Quantitative Chemical Proteomics Approach To Identify Post-translational Modification-Mediated Protein–Protein Interactions

Li, Xiang; Foley, Emily A.; Molloy, Kelly R.; Li, Yinyin; Chait, Brian T.; Kapoor, Tarun M.

doi:10.1021/ja210528v

Cited by 119 publications

(111 citation statements)

References 23 publications

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“…S5B). The MORC3 CW domain exhibited a significant preference for H3K4me2 and H3K4me3 over H3K4me0, consistent with previous reports of human MORC3 binding preferences (12,13). We used isothermal titration calorimetry (ITC) to calculate the binding affinity of the MORC3 CW domain for both unmodified H3K4me0 and H3K4me3 H3 peptides (Fig.…”

Section: Resultssupporting

confidence: 82%

“…1A). The CW-type zinc finger has been identified as an H3K4me reader module as assayed by structural and biochemical methods (11)(12)(13)15). To gain insight into the molecular mechanism of MORC3, we solved the crystal structure of the MORC3 ATPase-CW domain cassette in complex with the nonhydrolyzable ATP analog AMPPNP and H3(1-15)K4me3 peptide by singlewavelength anomalous diffraction.…”

Section: Resultsmentioning

confidence: 99%

“…Animal MORCs also carry a CW-type zinc finger domain, which has been proposed to read histone H3 lysine 4 (H3K4) dimethylation and trimethylation marks (H3K4me2 and H3K4me3) (11)(12)(13). Consistent with this hypothesis, human and murine MORC3 have been identified in mass spectrometry screens as H3K4me3 readers (12)(13)(14).…”

mentioning

confidence: 84%

“…Consistent with this hypothesis, human and murine MORC3 have been identified in mass spectrometry screens as H3K4me3 readers (12)(13)(14). Mutation of a critical tryptophan residue in the human MORC3 CW domain disrupts localization in the nucleus, suggesting that CW domain-mediated recognition of H3K4me3 is also critical for MORC3 targeting to chromatin (6).…”

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confidence: 93%

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Mouse MORC3 is a GHKL ATPase that localizes to H3K4me3 marked chromatin

Yen

Pastor

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Microrchidia (MORC) proteins are GHKL (gyrase, heat-shock protein 90, histidine kinase, MutL) ATPases that function in gene regulation in multiple organisms. Animal MORCs also contain CW-type zinc finger domains, which are known to bind to modified histones. We solved the crystal structure of the murine MORC3 ATPase-CW domain bound to the nucleotide analog AMPPNP (phosphoaminophosphonic acidadenylate ester) and in complex with a trimethylated histone H3 lysine 4 (H3K4) peptide (H3K4me3). We observed that the MORC3 N-terminal ATPase domain forms a dimer when bound to AMPPNP. We used native mass spectrometry to show that dimerization is ATPdependent, and that dimer formation is enhanced in the presence of nonhydrolyzable ATP analogs. The CW domain uses an aromatic cage to bind trimethylated Lys4 and forms extensive hydrogen bonds with the H3 tail. We found that MORC3 localizes to promoters marked by H3K4me3 throughout the genome, consistent with its binding to H3K4me3 in vitro. Our work sheds light on aspects of the molecular dynamics and function of MORC3.X-ray crystallography | histone mark reader | ATPase | native mass spectrometry T he Microrchidia (MORC) family of ATPase proteins has been shown to be an important regulator of gene silencing in multiple organisms. This family was first described in mice, when it was discovered that morc1 null males showed arrested spermatogenesis (1). This arrest was later shown to be associated with transposon derepression, implicating murine MORC1 as a crucial mediator of transposon silencing (2). Arabidopsis thaliana MORC1 and MORC6 were shown to mediate silencing of transposons in a manner largely independent of changes in DNA methylation (3)(4)(5). Studies in Caenorhabditis elegans, which lack DNA methylation, also concluded that the single MORC gene in this organism plays a role in transgene silencing (4). Although the biological importance of MORC ATPases in enforcing gene silencing across multiple organisms is clear, how they are targeted and how they function are poorly understood.The MORC ATPases share a similar domain arrangement. The N terminus contains a GHKL (gyrase, heat-shock protein 90, histidine kinase, MutL) type ATPase domain, and at the C terminus is a coiled-coil segment. MORCs have been reported to form functional homomultimers or heteromultimers, where multimerization is likely mediated by the N-and/or the C-terminal domains (4, 6, 7). The coiled-coil region has been proposed to promote constitutive dimerization, whereas N-terminal ATPase head dimerization occurs only on ATP binding (6). This is consistent with other GHKL ATPases described in the literature, many of which have been reported to undergo ATP-dependent dimerization (8-10). Both plant and animal MORCs are capable of forming nuclear bodies, and mutations that impair ATP binding and/or hydrolysis disrupt nuclear body formation of human MORC3 (6).Animal MORCs also carry a CW-type zinc finger domain, which has been proposed to read histone H3 lysine 4 (H3K4) dimethylation and trimethylation mark...

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Section: Resultssupporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 84%

mentioning

confidence: 93%

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Mouse MORC3 is a GHKL ATPase that localizes to H3K4me3 marked chromatin

Yen

Pastor

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Prior studies have shown that the CW domains of ZCWPW1 (8), MORC3 (10), and MORC4 (9) are readers of H3K4 3 methylated histones with differing preferences for histone H3K4 methylation states (i.e. ZCWPW1 and MORC3 preferentially recognize histone H3K4me3 (8,10), whereas the CW domain of human MORC4 prefers dimethylated H3K4 (9)). The LSD2 CW domain is required for the demethylation function of LSD2 but does not bind to any H3K4 peptides (13).…”

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confidence: 99%

Family-wide Characterization of Histone Binding Abilities of Human CW Domain-containing Proteins

Liu

Tempel

Zhang

et al. 2016

Journal of Biological Chemistry

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Covalent modifications of histone N-terminal tails play a critical role in regulating chromatin structure and controlling gene expression. These modifications are controlled by histone-modifying enzymes and read out by histone-binding proteins. Numerous proteins have been identified as histone modification readers. Here we report the family-wide characterization of histone binding abilities of human CW domain-containing proteins. We demonstrate that the CW domains in ZCWPW2 and MORC3/4 selectively recognize histone H3 trimethylated at Lys-4, similar to ZCWPW1 reported previously, while the MORC1/2 and LSD2 lack histone H3 Lys-4 binding ability. Our crystal structures of the CW domains of ZCWPW2 and MORC3 in complex with the histone H3 trimethylated at Lys-4 peptide reveal the molecular basis of this interaction. In each complex, two tryptophan residues in the CW domain form the "floor" and "right wall," respectively, of the methyllysine recognition cage. Our mutation results based on ZCWPW2 reveal that the right wall tryptophan residue is essential for binding, and the floor tryptophan residue enhances binding affinity. Our structural and mutational analysis highlights the conserved roles of the cage residues of CW domain across the histone methyllysine binders but also suggests why some CW domains lack histone binding ability.Chromatin structure is dynamically regulated by histone post-translational modifications, such as methylation, acetylation, phosphorylation, ubiquitination, and sumoylation (1). These post-translational modifications constitute the "histone code," which is written or erased by histone-modifying enzymes and recognized by histone code "reader" proteins (2-4).Histone methylation, such as lysine methylation at the ⑀-amino group at levels from mono-to trimethylation (me1-me3), has received extensive attention (5). A number of domains bind methylated histone tails. Prominent examples include the chromodomain, Tudor domain, MBT domain, PWWP domain, and PHD domain (4, 6, 7). The CW domain has recently been identified as a new member of the lysine methylation reader family (8 -11).The CW domain is a zinc binding domain, composed of ϳ50 amino acid residues with four conserved cysteine (C) and two conserved tryptophan (W) residues, and its name was derived from these conserved residues. CW domains are found in chromatin-associated proteins in animals and plants and grouped into 12 families based on sequence similarity (12). There are seven CW domain-containing proteins in humans, namely ZCWPW1, ZCWPW2, MORC1, MORC2, MORC3, MORC4, and LSD2 (Fig. 1A). Prior studies have shown that the CW domains of ZCWPW1 (8), MORC3 (10), and MORC4 (9) are readers of H3K4 3 methylated histones with differing preferences for histone H3K4 methylation states (i.e. ZCWPW1 and MORC3 preferentially recognize histone H3K4me3 (8, 10), whereas the CW domain of human MORC4 prefers dimethylated H3K4 (9)). The LSD2 CW domain is required for the demethylation function of LSD2 but does not bind to any H3K4 peptides (13). However, th...

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