Using NMR to Monitor TET-Dependent Methylcytosine Dioxygenase Activity and Regulation.

The active removal of DNA methylation marks is governed by the ten-eleven translocation (TET) family of enzymes (TET1-3), which iteratively oxidize 5-methycytosine (5mC) into 5-hydroxymethycytosine (5hmC), and then 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TET proteins are frequently mutated in myeloid malignancies or inactivated in solid tumors. These methylcytosine dioxygenases are α-ketoglutarate (αKG)-dependent and are, therefore, sensitive to metabolic homeostasis. For example, TET2 is activated by vitamin C (VC) and inhibited by specific oncometabolites. However, understanding the regulation of the TET2 enzyme by different metabolites and its activity remains challenging because of limitations in the methods used to simultaneously monitor TET2 substrates, products, and cofactors during catalysis. Here, we measure TET2-dependent activity in real time using NMR. Additionally, we demonstrate that in vitro activity of TET2 is highly dependent on the presence of VC in our system and is potently inhibited by an intermediate metabolite of the TCA cycle, oxaloacetate (OAA). Despite these opposing effects on TET2 activity, the binding sites of VC and OAA on TET2 are shared with αKG. Overall, our work suggests that NMR can be effectively used to monitor TET2 catalysis and illustrates how TET activity is regulated by metabolic and cellular conditions at each oxidation step.

MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis.

Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS-STING-mediated signalling and tumour suppression1-3. However, the precise mechanism of cGAS activation by endogenous DNA damage remains enigmatic, particularly given that high-affinity histone acidic patch (AP) binding constitutively inhibits cGAS by sterically hindering its activation by double-stranded DNA (dsDNA)4-10. Here we report that the DNA double-strand break sensor MRE11 suppresses mammary tumorigenesis through a pivotal role in regulating cGAS activation. We demonstrate that binding of the MRE11-RAD50-NBN complex to nucleosome fragments is necessary to displace cGAS from acidic-patch-mediated sequestration, which enables its mobilization and activation by dsDNA. MRE11 is therefore essential for cGAS activation in response to oncogenic stress, cytosolic dsDNA and ionizing radiation. Furthermore, MRE11-dependent cGAS activation promotes ZBP1-RIPK3-MLKL-mediated necroptosis, which is essential to suppress oncogenic proliferation and breast tumorigenesis. Notably, downregulation of ZBP1 in human triple-negative breast cancer is associated with increased genome instability, immune suppression and poor patient prognosis. These findings establish MRE11 as a crucial mediator that links DNA damage and cGAS activation, resulting in tumour suppression through ZBP1-dependent necroptosis.

Histone H3 proline 16 hydroxylation regulates mammalian gene expression.

Histone post-translational modifications (PTMs) are important for regulating various DNA-templated processes. Here, we report the existence of a histone PTM in mammalian cells, namely histone H3 with hydroxylation of proline at residue 16 (H3P16oh), which is catalyzed by the proline hydroxylase EGLN2. We show that H3P16oh enhances direct binding of KDM5A to its substrate, histone H3 with trimethylation at the fourth lysine residue (H3K4me3), resulting in enhanced chromatin recruitment of KDM5A and a corresponding decrease of H3K4me3 at target genes. Genome- and transcriptome-wide analyses show that the EGLN2-KDM5A axis regulates target gene expression in mammalian cells. Specifically, our data demonstrate repression of the WNT pathway negative regulator DKK1 through the EGLN2-H3P16oh-KDM5A pathway to promote WNT/ß-catenin signaling in triple-negative breast cancer (TNBC). This study characterizes a regulatory mark in the histone code and reveals a role for H3P16oh in regulating mammalian gene expression.

Visualizing a protonated RNA state that modulates microRNA-21 maturation.

MicroRNAs are evolutionarily conserved small, noncoding RNAs that regulate diverse biological processes. Due to their essential regulatory roles, microRNA biogenesis is tightly regulated, where protein factors are often found to interact with specific primary and precursor microRNAs for regulation. Here, using NMR relaxation dispersion spectroscopy and mutagenesis, we reveal that the precursor of oncogenic microRNA-21 exists as a pH-dependent ensemble that spontaneously reshuffles the secondary structure of the entire apical stem-loop region, including the Dicer cleavage site. We show that the alternative excited conformation transiently sequesters the bulged adenine into a noncanonical protonated A+-G mismatch, conferring a substantial enhancement in Dicer processing over its ground conformational state. These results indicate that microRNA maturation efficiency may be encoded in the intrinsic dynamic ensemble of primary and precursor microRNAs, providing a potential means of regulating microRNA biogenesis in response to environmental and cellular stimuli.

Structural basis of nucleosome-dependent cGAS inhibition.

Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) recognizes cytosolic foreign or damaged DNA to activate the innate immune response to infection, inflammatory diseases, and cancer. By contrast, cGAS reactivity against self-DNA in the nucleus is suppressed by chromatin tethering. We report a 3.3-angstrom-resolution cryo-electron microscopy structure of cGAS in complex with the nucleosome core particle. The structure reveals that cGAS uses two conserved arginines to anchor to the nucleosome acidic patch. The nucleosome-binding interface exclusively occupies the strong double-stranded DNA (dsDNA)-binding surface on cGAS and sterically prevents cGAS from oligomerizing into the functionally active 2:2 cGAS-dsDNA state. These findings provide a structural basis for how cGAS maintains an inhibited state in the nucleus and further exemplify the role of the nucleosome in regulating diverse nuclear protein functions.

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions.

Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg(2+) ion concentrations are low, K(+) concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo-like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg(2+) (0.5-2 mM) and K(+) (140 mM) if the solution is supplemented with physiological amounts (∼ 20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperature-dependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution.

Colocalization of fast and slow timescale dynamics in the allosteric signaling protein CheY.

It is now widely recognized that dynamics are important to consider for understanding allosteric protein function. However, dynamics occur over a wide range of timescales, and how these different motions relate to one another is not well understood. Here, we report an NMR relaxation study of dynamics over multiple timescales at both backbone and side-chain sites upon an allosteric response to phosphorylation. The response regulator, Escherichia coli CheY, allosterically responds to phosphorylation with a change in dynamics on both the microsecond-to-millisecond (µs-ms) timescale and the picosecond-to-nanosecond (ps-ns) timescale. We observe an apparent decrease and redistribution of µs-ms dynamics upon phosphorylation (and accompanying Mg(2+) saturation) of CheY. Additionally, methyl groups with the largest changes in ps-ns dynamics localize to the regions of conformational change measured by µs-ms dynamics. The limited spread of changes in ps-ns dynamics suggests a distinct relationship between motions on the µs-ms and ps-ns timescales in CheY. The allosteric mechanism utilized by CheY highlights the diversity of roles dynamics play in protein function.

Segmental motions, not a two-state concerted switch, underlie allostery in CheY.

The switch between an inactive and active conformation is an important transition for signaling proteins, yet the mechanisms underlying such switches are not clearly understood. Escherichia coli CheY, a response regulator protein from the two-component signal transduction system that regulates bacterial chemotaxis, is an ideal protein for the study of allosteric mechanisms. By using 15N CPMG relaxation dispersion experiments, we monitored the inherent dynamic switching of unphosphorylated CheY. We show that CheY does not undergo a two-state concerted switch between the inactive and active conformations. Interestingly, partial saturation of Mg2+ enhances the intrinsic allosteric motions. Taken together with chemical shift perturbations, these data indicate that the µs-ms timescale motions underlying CheY allostery are segmental in nature. We propose an expanded allosteric network of residues, including W58, that undergo asynchronous, local switching between inactive and active-like conformations as the primary basis for the allosteric mechanism.

Detection of native-state nonadditivity in double mutant cycles via hydrogen exchange.

Proteins have evolved to exploit long-range structural and dynamic effects as a means of regulating function. Understanding communication between sites in proteins is therefore vital to our comprehension of such phenomena as allostery, catalysis, and ligand binding/ejection. Double mutant cycle analysis has long been used to determine the existence of communication between pairs of sites, proximal or distal, in proteins. Typically, nonadditivity (or "thermodynamic coupling") is measured from global transitions in concert with a single probe. Here, we have applied the atomic resolution of NMR in tandem with native-state hydrogen exchange (HX) to probe the structure/energy landscape for information transduction between a large number of distal sites in a protein. Considering the event of amide proton exchange as an energetically quantifiable structural perturbation, m n-dimensional cycles can be constructed from mutation of n-1 residues, where m is the number of residues for which HX data is available. Thus, efficient mapping of a large number of couplings is made possible. We have applied this technique to one additive and two nonadditive double mutant cycles in a model system, eglin c. We find heterogeneity of HX-monitored couplings for each cycle, yet averaging results in strong agreement with traditionally measured values. Furthermore, long-range couplings observed at locally exchanging residues indicate that the basis for communication can occur within the native state ensemble, a conclusion not apparent from traditional measurements. We propose that higher-order couplings can be obtained and show that such couplings provide a mechanistic basis for understanding lower-order couplings via "spheres of perturbation". The method is presented as an additional tool for identifying a large number of couplings with greater coverage of the protein of interest.

Regulation of PKR by HCV IRES RNA: importance of domain II and NS5A.

Protein kinase R (PKR) is an essential component of the innate immune response. In the presence of double-stranded RNA (dsRNA), PKR is autophosphorylated, which enables it to phosphorylate its substrate, eukaryotic initiation factor 2alpha, leading to translation cessation. Typical activators of PKR are long dsRNAs produced during viral infection, although certain other RNAs can also activate. A recent study indicated that full-length internal ribosome entry site (IRES), present in the 5'-untranslated region of hepatitis C virus (HCV) RNA, inhibits PKR, while another showed that it activates. We show here that both activation and inhibition by full-length IRES are possible. The HCV IRES has a complex secondary structure comprising four domains. While it has been demonstrated that domains III-IV activate PKR, we report here that domain II of the IRES also potently activates. Structure mapping and mutational analysis of domain II indicate that while the double-stranded regions of the RNA are important for activation, loop regions contribute as well. Structural comparison reveals that domain II has multiple, non-Watson-Crick features that mimic A-form dsRNA. The canonical and noncanonical features of domain II cumulate to a total of approximately 33 unbranched base pairs, the minimum length of dsRNA required for PKR activation. These results provide further insight into the structural basis of PKR activation by a diverse array of RNA structural motifs that deviate from the long helical stretches found in traditional PKR activators. Activation of PKR by domain II of the HCV IRES has implications for the innate immune response when the other domains of the IRES may be inaccessible. We also study the ability of the HCV nonstructural protein 5A (NS5A) to bind various domains of the IRES and alter activation. A model is presented for how domain II of the IRES and NS5A operate to control host and viral translation during HCV infection.

Comparison of the folding mechanism of highly homologous proteins in the lipid-binding protein family.

The folding mechanism of two closely related proteins in the intracellular lipid-binding protein family, human bile acid-binding protein (hBABP), and rat bile acid-binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence. Both of these single domain proteins fit well to a two-state model for unfolding by fluorescence and circular dichroism at equilibrium. Three phases were observed during the unfolding of rBABP by fluorescence but only one phase was observed during the unfolding of hBABP, suggesting that at least two kinetic intermediates accumulate during the unfolding of rBABP that are not observed during the unfolding of hBABP. Fluorine NMR was used to examine the equilibrium unfolding behavior of the W49 side chain in 6-fluorotryptophan-labeled rBABP and hBABP. The structure of rBABP appears to be more dynamic than that of hBABP in the vicinity of W49 in the absence of denaturant, and urea has a greater effect on this dynamic behavior for rBABP than for hBABP. As such, the folding behavior of highly sequence related proteins in this family can be quite different. These differences imply that moderately sized proteins with high sequence and structural similarity can still populate quite different structures during folding.

Monitoring aromatic picosecond to nanosecond dynamics in proteins via 13C relaxation: expanding perturbation mapping of the rigidifying core mutation, V54A, in eglin c.

Long-range effects, such as allostery, have evolved in proteins as a means of regulating function via communication between distal sites. An NMR-based perturbation mapping approach was used to more completely probe the dynamic response of the core mutation V54A in the protein eglin c by monitoring changes in picosecond to nanosecond aromatic side-chain dynamics and H/D exchange stabilities. Previous side-chain dynamics studies on this mutant were limited to methyl-bearing residues, most of which were found to rigidify on the picosecond to nanosecond time scale in the form of a contiguous "network". Here, high precision (13)C relaxation data from 13 aromatic side chains were acquired by applying canonical relaxation experiments to a newly developed carbon labeling scheme [Teilum et al. (2006) J. Am. Chem. Soc. 128, 2506-2507]. The fitting of model-free parameters yielded S (2) variability which is intermediate with respect to backbone and methyl-bearing side-chain variability and tau e values that are approximately 1 ns. Inclusion of the aromatic dynamic response results in an expanded network of dynamically coupled residues, with some aromatics showing increases in flexibility, which partially offsets the rigidification in methyl side chains. Using amide hydrogen exchange, dynamic propagation on a slower time scale was probed in response to the V54A perturbation. Surprisingly, regional stabilization (slowed exchange) 10-12 A from the site of mutation was observed despite a global destabilization of 1.5 kcal x mol (-1). Furthermore, this unlikely pocket of stabilized residues colocalizes with increases in aromatic flexibility on the faster time scale. Because the converse is also true (destabilized residues colocalize with rigidification on the fast time scale), a plausible entropy-driven mechanism is discussed for relating colocalization of opposing dynamic trends on vastly different time scales.

A residual structure in unfolded intestinal fatty acid binding protein consists of amino acids that are neighbors in the native state.

Much of the recent effort in protein folding has focused on the possibility that residual structures in the unfolded state may provide an initiating site for protein folding. This hypothesis is difficult to test because of the weak stability and dynamic behavior of these structures. This problem has been simplified for intestinal fatty acid binding protein (IFABP) by incorporating fluorinated aromatic amino acids during synthesis in Escherichia coli. Only the labeled residues give signals by (19)F NMR, and the 1D spectra can be assigned in both the native and unfolded states by site-directed mutagenesis. One of the two tryptophans (W82), one of the four tyrosines (Y70), and at least four of the eight phenylalanines (including F68 and F93) of IFABP are involved in a structure that is significantly populated at concentrations of urea that unfold the native structure by fluorescence and CD criteria. These residues are nonlocal in sequence and also contact each other in the native structure. Thus, a template of nativelike hydrophobic contacts in the unfolded state may serve as an initiating site for folding this beta-sheet protein.

Swapping core residues in homologous proteins swaps folding mechanism.

Rat intestinal fatty acid binding protein (IFABP) displays an intermediate with little if any secondary structure during unfolding, while the structurally homologous rat ileal lipid binding protein (ILBP) displays an intermediate during unfolding with nativelike secondary structure. Double-jump experiments indicate that these intermediates are on the folding path for each protein. To test the hypothesis that differences in the number of buried hydrophobic atoms in a folding initiating site are responsible for the different types of intermediates observed for these proteins, two mutations (F68C-IFABP and C69F-ILBP) were made that swapped a more hydrophobic residue for a more hydrophilic residue in the respective cores of these two proteins. F68C-IFABP followed an unfolding path identical to that of WT-ILBP with an intermediate that showed nativelike secondary structure, whereas C69F-ILBP followed an unfolding path that was identical to that of WT-IFABP with an intermediate that lacked secondary structure. Further, a hydrophilic residue was introduced at an identical hydrophobic structural position in both proteins (F93S-IFABP and F94S-ILBP). Replacement of phenylalanine with serine at this site led to the appearance of an intermediate during refolding that lacked secondary structure for both proteins that was not detected for either parental protein. Altering the chemical characteristics and/or size of residues within an initiating core of hydrophobic interactions is critical to the types of intermediates that are observed during the folding of these proteins.