Mechanism for autoinhibition and activation of the MORC3 ATPase

Abstract

Microrchidia 3 (MORC3) is a human protein linked to autoimmune disorders, Down syndrome, and cancer. It is a member of a newly identified family of human ATPases with an uncharacterized mechanism of action. Here, we elucidate the molecular basis for inhibition and activation of MORC3. The crystal structure of the MORC3 region encompassing the ATPase and CW domains in complex with a nonhydrolyzable ATP analog demonstrates that the two domains are directly coupled. The extensive ATPase:CW interface stabilizes the protein fold but inhibits the catalytic activity of MORC3. Enzymatic, NMR, mutational, and biochemical analyses show that in the autoinhibited, off state, the CW domain sterically impedes binding of the ATPase domain to DNA, which in turn is required for the catalytic activity. MORC3 autoinhibition is released by disrupting the intramolecular ATPase:CW coupling through the competitive interaction of CW with histone H3 tail or by mutating the interfacial residues. Binding of CW to H3 leads to a marked rearrangement in the ATPase-CW cassette, which frees the DNA-binding site in active MORC3 (on state). We show that ATP-induced dimerization of the ATPase domain is strictly required for the catalytic activity and that the dimeric form of ATPase-CW might cooperatively bind to dsDNA. Together, our findings uncovered a mechanism underlying the fine-tuned regulation of the catalytic domain of MORC3 by the epigenetic reader, CW.

Keywords: ATPase; CW; MORC3; chromatin; histone.

Fig. 1.

Structural basis for MORC3 autoinhibition. (A) MORC3 architecture. (B) Rates of ATP hydrolysis by the indicated domains of MORC3. Error represents SD of at least three separate experiments. To eliminate binding of the ATPase domain to bacterial DNA during sample preparation, the purified proteins were subjected to DNase treatment followed by extensive washes with high-salt buffer. We note that without the additional treatment with DNase and high salts, the ATPase domain shows a high background-level activity due to stimulation by bound bacterial DNA (4). (C) A schematic showing that DNA and H3 are required to activate the MORC3 ATPase–CW cassette. (D) The crystal structure of the dimeric MORC3 ATPase–CW/AMPPNP complex. The ATPase and CW domains are shown as a ribbon with the ATPase domain colored blue in monomer A and white in monomer B, and the CW domain colored wheat. The magnesium (green) and zinc (gray) atoms are shown as spheres. The AMPPNP molecules are in stick representation and colored dark salmon.

Fig. 2.

Autoinhibition is released through disrupting the ATPase:CW interface. (A) A ribbon diagram of the monomer A of ATPase–CW as in Fig. 1F. The ATPase:CW interface residues are shown in stick representation. Dashed lines indicate hydrogen bonds and salt bridges. (B) A zoomed-in view of the ATPase:CW interface shown in A. (C) Superimposed ¹H,¹⁵N TROSY-HSQC spectra of the ¹⁵N-labeled WT and mutated MORC3 CW domain collected upon titration with the unlabeled ATPase domain. Spectra are color-coded according to the protein-to-protein molar ratio. A schematic (Left) specifies that the interfacial residues in CW were mutated. D424H mutant, control. (D and E) Rates of ATP hydrolysis by the MORC3 ATPase–CW cassette, WT and W419A mutant. Error represents SD of at least three separate experiments. (F) Superimposed ¹H,¹⁵N TROSY-HSQC spectra of ¹⁵N-labeled MORC3 CW collected upon titration with the unlabeled K274E/L277E/K280E mutant of His-ATPase (residues 1 to 392 of MORC3). Spectra are color-coded according to the protein-to-protein molar ratio. A schematic (Left) specifies that the interfacial residues in the ATPase domain were mutated. (G and H) Rates of ATP hydrolysis by the MORC3 ATPase–CW cassette, WT and KLK mutant. Error represents SD of three separate experiments.

Fig. 3.

Dimerization of MORC3 is required for ATPase activity. (A) T_ms derived from DSF for the ATPase domain, ATPase–CW cassette, and N-terminally truncated ATPase–CW of MORC3 in the absence (black) or presence (red) of 1 mM AMPPNP or ADP (gray). Error bars represent an SEM based on two separate experiments. (B and C) AMPPNP-induced MORC3 ATPase–CW (B) or ATPase (C) dimerization monitored by DSF. (D) MST binding curve used to determine K_d for the MORC3 ATPase dimerization. Error represents SD between three separate experiments; K_d values ± SD are shown. (E) MST binding curves used to determine K_ds for the MORC3 ATPase–CW dimerization (solid line) and ATPase–CW/H3 interaction (dashed line). Error represents SD between three separate experiments; K_d values ± SD are shown. (F) Ribbon diagram of the ATPase–CW dimer as in Fig. 1E with the N terminus (residues 9 to 16) colored red and encircled by a red oval. (G) Rates of ATP hydrolysis by the MORC3 ATPase–CW cassette, WT and ∆N mutant. Error represents SD of at least three separate experiments.

Fig. 4.

DNA-binding property of MORC3 ATPase–CW. (A) MORC3 ATPase binding to the indicated double-stranded DNA fragments in the presence of AMPPNP monitored by DSF. (B) EMSA with 37-bp dsDNA in the presence of increasing amounts of ATPase with or without AMPPNP. (C) Binding curves used to determine binding affinities of the MORC3 ATPase–CW W419A mutant for DNA by MST. Error represents SD between three separate experiments. (D) Binding affinities of the MORC3 ATPase or ATPase–CW cassette, WT and mutants, to the indicated dsDNAs; K_d values ± SD are shown. ND, not detected. (E) Electrostatic surface potential of the ATPase domain (in the ATPase–CW cassette) is colored blue and red for positive and negative charges, respectively. The CW domain is shown as a ribbon with selected negatively charged D and E residues depicted in stick form. (F) A model of MORC3 activation through the bivalent synergistic interactions with H3 and DNA.

Fig. 5.

MORC3 ATPase–CW binds to the nucleosome. (A) Structural overlay of the autoinhibited (this study; light blue and wheat) and active (PDB ID code 5ix1; white and magenta) (11) states of the MORC3 ATPase–CW cassette. The ATPase and CW domains are shown in ribbon and surface representations, respectively. H3K4me3 peptide in the active state of MORC3 is shown in stick form (yellow). (B) Structural overlay of the CW domain in the autoinhibited state (wheat) bound to the α8–β8 and β8–β9 loops of the ATPase domain (light blue) with the CW domain in the active state (PDB ID code 5ix1; magenta) bound to the H3K4me3 peptide (yellow). (C) EMSA with NCPs in the presence of increasing amounts of ATPase–CW. (D–F) Binding affinities (D) and binding curves (E and F) for the interactions of the indicated MORC3 constructs with NCP as measured by fluorescence polarization. Error bars represent an SD based on three separate experiments.