Identifying ligands for the PHD1 finger of KDM5A through high-throughput screening.

PHD fingers are a type of chromatin reader that primarily recognize chromatin as a function of lysine methylation state. Dysregulated PHD fingers are implicated in various human diseases, including acute myeloid leukemia. Targeting PHD fingers with small molecules is considered challenging as their histone tail binding pockets are often shallow and surface-exposed. The KDM5A PHD1 finger regulates the catalytic activity of KDM5A, an epigenetic enzyme often misregulated in cancers. To identify ligands that disrupt the PHD1-histone peptide interaction, we conducted a high-throughput screen and validated hits by orthogonal methods. We further elucidated structure-activity relationships in two classes of compounds to identify features important for binding. Our investigation offers a starting point for further optimization of small molecule PHD1 ligands.

Recognition of Histone H3 Methylation States by the PHD1 Domain of Histone Demethylase KDM5A.

PHD reader domains are chromatin binding modules often responsible for the recruitment of large protein complexes that contain histone modifying enzymes, chromatin remodelers, and DNA repair machinery. A majority of PHD domains recognize N-terminal residues of histone H3 and are sensitive to the methylation state of Lys4 in histone H3 (H3K4). Histone demethylase KDM5A, an epigenetic eraser enzyme that contains three PHD domains, is often overexpressed in various cancers, and its demethylation activity is allosterically enhanced when its PHD1 domain is bound to the H3 tail. The allosteric regulatory function of PHD1 expands roles of reader domains, suggesting unique features of this chromatin interacting module. Our previous studies determined the H3 binding site of PHD1, although it remains unclear how the H3 tail interacts with the N-terminal residues of PHD1 and how PHD1 discriminates against H3 tails with varying degrees of H3K4 methylation. Here, we have determined the solution structure of apo and H3 bound PHD1. We observe conformational changes occurring in PHD1 in order to accommodate H3, which interestingly binds in a helical conformation. We also observe differential interactions of binding residues with differently methylated H3K4 peptides (me0, me1, me2, or me3), providing a rationale for PHD1's preference for lower methylation states of H3K4. We further assessed the contributions of various H3 interacting residues in the PHD1 domain to the binding of H3 peptides. The structural details of the H3 binding site could provide useful information to aid the development of allosteric small molecule modulators of KDM5A.

Viral E protein neutralizes BET protein-mediated post-entry antagonism of SARS-CoV-2.

Inhibitors of bromodomain and extraterminal domain (BET) proteins are possible anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here we show that BET proteins should not be inactivated therapeutically because they are critical antiviral factors at the post-entry level. Depletion of BRD3 or BRD4 in cells overexpressing ACE2 exacerbates SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

Viral E Protein Neutralizes BET Protein-Mediated Post-Entry Antagonism of SARS-CoV-2.

Inhibitors of Bromodomain and Extra-terminal domain (BET) proteins are possible anti-SARS-CoV-2 prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here, we show that BET proteins should not be inactivated therapeutically as they are critical antiviral factors at the post-entry level. Knockouts of BRD3 or BRD4 in cells overexpressing ACE2 exacerbate SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection, and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

Exploring the Ligand Preferences of the PHD1 Domain of Histone Demethylase KDM5A Reveals Tolerance for Modifications of the Q5 Residue of Histone 3.

Understanding the ligand preferences of epigenetic reader domains enables identification of modification states of chromatin with which these domains associate and can yield insight into recruitment and catalysis of chromatin-acting complexes. However, thorough exploration of the ligand preferences of reader domains is hindered by the limitations of traditional protein-ligand binding assays. Here, we evaluate the binding preferences of the PHD1 domain of histone demethylase KDM5A using the protein interaction by SAMDI (PI-SAMDI) assay, which measures protein-ligand binding in a high-throughput and sensitive manner via binding-induced enhancement in the activity of a reporter enzyme, in combination with fluorescence polarization. The PI-SAMDI assay was validated by confirming its ability to accurately profile the relative binding affinity of a set of well-characterized histone 3 (H3) ligands of PHD1. The assay was then used to assess the affinity of PHD1 for 361 H3 mutant ligands, a select number of which were further characterized by fluorescence polarization. Together, these experiments revealed PHD1's tolerance for H3Q5 mutations, including an unexpected tolerance for aromatic residues in this position. Motivated by this finding, we further demonstrate a high-affinity interaction between PHD1 and recently identified Q5-serotonylated H3. This work yields interesting insights into permissible PHD1-H3 interactions and demonstrates the value of interfacing PI-SAMDI and fluorescence polarization in investigations of protein-ligand binding.

Domain cross-talk in regulation of histone modifications: Molecular mechanisms and targeting opportunities.

Functional cross-talk between the catalytic and reader domains in chromatin-modifying enzymes and protein complexes enable coordinated regulation of chromatin modification status, and consequently impacts chromatin-associated processes. ZZ domains are a recently identified class of chromatin readers that recognize the N-terminal region of histone H3 to direct and regulate acetylation activity of several histone acetylation complexes. Cross-talk between chromatin readers sensitive to methylation, and catalytic domains of methyltransferases and demethylases impacts substrate specificity, catalytic activity, and propagation of chromatin marks. Recently described allosteric ligands that target domain communication highlight the potential of domain cross-talk in the development of the next-generation of chromatin-directed therapeutics.

Dissecting contributions of catalytic and reader domains in regulation of histone demethylation.

Histone demethylases catalyze the removal of methyl marks from histones, an activity associated with transcriptional regulation and DNA damage repair. As these processes are critical for normal physiology, deregulation of histone demethylases is disease causative, and their function and regulation are targets for therapeutic intervention. The larger of two histone demethylase families are Jumonji C (JmjC) demethylases. The members of the JmjC family share a conserved catalytic domain, and often contain non-catalytic domains that "read" the modification state of chromatin. By binding to specific histone modifications, reader domains assist in recruitment and promote accumulation of demethylases at their targets, as well as regulate their activity and substrate specificity. Here, we present protocols for the investigation of this functional coupling between reader and catalytic domains in human histone demethylase KDM5A. Although we use KDM5A and its PHD1 domain as our model system, the procedures presented herein can be applied for the biochemical characterization of other JmjC demethylases and chromatin readers.

Extended Recognition of the Histone H3 Tail by Histone Demethylase KDM5A.

Human lysine demethylase KDM5A is a chromatin-modifying enzyme associated with transcriptional regulation, because of its ability to catalyze removal of methyl groups from methylated lysine 4 of histone H3 (H3K4me3). Amplification of KDM5A is observed in many cancers, including breast cancer, prostate cancer, hepatocellular carcinoma, lung cancer, and gastric cancer. In this study, we employed alanine scanning mutagenesis to investigate substrate recognition of KDM5A and identify the H3 tail residues necessary for KDM5A-catalyzed demethylation. Our data show that the H3Q5 residue is critical for substrate recognition by KDM5A. Our data also reveal that the protein-protein interactions between KDM5A and the histone H3 tail extend beyond the amino acids proximal to the substrate mark. Specifically, demethylation activity assays show that deletion or mutation of residues at positions 14-18 on the H3 tail results in an 8-fold increase in the KMapp, compared to wild-type 18mer peptide, suggesting that this distal epitope is important in histone engagement. Finally, we demonstrate that post-translational modifications on this distal epitope can modulate KDM5A-dependent demethylation. Our findings provide insights into H3K4-specific recognition by KDM5A, as well as how chromatin context can regulate KDM5A activity and H3K4 methylation status.

Convergence of theory and experiment on the role of preorganization, quantum tunneling and enzyme motions into flavoenzyme-catalyzed hydride transfer.

Hydride transfer is one of the most common reactions catalyzed by enzymatic systems and it has become an object of study due to possible significant quantum tunneling effects. In the present work, we provide a combination of theoretical QM/MM simulations and experimental measurements of the rate constants and kinetic isotopic effects (KIEs) for the hydride transfer reaction catalyzed by morphinone reductase, MR. Quantum mechanical tunneling coefficients, computed in the framework of variational transition-state theory, play a significant role in this reaction, reaching values of 23.8 ± 5.5 for the lightest isotopologue; one of the largest values reported for enzymatic systems. This prediction is supported by the agreement between the theoretically predicted rate constants and the corresponding experimental values. Simulations indicate that the role of protein motions can be satisfactorily described as equilibrium fluctuations along the reaction coordinate, in line with a high degree of preorganization displayed by this enzyme.

Histone H3 binding to the PHD1 domain of histone demethylase KDM5A enables active site remodeling.

Histone demethylase KDM5A removes methyl marks from lysine 4 of histone H3 and is often overexpressed in cancer. The in vitro demethylase activity of KDM5A is allosterically enhanced by binding of its product, unmodified H3 peptides, to its PHD1 reader domain. However, the molecular basis of this allosteric enhancement is unclear. Here we show that saturation of the PHD1 domain by the H3 N-terminal tail peptides stabilizes binding of the substrate to the catalytic domain and improves the catalytic efficiency of demethylation. When present in saturating concentrations, differently modified H3 N-terminal tail peptides have a similar effect on demethylation. However, they vary greatly in their affinity towards the PHD1 domain, suggesting that H3 modifications can tune KDM5A activity. Furthermore, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) experiments reveal conformational changes in the allosterically enhanced state. Our findings may enable future development of anti-cancer therapies targeting regions involved in allosteric regulation.

Untangling Heavy Protein and Cofactor Isotope Effects on Enzyme-Catalyzed Hydride Transfer.

"Heavy" (isotopically labeled) enzyme isotope effects offer a direct experimental probe of the role of protein vibrations on enzyme-catalyzed reactions. Here we have developed a strategy to generate isotopologues of the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) where the protein and/or intrinsic flavin mononucleotide (FMN) cofactor are isotopically labeled with 2H, 15N, and 13C. Both the protein and cofactor contribute to the enzyme isotope effect on the reductive hydride transfer reaction, but their contributions are not additive and may partially cancel each other out. However, the isotope effect specifically arising from the FMN suggests that vibrations local to the active site play a role in the hydride transfer chemistry, while the protein-only "heavy enzyme" effect demonstrates that protein vibrations contribute to catalysis in PETNR. In all cases, enthalpy-entropy compensation plays a major role in minimizing the magnitude of "heavy enzyme" isotope effects. Fluorescence lifetime measurements of the intrinsic flavin mononucleotide show marked differences between "light" and "heavy" enzymes on the nanosecond-picosecond time scale, suggesting relevant time scale(s) for those vibrations implicated in the "heavy enzyme" isotope effect on the PETNR reaction.

Structure and Mechanism of a Viral Collagen Prolyl Hydroxylase.

The Fe(II)- and 2-oxoglutarate (2-OG)-dependent dioxygenases comprise a large and diverse enzyme superfamily the members of which have multiple physiological roles. Despite this diversity, these enzymes share a common chemical mechanism and a core structural fold, a double-stranded ß-helix (DSBH), as well as conserved active site residues. The prolyl hydroxylases are members of this large superfamily. Prolyl hydroxylases are involved in collagen biosynthesis and oxygen sensing in mammalian cells. Structural-mechanistic studies with prolyl hydroxylases have broader implications for understanding mechanisms in the Fe(II)- and 2-OG-dependent dioxygenase superfamily. Here, we describe crystal structures of an N-terminally truncated viral collagen prolyl hydroxylase (vCPH). The crystal structure shows that vCPH contains the conserved DSBH motif and iron binding active site residues of 2-OG oxygenases. Molecular dynamics simulations are used to delineate structural changes in vCPH upon binding its substrate. Kinetic investigations are used to report on reaction cycle intermediates and compare them to the closest homologues of vCPH. The study highlights the utility of vCPH as a model enzyme for broader mechanistic analysis of Fe(II)- and 2-OG-dependent dioxygenases, including those of biomedical interest.