Catalytic Loop Research Articles

Structural studies of human muscle FBPase.

Muscle fructose-1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate (F6P) and inorganic phosphate, regulates glucose homeostasis by controlling the glyconeogenic pathway. FBPase requires divalent cations, such as Mg2+, Mn2+, or Zn2+, for its catalytic activity; however, calcium ions inhibit the muscle isoform of FBPase by interrupting the movement of the catalytic loop. It has been shown that residue E69 in this loop plays a key role in the sensitivity of muscle FBPase towards calcium ions. The study presented here is based on five crystal structures of wild-type human muscle FBPase and its E69Q mutant in complexes with the substrate and product of the enzymatic reaction, namely F1,6BP and F6P. The ligands are bound in the active site of the studied proteins in the same manner and have excellent definition in the electron density maps. In all studied crystals, the homotetrameric enzyme assumes the same cruciform quaternary structure, with the κ angle, which describes the orientation of the upper dimer with respect to the lower dimer, of -85o. This unusual quaternary arrangement of the subunits, characteristic of the R-state of muscle FBPase, is also observed in solution by small-angle X-ray scattering (SAXS).

Identification of Thermal Conduits That Link the Protein-Water Interface to the Active Site Loop and Catalytic Base in Enolase.

We report here on the salient role of protein mobility in accessing conformational landscapes that enable efficient enzyme catalysis. We are focused on yeast enolase, a highly conserved lyase with a TIM barrel domain and catalytic loop, as part of a larger study of the relationship of site selective protein motions to chemical reactivity within superfamilies. Enthalpically hindered variants were developed by replacement of a conserved hydrophobic side chain (Leu 343) with smaller side chains. Leu343 is proximal to the active site base in enolase, and comparative pH rate profiles for the valine and alanine variants indicate a role for side chain hydrophobicity in tuning the pKa of the catalytic base. However, the magnitude of a substrate deuterium isotope effect is almost identical for wild-type (WT) and Leu343Ala, supporting an unchanged rate-determining proton abstraction step. The introduced hydrophobic side chains at position 343 lead to a discontinuous break in both activity and activation energy as a function of side chain volume. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments were performed as a function of time and temperature for WT and Leu343Ala, and provide a spatially resolved map of changes in protein flexibility following mutation. Impacts on protein flexibility are localized to specific networks that arise at the protein-solvent interface and terminate in a loop that has been shown by X-ray crystallography to close over the active site. These interrelated effects are discussed in the context of long-range, solvent-accessible and thermally activated networks that play key roles in tuning the precise distances and interactions among reactants.

Structure and mechanism of Staphylococcus aureus oleate hydratase (OhyA)

Flavin adenine dinucleotide (FAD)-dependent bacterial oleate hydratases (OhyAs) catalyze the addition of water to isolated fatty acid carbon–carbon double bonds. Staphylococcus aureus uses OhyA to counteract the host innate immune response by inactivating antimicrobial unsaturated fatty acids. Mechanistic information explaining how OhyAs catalyze regiospecific and stereospecific hydration is required to understand their biological functions and the potential for engineering new products. In this study, we deduced the catalytic mechanism of OhyA from multiple structures of S. aureus OhyA in binary and ternary complexes with combinations of ligands along with biochemical analyses of relevant mutants. The substrate-free state shows Arg81 is the gatekeeper that controls fatty acid entrance to the active site. FAD binding engages the catalytic loop to simultaneously rotate Glu82 into its active conformation and Arg81 out of the hydrophobic substrate tunnel, allowing the fatty acid to rotate into the active site. FAD binding also dehydrates the active site, leaving a single water molecule connected to Glu82. This active site water is a hydronium ion based on the analysis of its hydrogen bond network in the OhyA•PEG400•FAD complex. We conclude that OhyA accelerates acid-catalyzed alkene hydration by positioning the fatty acid double bond to attack the active site hydronium ion, followed by the addition of water to the transient carbocation intermediate. Structural transitions within S. aureus OhyA channel oleate to the active site, curl oleate around the substrate water, and stabilize the hydroxylated product to inactivate antimicrobial fatty acids.

Synthesis, crystal structure and ligand based catalytic activity of octahedral salen Schiff base Co(III) compounds

Two octahedral cobalt(III) compounds ([CoL1CN(H2O)]-DMF (1) and [CoL2L3] - H2O (2)) were synthesized by using two Schiff bases, H2L1 and H2L2 and are well-characterized by different spectral methods. Catalytic ability of the compounds were studied by the catecholase oxidation reaction. Among the two compounds, compound 2 with bare aldehydic type coligand catalysed the 3,5-di-tertiarybutylcatechol (3,5-DTBC) into corresponding quinone. Here, the nature of the ligand exhibited a significant role in the oxidation of the substrate. A catalytic loop is proposed to explain the mechanistic aspect of the reaction and different kinetic parameters (Vmax (3.31 × 10−3 M) and KM (3.43 × 10−3 M min−1) were also determined.

Identification of fusarielin M as a novel inhibitor of Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB)

The secreted Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase B (MptpB) is an essential virulence factor required for the intracellular survival of Mtb within host macrophages. MptpB has become a promising target for the development of novel anti-tuberculosis (TB) drugs. In this study, two new fusarielins, fusarielins M (1) and N (2), and a biogenetically related known compound, fusarielin G (3) were isolated from the marine-derived fungus Fusarium graminearum SYSU-MS5127. Their inhibitory effects on MptpB were evaluated. Among these compounds, fusarielin M substantially inhibited MptpB with a half-maximal inhibitory concentration (IC50) of 1.05 ± 0.08 μM, and an inhibition constant (Ki) of 1.03 ± 0.39 μM. Surface plasmon resonance analysis was used to characterize the interaction between fusarielin M and MptpB in vitro. Fusarielin M also exhibited cellular activity in blocking MptpB-mediated Erk1/2 and p38 inactivation in macrophages. Importantly, fusarielin M (20 μM) substantially reduced intracellular mycobacterial growth within macrophages, causing a 62% reduction in the bacterial burden. The binding mode of fusarielin M was further explored via molecular docking which suggested that fusarielin M binds to the active site of MptpB, forming a hydrogen bond with the side chain of Asp165; this is unique in the P-loop of MptpB compared to conventional human PTPs. The contact between fusarielin M and Asp165 in the catalytic loop provides a potential basis for inhibitor selectivity. Therefore, fusarielin M shows great potential as an anti-TB drug candidate.

K27-Linked Diubiquitin Inhibits UCHL3 via an Unusual Kinetic Trap

Functional analysis of lysine 27-linked ubiquitin chains (K27Ub) is difficult due to the inability to make them through enzymatic methods and due to a lack of model tools and substrates. Here we generate a series of ubiquitin (Ub) tools to study how the deubiquitinase UCHL3 responds to K27Ub chains in comparison to lysine 63-linked chains and mono-Ub. From a crystal structure of a complex between UCHL3 and synthetic K27Ub2, we unexpectedly discover that free K27Ub2 and K27Ub2-conjugated substrates are natural inhibitors of UCHL3. Using our Ub tools to profile UCHL3's activity, we generate a quantitative kinetic model of the inhibitory mechanism and we find that K27Ub2 can inhibit UCHL3 covalently, by binding to its catalytic cysteine, and allosterically, by locking its catalytic loop tightly in place. Based on this inhibition mechanism, we propose that UCHL3 and K27Ub chains likely sense and regulate each other in cells.

Crystallographic Snapshots of the Dunathan and Quinonoid Intermediates provide Insights into the Reaction Mechanism of Group II Decarboxylases

PLP-dependent enzymes catalyze a plethora of chemical reactions affecting diverse physiological functions. Here we report the structural determinants of the reaction mechanism in a Group II PLP-dependent decarboxylase by assigning two early intermediates. The in-crystallo complexes of the PLP bound form, and the Dunathan and quinonoid intermediates, allowed direct observation of the active site interactions. The structures reveal that a subtle rearrangement of a conserved Arg residue in concert with a water-mediated interaction with the carboxylate of the Dunathan intermediate, appears to directly stabilize the alignment and facilitate the release of CO2 to yield the quinonoid. Modeling indicates that the conformational change of a dynamic catalytic loop to a closed form controls a conserved network of hydrogen bond interactions between catalytic residues to protonate the quinonoid. Our results provide a structural framework to elucidate mechanistic roles of residues that govern reaction specificity and catalysis in PLP-dependent decarboxylation.

Understanding the Redox Reaction of Self-Assembled Ferrocence-Containing Polymer Particle Comprising Ferrocenylmethyl Methacrylate–Methacrylic Acid Random Copolymers

In this study, the electrochemistry of Ferrocene-containing polymer particles (Fc-PPs) dispersed in an aqueous solution is investigated owing to the importance of Fc in drug-delivery systems, wherein drug release is triggered by reactive oxygen species (ROS). The mechanism of Fc+/Fc redox reaction in an aqueous solution containing Fc-PP was evaluated, where Fc-PP was self-assembled from Fc-containing polymers (Fc-Ps) synthesized via random copolymerization of ferrocenylmethyl methacrylate (FMMA) and methacrylic acid (MA). The cyclic voltammogram (CV) of the aqueous solution containing Fc-PP on a Pt ultramicroelectrode (UME) was sigmoidal, which is the typical shape observed when dissolved redox-active species are electrochemically oxidized or reduced under mass-transfer controlled conditions. Chronoamperometric analysis of the reaction of Fc-PP dispersed in solution on the Pt UME showed no stochastic current spikes associated with the direct electro-oxidation of Fc to Fc+ in the Fc-PP via collision. The absence of stochastic current spikes mainly resulted from slow e‒ transport through Fc-PP, which indicates its nature as an e‒ insulator. Thus, the voltammetric current was not directly attributed to the electrochemical redox reaction of Fc-PP on the Pt UME. It is proposed that the Faradaic current can be attributed to Fc-P species that were slightly dissociated from Fc-PP at equilibrium, where the former have a higher degree of freedom in the aqueous phase, which promotes facilitated e‒ transport from Fc-P to Pt UME or vice versa. Finite element analysis based on this model adequately explained the experimentally obtained voltammetric curves. From the simulation, redox-active Fc-P possibly reacted with Fc-PP via galvanic exchange, resulting in a catalytic loop and consequent current amplification. Nonetheless, the data revealed that this catalytic current induced by the galvanic exchange reaction would not be significant. The electrochemical analyses strongly indicate that charge transfer between Fc in Fc-PP and ROS (such as H2O2) would be the main rate determining step for the drug-release system, as previously reported (J. Mater. Chem. B 2020, 8, 1906).

TGFβ acts through PDGFRβ to activate mTORC1 via the Akt/PRAS40 axis and causes glomerular mesangial cell hypertrophy and matrix protein expression

Interaction of transforming growth factor-β (TGFβ)-induced canonical signaling with the noncanonical kinase cascades regulates glomerular hypertrophy and matrix protein deposition, which are early features of glomerulosclerosis. However, the specific target downstream of the TGFβ receptor involved in the noncanonical signaling is unknown. Here, we show that TGFβ increased the catalytic loop phosphorylation of platelet-derived growth factor receptor β (PDGFRβ), a receptor tyrosine kinase expressed abundantly in glomerular mesangial cells. TGFβ increased phosphorylation of the PI 3-kinase-interacting Tyr-751 residue of PDGFRβ, thus activating Akt. Inhibition of PDGFRβ using a pharmacological inhibitor and siRNAs blocked TGFβ-stimulated phosphorylation of proline-rich Akt substrate of 40 kDa (PRAS40), an intrinsic inhibitory component of mTORC1, and prevented activation of mTORC1 in the absence of any effect on Smad 2/3 phosphorylation. Expression of constitutively active myristoylated Akt reversed the siPDGFRβ-mediated inhibition of mTORC1 activity; however, co-expression of the phospho-deficient mutant of PRAS40 inhibited the effect of myristoylated Akt, suggesting a definitive role of PRAS40 phosphorylation in mTORC1 activation downstream of PDGFRβ in mesangial cells. Additionally, we demonstrate that PDGFRβ-initiated phosphorylation of PRAS40 is required for TGFβ-induced mesangial cell hypertrophy and fibronectin and collagen I (α2) production. Increased activating phosphorylation of PDGFRβ is also associated with enhanced TGFβ expression and mTORC1 activation in the kidney cortex and glomeruli of diabetic mice and rats, respectively. Thus, pursuing TGFβ noncanonical signaling, we identified how TGFβ receptor I achieves mTORC1 activation through PDGFRβ-mediated Akt/PRAS40 phosphorylation to spur mesangial cell hypertrophy and matrix protein accumulation. These findings provide support for targeting PDGFRβ in TGFβ-driven renal fibrosis.

Unliganded and CMP-Neu5Ac bound structures of human α-2,6-sialyltransferase ST6Gal I at high resolution

Sialic acid residues found as terminal monosaccharides in various types of glycan chains in cell surface glycoproteins and glycolipids have been identified as important contributors of cell-cell interactions in normal vs. abnormal cellular behavior and are pivotal in diseases such as cancers. In vertebrates, sialic acids are attached to glycan chains by a conserved subset of sialyltransferases with different enzymatic and substrate specificities. ST6Gal I is a sialyltransferase using activated CMP-sialic acids as donor substrates to catalyze the formation of a α2,6-glycosidic bond between the sialic acid residue and the acceptor disaccharide LacNAc. Understanding sialyltransferases at the molecular and structural level shed light into their function. We present here two human ST6Gal I structures, which show for the first time the enzyme in the unliganded state and with the full donor substrate CMP-Neu5Ac bound. Comparison of these structures reveal flexibility of the catalytic loop, since in the unliganded structure Tyr354 adopts a conformation seen also as an alternate conformation in the substrate bound structure. CMP-Neu5Ac is bound with the side chain at C5 of the sugar residue directed outwards at the surface of the protein. Furthermore, the exact binding mode of the sialic acid moiety of the substrate directly involves sialylmotifs L, S and III and positions the sialylmotif VS in the immediate vicinity. We also present a model for the ternary complex of ST6Gal I with both the donor and the acceptor substrates.

Probing the Highly Disparate Dual Inhibitory Mechanisms of Novel Quinazoline Derivatives against Mycobacterium tuberculosis Protein Kinases A and B.

Mycobacterium tuberculosis (Mtb) serine/threonine (Ser/Thr) Protein kinases A (PknA) and B (PknB) have been identified as highly attractive targets for overcoming drug resistant tuberculosis. A recent lead series optimization study yielded compound 33 which exhibited potencies ~1000 times higher than compound 57. This huge discrepancy left us curious to investigate the mechanistic ‘dual’ (in)activities of the compound using computational methods, as carried out in this study. Findings revealed that 33 stabilized the PknA and B conformations and reduced their structural activities relative to 57. Optimal stability of 33 in the hydrophobic pockets further induced systemic alterations at the P-loops, catalytic loops, helix Cs and DFG motifs of PknA and B. Comparatively, 57 was more surface-bound with highly unstable motions. Furthermore, 33 demonstrated similar binding patterns in PknA and B, involving conserved residues of their binding pockets. Both π and hydrogen interactions played crucial roles in the binding of 33, which altogether culminated in high ΔGs for both proteins. On the contrary, the binding of 57 was characterized by unfavorable interactions with possible repulsive effects on its optimal dual binding to both proteins, as evidenced by the relatively lowered ΔGs. These findings would significantly contribute to the rational structure-based design of novel and highly selective dual inhibitors of Mtb PknA and B.

Snapshots of ADP-ribose bound to Getah virus macro domain reveal an intriguing choreography

Alphaviruses are (re-)emerging arboviruses of public health concern. The nsP3 gene product is one of the key players during viral replication. NsP3 comprises three domains: a macro domain, a zinc-binding domain and a hypervariable region. The macro domain is essential at both early and late stages of the replication cycle through ADP-ribose (ADPr) binding and de-ADP-ribosylation of host proteins. However, both its specific role and the precise molecular mechanism of de-ADP-ribosylation across specific viral families remains to be elucidated. Here we investigate by X-ray crystallography the mechanism of ADPr reactivity in the active site of Getah virus macro domain, which displays a peculiar substitution of one of the conserved residues in the catalytic loop. ADPr adopts distinct poses including a covalent bond between the C′′1 of the ADPr and a conserved Togaviridae-specific cysteine. These different poses observed for ADPr may represent snapshots of the de-ADP-ribosylation mechanism, highlighting residues to be further characterised.

Reduction of hazardous SO2 into elemental sulphur over chicken eggshells-derived calcium-based redox agent: A systematic step-by-step thermodynamic analysis and process validations

Current work demonstrated the thermodynamic feasibility of SO2 reduction into elemental S via catalytic reaction-regeneration loop over chicken eggshells-derived Ca-based redox agent, while the experimental results were presented subsequent to thermodynamics analysis for validating purpose. Ideally, CaS, engineered from chicken eggshells, would facilitate the SO2 reduction upon oxygen-transferring process, resulting in CaSO4 and sulphur solid. Thermodynamic investigations via minimization of Gibb’s Energy (ΔGmin) were initiated with the development of Ca-based redox agent from modelled chicken eggshells, followed by pre-treatment of redox agent and finally the SO2 reduction reaction. Based on the thermodynamic analysis, Chicken eggshell, modelled by CaCO3, is mainly converted into functionalized CaS through dual-sulphurization process, favorably at mild temperature of 623–1023 K. At optimum 1023 K, the obtained product thermodynamically consists of CaSO4 (74.97%) and CaS (24.79%), which required further treatment to maximize CaS content prior to SO2 reduction reaction. H2- and CO-reductions indicate that conversion into 100% of desirable CaS are thermodynamically feasible, with H2-reduction preferred due to its lesser exothermic behavior. As for the SO2 reduction, CaS exhibits an excellent activity in reducing SO2 to elemental S (S2–S8) at relatively low temperature of 723–973 K, with exceptionally high yield (>99%). Upon increasing temperature (>1173 K), adverse effects to both SO2 conversion and sulphur yield were observed, implying the impracticality in laboratory executions. Simple, yet powerful experiments were conducted, subsequently validated the thermodynamic estimations in current study. This systematic study comprehensively investigated and justified the feasibility of entire process, hence providing provisional guidelines for the future execution of laboratory works.

Genome-wide and structural analyses of pseudokinases encoded in the genome of Arabidopsis thaliana provide functional insights.

Protein Kinase-Like Non-Kinases (PKLNKs), commonly known as "pseudokinases", are homologous to eukaryotic Ser/Thr/Tyr protein kinases (PKs) but lack the crucial aspartate residue in the catalytic loop, indispensable for phosphotransferase activity. Therefore, they are predicted to be "catalytically inactive" enzyme homologs. Analysis of protein-kinase like sequences from Arabidopsis thaliana led to the identification of more than 120 pseudokinases lacking catalytic aspartate, majority of which are closely related to the plant-specific receptor-like kinase family. These pseudokinases engage in different biological processes, enabled by their diverse domain architectures and specific subcellular localizations. Structural comparison of pseudokinases with active and inactive conformations of canonical PKs, belonging to both plant and animal origin, revealed unique structural differences. The currently available crystal structures of pseudokinases show that the loop topologically equivalent to activation segment of PKs adopts a distinct-folded conformation, packing against the pseudoenzyme core, in contrast to the extended and inhibitory geometries observed for active and inactive states, respectively, of catalytic PKs. Salt-bridge between ATP-binding Lys and DFG-Asp as well as hydrophobic interactions between the conserved nonpolar residue C-terminal to the equivalent DFG motif and nonpolar residues in C-helix mediate such a conformation in pseudokinases. This results in enhanced solvent accessibility of the pseudocatalytic loop in pseudokinases that can possibly serve as an interacting surface while associating with other proteins. Specifically, our analysis identified several residues that may be involved in pseudokinase regulation and hints at the repurposing of pseudocatalytic residues to achieve mechanistic control over noncatalytic functions of pseudoenzymes.

Diarylurea derivatives comprising 2,4-diarylpyrimidines: Discovery of novel potential anticancer agents via combined failed-ligands repurposing and molecular hybridization approaches

A series of diarylurea derivatives comprising 2,4-diarylpyrimidines were synthesized based on a combination of postulated molecular hybridization design and failed-ligands repurposing approaches, which enabled the discovery of novel potential antiproliferative agents. Towards credible biological evaluation, an in vitro anticancer activity assay was conducted employing a library of 60 cancer cell lines constituting nine panels representing blood, lung, colon, CNS, skin, ovary, renal, prostate, and breast cancers. The results revealed high effectiveness and broad-spectrum anticancer activity of compounds 4m and 4g. Five-dose assay of compounds 4m and 4g proved their high potency that surpassed that of four standard kinase inhibitors FDA-approved anticancer drugs against many cancer cells. Towards the identification of their molecular target, screening of kinase inhibitory profile employing a panel of 51 kinases involved in cancer revealed inhibition of several kinases from the platelet-derived growth factor/vascular endothelial growth factor receptor (PVR) kinase family, which might mediate, at least in part, the antiproliferative activity. Molecular docking of 4g into the crystal structure of the Feline McDonough Sarcoma (FMS) kinase predicted that it binds to a pocket formed by the juxtamembrane domain, the catalytic loop, and the αE helix, thus stabilizing the inhibited conformation of the kinase. Flow cytometric study of the cytotoxic effects of compound 4g in A549 cells showed it induces dose- and time-dependent apoptotic events leading to cell death. Collectively, this work presents compound 4g as a potential broad-spectrum anticancer agent against multiple cancer types.

Structure-Based Virtual Screening to Discover Potential Lead Molecules for the SARS-CoV-2 Main Protease.

The COVID-19 disease is caused by a new strain of the coronavirus family (SARS-CoV-2), and it has affected at present millions of people all over the world. The indispensable role of the main protease (Mpro) in viral replication and gene expression makes this enzyme an attractive drug target. Therefore, inhibition of SARS-CoV-2 Mpro as a proposition to halt virus ingression is being pursued by scientists globally. Here we carried out a study with two objectives: the first being to perform comparative protein sequence and 3D structural analysis to understand the effect of 12 point mutations on the active site. Among these, two mutations, viz., Ser46 and Phe134, were found to cause a significant change at the active sites of SARS-CoV-2. The Ser46 mutation present at the entrance of the S5 subpocket of SARS-CoV-2 increases the contribution of other two hydrophilic residues, while the Phe134 mutation, present in the catalytic cysteine loop, can cause an increase in catalytic efficiency of Mpro by facilitating fast proton transfer from the Cys145 to His41 residue. It was observed that active site remained conserved among Mpro of both SARS-CoVs, except at the entrance of the S5 subpocket, suggesting sustenance of substrate specificity. The second objective was to screen the inhibitory effects of three different data sets (natural products, coronaviruses main protease inhibitors, and FDA-approved drugs) using a structure-based virtual screening approach. A total of 73 hits had a combo score >2.0. Eight different structural scaffold classes were identified, such as one/two tetrahydropyran ring(s), dipeptide/tripeptide/oligopeptide, large (approximately 20 atoms) cyclic peptide, and miscellaneous. The screened hits showed key interactions with subpockets of the active site. Further, molecular dynamics studies of selected screened compounds confirmed their perfect fitting into the subpockets of the active site. This study suggests promising structures that can fit into the SARS-CoV-2 Mpro active site and also offers direction for further lead optimization and rational drug design.

Molecular Features and Metal Ions That Influence 10-23 DNAzyme Activity.

Deoxyribozymes (DNAzymes) with RNA hydrolysis activity have a tremendous potential as gene suppression agents for therapeutic applications. The most extensively studied representative is the 10-23 DNAzyme consisting of a catalytic loop and two substrate binding arms that can be designed to bind and cleave the RNA sequence of interest. The RNA substrate is cleaved between central purine and pyrimidine nucleotides. The activity of this DNAzyme in vitro is considerably higher than in vivo, which was suggested to be related to its divalent cation dependency. Understanding the mechanism of DNAzyme catalysis is hindered by the absence of structural information. Numerous biological studies, however, provide comprehensive insights into the role of particular deoxynucleotides and functional groups in DNAzymes. Here we provide an overview of the thermodynamic properties, the impact of nucleobase modifications within the catalytic loop, and the role of different metal ions in catalysis. We point out features that will be helpful in developing novel strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. Consideration of these features will enable to develop improved strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. These insights provide the basis for improving activity in cells and pave the way for developing DNAzyme applications.

Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms

Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases, DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterization of DNMT3A and DNMT3B, we here report a multi-layered substrate-recognition mechanism underpinning their divergent genomic methylation activities. A hydrogen bond in the catalytic loop of DNMT3B causes a lower CpG specificity than DNMT3A, while the interplay of target recognition domain and homodimeric interface fine-tunes the distinct target selection between the two enzymes, with Lysine 777 of DNMT3B acting as a unique sensor of the +1 flanking base. The divergent substrate preference between DNMT3A and DNMT3B provides an explanation for site-specific epigenomic alterations seen in ICF syndrome with DNMT3B mutations. Together, this study reveals distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis.

Allosteric Impact of the Variable Insert Loop in Vaccinia H1-Related (VHR) Phosphatase.

Dynamics and conformational motions are important to the activity of enzymes, including protein tyrosine phosphatases. These motions often extend to regions outside the active site, called allosteric regions. In the tyrosine phosphatase Vaccinia H1-related (VHR) enzyme, we demonstrate the importance of the allosteric interaction between the variable insert region and the active-site loops in VHR. These studies include solution nuclear magnetic resonance, computation, steady-state, and rapid kinetic measurements. Overall, the data indicate concerted millisecond motions exist between the variable insert and the catalytic acid loop in wild-type (WT) VHR. The 150 ns computation studies show a flexible acid loop in WT VHR that opens during the simulation from its initial closed structure. Mutation of the variable insert residue, asparagine 74, to alanine results in a rigidification of the acid loop as observed by molecular dynamics simulations and a disruption of crucial active-site hydrogen bonds. Moreover, enzyme kinetic analysis shows a weakening of substrate affinity in the N74A mutant and a >2-fold decrease in substrate cleavage and hydrolysis rates. These data show that despite being nearly 20 Å from the active site, the variable insert region is linked to the acid loop by coupled millisecond motions, and that disruption of the communication between the variable insert and active site alters the normal catalytic function of VHR and perturbs the active-site environment.

Functional analysis of an R311C variant of Ca2+ -calmodulin-dependent protein kinase kinase-2 (CaMKK2) found as a de novo mutation in a patient with bipolar disorder.

Loss-of-function mutations in the gene encoding the calcium-calmodulin (Ca2+ -CaM)-dependent protein kinase kinase-2 (CaMKK2) enzyme are linked to bipolar disorder. Recently, a de novo arginine to cysteine (R311C) mutation in CaMKK2 was identified from a whole exome sequencing study of bipolar patients and their unaffected parents. The aim of the present study was to determine the functional consequences of the R311C mutation on CaMKK2 activity and regulation by Ca2+ -CaM. The effects of the R311C mutation on CaMKK2 activity and Ca2+ -CaM activation were examined using a radiolabeled adenosine triphosphate (ATP) kinase assay. We performed immunoblot analysis to determine whether the R311C mutation impacts threonine-85 (T85) autophosphorylation, an activating phosphorylation site on CaMKK2 that has also been implicated in bipolar disorder. We also expressed the R311C mutant in CaMKK2 knockout HAP1 cells and used immunoblot analysis and an MTS reduction assay to study its effects on Ca2+ -dependent downstream signaling and cell viability, respectively. The R311C mutation maps to the conserved HRD motif within the catalytic loop of CaMKK2 and caused a marked reduction in kinase activity and Ca2+ -CaM activation. The R311C mutation virtually abolished T85 autophosphorylation in response to Ca2+ -CaM and exerted a dominant-negative effect in cells as it impaired the ability of wild-type CaMKK2 to initiate downstream signaling and maintain cell viability. The highly disruptive, loss-of-function impact of the de novo R311C mutation in human CaMKK2 provides a compelling functional rationale for being considered a potential rare monogenic cause of bipolar disorder.