Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional 1H-detected solid-state NMR.

NMR chemical shifts provide detailed information on the chemical properties of molecules, thereby complementing structural data from techniques like X-ray crystallography and electron microscopy. Detailed analysis of protein NMR data, however, often hinges on comprehensive, site-specific assignment of backbone resonances, which becomes a bottleneck for molecular weights beyond 40 to 45 kDa. Here, we show that assignments for the (2x)72-kDa protein tryptophan synthase (665 amino acids per asymmetric unit) can be achieved via higher-dimensional, proton-detected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atom-specific characterization of chemical properties and relaxation for the backbone and side chains, including those residues important for the catalytic turnover. Combined with first-principles calculations, the chemical shifts in the ß-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the ß-subunit from solution.

NMRtist: an online platform for automated biomolecular NMR spectra analysis.

SUMMARY: We present NMRtist, an online platform that combines deep learning, large-scale optimization and cloud computing to automate protein NMR spectra analysis. Our website provides virtual storage for NMR spectra deposition together with a set of applications designed for automated peak picking, chemical shift assignment and protein structure determination. The system can be used by non-experts and allows protein assignments and structures to be determined within hours after the measurements, strictly without any human intervention. AVAILABILITY AND IMPLEMENTATION: NMRtist is freely available to non-commercial users at https://nmrtist.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The CUE Domain of Cue1 Aligns Growing Ubiquitin Chains with Ubc7 for Rapid Elongation.

Ubiquitin conjugation is an essential process modulating protein function in eukaryotic cells. Surprisingly, little is known about how the progressive assembly of ubiquitin chains is managed by the responsible enzymes. Only recently has ubiquitin binding activity emerged as an important factor in chain formation. The Ubc7 activator Cue1 carries a ubiquitin binding CUE domain that substantially stimulates K48-linked polyubiquitination mediated by Ubc7. Our results from NMR-based analysis and in vitro ubiquitination reactions point out that two parameters accelerate ubiquitin chain assembly: the increasing number of CUE binding sites and the position of CUE binding within a growing chain. In particular, interactions with a ubiquitin moiety adjacent to the acceptor ubiquitin facilitate chain elongation. These data indicate a mechanism for ubiquitin binding in which Cue1 positions Ubc7 and the distal acceptor ubiquitin for rapid polyubiquitination. Disrupting this mechanism results in dysfunction of the ERAD pathway by a delayed turnover of substrates.

Conformations of Macrocyclic Peptides Sampled by Nuclear Magnetic Resonance: Models for Cell-Permeability.

The biological activities and pharmacological properties of peptides and peptide mimetics are determined by their conformational states. Therefore, a detailed understanding of the conformational landscape is crucial for rational drug design. Nuclear magnetic resonance (NMR) is the only method for structure determination in solution. However, it remains challenging to determine the structures of peptides using NMR because of very weak nuclear Overhauser effects (NOEs), the semiquantitative nature of the rotating frame Overhauser effect (ROE), and the low number of NOEs/ROEs in N-methylated peptides. In this study, we introduce a new approach to investigating the structures of modified macrocyclic peptides. We utilize exact NOEs (eNOEs) in viscous solvent mixtures to replicate various cellular environments. eNOEs provide detailed structural information for highly dynamic modified peptides. Structures of high precision were obtained for cyclosporin A, with a backbone atom rmsd of 0.10 Å. Distinct conformational states in different environments were identified for omphalotin A (OmphA), a fungal nematotoxic and multiple backbone N-methylated macrocyclic peptides. A model for cell-permeation is presented for OmphA, based on its structures in polar, apolar, and mixed polarity solvents. During the transition from a polar to an apolar environment, OmphA undergoes a rearrangement of its H-bonding network, accompanied by a cis to trans isomerization of the ω torsion angle within a type VIa ß-turn. We hypothesize that the kinetics of these conformational transitions play a crucial role in determining the membrane-permeation capabilities of OmphA.

An Analysis of Nucleotide-Amyloid Interactions Reveals Selective Binding to Codon-Sized RNA.

Interactions between RNA and proteins are the cornerstone of many important biological processes from transcription and translation to gene regulation, yet little is known about the ancient origin of said interactions. We hypothesized that peptide amyloids played a role in the origin of life and that their repetitive structure lends itself to building interfaces with other polymers through avidity. Here, we report that short RNA with a minimum length of three nucleotides binds in a sequence-dependent manner to peptide amyloids. The 3'-5' linked RNA backbone appears to be well-suited to support these interactions, with the phosphodiester backbone and nucleobases both contributing to the affinity. Sequence-specific RNA-peptide interactions of the kind identified here may provide a path to understanding one of the great mysteries rooted in the origin of life: the origin of the genetic code.

Efficient determination of the accessible conformation space of multi-domain complexes based on EPR PELDOR data.

Many proteins can adopt multiple conformations which are important for their function. This is also true for proteins and domains that are covalently linked to each other. One important example is ubiquitin, which can form chains of different conformations depending on which of its lysine side chains is used to form an isopeptide bond with the C-terminus of another ubiquitin molecule. Similarly, ubiquitin gets covalently attached to active-site residues of E2 ubiquitin-conjugating enzymes. Due to weak interactions between ubiquitin and its interaction partners, these covalent complexes adopt multiple conformations. Understanding the function of these complexes requires the characterization of the entire accessible conformation space and its modulation by interaction partners. Long-range (1.8-10 nm) distance restraints obtained by EPR spectroscopy in the form of probability distributions are ideally suited for this task as not only the mean distance but also information about the conformation dynamics is encoded in the experimental data. Here we describe a computational method that we have developed based on well-established structure determination software using NMR restraints to calculate the accessible conformation space using PELDOR/DEER data.

Fibrils Emerging from Droplets: Molecular Guiding Principles behind Phase Transitions of a Short Peptide-Based Condensate Studied by Solid-State NMR.

Biochemical reactions occurring in highly crowded cellular environments require different means of control to ensure productivity and specificity. Compartmentalization of reagents by liquid-liquid phase separation is one of these means. However, extremely high local protein concentrations of up to 400 mg/ml can result in pathological aggregation into fibrillar amyloid structures, a phenomenon that has been linked to various neurodegenerative diseases. Despite its relevance, the process of liquid-to-solid transition inside condensates is still not well understood at the molecular level. We thus herein use small peptide derivatives that can undergo both liquid-liquid and subsequent liquid-to-solid phase transition as model systems to study both processes. Using solid-state nuclear magnetic resonance (NMR) and transmission electron microscopy (TEM), we compare the structure of condensed states of leucine, tryptophan and phenylalanine containing derivatives, distinguishing between liquid-like condensates, amorphous aggregates and fibrils, respectively. A structural model for the fibrils formed by the phenylalanine derivative was obtained by an NMR-based structure calculation. The fibrils are stabilised by hydrogen bonds and side-chain π-π interactions, which are likely much less pronounced or absent in the liquid and amorphous state. Such noncovalent interactions are equally important for the liquid-to-solid transition of proteins, particularly those related to neurodegenerative diseases.

Optimization and validation of multi-state NMR protein structures using structural correlations.

Recent advances in the field of protein structure determination using liquid-state NMR enable the elucidation of multi-state protein conformations that can provide insight into correlated and non-correlated protein dynamics at atomic resolution. So far, NMR-derived multi-state structures were typically evaluated by means of visual inspection of structure superpositions, target function values that quantify the violation of experimented restraints and root-mean-square deviations that quantify similarity between conformers. As an alternative or complementary approach, we present here the use of a recently introduced structural correlation measure, PDBcor, that quantifies the clustering of protein states as an additional measure for multi-state protein structure analysis. It can be used for various assays including the validation of experimental distance restraints, optimization of the number of protein states, estimation of protein state populations, identification of key distance restraints, NOE network analysis and semiquantitative analysis of the protein correlation network. We present applications for the final quality analysis stages of typical multi-state protein structure calculations.

An automated iterative approach for protein structure refinement using pseudocontact shifts.

NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YM4A, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing.

Paramagnetic Solid-State NMR to Localize the Metal-Ion Cofactor in an Oligomeric DnaB Helicase.

Paramagnetic metal ions can be inserted into ATP-fueled motor proteins by exchanging the diamagnetic Mg2+ cofactor with Mn2+ or Co2+ . Then, paramagnetic relaxation enhancement (PRE) or pseudo-contact shifts (PCSs) can be measured to report on the localization of the metal ion within the protein. We determine the metal position in the oligomeric bacterial DnaB helicase from Helicobacter pylori complexed with the transition-state ATP-analogue ADP:AlF4 - and single-stranded DNA using solid-state NMR and a structure-calculation protocol employing CYANA. We discuss and compare the use of Mn2+ and Co2+ in localizing the ATP cofactor in large oligomeric protein assemblies. 31 P PCSs induced in the Co2+ -containing sample are then used to localize the DNA phosphate groups on the Co2+ PCS tensor surface enabling structural insights into DNA binding to the DnaB helicase.

Protein aggregation of the p63 transcription factor underlies severe skin fragility in AEC syndrome.

The p63 gene encodes a master regulator of epidermal commitment, development, and differentiation. Heterozygous mutations in the C-terminal domain of the p63 gene can cause ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, a life-threatening disorder characterized by skin fragility and severe, long-lasting skin erosions. Despite deep knowledge of p63 functions, little is known about mechanisms underlying disease pathology and possible treatments. Here, we show that multiple AEC-associated p63 mutations, but not those causative of other diseases, lead to thermodynamic protein destabilization, misfolding, and aggregation, similar to the known p53 gain-of-function mutants found in cancer. AEC mutant proteins exhibit impaired DNA binding and transcriptional activity, leading to dominant negative effects due to coaggregation with wild-type p63 and p73. Importantly, p63 aggregation occurs also in a conditional knock-in mouse model for the disorder, in which the misfolded p63 mutant protein leads to severe epidermal defects. Variants of p63 that abolish aggregation of the mutant proteins are able to rescue p63's transcriptional function in reporter assays as well as in a human fibroblast-to-keratinocyte conversion assay. Our studies reveal that AEC syndrome is a protein aggregation disorder and opens avenues for therapeutic intervention.

Evaluation of Multi-Objective Optimization Algorithms for NMR Chemical Shift Assignment.

An automated NMR chemical shift assignment algorithm was developed using multi-objective optimization techniques. The problem is modeled as a combinatorial optimization problem and its objective parameters are defined separately in different score functions. Some of the heuristic approaches of evolutionary optimization are employed in this problem model. Both, a conventional genetic algorithm and multi-objective methods, i.e., the non-dominated sorting genetic algorithms II and III (NSGA2 and NSGA3), are applied to the problem. The multi-objective approaches consider each objective parameter separately, whereas the genetic algorithm followed a conventional way, where all objectives are combined in one score function. Several improvement steps and repetitions on these algorithms are performed and their combinations are also created as a hyper-heuristic approach to the problem. Additionally, a hill-climbing algorithm is also applied after the evolutionary algorithm steps. The algorithms are tested on several different datasets with a set of 11 commonly used spectra. The test results showed that our algorithm could assign both sidechain and backbone atoms fully automatically without any manual interactions. Our approaches could provide around a 65% success rate and could assign some of the atoms that could not be assigned by other methods.

On the Entropy of a One-Dimensional Gas with and without Mixing Using Sinai Billiard.

A one-dimensional gas comprising N point particles undergoing elastic collisions within a finite space described by a Sinai billiard generating identical dynamical trajectories are calculated and analyzed with regard to strict extensivity of the entropy definitions of Boltzmann-Gibbs. Due to the collisions, trajectories of gas particles are strongly correlated and exhibit both chaotic and periodic properties. Probability distributions for the position of each particle in the one-dimensional gas can be obtained analytically, elucidating that the entropy in this special case is extensive at any given number N. Furthermore, the entropy obtained can be interpreted as a measure of the extent of interactions between molecules. The results obtained for the non-mixable one-dimensional system are generalized to mixable one- and two-dimensional systems, the latter by a simple example only providing similar findings.

Automated Backbone NMR Resonance Assignment of Large Proteins Using Redundant Linking from a Single Simultaneous Acquisition.

Thanks to magic-angle spinning (MAS) probes with frequencies of 60-100 kHz, the benefit of high-sensitivity 1H detection can now be broadly realized in biomolecular solid-state NMR for the analysis of microcrystalline, sedimented, or lipid-embedded preparations. Nonetheless, performing the assignment of all resonances remains a rate-limiting step in protein structural studies, and even the latest optimized protocols fail to perform this step when the protein size exceeds ∼20 kDa. Here, we leverage the benefits of fast (100 kHz) MAS and high (800 MHz) magnetic fields to design an approach that lifts this limitation. Through the creation, conservation, and acquisition of independent magnetization pathways within a single triple-resonance MAS NMR experiment, a single self-consistent data set can be acquired, providing enhanced sensitivity, reduced vulnerability to machine or sample instabilities, and highly redundant linking that supports fully automated peak picking and resonance assignment. The method, dubbed RAVASSA (redundant assignment via a single simultaneous acquisition), is demonstrated with the assignment of the largest protein to date in the solid state, the 42.5 kDa maltose binding protein, using a single fully protonated microcrystalline sample and 1 week of spectrometer time.

Backbone and methyl assignment of bacteriorhodopsin incorporated into nanodiscs.

Resonance assignments are challenging for membrane proteins due to the size of the lipid/detergent-protein complex and the presence of line-broadening from conformational exchange. As a consequence, many correlations are missing in the triple-resonance NMR experiments typically used for assignments. Herein, we present an approach in which correlations from these solution-state NMR experiments are supplemented by data from 13C unlabeling, single-amino acid type labeling, 4D NOESY data and proximity of moieties to lipids or water in combination with a structure of the protein. These additional data are used to edit the expected peaklists for the automated assignment protocol FLYA, a module of the program package CYANA. We demonstrate application of the protocol to the 262-residue proton pump from archaeal bacteriorhodopsin (bR) in lipid nanodiscs. The lipid-protein assembly is characterized by an overall correlation time of 44 ns. The protocol yielded assignments for 62% of all backbone (H, N, Cα, Cß, C') resonances of bR, corresponding to 74% of all observed backbone spin systems, and 60% of the Ala, Met, Ile (δ1), Leu and Val methyl groups, thus enabling to assign a large fraction of the protein without mutagenesis data. Most missing resonances stem from the extracellular half, likely due intermediate exchange line-broadening. Further analysis revealed that missing information of the amino acid type of the preceding residue is the largest problem, and that 4D NOESY experiments are particularly helpful to compensate for that information loss.

Dynamics of Bacteriorhodopsin in the Dark-Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy.

To achieve efficient proton pumping in the light-driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark-adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side-chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground-state bR.

Protein Allostery at Atomic Resolution.

Protein allostery is a phenomenon involving the long range coupling between two distal sites in a protein. In order to elucidate allostery at atomic resoluion on the ligand-binding WW domain of the enzyme Pin1, multistate structures were calculated from exact nuclear Overhauser effect (eNOE). In its free form, the protein undergoes a microsecond exchange between two states, one of which is predisposed to interact with its parent catalytic domain. In presence of the positive allosteric ligand, the equilibrium between the two states is shifted towards domain-domain interaction, suggesting a population shift model. In contrast, the allostery-suppressing ligand decouples the side-chain arrangement at the inter-domain interface thereby reducing the inter-domain interaction. As such, this mechanism is an example of dynamic allostery. The presented distinct modes of action highlight the power of the interplay between dynamics and function in the biological activity of proteins.

The Solution Structure and Dynamics of Cd-Metallothionein from Helix pomatia Reveal Optimization for Binding Cd over Zn.

Metallothioneins (MTs) are cysteine-rich polypeptides that are naturally found coordinated to monovalent and/or divalent transition metal ions. Three metallothionein isoforms from the Roman snail Helix pomatia are known. They differ in their physiological metal load and in their specificity for transition metal ions such as Cd2+ (HpCdMT isoform) and Cu+ (HpCuMT isoform) or in the absence of a defined metal specificity (HpCd/CuMT isoform). We have determined the solution structure of the Cd-specific isoform (HpCdMT) by nuclear magnetic resonance spectroscopy using recombinant isotopically labeled protein loaded with Zn2+ or Cd2+. Both structures display two-domain architectures, where each domain comprises a characteristic three-metal cluster similar to that observed in the ß-domains of vertebrate MTs. The polypeptide backbone is well-structured over the entire sequence, including the interdomain linker. Interestingly, the two domains display mutual contacts, as observed before for the metallothionein of the snail Littorina littorea, to which both N- and C-terminal domains are highly similar. Increasing the length of the linker motionally decouples both domains and removes mutual contacts between them without having a strong effect on the stability of the individual domains. The structures of Cd6- and Zn6-HpCdMT are nearly identical. However, 15N relaxation, in particular 15N R2 rates, is accelerated for many residues of Zn6-HpCdMT but not for Cd6-HpCdMT, revealing the presence of conformational exchange effects. We suggest that this snail MT isoform is evolutionarily optimized for binding Cd rather than Zn.

Noise peak filtering in multi-dimensional NMR spectra using convolutional neural networks.

Motivation: Multi-dimensional NMR spectra are generally used for NMR signal assignment and structure analysis. There are several programs that can achieve highly automated NMR signal assignments and structure analysis. On the other hand, NMR spectra tend to have a large number of noise peaks even for data acquired with good sample and machine conditions, and it is still difficult to eliminate these noise peaks. Results: We have developed a method to eliminate noise peaks using convolutional neural networks, implemented in the program package Filt_Robot. The filtering accuracy of Filt_Robot was around 90-95% when applied to 2D and 3D NMR spectra, and the numbers of resulting non-noise peaks were close to those in corresponding manually prepared peaks lists. The filtering can strongly enhance automated NMR spectra analysis. Availability and implementation: The full package of the program, documents and example data are available from http://bmrbdep.pdbj.org/en/nmr_tool_box/Filt_Robot.html. Supplementary information: Supplementary data are available at Bioinformatics online.

Proteome-wide analysis of phospho-regulated PDZ domain interactions.

A key function of reversible protein phosphorylation is to regulate protein-protein interactions, many of which involve short linear motifs (3-12 amino acids). Motif-based interactions are difficult to capture because of their often low-to-moderate affinities. Here, we describe phosphomimetic proteomic peptide-phage display, a powerful method for simultaneously finding motif-based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C-terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD-95/Dlg/ZO-1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR We uncover site-specific phospho-regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho-regulation of motif-based interactions on a large scale.