Xiangshi Tan

Structural insight into substrate preference for TET-mediated oxidation

DNA methylation is an important epigenetic modification. Ten-eleven translocation (TET) proteins are involved in DNA demethylation through iteratively oxidizing 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Here we show that human TET1 and TET2 are more active on 5mC-DNA than 5hmC/5fC-DNA substrates. We determine the crystal structures of TET2–5hmC-DNA and TET2–5fC-DNA complexes at 1.80 Å and 1.97 Å resolution, respectively. The cytosine portion of 5hmC/5fC is specifically recognized by TET2 in a manner similar to that of 5mC in the TET2–5mC-DNA structure, and the pyrimidine base of 5mC/5hmC/5fC adopts an almost identical conformation within the catalytic cavity. However, the hydroxyl group of 5hmC and carbonyl group of 5fC face towards the opposite direction because the hydroxymethyl group of 5hmC and formyl group of 5fC adopt restrained conformations through forming hydrogen bonds with the 1-carboxylate of NOG and N4 exocyclic nitrogen of cytosine, respectively. Biochemical analyses indicate that the substrate preference of TET2 results from the different efficiencies of hydrogen abstraction in TET2-mediated oxidation. The restrained conformation of 5hmC and 5fC within the catalytic cavity may prevent their abstractable hydrogen(s) adopting a favourable orientation for hydrogen abstraction and thus result in low catalytic efficiency. Our studies demonstrate that the substrate preference of TET2 results from the intrinsic value of its substrates at their 5mC derivative groups and suggest that 5hmC is relatively stable and less prone to further oxidation by TET proteins. Therefore, TET proteins are evolutionarily tuned to be less reactive towards 5hmC and facilitate the generation of 5hmC as a potentially stable mark for regulatory functions.

Antitumor Activity of cGAMP via Stimulation of cGAS-cGAMP-STING-IRF3 Mediated Innate Immune Response.

Immunotherapy is one of the key strategies for cancer treatment. The cGAS-cGAMP-STING-IRF3 pathway of cytosolic DNA sensing plays a pivotal role in antiviral defense. We report that the STING activator cGAMP possesses significant antitumor activity in mice by triggering the STING-dependent pathway directly. cGAMP enhances innate immune responses by inducing production of cytokines such as interferon-β, interferon-γ, and stimulating dendritic cells activation, which induces the cross-priming of CD8+ T cells. The antitumor mechanism of cGAMP was verified by STING and IRF3, which were up-regulated upon cGAMP treatment. STING-deficiency dramatically reduced the antitumor effect of cGAMP. Furthermore, cGAMP improved the antitumor activity of 5-FU, and clearly reduced the toxicity of 5-FU. These results demonstrated that cGAMP is a novel antitumor agent and has potential applications in cancer immunotherapy.

Converting cytochrome C into a peroxidase-like metalloenzyme by molecular design.

Hemoproteins, which have a heme prosthetic group, are excellent natural models for artificial design of desired metalloproteins. Various hemoproteins can perform a range of functions by means of combining heme with a different protein scaffold, especially the coordination environment of the heme, which includes the axial ligand, coordination number, and coordination sphere. Consequently, the de novo design of a heme pocket could be a useful strategy for constructing new hemoproteins with desired or novel properties and functions. Although cytochrome c (Cyt c), an electron transfer protein, has no peroxidase activity in living systems, previous studies have shown that it can catalyze a variety of oxidation reactions in the presence of H2O2. [3] Furthermore, characterized by the covalent attachment of a heme to the polypeptide, Cyt c is a very stable protein and has several advantages for use as a peroxidase mimic. 4] In addition, it was recently discovered that the release of Cyt c from mitochondria in the initial stages of apoptosis is related to the cardiolipin oxygenase activity of the cytochrome. This provides a compelling basis for understanding the structural features of the protein that dictates the chemical reactivity of the heme. However, the intrinsic peroxidase activity of Cyt c is suppressed by the protein matrix when compared to typical peroxidases, such as horseradish peroxidase (HRP) and cytochrome c peroxidase (CcP), which have a penta-coordination heme iron and a distal histidine in the heme pocket. Therefore, replacing the sixth axial ligand (Met80) with a non-coordination amino acid and introducing a distal histidine in the heme pocket could convert Cyt c into a peroxidase-like metalloenzyme. To achieve the above-mentioned purpose, we investigated the peroxidase activity of yeast iso-1-cytochrome c (PDB ID: 2YCC) variants in which selected substitutions at the active site had been introduced in an initial effort to mimic some of the structural features of classic peroxidases, such as HRP and CcP. Molecular modeling suggests that replacement of Tyr67 with histidine should place the Ne of His67 at ~5.2 B from the heme iron. This distance was obtained from an energy-minimized simulation of a molecular model based on the PDB file 2YCC, which was created with VMD and NAMD. This distance approximates to that between the Ne of the distal histidine and the heme iron in HRP (PDB ID: 1H5A) and CcP (PDB ID: 2CYP; 5.84 and 5.55 B, respectively). This result, combined with the lack of a coordinated axial ligand in either HRP or CcP, led us to construct and evaluate the peroxidase activity of Cyt c Met80Val, Tyr67His, and Tyr67His/Met80Val variants in which either the sixth axial ligand (Met80) was eliminated and/ or a distal histidine at position 67 was introduced in the heme pocket (Figure 1).

Structural and functional insights into polymorphic enzymes of cytochrome P450 2C8

The cytochrome P450 (CYP) superfamily plays a key role in the oxidative metabolism of a wide range of drugs and exogenous chemicals. CYP2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel in the human liver. Nearly all previous works about polymorphic variants of CYP2C8 were focused on unpurified proteins, either cells or human liver microsomes; therefore their structure–function relationships were unclear. In this study, two polymorphic enzymes of CYP2C8 (CYP2C8.4 (I264M) and CYP2C8 P404A) were expressed in E. coli and purified. Metabolic activities of paclitaxel by the two purified polymorphic enzymes were observed. The activity of CYP2C8.4 was 25% and CYP2C8 P404A was 30% of that of WT CYP2C8, respectively. Their structure–function relationships were systematically investigated for the first time. Paclitaxel binding ability of CYP2C8.4 increased about two times while CYP2C8 P404A decreased about two times than that of WT CYP2C8. The two polymorphic mutant sites of I264 and P404, located far from active site and substrate binding sites, significantly affect heme and/or substrate binding. This study indicated that two important nonsubstrate recognition site (SRS) residues of CYP2C8 are closely related to heme binding and/or substrate binding. This discovery could be valuable for explaining clinically individual differences in the metabolism of drugs and provides instructed information for individualized medication.

Regulating the coordination state of a heme protein by a designed distal hydrogen-bonding network.

Heme coordination state determines the functional diversity of heme proteins. Using myoglobin as a model protein, we designed a distal hydrogen-bonding network by introducing both distal glutamic acid (Glu29) and histidine (His43) residues and regulated the heme into a bis-His coordination state with native ligands His64 and His93. This resembles the heme site in natural bis-His coordinated heme proteins such as cytoglobin and neuroglobin. A single mutation of L29E or F43H was found to form a distinct hydrogen-bonding network involving distal water molecules, instead of the bis-His heme coordination, which highlights the importance of the combination of multiple hydrogen-bonding interactions to regulate the heme coordination state. Kinetic studies further revealed that direct coordination of distal His64 to the heme iron negatively regulates fluoride binding and hydrogen peroxide activation by competing with the exogenous ligands. The new approach developed in this study can be generally applicable for fine-tuning the structure and function of heme proteins.

Evolutionary alkaline transition in human cytochrome c

Conformational transitions in cytochrome c (cyt c) are being realized to be responsible for its multi-functions. Among a number of conformational transitions in cyt c, the alkaline transition has attracted much attention. The cDNA of human cyt c is cloned by RT-PCR and a high-effective expression system for human cyt c has been developed in this study. The equilibrium and kinetics of the alkaline transition of human cyt c have been systematically investigated for the first time, and compared with those of yeast and horse cyt c from an evolutionary perspective. The pKa value for the alkaline transition of human cyt c is apparently higher than that of yeast and horse. Kinetic studies suggest that it is increasingly difficult for the alkaline transition of cyt c from yeast, horse and human. Molecular modeling of human cyt c shows that the omega loop where the lysine residue is located apparently further away from heme in human cyt c than in yeast iso-1 and horse heart cyt c. These results regarding alkaline conformational transition provide valuable information for understanding the molecular basis for the biological multi-functions of cyt c.

Mössbauer Evidence for an Exchange-Coupled {[Fe4S4]1+ Nip1+} A-Cluster in Isolated α Subunits of Acetyl-Coenzyme A Synthase/Carbon Monoxide Dehydrogenase

The active site A-cluster in the alpha subunit of the title enzyme consists of an Fe4S4 cluster coordinated to a [Nip Nid] subcomponent. The cluster must be activated for catalysis using low-potential reductants such as Ti(III) citrate. Relative to the inactive {[Fe4S4]2+ Nip2+ Nid2+} state, the activated state appears to be 2-electrons more reduced, but the location of these electrons within the A-cluster is uncertain, with {[Fe4S4]2+ Nip0 Nid2+} and {[Fe4S4]1+ Nip1+ Nid2+} configurations proposed. Recombinant apo-alpha subunits oligomerize after activation with NiCl2. The dimer fraction, upon reduction with excess Ti(III)citrate, exhibited Mössbauer spectra consisting of two quadrupole doublets representing 51% and 21% of the Fe, with parameters indicating [Fe4S4]1+ states. Spectra recorded in strong magnetic fields were typical of diamagnetic systems, indicating an exchange-coupled S = 0 {[Fe4S4]1+ Nip1+} state. Additional treatment with CO altered the doublet Mössbauer parameters, suggesting an interaction with CO, but maintaining the cluster in the {[Fe4S4]1+ Nip1+} state. Reduction with substoichiometric equivalents of Ti(III) citrate afforded an EPR signal typical of Ni1+ ions, with g parallel = 2.10 and g perpendicular = 2.02. Addition of more Ti caused the signal intensity to decline, suggesting that it arises from the semireduced {[Fe4S4]2+ Nip1+} state.

The mechanism for heme to prevent Aβ1–40 aggregation and its cytotoxicity

The β-amyloid peptide (Aβ) aggregation in the brain, known as amyloid plaques, is a hallmark of Alzheimer’s disease (AD). The aberrant interaction of Cu2+ ion with Aβ potentiates AD by inducing Aβ aggregation and generating neurotoxic reactive oxygen species (ROS). In this study, the biosynthesized recombinant Aβ1–40 was, for the first time, used to investigate the mechanism for heme to prevent Aβ1–40 aggregation and its cytotoxicity. Cell viability studies of SH-SY5Y cells and rat primary hippocampal neurons showed that exogenous heme can protect the cells by reducing cytotoxicity in the presence of Cu2+ and/or Aβ1–40. UV–vis spectroscopy, circular dichroism spectroscopy, and differential pulse voltammetry were applied to examine the interaction between heme and Aβ1–40. It was proven that a heme–Aβ1–40 complex is formed and can stabilize the α-helix structure of Aβ1–40 to inhibit Aβ1–40 aggregation. The heme–Aβ1–40 complex possesses peroxidase activity and it may catalyze the decomposition of H2O2, reduce the generation of ROS downstream, and ultimately protect the cells. These results indicated that exogenous heme is able to alleviate the cytotoxicity induced by Aβ1–40 and Cu2+. This information may be a foundation to develop a potential strategy to treat AD.

Mössbauer and EPR Study of Recombinant Acetyl-CoA Synthase from Moorella thermoacetica†

Mössbauer and EPR spectroscopies were used to study the electronic structure of the A-cluster from recombinant acetyl-CoA synthase (the alpha subunit of the alpha2beta2 acetyl-CoA synthase/CO dehydrogenase). Once activated with Ni, these subunits have properties mimicking those associated with the alpha2beta2 tetramer, including structural heterogeneities. The Fe4S4 portion of the A-cluster in oxidized, methylated, and acetylated states was in the 2+ core oxidation state. Upon reduction with dithionite or Ti3+ citrate, samples of Ni-activated alpha developed the ability to accept a methyl group. Corresponding Mössbauer spectra exhibited two populations of A-clusters; roughly, 70% contained [Fe4S4]1+ cubanes, while approximately 30% contained [Fe4S4]2+ cubanes, suggesting an extremely low [Fe4S4](1+/2+) reduction potential for the 30% portion (perhaps <-800 mV vs NHE). The same population ratio was observed when Ni-free unactivated alpha was used. The 70% fraction exhibited paramagnetic hyperfine structure in the absence of an applied magnetic field, excluding the possibility that it represents an [Fe4S4]1+ cluster coupled to a (proximal) Ni(p)1+. EPR spectra of dithionite-reduced, Ni-activated alpha exhibited features at g = 5.8 and g(ave) approximately 1.93, consistent with a physical mixture of {S = 3/2; S = 1/2} spin-states for A-clusters containing [Fe4S4]1+ clusters. Incubation of Ni-activated alpha with dithionite and CO converted 25% of alpha subunits into the S = 1/2 A(red)-CO state. Previous correlation of this state to functional A-clusters suggests that only the 30% fraction not reduced by dithionite or Ti3+ citrate represents functional A-clusters. Comparison of spin states in oxidized and methylated states suggests that two electrons are required for reductive activation, starting from the oxidized state containing Ni(p)2+. Refitting published activity-vs-potential data supports an n = 2 reductive activation. Enzyme starting in the methylated state exhibited catalytic activity in the absence of an external reductant, suggesting that the two electrons used in reductive activation are retained by the enzyme after each catalytic cycle and that the enzyme does not have to pass through the A(red)-CO state during catalysis. Taken together, our results suggest that a Ni(p)0 state may form upon reductive activation and reform after each catalytic cycle.

A Novel Tyrosine-Heme C-O Covalent Linkage in F43Y Myoglobin: A New Post-translational Modification of Heme Proteins

Heme post‐translational modification plays a key role in tuning the structure and function of heme proteins. We herein report a novel tyrosine–heme covalent CO bond in an artificially produced sperm whale myoglobin (Mb) mutant, F43Y Mb, which formed spontaneously in vivo between the Tyr43 hydroxy group and the heme 4‐vinyl group. This highlights the diverse chemistry of heme post‐translational modifications, and lays groundwork for further investigation of the structural and functional diversity of covalently‐bound heme proteins.