Solution-state methyl NMR spectroscopy of large non-deuterated proteins enabled by deep neural networks.

Methyl-TROSY nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for characterising large biomolecules in solution. However, preparing samples for these experiments is demanding and entails deuteration, limiting its use. Here we demonstrate that NMR spectra recorded on protonated, uniformly 13C labelled samples can be processed using deep neural networks to yield spectra that are of similar quality to typical deuterated methyl-TROSY spectra, potentially providing information for proteins that cannot be produced in bacterial systems. We validate the methodology experimentally on three proteins with molecular weights in the range 42-360 kDa. We further demonstrate the applicability of our methodology to 3D NOESY spectra of Escherichia coli Malate Synthase G (81 kDa), where observed NOE cross-peaks are in good agreement with the available structure. The method represents an advance in the field of using deep learning to analyse complex magnetic resonance data and could have an impact on the study of large biomolecules in years to come.

Picosecond Dynamics of a Small Molecule in Its Bound State with an Intrinsically Disordered Protein.

Intrinsically disordered proteins (IDPs) are highly dynamic biomolecules that rapidly interconvert among many structural conformations. These dynamic biomolecules are involved in cancers, neurodegeneration, cardiovascular illnesses, and viral infections. Despite their enormous therapeutic potential, IDPs have generally been considered undruggable because of their lack of classical long-lived binding pockets for small molecules. Currently, only a few instances are known where small molecules have been observed to interact with IDPs, and this situation is further exacerbated by the limited sensitivity of experimental techniques to detect such binding events. Here, using experimental nuclear magnetic resonance (NMR) spectroscopy 19F transverse spin-relaxation measurements, we discovered that a small molecule, 5-fluoroindole, interacts with the disordered domains of non-structural protein 5A from hepatitis C virus with a Kd of 260 ± 110 µM. Our analysis also allowed us to determine the rotational correlation times (τc) for the free and bound states of 5-fluoroindole. In the free state, we observed a rotational correlation time of 27.0 ± 1.3 ps, whereas in the bound state, τc only increased to 46 ± 10 ps. Our findings imply that it is possible for small molecules to engage with IDPs in exceptionally dynamic ways, in sharp contrast to the rigid binding modes typically exhibited when small molecules bind to well-defined binding pockets within structured proteins.

Biomolecular NMR spectroscopy in the era of artificial intelligence.

Biomolecular nuclear magnetic resonance (NMR) spectroscopy and artificial intelligence (AI) have a burgeoning synergy. Deep learning-based structural predictors have forever changed structural biology, yet these tools currently face limitations in accurately characterizing protein dynamics, allostery, and conformational heterogeneity. We begin by highlighting the unique abilities of biomolecular NMR spectroscopy to complement AI-based structural predictions toward addressing these knowledge gaps. We then highlight the direct integration of deep learning approaches into biomolecular NMR methods. AI-based tools can dramatically improve the acquisition and analysis of NMR spectra, enhancing the accuracy and reliability of NMR measurements, thus streamlining experimental processes. Additionally, deep learning enables the development of novel types of NMR experiments that were previously unattainable, expanding the scope and potential of biomolecular NMR spectroscopy. Ultimately, a combination of AI and NMR promises to further revolutionize structural biology on several levels, advance our understanding of complex biomolecular systems, and accelerate drug discovery efforts.

Intrinsic structural dynamics dictate enzymatic activity and inhibition.

Enzymes are known to sample various conformations, many of which are critical for their biological function. However, structural characterizations of enzymes predominantly focus on the most populated conformation. As a result, single-point mutations often produce structures that are similar or essentially identical to those of the wild-type enzyme despite large changes in enzymatic activity. Here, we show for mutants of a histone deacetylase enzyme (HDAC8) that reduced enzymatic activities, reduced inhibitor affinities, and reduced residence times are all captured by the rate constants between intrinsically sampled conformations that, in turn, can be obtained independently by solution NMR spectroscopy. Thus, for the HDAC8 enzyme, the dynamic sampling of conformations dictates both enzymatic activity and inhibitor potency. Our analysis also dissects the functional role of the conformations sampled, where specific conformations distinct from those in available structures are responsible for substrate and inhibitor binding, catalysis, and product dissociation. Precise structures alone often do not adequately explain the effect of missense mutations on enzymatic activity and drug potency. Our findings not only assign functional roles to several conformational states of HDAC8 but they also underscore the paramount role of dynamics, which will have general implications for characterizing missense mutations and designing inhibitors.

Disorder in the Human Skp1 Structure is the Key to its Adaptability to Bind Many Different Proteins in the SCF Complex Assembly.

Skp1(S-phase kinase-associated protein 1 - Homo sapiens) is an adapter protein of the SCF(Skp1-Cullin1-Fbox) complex, which links the constant components (Cul1-RBX) and the variable receptor (F-box proteins) in Ubiquitin E3 ligase. It is intriguing how Skp1 can recognise and bind to a variety of structurally different F-box proteins. For practical reasons, previous efforts have used truncated Skp1, and thus it has not been possible to track the crucial aspects of the substrate recognition process. In this background, we report the solution structure of the full-length Skp1 protein determined by NMR spectroscopy for the first time and investigate the sequence-dependent dynamics in the protein. The solution structure reveals that Skp1 has an architecture: ß1-ß2-H1-H2-L1-H3-L2-H4-H5-H6-H7(partially formed) and a long tail-like disordered C-terminus. Structural analysis using DALI (Distance Matrix Alignment) reveals conserved domain structure across species for Skp1. Backbone dynamics investigated using NMR relaxation suggest substantial variation in the motional timescales along the length of the protein. The loops and the C-terminal residues are highly flexible, and the (R2/R1) data suggests µs-ms timescale motions in the helices as well. Further, the dependence of amide proton chemical shift on temperature and curved profiles of their residuals indicate that the residues undergo transitions between native state and excited state. The curved profiles for several residues across the length of the protein suggest that there are native-like low-lying excited states, particularly for several C-terminal residues. Our results provide a rationale for how the protein can adapt itself, bind, and get functionally associated with other proteins in the SCF complex by utilising its flexibility and conformational sub-states.

Aromatic side-chain flips orchestrate the conformational sampling of functional loops in human histone deacetylase 8.

Human histone deacetylase 8 (HDAC8) is a key hydrolase in gene regulation and an important drug-target. High-resolution structures of HDAC8 in complex with substrates or inhibitors are available, which have provided insights into the bound state of HDAC8 and its function. Here, using long all-atom unbiased molecular dynamics simulations and Markov state modelling, we show a strong correlation between the conformation of aromatic side chains near the active site and opening and closing of the surrounding functional loops of HDAC8. We also investigated two mutants known to allosterically downregulate the enzymatic activity of HDAC8. Based on experimental data, we hypothesise that I19S-HDAC8 is unable to release the product, whereas both product release and substrate binding are impaired in the S39E-HDAC8 mutant. The presented results deliver detailed insights into the functional dynamics of HDAC8 and provide a mechanism for the substantial downregulation caused by allosteric mutations, including a disease causing one.

Methodological advancements for characterising protein side chains by NMR spectroscopy.

The surface of proteins is covered by side chains of polar amino acids that are imperative for modulating protein functionality through the formation of noncovalent intermolecular interactions. However, despite their tremendous importance, the unique structures of protein side chains require tailored approaches for investigation by nuclear magnetic resonance spectroscopy and so have traditionally been understudied compared with the protein backbone. Here, we review substantial recent methodological advancements within nuclear magnetic resonance spectroscopy to address this issue. Specifically, we consider advancements that provide new insight into methyl-bearing side chains, show the potential of using non-natural amino acids and reveal the actions of charged side chains. Combined, the new methods promise unprecedented characterisations of side chains that will further elucidate protein function.

A distal regulatory region of a class I human histone deacetylase.

Histone deacetylases (HDACs) are key enzymes in epigenetics and important drug targets in cancer biology. Whilst it has been established that HDACs regulate many cellular processes, far less is known about the regulation of these enzymes themselves. Here, we show that HDAC8 is allosterically regulated by shifts in populations between exchanging states. An inactive state is identified, which is stabilised by a range of mutations and resembles a sparsely-populated state in equilibrium with active HDAC8. Computational models show that the inactive and active states differ by small changes in a regulatory region that extends up to 28 Å from the active site. The regulatory allosteric region identified here in HDAC8 corresponds to regions in other class I HDACs known to bind regulators, thus suggesting a general mechanism. The presented results pave the way for the development of allosteric HDAC inhibitors and regulators to improve the therapy for several disease states.

Intra-residue methyl-methyl correlations for valine and leucine residues in large proteins from a 3D-HMBC-HMQC experiment.

Methyl-TROSY based NMR experiments have over the last two decades become one of the most important means to characterise dynamics and functional mechanisms of large proteins and macromolecular machines in solution. The chemical shift assignment of methyl groups in large proteins is, however, still not trivial and it is typically performed using backbone-dependent experiments in a 'divide and conquer' approach, mutations, structure-based assignments or a combination of these. Structure-based assignment of methyl groups is an emerging strategy, which reduces the time and cost required as well as providing a method that is independent of a backbone assignment. One crucial step in available structure-based assignment protocols is linking the two prochiral methyl groups of leucine and valine residues. This has previously been achieved by recording NOESY spectra with short mixing times or by comparing NOESY spectra. Herein, we present a method based on through-bond scalar coupling transfers, a 3D-HMBC-HMQC experiment, to link the intra-residue methyl groups of leucine and valine. It is shown that the HMBC-HMQC method has several advantages over solely using NOESY spectra since a unique intra-residue cross-peak is observed. Moreover, overlap in the methyl-TROSY HMQC spectrum can easily be identified with the HMBC-HMQC experiment, thereby removing possible ambiguities in the assignment.

Backbone and side-chain resonance assignments of centromeric protein Scm3 from Saccharomyces cerevisiae.

The centromeric chromatin plays an essential role in regulating the attachment of microtubules and controlling the segregation of sister chromatids during mitosis. In budding yeast, the evolutionary conserved histone variant, Cse4 is a vital component of the multiprotein kinetochore complex and is recruited to the centromere through its chaperone, Suppressor of chromosome mis-segregation (Scm3). Scm3 is an inner kinetochore protein crucial for the formation of a functional inner kinetochore. Scm3 has been known to play an active role in the assembly of the centromeric nucleosome and its deletion has been found to have deleterious effects on the cells leading to chromosome segregation defects. However, structural details of monomeric full length Scm3 have remained elusive so far. Here, we report the backbone and side-chain resonance assignments of centromeric protein, Scm3. 1H, 13C and 15N chemical shifts of Scm3 have been obtained by various 2D and 3D heteronuclear NMR experiments at pH 7.4 and 283 K.

Solution structure and dynamics of glia maturation factor from Caenorhabditis elegans.

BACKGROUND: The GMF class of the ADF-H domain family proteins regulate actin dynamics by binding to the Arp2/3 complex and F-actin through their Site-1 and Site-2, respectively. CeGMF of C. elegans is analogous to GMFγ of human and mouse and is 138 amino acids in length. METHODS: We have characterized the solution structure and dynamics of CeGMF by solution NMR spectroscopy and its thermal stability by DSC. RESULTS: The solution structure of CeGMF shows canonical ADF-H fold with two additional ß-strands in the ß4-ß5 loop region. The Site-1 of CeGMF is well formed and residues of all three regions of Site-1 show dynamic flexibility. However, the ß4-ß5 loop of Site-2 is less inclined towards the C-terminal, as the latter is truncated by four residues in comparison to GMF isoforms of human and mouse. Regions of Site-2 show motions on ns-ps timescale, but dynamic flexibility of ß4-ß5 loop is low in comparison to corresponding F-loop region of ADF/cofilin UNC-60B. A general difference in packing of α3 and α1 between GMF and ADF/cofilins was noticed. Additionally, thermal stability of CeGMF was significantly higher than its ADF/cofilin homologs. CONCLUSION: We have presented the first solution structure of GMF from C. elegans, which highlights the structural differences between the Site-2 of CeGMF and mammalian GMF isoforms. Further, we have seen the differences in structure, dynamics, and thermal stability of GMF and ADF/cofilin. GENERAL SIGNIFICANCE: This study provides a useful insight to structural and dynamics factors that define the specificity of GMF towards Arp2/3 complex.

Structure, dynamics, and biochemical characterization of ADF/cofilin Twinstar from Drosophilamelanogaster.

BACKGROUND: Twinstar is an ADF/cofilin family protein, which is expressed by the tsr gene in Drosophila melanogaster. Twinstar is one of the main regulators of actin cytoskeleton remodelling and is essential for vital cellular processes like cytokinesis and endocytosis. METHODS: We have characterized the structure and dynamics of Twinstar by solution NMR spectroscopy, the interaction of Twinstar with rabbit muscle actin by ITC, and biochemical activities of Twinstar through different biochemical assays using fluorescence spectroscopy and ultra-centrifugation. RESULTS: The solution structure of Twinstar shows characteristic ADF-H fold with well-formed G/F-site and F-site for interaction with actin. The structure possesses an extended F-loop, which is rigid at the base, but flexible towards its apical region. Twinstar shares similar dynamics for the G/F-site with C. elegans homologs, UNC-60A and UNC-60B. However, the dynamics of its F-loop are different from its C. elegans homologs. Twinstar shows strong affinity for ADP-G-Actin and ATP-G-Actin with Kds of ~7.6 nM and ~0.4 µM, respectively. It shows mild F-actin depolymerizing activity and stable interaction with F-actin with a Kd of ~5.0 µM. It inhibits the rate of the nucleotide exchange in a dose dependent manner. CONCLUSION: On the basis of structure, dynamics, and biochemical activity, Twinstar can be taken to execute its biochemical role by facilitating directional growth and maintenance of length of actin filaments. GENERAL SIGNIFICANCE: This study characterizes the structure, backbone dynamics, and biochemical activities of Twinstar of Drosophila, which provides an insight into the regulation of actin dynamics in the member of phylum insecta.

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates.

Cyclophilin catalyzes the ubiquitous process "peptidyl-prolyl cis-trans isomerization," which plays a key role in protein folding, regulation, and function. Here, we present a detailed characterization of the unfolding of yeast mitochondrial cyclophilin (CPR3) induced by urea. It is seen that CPR3 unfolding is reversible and proceeds via two intermediates, I1 and I2. The I1 state has native-like secondary structure and shows strong anilino-8-naphthalenesulphonate binding due to increased exposure of the solvent-accessible cluster of non-polar groups. Thus, it has some features of a molten globule. The I2 state is more unfolded, but it retains some residual secondary structure, and shows weak anilino-8-naphthalenesulphonate binding. Chemical shift perturbation analysis by 1H-15N heteronuclear single quantum coherence spectra reveals disruption of the tertiary contacts among the regions close to the active site in the first step of unfolding, i.e., the N-I1 transition. Both of the intermediates, I1 and I2, showed a propensity to self-associate under stirring conditions, but their kinetic profiles are different; the native protein did not show any such tendency under the same conditions. All these observations could have significant implications for the function of the protein.

Backbone and side-chain resonance assignments of Plasmodium falciparum SUMO.

One of the most debilitating diseases Malaria, in its different forms, is caused by protozoan of Plasmodium species. Deadliest among these forms is the "cerebral malaria" which is afflicted upon by Plasmodium falciparum. Plasmodium adopts numerous strategies including various post-translational modifications (PTMs) to infect and survive in the human host. These PTMs have proven their critical requirement in the Plasmodium biology. Recently, sumoylation has been characterized as one of the important PTMs and many of its putative substrates have been identified in Plasmodium. Sumoylation is the covalent attachment of SUMO protein to the substrate protein, which is mediated by an enzyme cascade involving activating (E1), conjugating (E2), and ligating enzymes (E3). Here, we report resonance assignment for 1H, 13C and 15N of Plasmodium falciparum SUMO (Pf-SUMO) protein determined by various 2D and 3D heteronuclear NMR experiments along with predicted secondary structures.

A comparative study of fibrillation kinetics of two homologous proteins under identical solution condition.

Human lysozyme is homologous in the three-dimensional structure to hen lysozyme and the latter is commonly used to understand folding and amyloid aggregation pathway of the former. The fibrillation of the two proteins is known to occur via partial unfolding. A work dedicated to comparing the aggregation-prone conformations and their subsequent conversion into amyloid-like fibrils in an identical condition is not available. This has provided an opportunity to compare the fibrillation behaviors of the two homologous proteins under identical solution condition. In this work, we have shown that the temperature-induced unfolding of the two proteins at pH 1.5 occurred via a three states process. We found that temperature-unfolded states of the two proteins differ in contents of residual secondary and tertiary structures. The temperature-unfolded states of both proteins rapidly converted into well-defined amyloid-like fibrils on stirring at 230 RPM. We further observed that the kinetic parameters, lag time (tlag) and apparent rate constant (kapp) of aggregation of hen lysozyme were markedly enhanced than human lysozyme. Amyloid fibrils formed by the two proteins only slightly differ in their morphology and Tinctorial properties. Therefore, on the basis of our in vitro aggregation and in silico aggregation and α-helical propensities prediction studies, we concluded that the major determinant of acceleration of aggregation of hen lysozyme is the stabilization of amyloidogenic native α-helices in highly dynamics partially-folded state. Comparison of aggregation-prone conformations and their aggregation kinetics parameters also with other protein systems can serve as a useful model to understand the factors promoting amyloid aggregation.

NMR assignments of mitochondrial cyclophilin Cpr3 from Saccharomyces cerevisiae.

Cyclophilins regulate protein folding, transport and signalling through catalysis of proline isomerization, and are ubiquitously expressed in both prokaryotes and eukaryotes. Cpr3 is the yeast mitochondrial cyclophilin and it is structurally and biophysically uncharacterized so far. Yeast cyclophilin gene cpr3 is essential for the lactate metabolism. Here, we report (1)H, (13)C, and (15)N chemical shift assignments of Cpr3 protein determined by various 2D and 3D heteronuclear NMR experiments at pH 6.5, and temperature 298 K.

NMR assignments of actin depolymerizing factor (ADF) like UNC-60A and cofilin like UNC-60B proteins of Caenorhabditis elegans.

The actin filament dynamics in nematode, Caenorhabditis elegans, is regulated by differential activity of two proteins UNC-60A and UNC-60B. UNC-60A exhibits strong pointed end depolymerization on C. elegans actin (Ce-actin), strong inhibition of polymerization, strong monomer sequestering activity, weak severing activity, and low affinity for F-actin binding, while UNC-60B exhibits strong pointed end depolymerization on rabbit muscle actin, strong severing activity, and high affinity for F-actin binding. Structural characterization of these proteins will help to understand (1) molecular mechanism of actin dynamics regulation and (2) the differential activity of these proteins. Here, we report (1)H, (13)C, and (15)N chemical shift assignments of these two proteins as determined by heteronuclear NMR experiments (at pH 6.5 and temperature 298 K).

Solution structures and dynamics of ADF/cofilins UNC-60A and UNC-60B from Caenorhabditis elegans.

The nematode Caenorhabditis elegans has two ADF (actin-depolymerizing factor)/cofilin isoforms, UNC-60A and UNC-60B, which are expressed by the unc60 gene by alternative splicing. UNC-60A has higher activity to cause net depolymerization, and to inhibit polymerization, than UNC-60B. UNC-60B, on the other hand, shows much stronger severing activity than UNC-60A. To understand the structural basis of their functional differences, we have determined the solution structures of UNC-60A and UNC-60B proteins and characterized their backbone dynamics. Both UNC-60A and UNC-60B show a conserved ADF/cofilin fold. The G-actin (globular actin)-binding regions of the two proteins are structurally and dynamically conserved. Accordingly, UNC-60A and UNC-60B individually bind to rabbit muscle ADP-G-actin with high affinities, with Kd values of 32.25 nM and 8.62 nM respectively. The primary differences between these strong and weak severing proteins were observed in the orientation and dynamics of the F-actin (filamentous actin)-binding loop (F-loop). In the strong severing activity isoform UNC-60B, the orientation of the F-loop was towards the recently identified F-loop-binding region on F-actin, and the F-loop was relatively more flexible with 14 residues showing motions on a nanosecond-picosecond timescale. In contrast, in the weak severing protein isoform UNC-60A, the orientation of the F-loop was away from the F-loop-binding region and inclined towards its own C-terminal and strand ß6. It was also relatively less flexible with only five residues showing motions on a nanosecond-picosecond timescale. These differences in structure and dynamics seem to directly correlate with the differential F-actin site-binding and severing properties of UNC-60A and UNC-60B, and other related ADF/cofilin proteins.

Solution structure and dynamics of ADF from Toxoplasma gondii.

Toxoplasma gondii ADF (TgADF) belongs to a functional subtype characterized by strong G-actin sequestering activity and low F-actin severing activity. Among the characterized ADF/cofilin proteins, TgADF has the shortest length and is missing a C-terminal helix implicated in F-actin binding. In order to understand its characteristic properties, we have determined the solution structure of TgADF and studied its backbone dynamics from ¹5N-relaxation measurements. TgADF has conserved ADF/cofilin fold consisting of a central mixed ß-sheet comprised of six ß-strands that are partially surrounded by three α-helices and a C-terminal helical turn. The high G-actin sequestering activity of TgADF relies on highly structurally and dynamically optimized interactions between G-actin and G-actin binding surface of TgADF. The equilibrium dissociation constant for TgADF and rabbit muscle G-actin was 23.81 nM, as measured by ITC, which reflects very strong affinity of TgADF and G-actin interactions. The F-actin binding site of TgADF is partially formed, with a shortened F-loop that does not project out of the ellipsoid structure and a C-terminal helical turn in place of the C-terminal helix α4. Yet, it is more rigid than the F-actin binding site of Leishmania donovani cofilin. Experimental observations and structural features do not support the interaction of PIP2 with TgADF, and PIP2 does not affect the interaction of TgADF with G-actin. Overall, this study suggests that conformational flexibility of G-actin binding sites enhances the affinity of TgADF for G-actin, while conformational rigidity of F-actin binding sites of conventional ADF/cofilins is necessary for stable binding to F-actin.