The thermodynamic signature of ligand binding to histone deacetylase-like amidohydrolases is most sensitive to the flexibility in the L2-loop lining the active site pocket

BackgroundThe analysis of the thermodynamic driving forces of ligand-protein binding has been suggested to be a key component for the selection and optimization of active compounds into drug candidates. The binding enthalpy as deduced from isothermal titration calorimetry (ITC) is usually interpreted assuming single-step binding of a ligand to one conformation of the target protein. Although successful in many cases, these assumptions are oversimplified approximations of the reality with flexible proteins and complicated binding mechanism in many if not most cases. The relationship between protein flexibility and thermodynamic signature of ligand binding is largely understudied.MethodsDirected mutagenesis, X-ray crystallography, enzyme kinetics and ITC methods were combined to dissect the influence of loop flexibility on the thermodynamics and mechanism of ligand binding to histone deacetylase (HDAC)-like amidohydrolases.ResultsThe general ligand-protein binding mechanism comprises an energetically demanding gate opening step followed by physical binding. Increased flexibility of the L2-loop in HDAC-like amidohydrolases facilitates access of ligands to the binding pocket resulting in predominantly enthalpy-driven complex formation.ConclusionsThe study provides evidence for the great importance of flexibility adjacent to the active site channel for the mechanism and observed thermodynamic driving forces of molecular recognition in HDAC like enzymes.General significanceThe flexibility or malleability in regions adjacent to binding pockets should be given more attention when designing better drug candidates. The presented case study also suggests that the observed binding enthalpy of protein-ligand systems should be interpreted with caution, since more complicated binding mechanisms may obscure the significance regarding potential drug likeness.     

Structure, mechanism, and inhibition of histone deacetylases and related metalloenzymes

Metal-dependent histone deacetylases (HDACs) catalyze the hydrolysis of acetyl-L-lysine side chains in histone and nonhistone proteins to yield l-lysine and acetate. This chemistry plays a critical role in the regulation of numerous biological processes. Aberrant HDAC activity is implicated in various diseases, and HDACs are validated targets for drug design. Two HDAC inhibitors are currently approved for cancer chemotherapy, and other inhibitors are in clinical trials. To date, X-ray crystal structures are available for four human HDACs (2, 4, 7, and 8) and three HDAC-related deacetylases from bacteria (histone deacetylase-like protein (HDLP); histone deacetylase-like amidohydrolase (HDAH); acetylpolyamine amidohydrolase (APAH)). Structural comparisons among these enzymes reveal a conserved constellation of active site residues, suggesting a common mechanism for the metal-dependent hydrolysis of acetylated substrates. Structural analyses of HDACs and HDAC-related deacetylases guide the design of tight-binding inhibitors, and future prospects for developing isozyme-specific inhibitors are quite promising.     

Structural analysis of protein‐ligand interactions: the binding of endogenous compounds and of synthetic drugs

The large number of macromolecular structures deposited with the Protein Data Bank (PDB) describing complexes between proteins and either physiological compounds or synthetic drugs made it possible a systematic analysis of the interactions occurring between proteins and their ligands. In this work, the binding pockets of about 4000 PDB protein‐ligand complexes were investigated and amino acid and interaction types were analyzed. The residues observed with lowest frequency in protein sequences, Trp, His, Met, Tyr, and Phe, turned out to be the most abundant in binding pockets. Significant differences between drug‐like and physiological compounds were found. On average, physiological compounds establish with respect to drugs about twice as many hydrogen bonds with protein atoms, whereas drugs rely more on hydrophobic interactions to establish target selectivity. The large number of PDB structures describing homologous proteins in complex with the same ligand made it possible to analyze the conservation of binding pocket residues among homologous protein structures bound to the same ligand, showing that Gly, Glu, Arg, Asp, His, and Thr are more conserved than other amino acids. Also in the cases in which the same ligand is bound to unrelated proteins, the binding pockets showed significant conservation in the residue types. In this case, the probability of co‐occurrence of the same amino acid type in the binding pockets could be up to thirteen times higher than that expected on a random basis. The trends identified in this study may provide an useful guideline in the process of drug design and lead optimization. Copyright © 2014 John Wiley & Sons, Ltd.     

Ligand Clouds around Protein Clouds: A Scenario of Ligand Binding with Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) were found to be widely associated with human diseases and may serve as potential drug design targets. However, drug design targeting IDPs is still in the very early stages. Progress in drug design is usually achieved using experimental screening; however, the structural disorder of IDPs makes it difficult to characterize their interaction with ligands using experiments alone. To better understand the structure of IDPs and their interactions with small molecule ligands, we performed extensive simulations on the c-Myc_370–409 peptide and its binding to a reported small molecule inhibitor, ligand 10074-A4. We found that the conformational space of the apo c-Myc_370–409 peptide was rather dispersed and that the conformations of the peptide were stabilized mainly by charge interactions and hydrogen bonds. Under the binding of the ligand, c-Myc_370–409 remained disordered. The ligand was found to bind to c-Myc_370–409 at different sites along the chain and behaved like a ‘ligand cloud’. In contrast to ligand binding to more rigid target proteins that usually results in a dominant bound structure, ligand binding to IDPs may better be described as ligand clouds around protein clouds. Nevertheless, the binding of the ligand and a non-ligand to the c-Myc_370–409 target could be clearly distinguished. The present study provides insights that will help improve rational drug design that targets IDPs.     

Histone deacetylases: salesmen and customers in the post‐translational modification market

HDACs (histone deacetylases) are enzymes that remove the acetyl moiety from N‐?‐acetylated lysine residues in histones and non‐histone proteins. In recent years, it has turned out that HDACs themselves are also subject to post‐translational modification. Such structural alterations can determine the stability, localization, activity and protein—protein interactions of HDACs. This subsequently affects the modification of their substrates and the co‐ordination of cellular signalling networks. Intriguingly, physiologically relevant non‐histone proteins are increasingly found to be deacetylated by HDACs, and aberrant deacetylase activity contributes to several severe human diseases. Targeting the catalytic activity of these enzymes and their post‐translational modifications are therefore attractive targets for therapeutical intervention strategies. To achieve this ambitious goal, details on the molecular mechanisms regulating post‐translational modifications of HDACs are required. This review summarizes aspects of the current knowledge on the biological role and enzymology of the phosphorylation, acetylation, ubiquitylation and sumoylation of HDACs.     

Kinetic and structural insights into the binding of histone deacetylase 1 and 2 (HDAC1, 2) inhibitors

The structure–activity and structure–kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11 Å channel leading to the Zn²⁺ catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.     

Histone Deacetylases: A Saga of Perturbed Acetylation Homeostasis in Cancer

In the current era of genomic medicine, diseases are identified as manifestations of anomalous patterns of gene expression. Cancer is the principal example among such maladies. Although remarkable progress has been achieved in the understanding of the molecular mechanisms involved in the genesis and progression of cancer, its epigenetic regulation, particularly histone deacetylation, demands further studies. Histone deacetylases (HDACs) are one of the key players in the gene expression regulation network in cancer because of their repressive role on tumor suppressor genes. Higher expression and function of deacetylases disrupt the finely tuned acetylation homeostasis in both histone and non-histone target proteins. This brings about alterations in the genes implicated in the regulation of cell proliferation, differentiation, apoptosis and other cellular processes. Moreover, the reversible nature of epigenetic modulation by HDACs makes them attractive targets for cancer remedy. This review summarizes the current knowledge of HDACs in tumorigenesis and tumor progression as well as their contribution to the hallmarks of cancer. The present report also describes briefly various assays to detect histone deacetylase activity and discusses the potential role of histone deacetylase inhibitors as emerging epigenetic drugs to cure cancer.     

On the molecular basis of the high affinity binding of basic amino acids to LAOBP,a periplasmic binding protein from Salmonella typhimurium

The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high‐affinity binding of ligands to proteins is still limited; such is the case for l ‐lysine–l ‐arginine–l ‐ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l ‐arginine, l ‐lysine, and l ‐ornithine with nanomolar affinity and to l ‐histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l ‐histidine and l ‐arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of ~?300 cal mol^?1 K^?1, most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000‐fold higher affinity of LAOBP for l ‐arginine as compared with l ‐histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy‐driven micromolar affinity toward l ‐arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparison analysis of primary ligand‐binding sites in seven‐helix membrane proteins

Seven‐helix transmembrane proteins, including the G‐protein‐coupled receptors (GPCRs), mediate a broad range of fundamental cellular activities through binding to a wide range of ligands. Understanding the structural basis for the ligand‐binding selectivity of these proteins is of significance to their structure‐based drug design. Comparison analysis of proteins' ligand‐binding sites provides a useful way to study their structure‐activity relationships. Various computational methods have been developed for the binding‐site comparison of soluble proteins. In this work, we applied this approach to the analysis of the primary ligand‐binding sites of 92 seven‐helix transmembrane proteins. Results of the studies confirmed that the binding site of bacterial rhodopsins is indeed different from all GPCRs. In the latter group, further comparison of the binding sites indicated a group of residues that could be responsible for ligand‐binding selectivity and important for structure‐based drug design. Furthermore, unexpected binding‐site dissimilarities were observed among adrenergic and adenosine receptors, suggesting that the percentage of the overall sequence identity between a target protein and a template protein alone is not sufficient for selecting the best template for homology modeling of seven‐helix membrane proteins. These results provided novel insight into the structural basis of ligand‐binding selectivity of seven‐helix membrane proteins and are of practical use to the computational modeling of these proteins. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 31–38, 2011.     

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.     

SynPharm and the guide to pharmacology database: A toolset for conferring drug control on engineered proteins

Optimizing synthetic biological systems, for example novel metabolic pathways, becomes more complicated with more protein components. One method of taming the complexity and allowing more rapid optimization is engineering external control into components. Pharmacology is essentially the science of controlling proteins using (mainly) small molecules, and a great deal of information, spread between different databases, is known about structural interactions between these ligands and their target proteins. In principle, protein engineers can use an inverse pharmacological approach to include drug response in their design, by identifying ligand‐binding domains from natural proteins that are amenable to being included in a designed protein. In this context, “amenable” means that the ligand‐binding domain is in a relatively self‐contained subsequence of the parent protein, structurally independent of the rest of the molecule so that its function should be retained in another context. The SynPharm database is a tool, built on to the Guide to Pharmacology database and connected to various structural databases, to help protein engineers identify ligand‐binding domains suitable for transfer. This article describes the tool, and illustrates its use in seeking candidate domains for transfer. It also briefly describes already‐published proof‐of‐concept studies in which the CRISPR effectors Cas9 and Cpf1 were placed separately under the control of tamoxifen and mefipristone, by including ligand‐binding domains of the Estrogen Receptor and Progesterone Receptor in modified versions of Cas9 and Cpf1. The advantages of drug control or the rival protein‐control technology of optogenetics, for different purposes and in different situations, are also briefly discussed.     

Inhibitor-mediated stabilization of the conformational structure of a histone deacetylase-like amidohydrolase

Histone deacetylases are major regulators of eukaryotic gene expression. Not unexpectedly, histone deacetylases are among the most promising targets in cancer therapy. However, despite huge efforts in histone deacetylase inhibitor design, very little is known about the impact of histone deacetylase inhibitors on enzyme stability. In this study, the conformational stability of a well-established histone deacetylase homolog with high structural similarity (histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes species FB188) was investigated using denaturation titrations and stopped-flow kinetics. Based on the results of these complementary approaches, we conclude that the interconversion of native histone deacetylase-like amidohydrolase into its denatured form involves several intermediates possessing different enzyme activities and conformational structures. The refolding kinetics has shown to be strongly dependent on Zn(2+) and to a lesser extent on K(+), which underlines their importance not only for catalytic function but also for maintaining the correct conformational structure of the enzyme. Two main unfolding processes of histone deacetylase-like amidohydrolase were differentiated. The unfolding occurring at submolar concentrations of the denaturant guanidine hydrochloride was not affected by inhibitor binding, whereas the unfolding at higher concentrations of guanidine hydrochloride was strongly affected. It was shown that the known inhibitors suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate are capable of stabilizing the conformational structure of histone deacetylase-like amidrohydrolase. Judging from the free energies of unfolding, the protein stability was increased by 9.4 and 5.4 kJ.mol(-1) upon binding of suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate, respectively.     

Landscape of protein–small ligand binding modes

Elucidating the mechanisms of specific small‐molecule (ligand) recognition by proteins is a long‐standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein–ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all‐against‐all comparison of 20,040 protein–ligand complexes provided the landscape of the protein–ligand binding modes and its relationships with protein‐ and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R² = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein–ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them.