Order/Disorder Transitions Upon Protein Binding: A Unifying Perspective.

When two proteins bind to each other, this process is often accompanied by a change in their structural states (from disordered to ordered or vice versa). As it turns out, there are 10 distinct possibilities for such binding-related order/disorder transitions. Out of this number, seven scenarios have been experimentally observed, while another three remain hitherto unreported. As an example, we discuss the so-called mutual synergistic folding, whereby two disordered proteins come together to form a fully structured complex. Our bioinformatics analysis of the Protein Databank found potential new examples of this remarkable binding mechanism.

Using NMR diffusion data to validate MD models of disordered proteins: Test case of N-terminal tail of histone H4.

MD simulations can provide uniquely detailed models of intrinsically disordered proteins (IDPs). However, these models need careful experimental validation. The coefficient of translational diffusion Dtr, measurable by pulsed field gradient NMR, offers a potentially useful piece of experimental information related to the compactness of the IDP's conformational ensemble. Here, we investigate, both experimentally and via the MD modeling, the translational diffusion of a 25-residue N-terminal fragment from histone H4 (N-H4). We found that the predicted values of Dtr, as obtained from mean-square displacement of the peptide in the MD simulations, are largely determined by the viscosity of the MD water (which has been reinvestigated as a part of our study). Beyond that, our analysis of the diffusion data indicates that MD simulations of N-H4 in the TIP4P-Ew water give rise to an overly compact conformational ensemble for this peptide. In contrast, TIP4P-D and OPC simulations produce the ensembles that are consistent with the experimental Dtr result. These observations are supported by the analyses of the 15N spin relaxation rates. We also tested a number of empirical methods to predict Dtr based on IDP's coordinates extracted from the MD snapshots. In particular, we show that the popular approach involving the program HYDROPRO can produce misleading results. This happens because HYDROPRO is not intended to predict the diffusion properties of highly flexible biopolymers such as IDPs. Likewise, recent empirical schemes that exploit the relationship between the small-angle x-ray scattering-informed conformational ensembles of IDPs and the respective experimental Dtr values also prove to be problematic. In this sense, the first-principle calculations of Dtr from the MD simulations, such as demonstrated in this work, should provide a useful benchmark for future efforts in this area.

X-ray Crystallography Module in MD Simulation Program Amber 2023. Refining the Models of Protein Crystals.

The MD simulation package Amber offers an attractive platform to refine crystallographic structures of proteins: (i) state-of-the-art force fields help to regularize protein coordinates and reconstruct the poorly diffracting elements of the structure, such as flexible loops; (ii) MD simulations restrained by the experimental diffraction data provide an effective strategy to optimize structural models of protein crystals, including explicitly modeled interstitial solvent as well as crystal contacts. Here, we present the new crystallography module xray, released as a part of the Amber 2023 package. This module contains functions to calculate and scale structure factors (including the contributions from bulk solvent), evaluate the maximum-likelihood-type crystallographic potential, and compute its derivative forces. The X-ray functionality of Amber no longer relies on external dependencies so that the full advantage of GPU acceleration can be taken. This makes it possible to refine in a short time hundreds of crystal models, including supercell models comprised of multiple unit cells. The new automated Amber-based refinement procedure leads to an appreciable improvement in Rfree (in some cases, by as much as 0.067) as well as MolProbity scores.

Conformational and Interaction Landscape of Histone H4 Tails in Nucleosomes Probed by Paramagnetic NMR Spectroscopy.

The fundamental repeat unit of chromatin, the nucleosome, consists of approximately 147 base pairs of double-stranded DNA and a histone protein octamer containing two copies each of histones H2A, H2B, H3, and H4. Each histone possesses a dynamically disordered N-terminal tail domain, and it is well-established that the tails of histones H3 and H4 play key roles in chromatin compaction and regulation. Here we investigate the conformational ensemble and interactions of the H4 tail in nucleosomes by means of solution NMR measurements of paramagnetic relaxation enhancements (PREs) in recombinant samples reconstituted with 15N-enriched H4 and nitroxide spin-label tagged H3. The experimental PREs, which report on the proximities of individual H4 tail residues to the different H3 spin-label sites, are interpreted by using microsecond time-scale molecular dynamics simulations of the nucleosome core particle. Collectively, these data enable improved localization of histone H4 tails in nucleosomes and support the notion that H4 tails engage in a fuzzy complex interaction with nucleosomal DNA.

Simulating diffraction photographs based on molecular dynamics trajectories of a protein crystal: a new option to examine structure-solving strategies in protein crystallography.

A molecular dynamics (MD)-based pipeline has been designed and implemented to emulate the entire process of collecting diffraction photographs and calculating crystallographic structures of proteins from them. Using a structure of lysozyme solved in-house, a supercell comprising 125 (5 × 5 × 5) crystal unit cells containing a total of 1000 protein molecules and explicit interstitial solvent was constructed. For this system, two 300 ns MD trajectories at 298 and 250 K were recorded. A series of snapshots from these trajectories were then used to simulate a fully realistic set of diffraction photographs, which were further fed into the standard pipeline for structure determination. The resulting structures show very good agreement with the underlying MD model not only in terms of coordinates but also in terms of B factors; they are also consistent with the original experimental structure. The developed methodology should find a range of applications, such as optimizing refinement protocols to solve crystal structures and extracting dynamics information from diffraction data or diffuse scattering.

Aromatic ring flips in differently packed ubiquitin protein crystals from MAS NMR and MD.

Probing the dynamics of aromatic side chains provides important insights into the behavior of a protein because flips of aromatic rings in a protein's hydrophobic core report on breathing motion involving a large part of the protein. Inherently invisible to crystallography, aromatic motions have been primarily studied by solution NMR. The question how packing of proteins in crystals affects ring flips has, thus, remained largely unexplored. Here we apply magic-angle spinning NMR, advanced phenylalanine 1H-13C/2H isotope labeling and MD simulation to a protein in three different crystal packing environments to shed light onto possible impact of packing on ring flips. The flips of the two Phe residues in ubiquitin, both surface exposed, appear remarkably conserved in the different crystal forms, even though the intermolecular packing is quite different: Phe4 flips on a ca. 10-20 ns time scale, and Phe45 are broadened in all crystals, presumably due to µs motion. Our findings suggest that intramolecular influences are more important for ring flips than intermolecular (packing) effects.

Effect of rotation in NMR diffusion experiments on micron-sized particles: A generalized theoretical treatment.

We have recently developed an analytical framework to interpret the results of pulsed field gradient (PFG) NMR experiments on solution samples of micron-sized amyloid fibrils [Angew. Chem. Int. Ed. 60 (2021) 15445-15451. https://doi.org/10.1002/anie.202102408]. Here we generalize this result by reporting a rigorous theoretical model of such experiments, implemented in a form of efficient computational scheme. In particular, the new treatment fully accounts for the anisotropy of fibrils' translational diffusion and takes into consideration the finite length of the gradient pulses. The results hold not only for the historic spin-echo sequence, but also for the widely used stimulated echo experiment. We have found that fibrils' rotation can attenuate the echo by a factor comparable with that of translation. However, contrary to some recent claims, the rotational mechanism cannot lead to an apparent fast-diffusion situation.

Modeling a unit cell: crystallographic refinement procedure using the biomolecular MD simulation platform Amber.

A procedure has been developed for the refinement of crystallographic protein structures based on the biomolecular simulation program Amber. The procedure constructs a model representing a crystal unit cell, which generally contains multiple protein molecules and is fully hydrated with TIP3P water. Periodic boundary conditions are applied to the cell in order to emulate the crystal lattice. The refinement is conducted in the form of a specially designed short molecular-dynamics run controlled by the Amber ff14SB force field and the maximum-likelihood potential that encodes the structure-factor-based restraints. The new Amber-based refinement procedure has been tested on a set of 84 protein structures. In most cases, the new procedure led to appreciably lower R free values compared with those reported in the original PDB depositions or obtained by means of the industry-standard phenix.refine program. In particular, the new method has the edge in refining low-accuracy scrambled models. It has also been successful in refining a number of molecular-replacement models, including one with an r.m.s.d. of 2.15 Å. In addition, Amber-refined structures consistently show superior MolProbity scores. The new approach offers a highly realistic representation of protein-protein interactions in the crystal, as well as of protein-water interactions. It also offers a realistic representation of protein crystal dynamics (akin to ensemble-refinement schemes). Importantly, the method fully utilizes the information from the available diffraction data, while relying on state-of-the-art molecular-dynamics modeling to assist with those elements of the structure that do not diffract well (for example mobile loops or side chains). Finally, it should be noted that the protocol employs no tunable parameters, and the calculations can be conducted in a matter of several hours on desktop computers equipped with graphical processing units or using a designated web service.

The Role of Rotational Motion in Diffusion NMR Experiments on Supramolecular Assemblies: Application to Sup35NM Fibrils.

Pulsed-field gradient (PFG) NMR is an important tool for characterization of biomolecules and supramolecular assemblies. However, for micrometer-sized objects, such as amyloid fibrils, these experiments become difficult to interpret because in addition to translational diffusion they are also sensitive to rotational diffusion. We have constructed a mathematical theory describing the outcome of PFG NMR experiments on rod-like fibrils. To test its validity, we have studied the fibrils formed by Sup35NM segment of the prion protein Sup35. The interpretation of the PFG NMR data in this system is fully consistent with the evidence from electron microscopy. Contrary to some previously expressed views, the signals originating from disordered regions in the fibrils can be readily differentiated from the similar signals representing small soluble species (e.g. proteolytic fragments). This paves the way for diffusion-sorted NMR experiments on complex amyloidogenic samples.

Histone H4 Tails in Nucleosomes: a Fuzzy Interaction with DNA.

The interaction of positively charged N-terminal histone tails with nucleosomal DNA plays an important role in chromatin assembly and regulation, modulating their susceptibility to post-translational modifications and recognition by chromatin-binding proteins. Here, we report residue-specific 15 N NMR relaxation rates for histone H4 tails in reconstituted nucleosomes. These data indicate that H4 tails are strongly dynamically disordered, albeit with reduced conformational flexibility compared to a free peptide with the same sequence. Remarkably, the NMR observables were successfully reproduced in a 2-µs MD trajectory of the nucleosome. This is an important step toward resolving an apparent inconsistency where prior simulations were generally at odds with experimental evidence on conformational dynamics of histone tails. Our findings indicate that histone H4 tails engage in a fuzzy interaction with nucleosomal DNA, underpinned by a variable pattern of short-lived salt bridges and hydrogen bonds, which persists at low ionic strength (0-100 mM NaCl).

Insights into Ubiquitin Product Release in Hydrolysis Catalyzed by the Bacterial Deubiquitinase SdeA.

We report the co-crystal structure of the (catalytic Cys)-to-Ala mutant of the deubiquitinase domain of the Legionella pneumophila effector SdeA (SdeADUB) with its ubiquitin (Ub) product. Most of the intermolecular interactions are preserved in this product-bound structure compared to that of the previously characterized complex of SdeADUB with the suicide inhibitor ubiquitin vinylmethyl ester (Ub-VME), whose structure models the acyl-enzyme thioester intermediate. Nuclear magnetic resonance (NMR) titration studies show a chemical shift perturbation pattern that suggests that the same interactions also exist in solution. Isothermal titration calorimetry and NMR titration data reveal that the affinity of wild-type (WT) SdeADUB for Ub is significantly lower than that of the Cys-to-Ala mutant. This is potentially due to repulsive interaction between the thiolate ion of the catalytic Cys residue in WT SdeADUB and the carboxylate group of the C-terminal Gly76 residue in Ub. In the context of SdeADUB catalysis, this electrostatic repulsion arises after the hydrolysis of the scissile isopeptide bond in the acyl-enzyme intermediate and the consequent formation of the C-terminal carboxylic group in the Ub fragment. We hypothesize that this electrostatic repulsion may expedite the release of the Ub product by SdeADUB. We note that similar repulsive interactions may also occur in other deubiquitinases and hydrolases of ubiquitin-like protein modifiers and may constitute a fairly general mechanism of product release within this family. This is a potentially important feature for a family of enzymes that form extensive protein-protein interactions during enzyme-substrate engagement.

Structural and dynamic origins of ESR lineshapes in spin-labeled GB1 domain: the insights from spin dynamics simulations based on long MD trajectories.

Site-directed spin labeling (SDSL) ESR is a valuable tool to probe protein systems that are not amenable to characterization by x-ray crystallography, NMR or EM. While general principles that govern the shape of SDSL ESR spectra are known, its precise relationship with protein structure and dynamics is still not fully understood. To address this problem, we designed seven variants of GB1 domain bearing R1 spin label and recorded the corresponding MD trajectories (combined length 180 µs). The MD data were subsequently used to calculate time evolution of the relevant spin density matrix and thus predict the ESR spectra. The simulated spectra proved to be in good agreement with the experiment. Further analysis confirmed that the spectral shape primarily reflects the degree of steric confinement of the R1 tag and, for the well-folded protein such as GB1, offers little information on local backbone dynamics. The rotameric preferences of R1 side chain are determined by the type of the secondary structure at the attachment site. The rotameric jumps involving dihedral angles χ1 and χ2 are sufficiently fast to directly influence the ESR lineshapes. However, the jumps involving multiple dihedral angles tend to occur in (anti)correlated manner, causing smaller-than-expected movements of the R1 proxyl ring. Of interest, ESR spectra of GB1 domain with solvent-exposed spin label can be accurately reproduced by means of Redfield theory. In particular, the asymmetric character of the spectra is attributable to Redfield-type cross-correlations. We envisage that the current MD-based, experimentally validated approach should lead to a more definitive, accurate picture of SDSL ESR experiments.

Molecular Dynamics model of peptide-protein conjugation: case study of covalent complex between Sos1 peptide and N-terminal SH3 domain from Grb2.

We have investigated covalent conjugation of VPPPVPPRRRX' peptide (where X' denotes Nε-chloroacetyl lysine) to N-terminal SH3 domain from adapter protein Grb2. Our experimental results confirmed that the peptide first binds to the SH3 domain noncovalently before establishing a covalent linkage through reaction of X' with the target cysteine residue C32. We have also confirmed that this reaction involves a thiolate-anion form of C32 and follows the SN2 mechanism. For this system, we have developed a new MD-based protocol to model the formation of covalent conjugate. The simulation starts with the known coordinates of the noncovalent complex. When two reactive groups come into contact during the course of the simulation, the reaction is initiated. The reaction is modeled via gradual interpolation between the two sets of force field parameters that are representative of the noncovalent and covalent complexes. The simulation proceeds smoothly, with no appreciable perturbations to temperature, pressure or volume, and results in a high-quality MD model of the covalent complex. The validity of this model is confirmed using the experimental chemical shift data. The new MD-based approach offers a valuable tool to explore the mechanics of protein-peptide conjugation and build accurate models of covalent complexes.

What Drives 15N Spin Relaxation in Disordered Proteins? Combined NMR/MD Study of the H4 Histone Tail.

Backbone (15N) NMR relaxation is one of the main sources of information on dynamics of disordered proteins. Yet, we do not know very well what drives 15N relaxation in such systems, i.e., how different forms of motion contribute to the measurable relaxation rates. To address this problem, we have investigated, both experimentally and via molecular dynamics simulations, the dynamics of a 26-residue peptide imitating the N-terminal portion of the histone protein H4. One part of the peptide was found to be fully flexible, whereas the other part features some transient structure (a hairpin stabilized by hydrogen bonds). The following motional modes proved relevant for 15N relaxation. 1) Sub-picosecond librations attenuate relaxation rates according to S2 ∼0.85-0.90. 2) Axial peptide-plane fluctuations along a stretch of the peptide chain contribute to relaxation-active dynamics on a fast timescale (from tens to hundreds of picoseconds). 3) φ/ψ backbone jumps contribute to relaxation-active dynamics on both fast (from tens to hundreds of picoseconds) and slow (from hundreds of picoseconds to a nanosecond) timescales. The major contribution is from polyproline II (PPII) ↔ ß transitions in the Ramachandran space; in the case of glycine residues, the major contribution is from PPII ↔ (ß) ↔ rPPII transitions, in which rPPII is the mirror-image (right-handed) version of the PPII geometry, whereas ß geometry plays the role of an intermediate state. 4) Reorientational motion of certain (sufficiently long-lived) elements of transient structure, i.e., rotational tumbling, contributes to slow relaxation-active dynamics on ∼1-ns timescale (however, it is difficult to isolate this contribution). In conclusion, recent advances in the area of force-field development have made it possible to obtain viable Molecular Dynamics models of protein disorder. After careful validation against the experimental relaxation data, these models can provide a valuable insight into mechanistic origins of spin relaxation in disordered peptides and proteins.

A new structural arrangement in proteins involving lysine NH3+ group and carbonyl.

Screening of the Protein Data Bank led to identification of a recurring structural motif where lysine NH3+ group interacts with backbone carbonyl. This interaction is characterized by linear atom arrangement, with carbonyl O atom positioned on the three-fold symmetry axis of the NH3+ group (angle Cε-Nζ-O close to 180°, distance Nζ-O ca. 2.7-3.0 Å). Typically, this linear arrangement coexists with three regular hydrogen bonds formed by lysine NH3+ group (angle Cε-Nζ-acceptor atom close to 109°, distance Nζ-acceptor atom ca. 2.7-3.0 Å). Our DFT calculations using polarizable continuum environment suggest that this newly identified linear interaction makes an appreciable contribution to protein's energy balance, up to 2 kcal/mol. In the context of protein structure, linear interactions play a role in capping the C-termini of α-helices and 310-helices. Of note, linear interaction involving conserved lysine is consistently found in the P-loop of numerous NTPase domains, where it stabilizes the substrate-binding conformation of the P-loop. Linear interaction NH3+ - carbonyl represents an interesting example of ion-dipole interactions that has so far received little attention compared to ion-ion interactions (salt bridges) and dipole-dipole interactions (hydrogen bonds), but nevertheless represents a distinctive element of protein architecture.

Onset of disorder and protein aggregation due to oxidation-induced intermolecular disulfide bonds: case study of RRM2 domain from TDP-43.

We have investigated the behavior of second RNA-recognition motif (RRM2) of neuropathological protein TDP43 under the effect of oxidative stress as modeled in vitro. Toward this end we have used the specially adapted version of H/D exchange experiment, NMR relaxation and diffusion measurements, dynamic light scattering, controlled proteolysis, gel electrophoresis, site-directed mutagenesis and microsecond MD simulations. Under oxidizing conditions RRM2 forms disulfide-bonded dimers that experience unfolding and then assemble into aggregate particles (APs). These particles are strongly disordered, highly inhomogeneous and susceptible to proteolysis; some of them withstand the dithiothreitol treatment. They can recruit/release monomeric RRM2 through thiol-disulfide exchange reactions. By using a combination of dynamic light scattering and NMR diffusion data we were able to approximate the size distribution function for the APs. The key to the observed aggregation behavior is the diminished ability of disulfide-bonded RRM2 dimers to refold and their increased propensity to misfold, which makes them vulnerable to large thermal fluctuations. The emerging picture provides detailed insight on how oxidative stress can contribute to neurodegenerative disease, with unfolding, aggregation, and proteolytic cleavage as different facets of the process.

Simple MD-based model for oxidative folding of peptides and proteins.

Significant strides have been recently made to fold peptides and small proteins in silico using MD simulations. However, facilities are currently lacking to include disulfide bonding in the MD models of protein folding. To address this problem, we have developed a simple empirical protocol to model formation of disulfides, which is perturbation-free, retains the same speed as conventional MD simulations and allows one to control the reaction rate. The new protocol has been tested on 15-aminoacid peptide guanylin containing four cysteine residues; the net simulation time using Amber ff14SB force field was 61 µs. The resulting isomer distribution is in qualitative agreement with experiment, suggesting that oxidative folding of guanylin in vitro occurs under kinetic control. The highly stable conformation of the so-called isomer 2(B) has been obtained for full-length guanylin, which is significantly different from the poorly ordered structure of the truncated peptide PDB ID 1GNB. In addition, we have simulated oxidative folding of guanylin within the 94-aminoacid prohormone proguanylin. The obtained structure is in good agreement with the NMR coordinates 1O8R. The proposed modeling strategy can help to explore certain fundamental aspects of protein folding and is potentially relevant for manufacturing of synthetic peptides and recombinant proteins.

Slow conformational exchange and overall rocking motion in ubiquitin protein crystals.

Proteins perform their functions in solution but their structures are most frequently studied inside crystals. Here we probe how the crystal packing alters microsecond dynamics, using solid-state NMR measurements and multi-microsecond MD simulations of different crystal forms of ubiquitin. In particular, near-rotary-resonance relaxation dispersion (NERRD) experiments probe angular backbone motion, while Bloch-McConnell relaxation dispersion data report on fluctuations of the local electronic environment. These experiments and simulations reveal that the packing of the protein can significantly alter the thermodynamics and kinetics of local conformational exchange. Moreover, we report small-amplitude reorientational motion of protein molecules in the crystal lattice with an ~3-5° amplitude on a tens-of-microseconds time scale in one of the crystals, but not in others. An intriguing possibility arises that overall motion is to some extent coupled to local dynamics. Our study highlights the importance of considering the packing when analyzing dynamics of crystalline proteins.X-ray crystallography is the main method for protein structure determination. Here the authors combine solid-state NMR measurements and molecular dynamics simulations and show that crystal packing alters the thermodynamics and kinetics of local conformational exchange as well as overall rocking motion of protein molecules in the crystal lattice.

Origin of anti-tumor activity of the cysteine-containing GO peptides and further optimization of their cytotoxic properties.

Antitumor GO peptides have been designed as dimerization inhibitors of prominent oncoprotein mucin 1. In this study we demonstrate that activity of GO peptides is independent of the level of cellular expression of mucin 1. Furthermore, these peptides prove to be broadly cytotoxic, causing cell death also in normal cells such as dermal fibroblasts and endometrial mesenchymal stem cells. To explore molecular mechanism of their cytotoxicity, we have designed and tested a number of new peptide sequences containing the key CxC or CxxC motifs. Of note, these sequences bear no similarity to mucin 1 except that they also contain a pair of proximal cysteines. Several of the new peptides turned out to be significantly more potent than their GO prototypes. The results suggest that cytotoxicity of these peptides stems from their (moderate) activity as disulfide oxidoreductases. It is expected that such peptides, which we have termed DO peptides, are involved in disulfide-dithiol exchange reaction, resulting in formation of adventitious disulfide bridges in cell proteins. In turn, this leads to a partial loss of protein function and rapid onset of apoptosis. We anticipate that coupling DO sequences with tumor-homing transduction domains can create a potentially valuable new class of tumoricidal peptides.