Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION diversity and dynamics of the human proteome through Structural basis for substrate regulating protein three-dimensional (3D) structure, recognition by the human stability, activity, localization, and interaction with other cellular molecules, including proteins and nucleic acids. N-terminal methyltransferase 1 PTMs can occur on both the amino acid side chains and 1,5 2,3,5 1 backbones of proteins, often by covalent attachment of Cheng Dong, Yunfei Mao, Wolfram Tempel, α 1 1 1 2,3 functional groups such as the methyl group. -N-terminal Su Qin, Li Li, Peter Loppnau, Rong Huang, methylation is a ubiquitously present PTM that involves and Jinrong Min1,4 the methylation of the α-amino group of the newly ex- 1 posed N-terminal amino acid following cleavage of the Structural Genomics Consortium, University of Toronto, initiating methionine. Although α-N-terminal methyla- 2 Toronto, Ontaria M5G 1L7, Canada; Department of Medicinal tion was first identified almost 40 years ago (Brosius and 3 Chemistry, The Institute for Structural Biology, Drug Discovery Chen 1976; Wittmann-Liebold and Pannenbecker 1976; and Development, Virginia Commonwealth University, Stock et al. 1987) and is conserved from prokaryotes to hu- 4 Richmond, Virginia 23219, USA; Department of Physiology, mans, its function remains elusive and comparatively University of Toronto, Toronto, Ontario M5S 1A8, Canada underexplored. α-N-terminal methylation has been hy- α-N-terminal methylation represents a highly conserved pothesized to regulate protein stability via N-end rule – and prevalent post-translational modification, yet its bio- pathways or mediate protein protein interactions (Tooley and Schaner Tooley 2014). Intriguingly, α-N-termi- logical function has remained largely speculative. The re- – α nal methylation was recently found to mediate protein cent discovery of -N-terminal methyltransferase 1 DNA interactions between chromatin and regulator of (NTMT1) and its physiological substrates propels the elu- chromatin condensation (RCC1) (Chen et al. 2007; cidation of a general role of α-N-terminal methylation in Hitakomate et al. 2010), potentially through the posi- mediating DNA-binding ability of the modified proteins. tively charged and exhaustively methylated N terminus The phenotypes, observed from both NTMT1 knockdown of RCC1. RCC1 is the only known guanine nucleotide in breast cancer cell lines and knockout mouse models, exchange factor for the Ran GTPase, which plays in- suggest the potential involvement of α-N-terminal meth- dispensable roles in nucleocytoplasmic transport, mito- ylation in DNA damage response and cancer develop- sis, and nuclear envelope assembly (Chen et al. 2007). α ment. In this study, we report the first crystal structures Notably, -N-terminal methyltransferase 1 (NTMT1/ NRMT1/METTL11A) catalyzes the N-terminal methyla- of human NTMT1 in complex with cofactor S-adenosyl- tion of RCC1, representing a novel regulatory mechanism L-homocysteine (SAH) and six substrate peptides, respec- for the Ran GTPase activity (Tooley et al. 2010; Webb tively, and reveal that NTMT1 contains two characteris- et al. 2010). Abrogation of N-terminal methylation of tic structural elements (a β hairpin and an N-terminal RCC1 by mutations or knockdown of NTMT1 signifi- extension) that contribute to its substrate specificity. cantly diminishes the binding ability and association of Our complex structures, coupled with mutagenesis, bind- RCC1 to chromatin and causes spindle pole defects and ing, and enzymatic studies, also present the key elements aberrant mitosis, underlining the functional significance involved in locking the consensus substrate motif XPK (X of NTMT1-mediated α-N-methylation in mitotic spindle indicates any residue type other than D/E) into the cata- assembly and chromosome segregation (Chen et al. 2007). lytic pocket for α-N-terminal methylation and explain In addition to RCC1, several other proteins are subject to α-N-terminal methylation (Webb et al. 2010), including why NTMT1 prefers an XPK sequence motif. We propose α retinoblastoma protein RB1 and SET (Tooley et al. 2010), a catalytic mechanism for -N-terminal methylation. damaged DNA-binding protein 2 (DDB2) (Cai et al. 2014), Overall, this study gives us the first glimpse of the molec- centromere H3 variants CENP-A/B (Bailey et al. 2013; Dai ular mechanism of α-N-terminal methylation and poten- et al. 2015), Drosophila H2B (Villar-Garea et al. 2012), and tially contributes to the advent of therapeutic agents for poly(ADP-ribose) polymerase 3 (Dai et al. 2015), while human diseases associated with deregulated α-N-terminal data bank analysis of NTMT1/2’s consensus sequence methylation. predicts the existence of potentially >300 targets for α- N-terminal methylation (Tooley and Schaner Tooley Supplemental material is available for this article. 2014). NTMT1 is responsible for the α-N-terminal meth- Received August 18, 2015; revised version accepted ylation of DDB2 in response to the generation of UV-in- October 14, 2015. duced cyclobutane pyrimidine dimers (Cai et al. 2014). This methylation of DDB2 promotes its recruitment to form foci at the sites of DNA damage and facilitates nu- cleotide excision repair, possibly indicating a role of Post-translational modifications (PTMs) of proteins dur- NTMT1 in the DNA damage response (DDR) network ing or after their synthesis contribute to the functional (Cai et al. 2014). Moreover, knockdown of NTMT1 leads [Keywords: α-N-terminal methylation; methyltransferase; NTMT1; © 2015 Dong et al. This article is distributed exclusively by Cold Spring crystal structure] Harbor Laboratory Press for the first six months after the full-issue publi- 5These authors contributed equally to this work. cation date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After Corresponding authors: [email protected], [email protected] six months, it is available under a Creative Commons License (At- Article published online ahead of print. Article and publication date are tribution-NonCommercial 4.0 International), as described at http:// online at http://www.genesdev.org/cgi/doi/10.1101/gad.270611.115. creativecommons.org/licenses/by-nc/4.0/. GENES & DEVELOPMENT 29:2343–2348 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/15; www.genesdev.org 2343 Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Dong et al. to hypersensitivity of breast cancer cell lines to both eto- 2010). So far, all known substrates of NTMT1 contain poside and γ-irradiation treatments, further suggest- the N-terminal consensus sequence XPK (X = S/P/A/G) ing NTMT1 as a component of DDR (Bonsignore et al. (Fig. 2A), although NTMT1 can also methylate peptides 2015a). Interestingly, NTMT1 knockout mice suffer a with X being F, Y, C, M, K, R, N, Q, or H in vitro (Petkow- high mortality rate shortly after birth and exhibit prema- ski et al. 2012). In order to understand the substrate specif- ture aging and phenotypes characteristic of mouse models icity of NTMT1, we determined the crystal structures of deficient for DDR molecules, indicating the biological sig- the full-length human NTMT1 in complex with its cofac- nificance of NTMT1 in vivo (Bonsignore et al. 2015b). Ad- tor (SAH) and a peptide derived from either human (se- ditionally, NTMT1-mediated α-N-methylation of CENP- quence: SPKRIA) or mouse (sequence: PPKRIA) RCC1. B promotes its binding to centromeric DNA (Dai et al. Furthermore, we also generated crystals of NTMT1 in 2015). Overall, the above findings highlight a general complex with SAH and either the RPK or YPK peptide, and important role of NTMT1-mediated α-N-methylation which can be efficiently methylated by NTMT1 in vitro in facilitating interactions between methylated target pro- (Tooley et al. 2010). These crystal structures reveal that teins and DNA. the human NTMT1 folds as a single domain (Fig. 2B), Although some progress has been made in the field of and the root mean square deviation (RMSD) values be- α-N-terminal methylation, many questions remain to be tween these structures and the NTMT1-SAH binary answered. For instance, why does NTMT1 specifically structure (Protein Data Bank [PDB]: 2EX4) range from carry out the α-N-terminal methylation, and what is the 0.2 to 0.4 Å for the Cα atoms of NTMT1. The crystal dif- catalytic mechanism? Given that the majority of known fraction data and refinement statistics for all of these physiological substrates of NTMT1 contains an XPK (X structures are shown in Supplemental Table S1. = S/P/A/G) N-terminal sequence, what is the structural NTMT1 includes a typical methyltransferase Ross- basis for the requirement of this consensus sequence, mann fold that consists of a seven-strand β sheet and and is there any residue tolerance along the N-terminal five α helixes, of which two α helixes (α6 and α7) pack on consensus sequence? In an effort to address these ques- one side of the β sheet, and the other three α helixes (α3, tions, we determined the X-ray crystal structures of hu- α4, and α5) pack on the other side of the β sheet (Fig. 2B). man NTMT1 in ternary complexes with its cofactor In addition to the highly conserved Rossmann fold, product (S-adenosyl-L-homocysteine [SAH]) and six differ- NTMT1 contains two unique structural elements distinct ent hexapeptides as substrates, including the very N-ter- from other methyltransferases: a β hairpin inserted be- minal fragment of RCC1 and its mutant peptides. We tween strand β5 and helix α7 and an N-terminal extension deduced the molecular mechanism of NTMT1-mediated consisting of two α helixes ( α1 and α2) and one 310 helix α-N-methylation on its physiological substrate, RCC1, (η1) (Figs. 1, 2B). These two unique structural elements based on data obtained from structure-based mutagenesis are extensively involved in substrate binding (Fig.