. Here we report a simple strategy that enabled successful bacterial production of the isolated LUBAC LTM domain in high yield and at high purity. The strategy combines (1) structural analysis highlighting the possibility of tandem expression in the SHARPINL™ to HOIL-1LL™ direction; (2) bacterial expression downstream of EGFP to efficiently monitor expression and solubility; (3) gentle low-temperature folding using autoinduction. Formation of stably folded LTM was verified by size-exclusion chromatography and heteronuclear NMR spectroscopy. From 200-ml cultures sufficient quantities (~7 mg) of high-purity protein for structural studies could be obtained. The presented strategy will be beneficial for LUBAC LTM-based drug-screening efforts and likely serve as a useful primer for similar cases, i.e., whenever a smaller folded fragment is to be isolated from a larger protein complex for site-specific downstream applications.

Multiple-State Monitoring of SOD1 Amyloid Formation at Single-Residue Resolution by Rheo-NMR Spectroscopy.

Formation of protein aggregates or fibrils entails the conversion of soluble native protein monomers via multiple molecular states. No spectroscopic techniques have succeeded in capturing the transient molecular-scale events of fibrillation in situ. Here we report residue- and state-specific real-time monitoring of the fibrillation of amyotrophic lateral sclerosis-related SOD1 by rheology NMR (Rheo-NMR) spectroscopy. Under moderately denaturing conditions, where NMR signals of folded and unfolded monomeric SOD1 are simultaneously observable, the cross-peak intensities of folded monomeric SOD1 decreased faster than those of the unfolded species, and a 310-helix in folded SOD1 was deformed prior to global unfolding. Furthermore, real-time protein dynamics analysis identified residues involved in the core structure formation of SOD1 oligomers. Our findings provide insight into local and global unfolding events in SOD1 and fibril formation. This Rheo-NMR analysis will be applicable not only to atomic-level monitoring of other amyloidogenic proteins but also to quantification of shear-induced structural changes of non-amyloidogenic proteins and elucidation of shear-enhanced chemical phenomena such as viscosity increase and crystallization of various solution-state compounds.

Backbone resonance assignments of the A2 domain of mouse von Willebrand factor.

von Willebrand factor (vWF) is an adhesive plasma protein that is important for platelet adhesion in normal hemostasis in response to vascular injury. Although large vWF multimers are released from storage granules of platelets and (sub-)endothelial cells in response to hemostatic stimuli, for normal physiological function, vWF multimers are required to be cleaved into smaller multimeric forms. The plasma metalloproteinase ADAMTS13 specifically cleaves the peptide bond located in the middle of the A2 domain of vWF (vWF-A2), but the cleavage site is buried inside the structure of vWF and is difficult to access in the absence of elevated flow shear stress. On the other hand, in the presence of high vascular shear stress, the structure of vWF-A2 is supposed to be unfolded, thereby becoming accessible for proteolysis by ADAMTS13. However, the atomic-level mechanism underlying shear-induced structural changes of vWF-A2 remains unclear and to date no solution NMR information is available. In this study, we present the backbone 1H, 13C, and 15N resonance assignments of mouse vWF-A2; side chain assignments of 13Cß are also provided. Secondary structure propensity analysis based on the assigned chemical shifts showed that mouse vWF-A2 forms similar secondary structures in solution to the previously determined crystal structure of human vWF-A2. The obtained NMR assignment data will contribute to an atomic-level characterization of shear-induced unfolding of vWF-A2 in solution.

Molecular recognition and deubiquitination of cyclic K48-linked ubiquitin chains by OTUB1.

Conjugation of K48-linked ubiquitin chains to intracellular proteins mainly functions as a signal for proteasomal degradation. The conjugating enzyme E2-25K synthesizes not only canonical (noncyclic) but also cyclic K48-linked ubiquitin chains. Although the cyclic conformation is expected to repress molecular recognition by ubiquitin binding proteins due to restricting the flexibility of the ubiquitin subunits in a chain, multiple proteins are reported to associate with cyclic ubiquitin chains similar to noncyclic chains. However, the molecular mechanism of how cyclic ubiquitin chains are recognized remains unclear. Here we investigated the effect of cyclization on ubiquitin-chain cleavage and molecular recognition by a K48-linkage specific deubiquitinating enzyme OTUB1 for cyclic diubiquitin by NMR spectroscopic analyses. Compared to noncyclic diubiquitin, we observed slow but unambiguously detectable cleavage of cyclic diubiquitin to monoubiquitin by OTUB1. Intriguingly, upon ubiquitin chain cleavage, cyclic diubiquitin appeared to alter its "autoinhibited" conformation to an incompletely but partially accessible conformation, induced by interaction with OTUB1 via the ubiquitin-subunit specific recognition patches and adjacent surfaces. These data imply that cyclic ubiquitin chains may exist stably in cells in spite of the presence of deubiquitinating enzymes and that these chains can be recognized by intracellular proteins in a manner distinct from that of noncyclic ubiquitin chains.

Transient Diffusive Interactions with a Protein Crowder Affect Aggregation Processes of Superoxide Dismutase 1 ß-Barrel.

Aggregate formation of superoxide dismutase 1 (SOD1) inside motor neurons is known as a major factor in onset of amyotrophic lateral sclerosis. The thermodynamic stability of the SOD1 ß-barrel has been shown to decrease in crowded environments such as inside a cell, but it remains unclear how the thermodynamics of crowding-induced protein destabilization relate to SOD1 aggregation. Here we have examined the effects of a protein crowder, lysozyme, on fibril aggregate formation of the SOD1 ß-barrel. We found that aggregate formation of SOD1 is decelerated even in mildly crowded solutions. Intriguingly, transient diffusive interactions with lysozyme do not significantly affect the static structure of the SOD1 ß-barrel but stabilize an alternative excited "invisible" state. The net effect of crowding is to favor species off the aggregation pathway, thereby explaining the decelerated aggregation in the crowded environment. Our observations suggest that the intracellular environment may have a similar negative (inhibitory) effect on fibril formation of other amyloidogenic proteins in living cells. Deciphering how crowded intracellular environments affect aggregation and fibril formation of such disease-associated proteins will probably become central in understanding the exact role of aggregation in the etiology of these enigmatic diseases.

Structural Dynamic Heterogeneity of Polyubiquitin Subunits Affects Phosphorylation Susceptibility.

Polyubiquitin is a multifunctional protein tag formed by the covalent conjugation of ubiquitin molecules. Due to the high rigidity of the ubiquitin fold, the ubiquitin moieties in a polyubiquitin chain appear to be structurally equivalent to each other. It is therefore unclear how a specific ubiquitin moiety in a chain may be preferentially recognized by some proteins, such as the kinase PINK1. Here we show that there is structural dynamic heterogeneity in the two ubiquitin moieties of K48-linked diubiquitin by NMR spectroscopic analyses. Our analyses capture subunit-asymmetric structural fluctuations that are not directly related to the closed-to-open transition of the two ubiquitin moieties in diubiquitin. Strikingly, these newly identified heterogeneous structural fluctuations may be linked to an increase in susceptibility to phosphorylation by PINK1. Coupled with the fact that there are almost no differences in static tertiary structure among ubiquitin moieties in a chain, the observed subunit-specific structural fluctuations may be an important factor that distinguishes individual ubiquitin moieties in a chain, thereby aiding both efficiency and specificity in post-translational modifications.

Structural dynamics of double-stranded DNA with epigenome modification.

Modification of cytosine plays an important role in epigenetic regulation of gene expression and genome stability. Cytosine is converted to 5-methylcytosine (5mC) by DNA methyltransferase; in turn, 5mC may be oxidized to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation enzyme. The structural flexibility of DNA is known to affect the binding of proteins to methylated DNA. Here, we have carried out a semi-quantitative analysis of the dynamics of double-stranded DNA (dsDNA) containing various epigenetic modifications by combining data from imino 1H exchange and imino 1H R1ρ relaxation dispersion NMR experiments in a complementary way. Using this approach, we characterized the base-opening (kopen) and base-closing (kclose) rates, facilitating a comparison of the base-opening and -closing process of dsDNA containing cytosine in different states of epigenetic modification. A particularly striking result is the increase in the kopen rate of hemi-methylated dsDNA 5mC/C relative to unmodified or fully methylated dsDNA, indicating that the Watson-Crick base pairs undergo selective destabilization in 5mC/C. Collectively, our findings imply that the epigenetic modulation of cytosine dynamics in dsDNA mediates destabilization of the GC Watson-Crick base pair to allow base-flipping in living cells.

Quantitative monitoring of ubiquitination/deubiquitination reaction cycles by 18O-incorporation.

Ubiquitination is one of the major post-translational modifications and entails conjugation of ubiquitin molecules to target proteins. To make free ubiquitin molecules available for conjugation, in cells ubiquitin is not only synthesized de novo, but is also provided by cleaving off existing conjugated ubiquitin molecules, so-called deubiquitination reaction. Therefore, intracellular ubiquitin molecules are thought to be recycled, but the recycling frequency remains elusive. The main reason for the lack of such mechanistic details is that the original and recycled ubiquitin molecules are indistinguishable in their chemical and physical properties. To tackle this issue, here we applied 18O-labeling to trace how ubiquitin is recycled in a simultaneous ubiquitination/deubiquitination reaction (ubiquitin cycle reaction). Because deubiquitination is a hydrolysis reaction, the two 16O atoms of the C-terminal carboxy group of a ubiquitin molecule can be exchanged with 18O atoms by deubiquitination in 18O-labeled aqueous solution. By using quantitative mass spectrometry, we detected 18O atom incorporation into the C-terminal carboxy group of ubiquitin in the course of a deubiquitination reaction, in addition, we were able to quantify the 18O-incorporation in a ubiquitin cycle reaction. Unexpectedly, kinetic analysis suggested that ubiquitination reactivity was accelerated in the presence of a deubiquitinating enzyme. Collectively, we have established a quantitative method to trace ubiquitin cycle reactions by analyzing deubiquitination-associated 18O-incorporation into ubiquitin.

Tracking the 3D Rotational Dynamics in Nanoscopic Biological Systems.

The rotation of an object cannot be fully tracked without understanding a set of three angles, namely, roll, pitch, and yaw. Tracking these angles as a three-degrees-of-freedom (3-DoF) rotation is a fundamental measurement, facilitating, for example, attitude control of a ship, image stabilization to reduce camera shake, and self-driving cars. Until now, however, there has been no method to track 3-DoF rotation to measure nanometer-scale dynamics in biomolecules and live cells. Here we show that 3-DoF rotation of biomolecules can be visualized via nitrogen-vacancy centers in a fluorescent nanodiamond using a tomographic vector magnetometry technique. We demonstrate application of the method to three different types of biological systems. First, we tracked the rotation of a single molecule of the motor protein F1-ATPase by attaching a nanodiamond to the γ-subunit. We visualized the 3-step rotation of the motor in 3D space and, moreover, a delay of ATP binding or ADP release step in the catalytic reaction. Second, we attached a nanodiamond to a membrane protein in live cells to report on cellular membrane dynamics, showing that 3D rotational motion of the membrane protein correlates with intracellular cytoskeletal density. Last, we used the method to track nonrandom motions in the intestine of Caenorhabditis elegans. Collectively, our findings show that the method can record nanoscale 3-DoF rotation in vitro, in cells, and even in vivo. 3-DoF rotation tracking introduces a new perspective on microscopic biological samples, revealing in greater detail the functional mechanisms due to nanoscale dynamics in molecules and cells.

Pinpoint analysis of a protein in slow exchange using F1F2-selective ZZ-exchange spectroscopy: assignment and kinetic analysis.

ZZ-exchange spectroscopy is widely used to study slow exchange processes in biomolecules, especially determination of exchange rates and assignment of minor peaks. However, if the exchange cross peaks overlap or the populations are skewed, kinetic analysis is hindered. In order to analyze slow exchange protein dynamics under such conditions, here we have developed a new method by combining ZZ-exchange and F1F2-selective NMR spectroscopy. We demonstrate the utility of this method by examining the monomer-dimer transition of the ubiquitin-associated domain of p62, successfully assigning the minor (monomeric) peaks and obtaining the exchange rates, which cannot be achieved by ZZ-exchange alone.

Visualizing protein motion in Couette flow by all-atom molecular dynamics.

In living cells, biomacromolecules are exposed to a highly crowded environment. The cytoplasm, the nucleus, and other organelles are highly viscous fluids that differ from dilute in vitro conditions. Viscosity, a measure of fluid internal friction, directly affects the forces that act on immersed macromolecules. Although active motion of this viscous fluid - cytoplasmic streaming - occurs in many plant and animal cells, the effect of fluid motion (flow) on biomolecules is rarely discussed. Recently NMR experiments that apply a shearing flow in situ have been used for protein studies. While these NMR experiments have succeeded in spectroscopically tracking protein aggregation in real time, they do not provide a visual picture of protein motion under shear. To fill this gap, here we have used molecular dynamics simulations to study the motion of three proteins of different size and shape in a simple shearing flow. The proteins exhibit a superposition of random diffusion and shear-flow-induced rotational motion. Random rotational diffusion dominates at lower shear stresses, whereas an active "rolling motion" along the axis of the applied flow occurs at higher shear stress. Even larger shear stresses perturb protein secondary structure elements resulting in local and global unfolding. Apart from shear-induced unfolding, our results imply that, in an ideal Couette flow field biomolecules undergo correlated motion, which should enhance the probability of inter-molecular interaction and aggregation. Connecting biomolecular simulation with experiments applying shear flow in situ appears to be a promising strategy to study protein alignment, deformation, and dynamics under shear.