Identification of the YEATS domain of GAS41 as apH-dependent reader of histone succinylationYi Wanga,1, Jing Jina,1, Matthew Wai Heng Chunga,1, Ling Fengb, Hongyan Sunb,c, and Quan Haoa,2

aSchool of Biomedical Sciences, University of Hong Kong, Hong Kong, China; bDepartment of Chemistry, City University of Hong Kong, Hong Kong, China;and cKey Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research Institute of City University of Hong Kong, Shenzhen 518057, China

Edited by Dinshaw J. Patel, Memorial Sloan–Kettering Cancer Center, New York, NY, and approved January 29, 2018 (received for review October 10, 2017)

Lysine succinylation is a newly discovered posttranslational modifi-cation with distinctive physical properties. However, to date rarelyhave studies reported effectors capable of interpreting this modifi-cation on histones. Following our previous study of SIRT5 as aneraser of succinyl-lysine (Ksuc), here we identified the GAS41 YEATSdomain as a reader of Ksuc on histones. Biochemical studies showedthat the GAS41 YEATS domain presents significant binding affinitytoward H3K122suc upon a protonated histidine residue. Further-more, cellular studies showed that GAS41 had prominent interactionwith H3K122suc on histones and also demonstrated the coenrich-ment of GAS41 and H3K122suc on the p21 promoter. To investigatethe binding mechanism, we solved the crystal structure of the YEATSdomain of Yaf9, the GAS41 homolog, in complex with an H3K122sucpeptide that demonstrated the presence of a salt bridge formedwhen a protonated histidine residue (His39) recognizes the carboxylterminal of the succinyl group. We also solved the apo structure ofGAS41 YEATS domain, in which the conserved His43 residue super-imposes well with His39 in the Yaf9 structure. Our findings identifieda reader of succinyl-lysine, and the binding mechanism will provideinsight into the development of specific regulators targeting GAS41.

succinyl-lysine | reader | GAS41

Posttranslational modifications (PTMs) discovered on his-tones provide another layer of gene transcription regulation

which complement the cis-regulatory elements in our genome.Emerging evidence shows that these PTMs, also collectivelytermed “histone codes,” function individually or in combinationsto regulate nuclear processes such as transcription, DNA damagerepair, replication, and chromatin condensation (1). Lysine succi-nylation (Ksuc) was initially identified as a novel protein PTM onEscherichia coli isocitrate dehydrogenase, serine hydroxymethyl-transferase, and glyceraldehyde-3-phosphate dehydrogenase A(GAPDH) (2). Subsequently, Xie et al. (3) confirmed lysine suc-cinylation as part of the histone code conserved in yeast, fruit flies,mouse, and human. Succinyl-lysine carries a negative charge, whichdistinguishes it from acetyl- and other aliphatic forms of modifiedlysines, and provides the basis for more characteristic chemical in-teractions (Fig. 1A). The dramatic change of side-chain charge onhistone lysines contributed by succinylation may imply its uniquerole in nucleosomal functioning distinct from competing modifica-tions (2). The dynamic system of histone codes falls under the controlof “writer” and “eraser” enzymes dedicated to specific modificationsites and types (1, 4). Meanwhile, the “readers” translate the histonecode and disseminate the message into myriad cell phenotypes andbehaviors (4, 5). Succinyl-CoA is believed to be a cofactor forsuccinyl-transferase activity on various substrate proteins, analogousto the addition of acetyl groups, or alternatively, it may directly reactwith freely exposed lysines for succinylation to take place (2, 6, 7).Recently, acetyltransferase KAT2A was found to couple withnuclear-translocated α-Ketoglutarate Dehydrogenase complex towrite succinylation from the coenzyme onto H3K79 (8). Ourprevious study identified SIRT5, one of the NAD-dependentdeacylases, as a potent desuccinylase and demalonylase on ly-sines (9, 10). However, little is known about the readers for the

distinct Ksuc, which motivates the studies that discover potentialcandidates and advance our knowledge of its biological functions.YEATS domain was initially characterized in Yaf9 as a crucial

subunit of yeast histone acetyltransferase NuA4 (11). The YEATSdomain-containing proteins, including Yaf9, Taf14, ENL, AF9, andGlioma-Amplified Sequence-41 (GAS41), are involved in acetyl-transferase or methyltransferase complexes, which participate in theprocesses of chromatin remodeling and gene transcription (12). Shiand coworkers (13, 14) illustrated that human AF9 and ENLYEATS domains could recognize acetyl-lysine (Kac) to mediatenormal physiological processes, but the readout could also underliesevere pathological conditions. Subsequently, studies have reportedhuman AF9, YEATS2, and ENL and yeast Taf14 YEATS domainsas readers of crotonyl-lysine (Kcr), whereby AF9 YEATS domainsenses an enrichment of H3K18cr deposited by p300 to coordinatea mechanism for transcriptional activation of inflammation genes(15–18). YEATS domain adopts a classical immunoglobin foldstructure of eight antiparallel β-strands and two α-helices (19).Currently, all solved YEATS domain structures reveal the consis-tent positioning of the aliphatic modified-lysine ligand sandwichedbetween two aromatic residues, establishing a robust π-π-π stackingas the recognition mechanism underlying crotonyl-lysine readoutcapability of Taf14 and AF9 (15–18).

Results and DiscussionPrediction of the Readout of GAS41 or Yaf9 Toward Ksuc. In favor ofhydrophobic interactions, the extremely low binding affinity

Significance

Posttranslational modifications (PTMs) on histone lysines regulategene expression and physiological functions. Succinylation is anewly discovered PTM with distinctive features. However, rarelystudies have shown the function of succinylation on histone ly-sines. Our biochemical and structural studies demonstrate thatGAS41, an oncogene-coded protein, can act as the reader of suc-cinylation on histone H3K122. The functional significance of thepH-dependent histidine of GAS41 recognizing succinyl lysine (Ksuc)could have implications in local pH-dysregulated circumstances.The mechanism illustrated by the structures also provides an im-portant insight into the development of specific regulators tar-geting the GAS41 YEATS domain in the future.

Author contributions: Y.W., J.J., and Q.H. designed research; Y.W., J.J., M.W.H.C., and L.F.performed research; H.S. contributed new reagents/analytic tools; Y.W., J.J., M.W.H.C.,L.F., and Q.H. analyzed data; and Y.W., J.J., M.W.H.C., H.S., and Q.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.wwpdb.org (PDB ID codes 5WYI and 5Y8V).1Y.W., J.J., and M.W.H.C. contributed equally to this work.2To whom correspondence should be addressed. Email: qhao@hku.hk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1717664115/-/DCSupplemental.

Published online February 20, 2018.

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Fig. 1. The YEATS domains of Yaf9 and GAS41 are pH-dependent succinyl readers on histones. (A) Different chemical structures of acetyl-lysine (Kac),crotonyl-lysine (Kcr), and succinyl-lysine (Ksuc). The succinyl acyl group is colored in red. (B) Microarray screening assay of Yaf9 and GAS41 YEATS domainsagainst H3 and H4 peptides succinylated on previously identified succinyl-lysine sites. H3K122suc binds both Yaf9 and GAS41 (highlighted in dashed box). (C)Peptide pull-down assay of succinyl-lysine peptides against Yaf9 or GAS41 YEATS domain. (D) Peptide pull-down assays of GAS41 WT against different acylgroups on H3K122 peptides under pH 6.0, pH 7.0, and pH 7.4 conditions. GAS41 mutant (H43F) shows decreased binding affinity toward H3K122suc peptide.Images are representative of three independent experiments for each condition. (E) Statistical analysis of the efficiency of peptide pull-down assays in D. (F)Confirmation of GAS41 knockdown (KD) by Western blot. GAPDH was used as loading control. (G) Western blot for the identification of H3K122suc in anti-FLAG immunoprecipitates. FLAG-tagged GAS41 proteins were used as loading control. (H) The coenrichment of GAS41 and H3K122suc on p21 promoter. ChIP-qPCR analysis using anti-FLAG antibody and IgG control to probe the enrichment of FLAG-tagged WT or mutant (H43F) GAS41 on p21 promoter in cells (Left).ChIP-qPCR analysis using anti-H3K122suc antibody and IgG control to probe the enrichment of H3K122suc on p21 promoter in cells (Right). A Western blotwith anti-FLAG and anti-GAPDH antibodies shows that both constructs are expressed at appropriate levels.

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between AF9 and Ksuc was attributed to a key hydrophobicaromatic residue (Phe28) on loop 1 located at the end of theacyl-binding pocket of its YEATS domain (17). The residue atthis position may have a crucial role in the recognition of dif-ferent acyl groups, which has displayed a major discrepancyacross human YEATS domains with a phenylalanine (Phe) inAF9 and ENL, a serine (Ser) in YEATS2, and a histidine (His)in GAS41 (SI Appendix, Fig. S1) (15). As a comparative case,YEATS2 can favorably bind 2-hydroxyisobutyryl-lysine (Khib)while AF9 cannot, probably due to the contribution of a hydro-gen bond by the Ser254 hydroxyl group in YEATS2 (15, 17). In-triguingly, GAS41 has a histidine residue at this position. GAS41,otherwise known as YEATS4 (YEATS Domain-containing Pro-tein 4), is a newly discovered oncogene regulating the p53-p21pathway (20). However, despite the availability of crystal struc-tures of its homolog Yaf9, the role of GAS41 YEATS domain hasbeen left unexplored (19). In concordance with GAS41, Yaf9 hasa histidine residue that surrogates the key Phe residue recognizingKcr in human AF9 when superimposed (SI Appendix, Fig. S2 Aand B). As histidine could gain a positive charge when protonated(21), therefore, we proposed that human GAS41, as well as yeastYaf9, may have the preference for an acyl group with a negativecharge, such as the succinyl group.

Screening of the Potential Ksuc Sites Binding to GAS41 or Yaf9. Totest our hypothesis, we first applied peptide microarray assay toprofile the binding affinities of yeast Yaf9 or human GAS41YEATS domain against N-terminal biotinylated peptides bear-ing a subset of succinyl-lysine sites on histone H3 or H4 (Ksuc-H3/H4) (19). Different experimental methods have been used tomeasure pH of resting yeast at 5.8 and 6.35, and that of fer-menting yeast at 6.1 and 7.1 (22, 23); the intracellular pH of yeastcould be acidic, thus prompting us to choose pH 6.0 for testing.We also chose the succinyl-lysine sites that are physiologicallydetectable in HeLa cells (3). The screening results showed thatboth Yaf9 and GAS41 YEATS domains could interact with Ksucon H3 and H4 peptides with binding preferences for H3. BothYaf9 and GAS41 displayed strong binding affinity toward theH3K14suc, H3K56suc, H3K79suc, and H3K122suc peptides whileYaf9 could also bind the H4K12suc and H4K31suc peptides(Fig. 1B). GAS41 showed a stronger binding affinity toward theH3K122suc peptide than the others. Moreover, we coupledthese peptides onto streptavidin agarose to perform a peptidepull-down assay (24). The results confirmed that both Yaf9 andGAS41 YEATS domains were able to bind Ksuc on histoneH3 and H4 peptides upon a protonated histidine under pH 6.0,but their affinities with H3K122suc were relatively strongerthan the others (Fig. 1C). Therefore, H3K122suc stood out forfurther studies because of its similarly strong binding towardboth Yaf9 and GAS41.

Measurement of GAS41 or Yaf9 Binding Toward Acetyl-, Crotonyl-,Succinyl H3K122 Peptides. Next, we compared the binding capabil-ities of different acyl groups on H3K122 against GAS41 YEATSdomain under different pH conditions. As reported, the intracel-lular pH value varies in different cellular organelles (from ∼pH 4.7to ∼pH 7.4), owing to the activities of various metabolic pathwayssuch as ATP production in the cytoplasm (25). The regulation ofnuclear pH is hypothesized to have a mechanism similar to that inthe cytoplasm, but this requires experimental validation (25).Therefore, we chose pH 6.0, pH 7.0, or pH 7.4 for the followingexperiments. The results of peptide pull-down revealed that theH3K122suc peptide, rather than the others, showed prominentbinding ability toward GAS41 under pH 6.0 or pH 7.0 (Fig. 1D).Mutation of the proposed key residue from His to Phe (H43F)significantly decreased the binding affinity. When the pH reached7.4, H3K122suc demonstrated dramatically decreased binding to-ward GAS41, while H3K122cr and H3K122ac showed increased

affinity to GAS41 (Fig. 1D). Quantification by statistical analysissupported the scenario of the above peptide pull-down experiment(Fig. 1E). Together, the GAS41 YEATS domain can interact withH3K122suc presumably upon a protonated histidine. Furthermore,we measured the binding affinities of both GAS41 and Yaf9YEATS domains against different acylations on H3K122 peptidewith surface plasmon resonance spectroscopy (SPR). In line withthe peptide pull-down assay, the SPR experiment was also per-formed at pH 6.0, 7.0, and 7.4 (Table 1). Each measurement wasrepeated three times. At pH 7.4, affinity of H3K122suc binding wasnot determined, but at pH 7.0, it was calculated to be 48.34 ±3.8 μM. This suggests that the proportion of protonated His43 in-creases from pH 7.4 to 7.0. Affirmatively, a stronger binding affinitybetween GAS41 and the H3K122suc peptide was determined to be2.937 ± 0.21 μM at pH 6.0. The mutation H43F in GAS41 sharplydecreased its affinity toward the H3K122suc peptide (Table 1).Together, upon pH variation from pH 7.4 to pH 6.0, we observedan increase in binding affinity of GAS41 toward H3K122suc, incontrast to the corresponding decrease in affinity toward H3K122cr(Table 1). The affinities did not vary as much toward H3K122acfrom pH 7.4 to 7.0. Additionally, Yaf9 was able to bind H3K122sucwith an affinity of 27.44 ± 3.4 μM at pH 6.0 while it has no de-tectable binding toward H3K122cr and H3K122ac. Altogether, theresults suggest that GAS41 and Yaf9 YEATS domains can interactwith H3K122suc peptide in a pH-dependent manner, probablythrough the protonation of His43, which facilitates recognition ofthe Ksuc carboxyl terminal.

Cellular Studies of GAS41 Binding H3K122suc on Histones. Subsequently,we investigated whether the interaction between GAS41 YEATSdomain and H3K122suc is physiologically relevant in cells. It ispreviously described that GAS41 could act as a transcription factorinvolved in cancer development, while H3K122suc was detected inHeLa cells (3, 20). Peptide dot-blot assays showed that the anti-H3K122suc antibody specifically recognizes H3K122suc, but notother modification types or sites including unmodified histoneH3K122, H3K122ac, H3K122cr, and other identified Ksuc sites onH3 (SI Appendix, Fig. S3). We then generated GAS41 knockdown(KD) HeLa cells (Fig. 1F), in which FLAG-tagged constructs offull-length GAS41 WT or mutant (H43F) were overexpressed.FLAG immunoprecipitation (FLAG-IP) was then carried out toanalyze the content of H3K122suc in FLAG immunoprecipitates.Western blot analysis showed that GAS41 WT, rather than themutant (H43F), had prominent interaction with H3K122suc in cells

Table 1. The binding affinities of GAS41 and Yaf9 YEATSdomains against different acylations on H3K122 peptidesmeasured by surface plasmon resonance spectroscopy

Peptides pH

Kd, μM

GAS41 Yaf9

H3K122suc 6.0 2.937 ± 0.21* (WT) 27.44 ± 3.4103.7 ± 16 (H43F)

7.0 48.34 ± 3.8* (WT) ND102.0 ± 1.5 (H43F)

7.4 ND (WT) NDND (H43F)

H3K122cr 6.0 79.82 ± 1.0 ND7.0 31.27 ± 2.67.4 18.23 ± 4.3

H3K122ac 6.0 ND ND7.0 51.94 ± 6.17.4 47.68 ± 6.7

Kd values are calculated with an equilibriummodel, except when marked withan *, the values are calculated with kinetic rate constants. ND, not detectable.Choice of analysis and the original rate constants are available in the SI Appendix.

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(Fig. 1G), suggesting the essential role of this key histidine residueduring the interaction. Furthermore, we also performed ChIP-qPCR analysis to investigate cellular coenrichment of GAS41and H3K122suc. As reported, GAS41 was located on p14ARF andthe p21 promoter in proliferating cells (26). Our data underscoredthe fact that GAS41 WT enriches on the p21 promoter andshowed the low occupancy of the mutant (H43F) accordingly (Fig.1H, Left histogram). H3K122ac was also detected on the p21promoter (26–29). Thus, H3K122suc is proposed to competitivelyoccupy the p21 promoter region. ChIP-qPCR results confirmedthe enrichment of H3K122suc on the p21 promoter in cells (Fig.1H, Right histogram). Therefore, GAS41, but not the mutant,could be coenriched with H3K122suc on the p21 promoter, im-plicating a relevant physiological interaction in cells. Collectively,

the cellular data supported that GAS41 could act as a reader ofhistone succinylation in cells.

Molecular Mechanism of H3K122suc Readout. To gain insight intothe molecular basis underlying Ksuc readout by GAS41, weperformed crystallographic studies of GAS41 and Yaf9 YEATSdomains in complex with the H3K122suc peptide. Crystals of theYaf9/H3K122suc complex could diffract to 2.0-Å resolution afteroptimization. Statistics of the crystallographic data collectionand refinement are summarized in SI Appendix, Table S1. Theoverall structure of Yaf9 shows a typical YEATS domain witheight antiparallel β-strands capped by two short α-helices (Fig.2A). Despite our efforts to cocrystallize GAS41 with H3K122sucpeptide, only the apo structure of GAS41 was obtained at 2.6-Åresolution (SI Appendix, Table S1). The structure of GAS41

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Fig. 2. Molecular basis of H3K122suc readout by Yaf9 and GAS41 YEATS domains. (A) The overall structure of Yaf9 YEATS domain (yellow) in complex withH3K122suc (green). (B) The apo structure of GAS41 YEATS domain (red). (C) The Ksuc readout by Yaf9 via the hydrophilic interactions between H39, E38, andT69 residues and carboxyl terminal of succinyl group. 2Fo-Fc omit map (1σ) shows that the succinyl-lysine is well-defined in the pocket. (D) Superimposition ofthe pockets of Yaf9/H3K122suc (yellow) and GAS41 (Red) YEATS domains. (E) The electrostatic potential map for the interactions between residues andsuccinyl group. The negative potential is colored in red while the positive potential is colored in blue. (F) Interactions between H3 peptide (green) and Yaf9YEATS domain (yellow).

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again demonstrates a classical YEATS domain that superimposeswell with Yaf9 (Fig. 2B and SI Appendix, Fig. S4). In the complex,the succinyl-lysine is anchored in the groove of Yaf9 formed be-tween loops 1 and 4 (Fig. 2A). 2Fo-Fc omit map (1σ) shows thecomplete density of Ksuc anchored inside the pocket (Fig. 2C).Remarkably, the carboxyl terminal of Ksuc could be recognized byHis39 via a salt bridge as well as by Glu38 and Thr69 throughhydrogen bonds (Fig. 2C). The pattern of H3K122suc bound toYaf9 YEATS domain is similar to that of H3K9cr being recog-nized by human AF9 (PDB ID code 5HJB) (SI Appendix, Fig. S5).The succinyl-lysine superimposes well with crotonyl-lysine in thepockets of both yeast Yaf9 and human AF9 YEATS domains (SIAppendix, Fig. S5). The His39 of Yaf9 aligns best with Phe28 ofhuman AF9, which distinguishes Yaf9 as a reader of Ksuc (SIAppendix, Fig. S5). Superimposition of the residues Trp89 andTyr70 of Yaf9 to Tyr78 and Phe59 of AF9, respectively, demon-strates the typical binding pocket constituents adopted by theYEATS domains for the readouts of different acyl-lysines (SIAppendix, Fig. S5). The distance between the imidazole ring ofHis39 and the carboxyl group is 2.6 Å, which is comparable to thatbetween Arg105 and H3K9suc (2.7 Å) in the SIRT5 complexstructure (10), indicating a strong electrostatic interaction.Notably, His43 of GAS41 is conserved and superimposes well

with His39 of Yaf9 in the pocket, demonstrating a similar layoutof the H3K122suc-binding pocket residues (Fig. 2D). As afore-mentioned, mutation of this His to Phe (H43F) significantlydecreased the binding affinity of GAS41 to the H3K122sucpeptide in both pull-down and SPR assays (Fig. 1 D and E andTable 1), which again emphasizes the key role of this histidine foracyl recognition. Additionally, the electrostatic potential wascalculated to illustrate charge distribution in the interactionenvironment within the binding pocket (Fig. 2E). Meanwhile, thestructure revealed an aromatic sandwiching cage, which specifi-cally accommodates the flat carbonyl group (C = O) on the li-gand by snugly clamping it via π stacking into a well-orientedposition and permits other associated intermolecular interac-tions (SI Appendix, Fig. S6). Moreover, 2Fo-Fc omit map (1σ)shows that the flanking residues of the H3K122suc peptide couldbe well-defined in the structure (SI Appendix, Fig. S7). They formintimate contacts with the surface residues of Yaf9 YEATSdomain (Fig. 2F). The hydrophobic interaction between theresidues of H3K122suc peptide, including M1, P2, I4, Q6, andL7, and the residues H67, W89, F92, and H118 of Yaf9 stabilizesthe binding (Fig. 2F). It may explain why H3K122suc couldstandout to bind both Yaf9 and GAS41, probably due to theadditional interactions available for the specific peptide se-quence. Moreover, hydrogen bonds formed between the residuesH67, E91, R116, and H118 of Yaf9 and the main chain of

H3K122suc peptide also contribute to the interaction (Fig. 2F).Therefore, GAS41 is able to recognize Ksuc with a mechanismsimilar to Yaf9, utilizing the characteristic salt bridge establishedwith a protonated histidine residue.

ConclusionWith a rapidly expanding library of PTM marks on histones, it isexciting to understand their interplay and the emerging impor-tant roles that lysine acylations play in epigenetic regulation onnucleosomes. Succinylation is a newly discovered PTM withdistinct structural and chemical properties. YEATS domain-containing proteins have been reported as readers of Kac or Kcron histones. Our cellular and structural studies revealed thatGAS41 is a lysine succinylation reader owing to a protonatedhistidine residue located inside the pocket. As reported, in-tracellular pH could be regulated by global histone acetylation ata genomic level by the relevant physiological functions in normal orcancer cells (30). The wax and wane of the binding affinities ofGAS41 toward H3K122suc and H3K122cr indicate the switchingrole of GAS41 upon pH variation. The functional significance ofthe pH-dependent histidine switch of GAS41 for Ksuc recognitioncould have implications in local pH-dysregulated circumstances(31). This cross-talk for GAS41 recognizing Kac, Kcr, or Ksuc uponintracellular pH variation would be an interesting area for furtherinvestigation. Currently, there are more than 20 Ksuc sites identi-fied on various histones in cancer cells (3), which urges furtherinvestigation on profiling GAS41 readouts of other potential his-tone Ksuc sites and other types of acyl-lysines with a terminalcarboxyl group including malonyl- and glutaryl-lysine, to compre-hensively elucidate GAS41-mediated epigenetic regulation.

Materials and MethodsFull details are provided in SI Materials and Methods. The in vitro screeningof the potential Ksuc sites binding to GAS41 or Yaf9 was achieved bymicroarray assay using proteins against N-terminal biotinylated peptidesthat bear a subset of succinyl-lysine sites on histone H3 or H4. The mea-surement of GAS41 or Yaf9 binding toward acetyl-, crotonyl-, succinyl H3K122was performed by peptide pull-down and SPR assay. For the cellular study, thecoenrichment of GAS41 and H3K122suc on the p21 promoter was detected byChIP-qPCR. Then, the crystallizations of GAS41 and Yaf9 with H3K122suc werecarried out and diffraction data were collected at the Shanghai and Taiwansynchrotron facilities.

ACKNOWLEDGMENTS. We thank Dr. Michael S. Kobor from the Centre forMolecular Medicine and Therapeutics, Canada, for providing the Yaf9 con-struct. The crystallographic data were collected on BL17U of the ShanghaiSynchrotron Radiation Facility and beamline BL13C1 at the Taiwan NationalSynchrotron Radiation Research Center. This work was supported by HongKong Research Grants Council Grants C7037-14G, AoE/P-705/16, and HKU2/CRF/13G and National Natural Science Foundation of China Grant 21572190.

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