Crystal structure of GCN5 PCAF N-terminal domain reveals atypical ubiquitin ligase structure

Abstract

General control nonderepressible 5 (GCN5, also known as Kat2a) and p300/CBP-associated factor (PCAF, also known as Kat2b) are two homologous acetyltransferases. Both proteins share similar domain architecture consisting of a PCAF N-terminal (PCAF_N) domain, acetyltransferase domain, and a bromodomain. PCAF also acts as a ubiquitin E3 ligase whose activity is attributable to the PCAF_N domain, but its structural aspects are largely unknown. Here, we demonstrated that GCN5 exhibited ubiquitination activity in a similar manner to PCAF and its activity was supported by the ubiquitin-conjugating enzyme UbcH5. Moreover, we determined the crystal structure of the PCAF_N domain at 1.8 Å resolution and found that PCAF_N domain folds into a helical structure with a characteristic binuclear zinc region, which was not predicted from sequence analyses. The zinc region is distinct from known E3 ligase structures, suggesting this region may form a new class of E3 ligase. Our biochemical and structural study provides new insight into not only the functional significance of GCN5 but also into ubiquitin biology. General control nonderepressible 5 (GCN5, also known as Kat2a) and p300/CBP-associated factor (PCAF, also known as Kat2b) are two homologous acetyltransferases. Both proteins share similar domain architecture consisting of a PCAF N-terminal (PCAF_N) domain, acetyltransferase domain, and a bromodomain. PCAF also acts as a ubiquitin E3 ligase whose activity is attributable to the PCAF_N domain, but its structural aspects are largely unknown. Here, we demonstrated that GCN5 exhibited ubiquitination activity in a similar manner to PCAF and its activity was supported by the ubiquitin-conjugating enzyme UbcH5. Moreover, we determined the crystal structure of the PCAF_N domain at 1.8 Å resolution and found that PCAF_N domain folds into a helical structure with a characteristic binuclear zinc region, which was not predicted from sequence analyses. The zinc region is distinct from known E3 ligase structures, suggesting this region may form a new class of E3 ligase. Our biochemical and structural study provides new insight into not only the functional significance of GCN5 but also into ubiquitin biology. Post-translational modification of proteins regulates many biological processes. Acetyltransferases transfer acetyl groups to lysine residues on target proteins and are one of the major types of post-translational enzymes. General control nonderepressible 5 (GCN5) is one of the best characterized histone acetyltransferases that promote transcriptional activity (1Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation.Cell. 1996; 84 (8601308): 843-85110.1016/S0092-8674(00)81063-6Abstract Full Text Full Text PDF PubMed Scopus (1282) Google Scholar, 2Sterner D.E. Berger S.L. Acetylation of histones and transcription-related factors.Microbiol. Mol. Biol. Rev. 2000; 64 (10839822): 435-45910.1128/mmbr.64.2.435-459.2000Crossref PubMed Scopus (1398) Google Scholar). GCN5 is the enzyme subunit of the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex, whose architecture was visualized by cryo-EM analyses (3Papai G. Frechard A. Kolesnikova O. Crucifix C. Schultz P. Ben-Shem A. Structure of SAGA and mechanism of TBP deposition on gene promoters.Nature. 2020; 577 (31969704): 711-71610.1038/s41586-020-1944-2Crossref PubMed Scopus (61) Google Scholar, 4Wang H. Dienemann C. Stutzer A. Urlaub H. Cheung A.C.M. Cramer P. Structure of the transcription coactivator SAGA.Nature. 2020; 577 (31969703): 717-72010.1038/s41586-020-1933-5Crossref PubMed Scopus (72) Google Scholar), and modifies multiple lysine residues on histone H3 in vitro (5Grant P.A. Eberharter A. John S. Cook R.G. Turner B.M. Workman J.L. Expanded lysine acetylation specificity of Gcn5 in native complexes.J. Biol. Chem. 1999; 274 (10026213): 5895-590010.1074/jbc.274.9.5895Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Recently, it was reported that GCN5 is phosphorylated by protein kinase A in a manner dependent on the transcriptional coregulator Cbp/p300-interacting transactivator 2 (CITED2) (6Sakai M. Tujimura-Hayakawa T. Yagi T. Yano H. Mitsushima M. Unoki-Kubota H. Kaburagi Y. Inoue H. Kido Y. Kasuga M. Matsumoto M. The GCN5-CITED2-PKA signaling module controls hepatic glucose metabolism through a cAMP-induced substrate switch.Nat. Commun. 2016; 7 (27874008): 1314710.1038/ncomms13147Crossref PubMed Scopus (24) Google Scholar), thereby increasing its acetyltransferase activity for histone and attenuating that for peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). This suggests that GCN5 functions in multiple situations. Metazoans possess two GCN5 isoforms that arise from alternative splicing (7Smith E.R. Belote J.M. Schiltz R.L. Yang X.J. Moore P.A. Berger S.L. Nakatani Y. Allis C.D. Cloning of Drosophila GCN5: conserved features among metazoan GCN5 family members.Nucleic Acids Res. 1998; 26 (9611240): 2948-295410.1093/nar/26.12.2948Crossref PubMed Scopus (92) Google Scholar). The lower molecular weight isoform (isoform 2) is similar in size and function to yeast GCN5, consisting of an acetyltransferase (AT) domain and a bromodomain at the N and C termini, respectively (2Sterner D.E. Berger S.L. Acetylation of histones and transcription-related factors.Microbiol. Mol. Biol. Rev. 2000; 64 (10839822): 435-45910.1128/mmbr.64.2.435-459.2000Crossref PubMed Scopus (1398) Google Scholar, 8Xu W. Edmondson D.G. Roth S.Y. Mammalian GCN5 and P/CAF acetyltransferases have homologous amino-terminal domains important for recognition of nucleosomal substrates.Mol. Cell. Biol. 1998; 18 (9742083): 5659-566910.1128/mcb.18.10.5659Crossref PubMed Scopus (136) Google Scholar) (Fig. 1A). The higher molecular weight isoform (isoform 1) contains an N-terminal extension that has high similarity to the N-terminal domain of PCAF (8Xu W. Edmondson D.G. Roth S.Y. Mammalian GCN5 and P/CAF acetyltransferases have homologous amino-terminal domains important for recognition of nucleosomal substrates.Mol. Cell. Biol. 1998; 18 (9742083): 5659-566910.1128/mcb.18.10.5659Crossref PubMed Scopus (136) Google Scholar), termed PCAF_N domain, which is conserved only among vertebrate (Fig. 1A) (9Nagy Z. Tora L. Distinct GCN5/PCAF-containing complexes function as co-activators and are involved in transcription factor and global histone acetylation.Oncogene. 2007; 26 (17694077): 5341-535710.1038/sj.onc.1210604Crossref PubMed Scopus (301) Google Scholar). According to the Pfam database (10El-Gebali S. Mistry J. Bateman A. Eddy S.R. Luciani A. Potter S.C. Qureshi M. Richardson L.J. Salazar G.A. Smart A. Sonnhammer E.L.L. Hirsh L. Paladin L. Piovesan D. Tosatto S.C.E. et al.The Pfam protein families database in 2019.Nucleic Acids Res. 2019; 47 (30357350): D427-D43210.1093/nar/gky995Crossref PubMed Scopus (2563) Google Scholar), there are 695 eukaryotic proteins harboring the PCAF_N domain, categorized into 18 architectures. Ubiquitination is also a post-translational modification that targets lysine residues. This modification regulates many cellular processes, including cell division and immune responses, among others. Ubiquitination is achieved by the sequential reaction of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3), which is responsible for the ligation of ubiquitin onto a substrate in conjunction with the E2 (11Pickart C.M. Eddins M.J. Ubiquitin: structures, functions, mechanisms.Biochim. Biophys. Acta. 2004; 1695 (15571809): 55-7210.1016/j.bbamcr.2004.09.019Crossref PubMed Scopus (1021) Google Scholar). The human genome encodes two E1s, ∼38 E2s, and more than 600 E3s (12Jin J. Li X. Gygi S.P. Harper J.W. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging.Nature. 2007; 447 (17597759): 1135-113810.1038/nature05902Crossref PubMed Scopus (263) Google Scholar, 13Li W. Bengtson M.H. Ulbrich A. Matsuda A. Reddy V.A. Orth A. Chanda S.K. Batalov S. Joazeiro C.A. Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle's dynamics and signaling.PLoS One. 2008; 3 (18213395): e148710.1371/journal.pone.0001487Crossref PubMed Scopus (572) Google Scholar, 14Ye Y. Rape M. Building ubiquitin chains: E2 enzymes at work.Nat. Rev. Mol. Cell Biol. 2009; 10 (19851334): 755-76410.1038/nrm2780Crossref PubMed Scopus (693) Google Scholar). E3s plays a pivotal role in selecting substrates and to date, three classes of E3 have been identified (15Buetow L. Huang D.T. Structural insights into the catalysis and regulation of E3 ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2016; 17 (27485899): 626-64210.1038/nrm.2016.91Crossref PubMed Scopus (297) Google Scholar): RING (really interesting new gene), HECT (homologous to E6AP C terminus), and RBR (RING-between-RING). It has been reported that in addition to acetyltransferase activity, PCAF also harbors ubiquitination activity (16Linares L.K. Kiernan R. Triboulet R. Chable-Bessia C. Latreille D. Cuvier O. Lacroix M. Le Cam L. Coux O. Benkirane M. Intrinsic ubiquitination activity of PCAF controls the stability of the oncoprotein Hdm2.Nat. Cell Biol. 2007; 9 (17293853): 331-33810.1038/ncb1545Crossref PubMed Scopus (147) Google Scholar) and it has been demonstrated that PCAF acts as an E3 ligase targeting human Hdm2, human Gli1, and human CIITA and promotes self-ubiquitination (16Linares L.K. Kiernan R. Triboulet R. Chable-Bessia C. Latreille D. Cuvier O. Lacroix M. Le Cam L. Coux O. Benkirane M. Intrinsic ubiquitination activity of PCAF controls the stability of the oncoprotein Hdm2.Nat. Cell Biol. 2007; 9 (17293853): 331-33810.1038/ncb1545Crossref PubMed Scopus (147) Google Scholar, 17Mazzà D. Infante P. Colicchia V. Greco A. Alfonsi R. Siler M. Antonucci L. Po A. De Smaele E. Ferretti E. Capalbo C. Bellavia D. Canettieri G. Giannini G. Screpanti I. et al.PCAF ubiquitin ligase activity inhibits Hedgehog/Gli1 signaling in p53-dependent response to genotoxic stress.Cell Death Differ. 2013; 20 (24013724): 1688-169710.1038/cdd.2013.120Crossref PubMed Scopus (76) Google Scholar, 18Morgan J.E. Greer S.F. Pulling a ligase out of a “HAT”: pCAF mediates ubiquitination of the class II transactivator.Int. J. Cell Biol. 2017; 2017 (28286521): 809381310.1155/2017/8093813Crossref PubMed Scopus (3) Google Scholar). PCAF_N is identified as a domain containing ubiquitin E3 ligase activity and the longer isoform of GCN5 possesses PCAF_N domain. However, whether GCN5 functions as an E3 enzyme remains to be determined. Here, we demonstrated that GCN5 exhibits ubiquitination activity in a similar manner to PCAF. We also performed a structural based study focusing on PCAF_N domain to elucidate any functions of GCN5 as an E3 enzyme. Our findings provide new avenues for both the functional study of GCN5 as well as that of ubiquitin biology. GCN5 and PCAF are highly homologous with ∼75% sequence identity (Fig. 1A) and both share the same domain architecture (Fig. 1A, Fig. S1). This strongly suggested that GCN5 was able to act as an E3 enzyme in a similar manner to that of PCAF. E3 capacity for ubiquitination is utilized as a means of assessing their potential to function with E2. Therefore, we evaluated whether GCN5 demonstrated autoubiquitination activity. Full-length human PCAF (hPCAF), full-length mouse PCAF (mPCAF), hGCN5 with 80 residues truncated at the N-terminal (hGCN5), and full-length mGCN5 (mGCN5) were each expressed in Escherichia coli. As a previous study used UbcH5b as an E2 enzyme in ubiquitination assays of PCAF (16Linares L.K. Kiernan R. Triboulet R. Chable-Bessia C. Latreille D. Cuvier O. Lacroix M. Le Cam L. Coux O. Benkirane M. Intrinsic ubiquitination activity of PCAF controls the stability of the oncoprotein Hdm2.Nat. Cell Biol. 2007; 9 (17293853): 331-33810.1038/ncb1545Crossref PubMed Scopus (147) Google Scholar), we further examined several E2 enzymes with a variety of identities ranging from 31 (UbcH2) to 97% (UbcH5c). Eight E2s (UbcH1, UbcH2, UbcH5a, UbcH5b, UbcH5c, UbcH7, UbcH8, and UbcH10) were tested to assess ubiquitination activity, UbcH5a and UbcH5c were likely to work as E2 enzymes for PCAF and GCN5, probably because UbcH5a, UbcH5b, and UbcH5c are highly homologous proteins. Next, we assessed ubiquitination activity using UbcH5b more precisely. Polyubiquitinated bands were increased as time passed for both GCN5 and PCAF, whereas the bands of purified proteins were decreased, demonstrating that GCN5 as well as PCAF exhibited E3 ligase activity (Fig. 1C). The result also revealed that UbcH5b was an effective E2 for PCAF and GCN5 (Fig. 1C). Linares et al. (16Linares L.K. Kiernan R. Triboulet R. Chable-Bessia C. Latreille D. Cuvier O. Lacroix M. Le Cam L. Coux O. Benkirane M. Intrinsic ubiquitination activity of PCAF controls the stability of the oncoprotein Hdm2.Nat. Cell Biol. 2007; 9 (17293853): 331-33810.1038/ncb1545Crossref PubMed Scopus (147) Google Scholar) identified two regions critical for PCAF autoubiquitination with amino acid (aa) residues 121–242 corresponding to the active ubiquitination domain of hPCAF and residues 350–445 corresponding to a motif or residues important for autoubiquitination. PCAF_N domain included residues 121–242, but based on sequence analysis could not be categorized with other known E3s. Although many crystal structures of ATs and bromodomains have previously been determined and are deposited in the Protein Data Bank (19Filippakopoulos P. Picaud S. Mangos M. Keates T. Lambert J.P. Barsyte-Lovejoy D. Felletar I. R. S. T. S. recognition and structural analysis of the human bromodomain Full Text Full Text PDF PubMed Scopus Google Scholar, A. G. A. T. H. P. A. structure of a human GCN5 histone acetyltransferase domain and acetyl 2007; PubMed Scopus Google Scholar), PCAF_N domain structure has been We mGCN5 and determined its crystal structure at 1.8 Å and were and to be Pfam database as PCAF_N domain, but crystal structure revealed folds into a domain. Therefore, this region be as were two PCAF_N domain in an (Fig. that were the same with a of the structure and in for PCAF_N domain, we performed size analyses. The molecular weight from analysis was which with the molecular weight of (Fig. demonstrating that mGCN5 PCAF_N domain as a in with the structure well with the crystal structure (Fig. C and The mGCN5 PCAF_N domain folds into a structure three The N-terminal region an binuclear zinc region in (Fig. Fig. S1). The region the N-terminal and regions with an structure with a in the N-terminal region, a This region was termed a The region folds into an using the L. Acids Res. 2016; PubMed Scopus Google Scholar) revealed that this region structural to the gene domain of 3 J. E. M. H. J. S. A. Structural for and into the by Mol. Biol. 18 PubMed Scopus Google Scholar). The from residues to of the domain well onto of the region of the PCAF_N domain with a of and of Å to the (Fig. its sequence identity is (Fig. This region was termed a regions are with and (Fig. and The zinc region with the region and has a of The region also with the region and has a of two and in the N-terminal region were the and that were the we performed the analysis was performed to whether the were zinc but by zinc were from PCAF_N domain Next, we two with a higher and lower to the of zinc and an were in the higher but the were in the lower (Fig. This that the two were zinc and revealed that the PCAF_N domain a zinc This structure was not predicted from the amino acid Moreover, a using this domain identified structures homologous to the RING E3 ligase. The zinc region has a binuclear structure The two zinc with residues and The of is used to both zinc (Fig. and Fig. S1). the residues of the zinc are conserved GCN5 and PCAF (Fig. The zinc region of PCAF_N domain is in its zinc and structure with the RING domain. The RING domains are characterized with the RING motif to two of zinc using a (Fig. this the and of zinc share and the and of zinc share PCAF_N sequence with the RING domain to the and because the two domains with the zinc in a manner (Fig. The zinc region of PCAF_N domain a but the structure of PCAF_N domain is distinct from that of the RING domain. sequence and structural similarity to the is reported to have ubiquitin ligase activity, but this region PCAF_N domain, of region, to the crystal We ubiquitin ligase using PCAF_N domain. PCAF_N domain exhibited ligase activity (Fig. further whether or not the zinc region or the zinc is for E3 ligase activity, we to the zinc region or of mGCN5 but we to the protein probably to the we the the zinc region This exhibited ligase activity (Fig. analysis used purified and (Fig. UbcH5b was 17 the of and UbcH5b was two proteins were at the same and UbcH5b was (Fig. suggesting that both proteins were This is the to a ligase activity of PCAF_N domain of GCN5 and the crystal structure of PCAF_N domain among PCAF_N family proteins. PCAF_N domain a zinc region that a of RING E3 ligase. RING E3s are highly in RING domains are active as whereas are active as or GCN5 is a protein and each domain A. G. A. T. H. P. A. structure of a human GCN5 histone acetyltransferase domain and acetyl 2007; PubMed Scopus Google Scholar), and bromodomain (19Filippakopoulos P. Picaud S. Mangos M. Keates T. Lambert J.P. Barsyte-Lovejoy D. Felletar I. R. S. T. S. recognition and structural analysis of the human bromodomain Full Text Full Text PDF PubMed Scopus Google is as a Moreover, the PCAF_N domain, which as a in exhibited ligase activity. it into GCN5 could act as E3 ligase. E3 ligases an domain. structures, have a of with the two which zinc a by as a for with E2. PCAF_N domain, two zinc a with and its structure and of PCAF_N domain were distinct from that of RING E3 However, the remains and further study is to E6AP C and E3 ligases among three classes of E3s a that ubiquitin from GCN5 PCAF_N domain and PCAF have three conserved in addition to the zinc but form a Therefore, residues are to The and ubiquitin including to be The region of the PCAF_N domain is similar to the domain in which is reported to activity of the histone acetyltransferase through with J. E. M. H. J. S. A. Structural for and into the by Mol. Biol. 18 PubMed Scopus Google Scholar). the domain is reported to with a of PCAF_N domain is likely to protein targets as including which was reported previously (8Xu W. Edmondson D.G. Roth S.Y. Mammalian GCN5 and P/CAF acetyltransferases have homologous amino-terminal domains important for recognition of nucleosomal substrates.Mol. Cell. Biol. 1998; 18 (9742083): 5659-566910.1128/mcb.18.10.5659Crossref PubMed Scopus (136) Google Scholar). It is that function of the RING E3 is to both the E2 and the substrate domain at the C work as a date, GCN5 substrate has been three substrates for PCAF have been the functions of ubiquitination and acetylation by GCN5 and PCAF target residues as This function of GCN5 and PCAF conserved in may provide a in higher further structural and functional are The gene of PCAF was by the through the of the of and mGCN5 were into The E. were with the and were at with to a cell and protein with at 18 were and with the cell zinc of for 20 on and and and on for 20 the was at for 20 at The was to and purified with a by using zinc The of each was with to the and the were to of The and of were by the on by The of were by the through the of the the UbcH5b gene was into protein harbors and recognition at the Protein was performed by the same as were with a The proteins were to and by with proteins used for the were purified with the The N-terminal of was with the proteins were purified with The of The of human ubiquitin was by Protein and were performed by the same reported previously Y. 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