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ApaH decaps Np4N-capped RNAs in two alternative orientations

Nature Chemical Biology (2025)Cite this article

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Abstract

Enigmatic dinucleoside tetraphosphates, known as ‘alarmones’ (Np4Ns), have recently been shown to function in bacteria as precursors to Np4 caps on transcripts, likely influencing RNA longevity and cellular adaptation to stress. In proteobacteria, ApaH is the predominant enzyme that hydrolyzes Np4Ns and decaps Np4-capped RNAs to initiate their 5′-end-dependent degradation. Here we conducted a biochemical and structural study to uncover the catalytic mechanism of Escherichia coli ApaH, a prototypic symmetric Np4N hydrolase, on various Np4Ns and Np4-capped RNAs. We found that the enzyme uses a unique combination of nonspecific and semispecific substrate recognition, enabling substrates to bind in two orientations with a slight orientational preference. Despite such exceptional recognition properties, ApaH efficiently decaps various Np4-capped mRNAs and sRNAs, thereby impacting their lifetimes. Our findings highlight the need to determine substrate orientation preferences before designing substrate-mimicking drugs, as enzymes may escape activity modulation with one of the alternative substrate orientations.

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Fig. 1: Cellular function of E. coli ApaH.
Fig. 2: Promiscuity of decapping of Np4-capped RNAs by E. coli ApaH.
Fig. 3: Structures of E. coli ApaH in the apo and NDP-bound states.
Fig. 4: Binding of Np4N substrates and the catalytic mechanism of ApaH.
Fig. 5: Bidirectional binding of RNA to ApaH.
Fig. 6: Summary of ApaH-mediated decapping of Np4-RNA.

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Data availability

Coordinates of the structures were deposited in the Protein Data Bank with PDB ID codes: 9OJD, apo ApaH; 9OJP, apo ApaH with Mn2+; 9OJX, cocrystallized with GDP; 9OK1, cocrystallized with CDP; 9ON7, cocrystallized with ppAG; 9OLN, cocrystallized with NAD+; 9OND, cocrystallized with ppAGG; 9OJQ, soaked ADP, active conformation; 9OJW, soaked ADP, inactive conformation; 9OK2, soaked UDP; 9OLZ, soaked Ap4A; 9OM9, soaked AppCH2ppA; 9OMC, soaked Gp4G; 9OMU, soaked Gp4A; 9OMW, soaked Up4A; 9OMX, soaked Up4U; 9ON0, D37A variant, soaked Up4A; 9OLY, apo D37A variant; 9ONG, soaked Ap4AG; 9OQ9, soaked Ap4AGG; 9OON, soaked Ap4GU; 9OOY, soaked Gp4AU; 9OPH, soaked Up4AG; 9OQB, soaked Up4AGG; 9OPG, soaked Ap4UG; 9OP2, soaked Ap4GUAA. The MD simulation results were deposited in Zenodo (https://doi.org/10.5281/zenodo.15594844 (ref. 77)). Source data are provided with this paper.

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Acknowledgements

This research used the Northeastern Collaborative Access Team beamlines, funded by the NIH (P30 GM124165), at the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated by Argonne National Laboratory under contract DE-AC02-06CH11357. This research also used beamlines 17-ID-1 (AMX) and 17-ID-2 (FMX) of the National Synchrotron Light Source II, a U.S. DOE Office of Science User Facility operated by Brookhaven National Laboratory under contract DE-SC0012704. Beamline operation is supported by the Center for BioMolecular Structure, funded by the NIH (P30 GM133893) and the DOE Office of Biological and Environmental Research (FWP BO070). R.S.B. is a Damon Runyon Dale F. Frey awardee supported by the Damon Runyon Cancer Research Foundation (DRG-50-22). This research was also supported by the Hirschfelder Professorship Fund and the Research Forward Fund from the University of Wisconsin–Madison (to X.H.) and by the NIH (grants R35GM145359 and R01GM035769 to J.G.B., R37CA289040 to R.S.B. and R01GM112940 and R21GM151508 to A.S.).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York City, NY, USA

    Ashok Nuthanakanti, Megan Korn, Nandhini Rajesh Babu, Robert S. Banh & Alexander Serganov

  2. Laura and Isaac Perlmutter Cancer Center, New York University Grossman School of Medicine, New York City, NY, USA

    Megan Korn, Nandhini Rajesh Babu & Robert S. Banh

  3. Department of Microbiology, New York University Grossman School of Medicine, New York City, NY, USA

    Rose Levenson-Palmer & Joel G. Belasco

  4. Department of Chemistry, University of Wisconsin—Madison, Madison, WI, USA

    Yue Wu & Xuhui Huang

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  1. Ashok Nuthanakanti
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Contributions

A.N. determined the X-ray structures and conducted biochemical experiments. M.K. and N.R.B. performed MS experiments under the supervision of R.S.B. R.L.-P. conducted decapping experiments with long RNAs under the supervision of J.G.B. Y.W. and X.H. conceived and carried out MD simulations. J.G.B. and A.S. wrote the manuscript with the help of A.N.

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Correspondence to Alexander Serganov.

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Nature Chemical Biology thanks Ping Yin and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Specificity of Np4N hydrolysis and RNA decapping by E. coli ApaH.

(a,b) Representative chromatograms showing the kinetics of Ap4A (a) and Ap4G (b) hydrolysis. (c) Sequences and expected secondary structures of long RNA substrates. Left, a representative RNA substrate bearing two RNA hairpins. RNAs used in the study shared the same stem–loop structures. Right, Ap4A8XL RNA used as an internal standard. This RNA contains three RNA hairpins and a long unpaired 5′ segment. (d) A representative gel showing the kinetics of Ap4G0 RNA decapping. Ap4G0 was mixed with the internal standard Ap4A8XL and treated with ApaH. Decapping of each RNA was monitored as a function of time by boronate gel electrophoresis and fluorescence. (e) Decapping of the invariant internal standard Ap4A8XL (Std). Left to right, decapping of Ap4A8XL in the reactions graphed in Fig. 2a–c, respectively. Time points and error bars represent mean ± s.d. of three independent measurements (n = 3). (f–h) Inhibition of ApaH by reaction products. IC50 values, calculated by fitting the data to the ‘inhibitor vs. response’ model of GraphPad Prism, are indicated for Ap4A + ppAG (f) and Ap4AGG + ppAGG (h). Data points and error bars are mean ± s.d. of three independent measurements (n = 3) for Ap4A+ppAG. Data points for all other reactions are from 2 independent measurements (n = 2). The reactions proceeded for 5 min. (f) Inhibition of Np4A hydrolysis by ppAG, ADP, and UDP. (g) Inhibition of Ap4AG hydrolysis by ADP. (h) Inhibition of Ap4AGG hydrolysis by ppAGG.

Source data

Extended Data Fig. 2 Details of cation and NDP recognition by ApaH.

Close-up views of the Mn2+ cations in the apo structure with supplemented Mn2+ (a), soaked with Mg2+ (b), and soaked with ADP (c). Red, violet, and green spheres represent water molecules, Mn2+ and Mg2+ cations, respectively. Final refined structures are shown with the unbiased simulated annealing composite omit maps (light blue mesh) contoured at 1.0–1.1σ levels. Magenta mesh represents the anomalous map at 4 (a) and 3 (b,c) σ levels. (d–g) Unbiased maps (0.8–1.0σ levels) for ApaH-bound ADP (d), GDP (e), CDP (f), and UDP (g). Amino acids contacting the adenine of ADP are in sticks. Dashed dark blue and red lines represent putative hydrogen bonds and CH–π interactions. (h) Superposition of the ADP-bound catalytically competent (gray) and incompetent (light orange) conformations. Top: the active site showing amino acids interacting with phosphates in the catalytically incompetent conformation. A yellow dashed line connects water molecule W1 with the β phosphorus atom. Magenta ovals indicate new or changed hydrogen bonds. Bottom, an arrangement involving the reactive water molecule, Mn2+ cations, and the β-phosphate. Blue arrows show different positions of W1 and the β-phosphate in the two structures. The distance between W1 and the phosphorus atom and the angles between W1, the phosphorus atom, and the non-bridging oxygen atoms are depicted in yellow. (i) Superposition of the apo (cyan) and ADP-bound (gray) structures. Red spheres represent water molecules in the apo structure, including Mn2+-coordinated waters and an extra water molecule (extra W) found in the place of the α-phosphate. Blue arrows show shifts upon ADP binding. (j) ADP binding improves the quality of the unbiased maps (1.0σ level). Trp249 from the apo and ADP-bound structures is in cyan and gray colors, respectively.

Extended Data Fig. 3 Structural and biochemical studies of ApaH targeting Np4N substrates and their analogs.

(a) Hydrolysis of 0.1 mM Ap4A and Ap4AG by 70 nM ApaH in 1 mM NaF for 5 min. The bars are means of two independent experiments (n = 2). (b) Hydrolysis of 0.5 mM AppCH2ppA (AppcppA) by 0.3 mM ApaH in 4 mM Mg2+ and 10 mM Ca2+ for 3 h. (c) Hydrolysis of 0.5 mM NAD+ by 0.3 mM ApaH in 4 mM Mg2+ and 10 mM Ca2+ for 3 h. (d,e) The active site bound to Ap4A (d) and Up4A (e). Refined structures are shown with unbiased simulated annealing composite omit maps (light blue mesh) contoured at 1.0σ level. The adenosine (magenta) and four phosphates are visible in the map and were built into the structure, while the second nucleoside (yellow) was only modeled to show the feasibility of fitting it into the structure. (f) Replacement of Mn1 by a Mg2+ cation in the Ap4A-bound structure. Mn1 has weaker unbiased (top, light blue mesh, 6.0σ level) and anomalous (bottom, magenta mesh, 4.0σ level) maps than Mn2 and was modeled as a mixture of Mn2+ and Mg2+. (g–i) Views of the bound Up4U (g), Gp4G (h), and AppCH2ppA (i) shown with an unbiased map. (j) Superposition of the Np4N-bound structures. Gp4A, orange; Ap4A, magenta; Up4A, purple; Up4U, cyan; Gp4G, dark green; and AppCH2ppA, green. (k) A structural diagram of NAD+. The shading corresponds to the visible (ADP) and disordered (nicotinamide ribose (NR)) moieties. (l) The bound NAD+ with an unbiased map (0.9σ level). The model in yellow represents a visible map and may account for one of the possible positions of the moiety. (m) Superposition of the NAD+-bound (in colors) and ADP-bound (gray) structures.

Extended Data Fig. 4 Effects of mutations on the catalytic activity of ApaH.

(a) Conservation of ApaH in bacteria. The E. coli ApaH sequence is shown with secondary structure elements. Levels of strict sequence conservation were determined from a multiple sequence alignment of a representative set of moderately similar ApaH sequences, as described in Methods. Amino acid identities are: 100%, magenta; 90%, red; 80%, orange; 70%, yellow. The residues involved in metal coordination are indicated in green boxes. (b) Projection of amino acid conservation onto the Ap4A-bound structure. Color code as in (a). (c) Kinetics of Ap4A hydrolysis by ApaH mutants. Data points are from two independent experiments (n = 2) with similar results. The full-time course and initial time points fitted to an exponential decay model and used to calculate rate constants (indicated) are shown in the left and right panels, respectively. (d) A close-up view of the catalytic site of the Asp37Ala mutant in the apo state, shown with unbiased simulated annealing omit maps (light blue mesh) contoured at the 1.0σ level. The red dashed circle indicates the loss of the map for Mn2. (e) Superposition of the catalytic sites from the wild type (gray) and Asp37Ala mutant (colored) bound to Ap4U. Please note that two alternative conformations of the phosphate moieties in the mutant do not align well with the corresponding moieties in the wild-type protein, as shown by a blue arrow. (f–i) Hydrolysis of Ap4A by ApaH mutants: Glu232Ala (f), Glu202Ala (g), Trp249Ala and Trp249Phe (h), and Ser230Gly (i). Left, full-time course, and right, initial rates of hydrolysis. The decapping curve for the WT protein is shown as a dashed line. Data points are from two independent experiments (n = 2) with similar results.

Extended Data Fig. 5 Structural evidence for bidirectional binding of RNA to ApaH.

(a–f) Close-up views of the active site bound to the ppAG (a), Ap4AGG (b), Up4AG (c), Ap4GU (d), Up4AGG (e), and Ap4GUAA (f) RNAs. The refined structures are shown with a simulated annealing composite omit map (light blue mesh) contoured at 0.9–1.0σ level. Dashed circles highlight the areas corresponding to the phosphate of the +2 nucleotide or, in the ppAG structure (a), the area for the γ-phosphate and δ-phosphate of Np4N. Double-headed arrows indicate the orientation of the RNAs in the catalytic site. Arrowhead size is proportional to 18O labeling in the mass spectrometry experiments (Fig. 5c). Blue arrows point to the alternative conformations of the β-phosphate in the ppAG structure (a).

Extended Data Fig. 6 Conformational variability of the disordered RNA regions assessed by MD simulations of the RNA–ApaH complexes.

(a,b) Structural models of the Ap4AG (cyan and magenta)-bound ApaH (green) in the downward (a) and upward (b) orientations used for MD simulations (e). (c) The binding free energy of Ap4AG to ApaH in the downward (cyan) and upward (magenta) orientations. Each bar is a mean ± s.d. derived from ten bootstrapped ensembles (n = 10, using resampled trajectories with replacement). A p-value was calculated using a two-sided t-test based on the average binding free energies from the ten bootstrapped ensembles, with no adjustments for multiple comparisons. (d) Stability of RNA–ApaH binding in MD simulations (e). The plots show time-dependent distance changes between the center of mass of the tetraphosphate group in Ap4AG and the combined center of mass of residues R41 and R184. Results from five independent MD simulations, separated by gray dashed lines, for each system are concatenated. (e) Conformational variability of the disordered RNA regions. Two-dimensional heatmaps show conformational ensembles sampled from restrained MD simulations of the indicated ApaH-bound RNAs. The axes correspond to the first two principal components (PCs) derived from principal component analysis of the Cartesian coordinates of the flexible RNA regions. The adenosine of each RNA was positioned in the nucleoside binding site so that the RNAs with adenosine in the cap (0) or (+1) position were modeled in the downward and upward conformations. The Ap4 moiety was restrained during simulations. The colors indicate conformational density estimated via kernel density estimation, with darker colors representing more frequent conformations. (f) Root mean square fluctuation (RMSF) for each nucleotide of the RNA for the simulations from (e). Each bar is RMSF from 4,000 frames, with a saving interval of 1 ns between two consecutive frames. The systems with RNA in the downward and upward orientations are teal and purple.

Extended Data Fig. 7 The frequency of contacts between RNA and ApaH in MD simulations.

(a–f) The contact analysis was performed based on the simulations in Extended Data Fig. 6e,f for the following RNAs: (a) Ap4GU, (b) Gp4AU, (c) Ap4GUC, (d) Gp4AUC, (e) Ap4GUCU, and (f) Gp4AUCU. The contacts are defined by the distance smaller than 4 Å between heavy protein atoms of amino acid side chains and unrestrained parts of the RNAs. Ap4 moieties were restrained during the MD simulations. The plots (left panels) show the contact frequency, calculated as the ratio of frames with a contact to the total number of frames. For each simulated nucleotide of the RNA (0, +1, +2, +3 or +4), the top ten protein residues are listed. The images (right panels) visualize the contact frequencies on representative protein–RNA complex structures from the MD simulations. RNAs are in cyan sticks. Protein residues selected for mutagenesis are shown as sticks, with darker colors representing higher contact frequencies in each system. The contact residues also contributing to the interactions with the Ap4 group are in red in the left panels and are shown as red lines in the right panels. Mn²⁺ ions are shown as spheres.

Extended Data Fig. 8 Analysis of a positively charged surface near the active site of ApaH.

(a) Multiple sequence alignment for the region corresponding to amino acids 75–88 of E. coli ApaH. The entire protein sequences were aligned well, as evidenced by the absence of gaps and the perfect alignment of the flanking regions, which contain highly conserved residues such as Asn65, His66, Asp67, and Trp101. The figure shows representative sequences with various numbers and distributions of positively charged residues, as well as the absence of these residues in the 75–88 region. (b–e). Effects of the mutations on the hydrolysis of various substrates by E. coli ApaH. Hydrolysis of Ap4A (b,c), Ap4AG (c), and Up4AG (d). Left, full-time course and right, initial rates of hydrolysis, shown with the initial rate constants (k, s−1). The decapping curve for the WT protein is shown as a dashed line. Data points are from two independent experiments (n = 2) with similar results.

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Supplementary Tables 1–7 and Supplementary Figs. 1–5.

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Source Data Fig. 2 and Extended Data Fig. 1

Unprocessed gel images for Fig. 2a–c and Extended Data Fig. 1e.

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Nuthanakanti, A., Korn, M., Levenson-Palmer, R. et al. ApaH decaps Np4N-capped RNAs in two alternative orientations. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-01991-4

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