Protein Side Chains Research Articles

Computational analysis of protein conformational heterogeneity

In this paper, we applied the molecular dynamics (MD) simulations and used thermolysin as the system to study the overall protein dynamics and side chain dihedral angles across the Arrhenius break. Simulations were performed at a high temperature of 36 °C which is above the previously observed Arrhenius break, and the lower temperature of 20 °C which is below the Arrhenius break. We observed different protein dynamics and conformational heterogeneity of side chain dihedral angles of thermolysin at the two temperatures. Our results indicated that certain regions of thermolysin have a higher level of fluctuation at lower temperature. A temperature dependent dihedral angles were also observed at the two temperatures. The changes observed in the protein indicated key areas of temperature sensitivity within the protein. Communicated by Ramaswamy H. Sarma

Correlated Response of Protein Side-Chain Fluctuations and Conformational Entropy to Ligand Binding.

The heterogeneous fast side-chain dynamics of proteins plays crucial roles in molecular recognition and binding. Site-specific NMR experiments quantify these motions by measuring the model-free order parameter (Oaxis2) on a scale of 0 (most flexible) to 1 (least flexible) for each methyl-containing residue of proteins. Here, we have examined ligand-induced variations in the fast side-chain dynamics and conformational entropy of calmodulin (CaM) using five different CaM-peptide complexes. Oaxis2 of CaM in the ligand-free (Oaxis,U2) and ligand-bound (Oaxis,B2) states are calculated from molecular dynamics trajectories and conformational energy surfaces obtained using the adaptive biasing force (ABF) method. ΔOaxis2 = Oaxis,B2 - Oaxis,U2 follows a Gaussian-like unimodal distribution whose second moment is a potential indicator of the binding affinity of these complexes. The probability for the binding-induced Oaxis,U2 → Oaxis,B2 transition decreases with increasing magnitude of ΔOaxis2, indicating that large flexibility changes are improbable for side chains of CaM after ligand binding. A linear correlation established between ΔOaxis2 and the conformational entropy change of the protein makes possible the determination of the conformational entropy of binding of protein-ligand complexes. The results not only underscore the functional importance of fast side-chain fluctuations but also highlight key motional and thermodynamic correlates of protein-ligand binding.

Site-Selective, Chemical Modification of Protein at Aromatic Side Chain and Their Emergent Applications.

Site-selective chemical modification of protein side chain has probed enormous opportunities in the fundamental understanding of cellular biology and therapeutic applications. Primarily, in the field of biopharmaceuticals, the formulation of bioconjugates has been found to have more potential than an individual constituent. In this regard, Lysine and Cysteine are the most widely used endogenous amino acid for these purposes. Recently, the aromatic side chain residues (Trp, Tyr, and His) that are low abundant in protein have gained more attention in therapeutic applications due to their advantages of chemical reactivity and specificity. This review discusses the site-selective bioconjugation methods for aromatic side chains (Trp, Tyr and His) and highlights the developed strategies in the last three years, along with their applications. Also, the review highlights the prevalent methods published earlier. We have examined that metal-catalyzed and photocatalytic reactions are gaining more attention for bioconjugation, though their practical operation is under development. The review has been summarized with the future perspective of protein and peptide conjugations contemplating therapeutic applications and challenges.

Axial Heme Coordination by the Tyr-His Motif in the Extracellular Hemophore HasAp Is Critical for the Release of Heme to the HasR Receptor of Pseudomonas aeruginosa.

Pseudomonas aeruginosa senses extracellular heme via an extra cytoplasmic function σ factor that is activated upon interaction of the hemophore holo-HasAp with the HasR receptor. Herein, we show Y75H holo-HasAp interacts with HasR but is unable to release heme for signaling and uptake. To understand this inhibition, we undertook a spectroscopic characterization of Y75H holo-HasAp by resonance Raman (RR), electron paramagnetic resonance (EPR), and X-ray crystallography. The RR spectra are consistent with a mixed six-coordinate high-spin (6cHS), six-coordinate low-spin (6cLS) heme configuration and an H218O exchangeable FeIII-O stretching frequency with 16O/18O and H/D isotope shifts that support a two-body Fe-OH2 oscillator with (iron-hydroxy)-like character as both hydrogen atoms are engaged in short hydrogen bond interactions with protein side chains. Further support comes from the EPR spectrum of Y75H holo-HasAp that shows a LS rhombic signal with ligand-field splitting values intermediate between those of His-hydroxy and bis-His ferric hemes. The crystal structure of Y75H holo-HasAp confirmed the coordinated solvent molecule hydrogen bonded through H75 and H83. The long-range conformational rearrangement of HasAp upon heme binding can still take place in Y75H holo-HasAp, because the intercalation of a hydroxy ligand between the heme iron and H75 allows the variant to reproduce the heme binding pocket observed in wild-type holo-HasAp. However, in the absence of a covalent linkage to the Y75 loop combined with the malleability provided by the bracketing H75 and H83 hydrogen bonds, either the hydroxy sixth ligand remains bound after complexation of Y75H holo-HasAp with HasR or rearrangement and coordination of H85 prevent heme transfer.

Exploring ligand dynamics in protein crystal structures with ensemble refinement.

Understanding the dynamics of ligands bound to proteins is an important task in medicinal chemistry and drug design. However, the dominant technique for determining protein-ligand structures, X-ray crystallography, does not fully account for dynamics and cannot accurately describe the movements of ligands in protein binding sites. In this article, an alternative method, ensemble refinement, is used on six protein-ligand complexes with the aim of understanding the conformational diversity of ligands in protein crystal structures. The results show that ensemble refinement sometimes indicates that the flexibility of parts of the ligand and some protein side chains is larger than that which can be described by a single conformation and atomic displacement parameters. However, since the electron-density maps are comparable and Rfree values are slightly increased, the original crystal structure is still a better model from a statistical point of view. On the other hand, it is shown that molecular-dynamics simulations and automatic generation of alternative conformations in crystallographic refinement confirm that the flexibility of these groups is larger than is observed in standard refinement. Moreover, the flexible groups in ensemble refinement coincide with groups that give high atomic displacement parameters or non-unity occupancy if optimized in standard refinement. Therefore, the conformational diversity indicated by ensemble refinement seems to be qualitatively correct, indicating that ensemble refinement can be an important complement to standard crystallographic refinement as a tool to discover which parts of crystal structures may show extensive flexibility and therefore are poorly described by a single conformation. However, the diversity of the ensembles is often exaggerated (probably partly owing to the rather poor force field employed) and the ensembles should not be trusted in detail.

Mutual information analysis of the dynamic correlation between side chains in proteins.

Protein dynamics play an essential role in function regulation. In recent years, many experimental and theoretical studies have shown that changes in protein fluctuations in the backbone and side chains fulfill a pivotal role associated with amino acid mutations, chemical modifications, and ligand binding. The dynamic correlations between protein side chains have not been sufficiently studied, and no reliable analysis method has been available so far. Therefore, we developed a method to evaluate the dynamic correlation between protein side chains using mutual information and molecular dynamics simulations. To eliminate the structural superposition errors dealing with conventional analysis methods, and to accurately extract the intrinsic fluctuation properties of the side chains, we employed distance principal component analysis (distPCA). The motion of the side chain was then projected onto the eigenvector space obtained by distPCA, and the mutual information between the projected motions was calculated. The proposed method was then applied to a small protein "eglin c" and the mutants. The results show that even a single mutation significantly changed the dynamic correlations and also suggest that the dynamic change is deeply related to the stability. Those results indicate that our developed method could be useful for analyzing the molecular mechanism of allosteric communication in proteins.

SidechainNet: An all‐atom protein structure dataset for machine learning

Despite recent advancements in deep learning methods for protein structure prediction and representation, little focus has been directed at the simultaneous inclusion and prediction of protein backbone and sidechain structure information. We present SidechainNet, a new dataset that directly extends the ProteinNet dataset. SidechainNet includes angle and atomic coordinate information capable of describing all heavy atoms of each protein structure and can be extended by users to include new protein structures as they are released. In this article, we provide background information on the availability of protein structure data and the significance of ProteinNet. Thereafter, we argue for the potentially beneficial inclusion of sidechain information through SidechainNet, describe the process by which we organize SidechainNet, and provide a software package (https://github.com/jonathanking/sidechainnet) for data manipulation and training with machine learning models.

Structural and dynamical heterogeneity of water trapped inside Na+-pumping KR2 rhodopsin in the dark state.

Photoisomerization in the retinal leads to a channel opening in rhodopsins that triggers translocation or pumping of ions/protons. Crystal structures of rhodopsins contain several structurally conserved water molecules. It has been suggested that water plays an active role in facilitating the ion pumping/translocation process by acting as a lubricant in these systems. In this paper, we systematically investigate the localization, structure, dynamics, and energetics of the water molecules along the channel for the resting/dark state of KR2 rhodopsin. By employing several microseconds long atomistic molecular dynamics simulation of this trans-membrane protein system, we demonstrate the presence of five distinct water containing pockets/cavities separated by gateways controlled by protein side-chains. There exists a strong hydrogen bonded network involving these buried water molecules and functionally important key residues. We present evidence of significant structural and dynamical heterogeneity in the water molecules present in these cavities, with very rare exchange between them. The exchange time scale of such buried water with the bulk has an extremely wide range, from tens of nanoseconds to >1.5µs. The translational and rotational dynamics of buried water are found to be strongly dependent on the protein cavity size and local interactions with a classic signature of trapped diffusion and rotational anisotropy.

Molecular Dynamics Free Energy Simulations Reveal the Mechanism for the Antiviral Resistance of the M66I HIV-1 Capsid Mutation.

While drug resistance mutations can often be attributed to the loss of direct or solvent-mediated protein−ligand interactions in the drug-mutant complex, in this study we show that a resistance mutation for the picomolar HIV-1 capsid (CA)-targeting antiviral (GS-6207) is mainly due to the free energy cost of the drug-induced protein side chain reorganization in the mutant protein. Among several mutations, M66I causes the most suppression of the GS-6207 antiviral activity (up to ~84,000-fold), and only 83- and 68-fold reductions for PF74 and ZW-1261, respectively. To understand the molecular basis of this drug resistance, we conducted molecular dynamics free energy simulations to study the structures, energetics, and conformational free energy landscapes involved in the inhibitors binding at the interface of two CA monomers. To minimize the protein−ligand steric clash, the I66 side chain in the M66I−GS-6207 complex switches to a higher free energy conformation from the one adopted in the apo M66I. In contrast, the binding of GS-6207 to the wild-type CA does not lead to any significant M66 conformational change. Based on an analysis that decomposes the absolute binding free energy into contributions from two receptor conformational states, it appears that it is the free energy cost of side chain reorganization rather than the reduced protein−ligand interaction that is largely responsible for the drug resistance against GS-6207.

Protein Loop Conformational Free Energy Changes via an Alchemical Path without Reaction Coordinates.

We introduce a method called restrain-free energy perturbation-release 2.0 (R-FEP-R 2.0) to estimate conformational free energy changes of protein loops via an alchemical path. R-FEP-R 2.0 is a generalization of the method called restrain-free energy perturbation-release (R-FEP-R) that can only estimate conformational free energy changes of protein side chains but not loops. The reorganization of protein loops is a central feature of many biological processes. Unlike other advanced sampling algorithms such as umbrella sampling and metadynamics, R-FEP-R and R-FEP-R 2.0 do not require predetermined collective coordinates and transition pathways that connect the two endpoint conformational states. The R-FEP-R 2.0 method was applied to estimate the conformational free energy change of a β-turn flip in the protein ubiquitin. The result obtained by R-FEP-R 2.0 agrees with the benchmarks very well. We also comment on problems commonly encountered when applying umbrella sampling to calculate protein conformational free energy changes.

Fic and non-Fic AMPylases: protein AMPylation in metazoans.

Protein AMPylation refers to the covalent attachment of an AMP moiety to the amino acid side chains of target proteins using ATP as nucleotide donor. This process is catalysed by dedicated AMP transferases, called AMPylases. Since this initial discovery, several research groups have identified AMPylation as a critical post-translational modification relevant to normal and pathological cell signalling in both bacteria and metazoans. Bacterial AMPylases are abundant enzymes that either regulate the function of endogenous bacterial proteins or are translocated into host cells to hijack host cell signalling processes. By contrast, only two classes of metazoan AMPylases have been identified so far: enzymes containing a conserved filamentation induced by cAMP (Fic) domain (Fic AMPylases), which primarily modify the ER-resident chaperone BiP, and SelO, a mitochondrial AMPylase involved in redox signalling. In this review, we compare and contrast bacterial and metazoan Fic and non-Fic AMPylases, and summarize recent technological and conceptual developments in the emerging field of AMPylation.

Crystallin Proteins from Aquatic Organisms: Stability and Function in the Lens and Beyond

The crystallins are the major structural and refractive proteins of the eye lens. They are extremely long-lived proteins that maintain their stability for years or decades in the near-absence of protein turnover in the lens. In vertebrates, the structural crystallins are characterized by a common structural motif, the double-Greek key βγ-crystallin fold that originated as a Ca2+-binding protein, a function that is still maintained by microbial crystallins. Ca2+-binding βγ-crystallins are characterized by a conserved N/D-N/D-#-I-S/T-S double-clamp metal ion binding motif located in a loop region. Despite the conservation of the overall fold, the ubiquitous vertebrate structural crystallins have mostly lost their cation-binding activity in favor of stabilization via other structural means. The βγ-crystallin from the tunicate Ciona intestinalis provides a fascinating example of a protein that is intermediate in function: it tightly binds two Ca2+ ions, and is greatly stabilized by doing so. At the same time, it is expressed in the sensory organs of the larval tunicate, and earlier work by our group has shown that its refractive index increment is higher than would be expected based on its amino acid sequence alone, suggesting an additional refractive function. Structurally, the tunicate crystallin resembles one half of a vertebrate βγ-crystallin, which evolved from a common ancestor of these proteins via gene duplication and selection for new functionality in the lens. Recent work from my laboratory has focused on investigating the structure and dynamics of the tunicate crystallin in the presence and absence of Ca2+ using solution-state NMR spectroscopy. Although a crystal structure (PDB ID:2BV2) was apreviously solved by Shimeld et al., NMR data enable us to probe the molecular motions on a site-by-site basis. Identification of protein sidechains or regions of the backbone with lower-amplitude dynamics in the holo form compared to the apo form highlight features that increase the stability of the protein upon Ca2+ binding. Comparison of these sequence and structural factors with those of the vertebrate structural crystallins point to features that are needed to provide stability in the absence of cation binding as well as higher refractivity in the vertebrate lens. I will present NMR spectra and other spectroscopic data illustrating the structural and motional differences between Ca2+-bound and apo tunicate crystallin, as well as interactions with the solvent. Finally, I will discuss similarities differences between the vertebrate structural crystallins and an evolutionarily unrelated protein that serves the same function, the J2-crystallin of the box jelly Tripedalia cystophora. Unlike C. intestinalis and most other non-cephalopod invertebrates, T. cystophora has camera-type eyes with a lens and a retina. J2-crystallin has a completely different fold than the vertebrate proteins, yet it must perform the same function. I will present structural and biophysical data for this protein and discuss general functional characteristics of structural and refractive lens proteins.

Probing Side-Chain Dynamics in Proteins by NMR Relaxation of Isolated 13C Magnetization Modes in 13CH3 Methyl Groups.

The dynamics of methyl-bearing side chains in proteins were probed by 13C relaxation measurements of a number of 13C magnetization modes in selectively 13CH3-labeled methyl groups of proteins. We first show how 13C magnetization modes in a 13CH3 spin-system can be isolated using acute-angle 1H radio-frequency pulses. The parameters of methyl-axis dynamics, a measure of methyl-axis ordering (Saxis2) and the correlation time of fast local methyl-axis motions (τf), derived from 13C relaxation in 13CH3 groups are compared with their counterparts obtained from 13C relaxation in 13CHD2 methyl isotopomers. We show that in high-molecular-weight proteins, excellent correlations are obtained between the [13CHD2]-derived Saxis2 values and those extracted from relaxation of the 13C magnetization of the I = 1/2 manifold in 13CH3 methyls. In smaller proteins, a certain degree of anticorrelation is observed between the Saxis2 and τf values obtained from 13C relaxation of the I = 1/2 manifold magnetization in 13CH3 methyls. These parameters can be partially decorrelated by inclusion in the analysis of relaxation data of the I = 3/2 manifold 13C magnetization.

Conservation of the HBV RNA element epsilon in nackednaviruses reveals ancient origin of protein-primed reverse transcription

Hepadnaviruses, with the human hepatitis B virus as prototype, are small, enveloped hepatotropic DNA viruses which replicate by reverse transcription of an RNA intermediate. Replication is initiated by a unique protein-priming mechanism whereby a hydroxy amino acid side chain of the terminal protein (TP) domain of the viral polymerase (P) is extended into a short DNA oligonucleotide, which subsequently serves as primer for first-strand synthesis. A key component in the priming of reverse transcription is the viral RNA element epsilon, which contains the replication origin and serves as a template for DNA primer synthesis. Here, we show that recently discovered non-enveloped fish viruses, termed nackednaviruses [C. Lauber et al., Cell Host Microbe 22, 387-399 (2017)], employ a fundamentally similar replication mechanism despite their huge phylogenetic distance and major differences in genome organization and viral lifestyle. In vitro cross-priming studies revealed that few strategic nucleotide substitutions in epsilon enable site-specific protein priming by heterologous P proteins, demonstrating that epsilon is functionally conserved since the two virus families diverged more than 400 Mya. In addition, other cis elements crucial for the hepadnavirus-typical replication of pregenomic RNA into relaxed circular double-stranded DNA were identified at conserved positions in the nackednavirus genomes. Hence, the replication mode of both hepadnaviruses and nackednaviruses was already established in their Paleozoic common ancestor, making it a truly ancient and evolutionary robust principle of genome replication that is more widespread than previously thought.

Peptide and protein engineering by modification of backbone and sidechain functional groups

AbstractModifications of the sidechain and backbone of peptides and proteins are commonly used to improve their stability, to enhance their biological function, to enable and improve imaging and detection capabilities via conjugation to imaging agents and to otherwise introduce new properties. The most common approaches for modification and conjugation typically target amino acids such as lysine and cysteine. However, less abundant residues such as tyrosine, methionine and tryptophan are also potential targets for orthogonal modification. Non‐traditional modifications of common amino acids such as cysteine can also be used to introduce novel functional groups in a peptide sidechain. Additionally, modification via sortase is also very precise and site specific in biological conditions. This mini‐review article describes recent advances in selective modification reactions of peptides and proteins via these less common approaches. © 2021 Society of Industrial Chemistry.

Lipid-Dependent Titration of Glutamic Acid at a Bilayer Membrane Interface.

The ionization properties of protein side chains in lipid-bilayer membranes will differ from the canonical values of side chains exposed to an aqueous solution. While the propensities of positively charged side chains of His, Lys, and Arg to release a proton in lipid membranes have been rather well characterized, the propensity for a negatively charged Glu side chain to receive a proton and achieve the neutral state in a bilayer membrane has been less well characterized. Indeed, the ionization of the glutamic acid side chain has been predicted to depend on its depth of burial in a lipid membrane but has been difficult to verify experimentally. To address the issue, we incorporated an interfacial Glu residue at position 4 of a distinct 23-residue transmembrane helix and used 2H NMR to examine the helix properties as a function of pH. We observe that the helix tilt and azimuthal rotation vary little with pH, but the extent of helix unraveling near residues 3 and 4 changes as the Glu residue E4 titrates. Remarkably, the 2H quadrupolar splitting for the side chain of alanine A3 responds to pH with an apparent pKa of 4.8 in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 6.3 in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), but is unchanged up to pH 8.0 in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of residue E4. With bilayers composed of alkali-stable ether-linked lipids, the side chain of A3 responds to pH with an apparent pKa of 11.0 in the ether analogue of DOPC. These results suggest that the depth dependence of Glu ionization in lipid-bilayer membranes may be steeper than previously predicted or envisioned.

Antioxidant and Antiglycation Effects of Polyphenol Compounds Extracted from Hazelnut Skin on Advanced Glycation End-Products (AGEs) Formation.

The advanced glycation end-products (AGEs) arise from non-enzymatic reactions of sugar with protein side chains, some of which are oxido-reductive in nature. Enhanced production of AGEs plays an important role in the pathogenesis of diabetic complications as well as in natural aging, renal failure, oxidative stress, and chronic inflammation. The aim of this work is to study antiglycation effects of polyphenol compounds extracted by hazelnut skin that represents an example of polyphenols-rich food industry by-product, on AGEs formation. AGEs derived from incubation of bovine serum albumin (BSA) and methylglyoxal (MGO) were characterized by fluorescence. The phenolics identification and total polyphenol content in hazelnut skin extracts were analyzed by HPLC-MS and the Folin–Ciocalteu method, respectively. Antioxidant efficacy was evaluated by monitoring total antioxidant activity to assess the ABTS radical scavenging activity of samples by TEAC assay and oxygen radical absorbance capacity (ORAC) assay, expressed as millimoles of Trolox equivalents per gram of sample. Data here presented suggest that phenolic compounds in hazelnut skin have an inhibitory effect on the BSA-AGEs model in vitro, and this effect is concentration-dependent. The putative role of the hazelnut skin antioxidative properties for hindering AGEs formation is also discussed. Because of AGEs contribution to the pathogenesis of several chronic diseases, foods enriched, or supplements containing natural bioactive molecules able to inhibit their production could be an interesting new strategy for supporting therapeutic approaches with a positive effect on human health.

Soy meal adhesive with high strength and water resistance via carboxymethylated wood fiber-induced crosslinking

Soy meal-based wood adhesive has received much interest for its environmentally friendly properties, as it is thought to provide an effective alternative to conventional formaldehyde-based wood adhesives. However, the abundant hydrophilic groups and weak internal structure result in poor water resistance and low bonding strength of soy meal-based wood adhesives. In this study, bio-based carboxymethylated wood fibers (CMWFs) were used to develop a high-performance soy meal-based adhesive. Wood fibers (WFs) were pre-treated via carboxymethylation to endow WFs with abundant carboxyl groups and facilitate their dispersion in soy protein matrix. CMWFs containing abundant carboxyl groups served as a multiple “crosslinking core” and effectively cross-linked with soy protein side-chain, resulting in the construction of a stable adhesive system. The modified soy meal-based adhesive exhibited excellent bonding strength and water resistance. In particular, tension tests indicated that the water-resistant bonding strength of the adhesive modified with CMWFs reached 1.69 MPa, which was 160% higher compared with the unmodified adhesive; the wood failure percentage of the plywood bonded by CMWF-modified soy meal-based adhesive was as high as 100%. Moisture uptake and residual rate tests indicated that high water resistance of the adhesive was achieved. This design provides a facile and sustainable strategy for developing high-performance soy meal-based adhesives.

Increased Histone Acetylation and Decreased Expression of Specific Histone Deacetylases in Ultraviolet-Irradiated and Intrinsically Aged Human Skin In Vivo.

Histone deacetylases (HDACs) are conserved enzymes that remove acetyl groups from lysine side chains in histones and other proteins and play a crucial role in epigenetic regulation. Previously, we showed that histone acetylation is implicated in ultraviolet (UV)-induced inflammation and matrix impairment. To elucidate the histone acetylation status and specific HDACs involved in skin aging, we examined the changes in histone acetylation, global HDAC activity, and the expression of HDACs and sirtuins (SIRTs) in intrinsically aged and photoaged human skin as well as in UV-irradiated human skin in vivo. Following acute UV irradiation, the acetylated histone H3 (AcH3) level was increased, but HDAC activity and the expression levels of HDAC4, HDAC11, and SIRT4 were significantly decreased. In intrinsically aged skin, AcH3 levels were increased, but HDAC activity and the expression levels of HDAC4, HDAC5, HDAC10, HDAC11, SIRT6, and SIRT7 were significantly decreased. However, histone acetylation and HDAC expression in photoaged skin were not significantly different from those in intrinsically aged skin. Collectively, HDAC4 and HDAC11 were decreased in both UV-irradiated and intrinsically aged skin, suggesting that they may play a universal role in increased histone acetylation associated with skin aging.

Dynamics of the HD regulatory subdomain of PARP-1; substrate access and allostery in PARP activation and inhibition.

PARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1’s F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed.