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Structural basis for recognition of centromere

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doi:10.1038/nature09854

Structural basis for recognition of centromere histone variant CenH3by the chaperone Scm3

Zheng Zhou 1*,Hanqiao Feng 1*,Bing-Rui Zhou 1,Rodolfo Ghirlando 2,Kaifeng Hu 3,Adam Zwolak 4,Lisa https://www.sodocs.net/doc/2817480805.html,ler Jenkins 5,Hua Xiao 1,Nico Tjandra 4,Carl Wu 1&Yawen Bai 1

The centromere is a unique chromosomal locus that ensures accurate segregation of chromosomes during cell division by directing the assembly of a multiprotein complex,the kinetochore 1.The centro-mere is marked by a conserved variant of conventional histone H3termed CenH3or CENP-A (ref.2).A conserved motif of CenH3,the CATD,defined by loop 1and helix 2of the histone fold,is necessary and sufficient for specifying centromere functions of CenH3(refs 3,4).The structural basis of this specification is of particular interest.Yeast Scm3and human HJURP are conserved non-histone proteins that interact physically with the (CenH3–H4)2heterotetramer and are required for the deposition of CenH3at centromeres in vivo 5–13.Here we have elucidated the structural basis for recognition of budding yeast (Saccharomyces cerevisiae )CenH3(called Cse4)by Scm3.We solved the structure of the Cse4-binding domain (CBD)of Scm3in complex with Cse4and H4in a single chain model.An a -helix and an irregular loop at the conserved amino terminus and a shorter a -helix at the carboxy terminus of Scm3(CBD)wraps around the Cse4–H4dimer.Four Cse4-specific residues in the N-terminal region of helix 2are sufficient for specific recognition by conserved and functionally important residues in the N-terminal helix of Scm3through formation of a hydrophobic cluster.Scm3(CBD)induces major conformational changes and sterically occludes DNA-binding sites in the structure of Cse4and H4.These findings have implications for the assembly and architecture of the centromeric nucleosome.

Unlike other eukaryotic species that have complex regional centro-meres with multiple centromeric nucleosomes 14,budding yeast has a single centromeric nucleosome that is necessary and sufficient to mediate the accurate segregation of chromosomes during mitosis and meiosis 15–18.The simple centromeres of budding yeast provide an attractive system for investigating outstanding topics in centromere biology,including the pathway of CenH3deposition and the architecture of the centromeric nucleosome 19,20.

Yeast Scm3and human HJURP are binding partners of CenH3–H4and are functionally required for their deposition at centromeres in vivo 5–13.A conserved domain of Scm3dictates specific and stoichi-ometric binding of CenH3–H4(Fig.1a),forming a (Scm3–Cse4–H4)2hexamer in 2M NaCl 5.This CBD of Scm3is mapped to residues 84–187(ref.5).To investigate the structural basis for the recognition of Cse4by Scm3,we first analysed the CBD of Scm3by NMR and found that it is intrinsically disordered (Supplementary Fig.1).To overcome instability inherent in complexes of individual Scm3,Cse4and H4fragments (Supplementary Figs 2–4),we engineered a single-chain molecule in which Scm3is inserted between Cse4and H4to assemble a stably folded molecule (Supplementary Fig.5).For convenience,we termed the single-chain molecule scSCH (Scm3,Cse4,H4).

The structure of scSCH was determined using multidimensional NMR and verified by structural analysis of its mutants (Fig.1b–d and Supplementary Fig.6).The structure of the folded core of scSCH,which includes residues 97–135of Scm3,157–202of Cse4and 50–99of H4,is

*These authors contributed equally to this work.

Laboratory of Biochemistry and Molecular Biology,National Cancer Institute,Bethesda,Maryland 20892,USA.2Laboratory of Molecular Biology,National Institute of Diabetes and Digestive and Kidney Diseases,Bethesda,Maryland 20892,USA.3National Magnetic Resonance Facility at Madison,University of Wisconsin,Madison,Wisconsin 53706,USA.4Laboratory of Molecular Biophysics,National Heart,Lung,and Blood Institute,NIH,Bethesda,Maryland 20892,USA.5Laboratory of Cell Biology,National Cancer Institute,NIH,Bethesda,Maryland 20892,USA. 90 DEVMERHKLADENMRKVWSNIISKYES--IEEQGDLVDLKTGEIVEDNGHIKTLTANNSTKDKRTKYTSVLRDIIDISDEED 169 36 DDVFCKRIESEKKYNDFLESLFKKYGR-DTSDIADEVDLATGEIIVNNGHLEALK 89 16 DQLLQKLRASRRRFQRRMQRLIEKYNQ--PFEDTPVVQMATLTYETPQGLRIWGG 68 DE DD DQ VM VF LL MR YN FQ KVW F RRM II LF LI KY KKY KYN EQ D ED KTG ATG ATL VD VD VQ LK LA MA NGH

NGH GL N

2′

3′

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L23′

2′

C 2

L2 3 2

1 2

3′

2′

IV II YE 32

Scm3

Cse4

135 99

201

157

Scm3

Cse4

S. cerevisiae S. pombe Human HJURP

Figure 1|Overall structure of scSCH.a ,The amino acid sequence and secondary structures of the Cse4-binding domain of Scm3in scSCH.Also shown are the conserved regions in Scm3of S.pombe and human HJURP.Highly conserved residues are highlighted in red.The region in the folded core

is shown in dark magenta (see e ).b –d ,Front,bottom and back view of the scSCH structure shown in ribbon representation.Scm3,Cse4and H4are in magenta,cyan and dark green,respectively.The full sequence of scSCH is M-His 6-KK-Cse4(150–227)-LVPRGS-Scm3(93–169)-GDK-H4(42–103).

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well defined with root mean squared deviations (r.m.s.d.)of 0.54A

?for backbone atoms and 1.06A

?for all heavy atoms (Fig.1e and Sup-plementary Table 1).Importantly,linker residues inserted between Scm3,Cse4and H4do not alter the structure of the folded region.Proteolytic cleavage of the two linkers in the folded scSCH only affects chemical shifts of neighbouring residues close to the cutting sites (Supplementary Fig.7).Moreover,the folded structure of the above tertiary complex is unchanged by refolding after denaturation in 6M GdmCl to liberate the three components as individual polypeptides (Supplementary Fig.8).Backbone amide 15N-{1H}heteronuclear Overhauser effects (NOE)reflect dynamic motions.The folded core shows small dynamic motions (NOE .0.7)except for loop 1of Cse4and the small loop region following the N-terminal a -helix (a N)in Scm3(Supplementary Fig.9a,b).In contrast,other regions display larger dynamic motions (NOE ,0.7),corresponding to less-well-defined structures (Supplementary Figs 9–11).

In the structure of scSCH,Scm3interacts broadly with both Cse4and H4.The a N helix of Scm3makes close contacts with both the a 2helix of Cse4and the a 3helix of H4through multiple hydrophobic interactions (Figs 1b and 2a–d and Supplementary Figs 9c and 13a).Following the a N helix,the loop region of Scm3(residues 121–144)mainly interacts with loop 1of Cse4,except that a bulge (Scm3residues 125–130)in the middle of the loop lies on top of loop 2of H4(Fig.1c and Supplementary Figs 9d and 13b–d).Scm3loop residues 140–144also interact with the C-terminal portion of the a 2helix of H4(Sup-plementary Figs 9d and 13e).Interestingly,Scm3residues 145–154are

completely disordered (Supplementary Figs 9b,e and 10).Finally,the C-terminal a -helix (a C)of Scm3(155–161)makes interactions with the N-terminal region of the a 2helix of H4(Supplementary Fig.13f).Next,we analysed the effects of mutations on the formation of Scm3–Cse4–H4complexes with isothermal titration calorimetry.The results reveal that the Scm3recognition motif resides in the N-terminal region (181–190)of the a 2helix of Cse4.Double muta-tions Met181Ser/Met184Gly and Ala189Ser/Ser190Val in Cse4that change the Cse4-specific residues to the corresponding residues in H3reduced the binding affinity by a factor of 5.5and 9,respectively (Fig.2a,b,Supplementary Table 2and Supplementary Fig.14).A double mutation Ile110Asp/Ile117Asn in the a N helix of Scm3decreased the binding affinity by a factor of 85(Fig.2a,Supplemen-tary Fig.14and Supplementary Table 2).These residues are important for cell growth:mutation of the three residues (Met184,Ala189,and Ser190)in Cse4to corresponding residues in H3leads to growth defect (small colony)21,and mutation Ile110Asp/Ile117Asn in Scm3abrogates cell viability 7,consistent with the effects of these mutations on the binding affinity between Scm3and Cse4/H4(Supplementary Table 2).Met 181should also be important for cell function because it inter-acts with Ile 117of Scm3(Fig.2a).It is possible that simultaneous mutation of the four residues in Cse4to the corresponding residues in H3would abrogate cell viability.

In contrast,deleting the three extra residues Lys 172,Asp 173and Gln 174and mutating Thr 170in loop 1(to Lys,as in H3),all residues specific to Cse4(Supplementary Fig.12),had little effect on the bind-ing affinity (a factor of 1.1)(Supplementary Table 2).Mutation of four residues (Val 165,Thr 166,Asp 167,Glu 168)at the C-terminal region of the a 1helix of Cse4to corresponding residues in H3(Ile,Ala,Gln and Asp)also showed little effect on the binding affinity (a factor of 1.4)(Supplementary Table 2).In addition,we found that Scm3is capable of pulling down the H3CATD –H4chimaera,in which the CATD of Cse4is swapped to the corresponding region of H3(ref.22(Supplementary Fig.15).Furthermore,Scm3can pull down an H3mutant with only four residues replaced by the corresponding residues in the a 2helix of Cse4(Met 181,Met 184,Ala 188and Ser 189)as well (Fig.2c,d).Importantly,Scm3residues that interact with the four Cse4-specific residues are well conserved in human HJURP (Fig.1a).Indeed,Cse4can also pull down the N-terminal region (residues 2–81)of HJURP (homologous to the Cse4-binding motif of Scm3(refs 9,22))and human H4(Supplementary Fig.16).This result is consistent with the ability of Cse4to replace human CENP-A at centromeres and maintain centromere function in human cells 23.In addition,the CENP-A residues that correspond to the four Cse4-specific residues are reasonably conserved (Fig.2d).

The structure of scSCH reveals the induction of major local con-formational changes in the structure of Cse4and H4relative to the (CENP-A–H4)2tetramer.First,the packing between the central a 2helices of Cse4and H4in the structure of scSCH is loose in comparison to tight hydrophobic interactions in the homology-modelled dimer based on the H3–H4structure in the nucleosome (Fig.3a,b),or in the CENP-A–H4dimer in the (CENP-A–H4)2tetramer 24.Hydro-phobic residues Leu 59,Phe 62and Val 66in the a 2helix of H4lose interacting partners Tyr 193,Ser 192and Leu 186in the a 2helix of Cse4(Fig.3a,b;Phe 101,Ala 98and Leu 94in human CENP-A (ref.24)).Second,owing to the insertion of the Scm3loop,loop 1in Cse4loses close contact with loop 2of H4(Supplementary Fig.13b)when com-pared with the corresponding loops in the canonical histone octamer 25(Fig.3c,d)or in the human (CENP-A–H4)2tetramer 24(Supplemen-tary Fig.17).Third,the a 2helix of Cse4kinks in the middle in scSCH (Fig.4a),as forced by the side chain of residue Met 103of the a N helix of Scm3(Fig.4a).In contrast,the a 2helix of CENP-A or H3is relatively straight in the CENP-A–H4(ref.24)(Fig.4b)or H3–H4tetramer 25(Supplementary Fig.18).Fourth,the C-terminal region (94–99)of H4in scSCH adopts a striking helical conformation and extends the a 3helix of H4.The helical conformation is induced by the side chain

H3 mutant His6–Scm3H3 mutant H4H4c

H 3M u

t _5

+ H 4H 3M u

t _4

+ H 4H 3L 1 + H 4

I110

I111

Y114

I117

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M181

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TDE F T T KDQ DLR W QS M AI M ALQE AS EAYLV G L L E H TNLLA L 20175AQD F K T---DLR F QS S AI G ALQE SV EAYLV S L F E D TNLAA I 112

M AI M ALQE AS

W QS M AI M ALQE AS

7575 TDE F T T KDQ DLR W CVKFT-RGVDFNWQA Q AL L ALQE AA EAFLVHLFEDAYLLTL 114

.CATD H3122′3′

L1Cse4

CENP-A

H3L1

H3Mut_4H3Mut_5..

Cse4

Scm3d

Scm3

Scm3Cse4Cse4

Figure 2|The N-terminal region (181–190)of the a 2helix of Cse4is the

Scm3recognition motif.a ,Ile 110,Ile 111,Tyr 114and Ile 117(balls in magenta)in the a N helix of Scm3form a hydrophobic cluster with Cse4-specific residues Met 181and Met 184(balls in cyan)in the a 2helix of Cse4.b ,Trp 107(balls in magenta)in the a N helix of Scm3has close interactions with the Cse4-specific residue Ala 189(balls in cyan)in the a 2helix of Cse4.Ser 190is also a Cse4-specific residue (balls in cyan).c ,SDS–PAGE gels showing the pull-down results with mutants of H3.The top panel shows the input of H3mutants and H4.The bottom panel shows the molecules eluted from His 6–Scm3(Scm3(65–169))-bound beads with 250mM imidazole.H3Mut_4,H3Mut_5and H3L1are the mutants of H3(see d ).d ,Illustration of the secondary

structures in Cse4and the Scm3recognition motif (dark cyan),CATD,and the mutants used in the pull-down experiments.The red squares indicate the four residues that are sufficient for specific recognition of Cse4by Scm3.The sequences swapped from Cse4to H3in the mutations are shown.The sequences that are not changed in the swap are omitted.

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of Leu 98in the a N helix of Scm3through hydrophobic interactions with the side chains of Leu 98and Tyr 99of H4(Fig.4a,Supplemen-tary Fig.14and Supplementary Table 2).This region is presumably disordered in the (CENP-A–H4)2tetramer 24(Fig.4b).Interestingly,the same region forms a mini b -strand that pairs with a b -strand of H2A in the canonical histone octamer or with a b -strand of histone

chaperone Asf1in the Asf1–H3–H4complex 26,27(Supplementary Fig.18).

Furthermore,the C-terminal region of Cse4has considerable dis-order in scSCH (Figs 4a,b).The same region is also disordered in the Cse4–H4dimer and is not required for Cse4–H4binding to Scm3(Supplementary Fig.5and Supplementary Fig.19).This ‘tetrameriza-tion domain’is well folded in the (CENP-A–H4)2tetramer.Structure modelling shows that imposing this folded domain on the correspond-ing region of Cse4in scSCH allows association as a (Scm3–Cse4–H4)2hexamer without major structural incompatibility (Supplementary Fig.20),consistent with the existence of (Scm3–Cse4–H4)2hexamers in 2M NaCl (ref.5).However,in this context,histone topography in the scSCH structure outside the tetramerization domain displays dramatic global conformational changes when compared with the (CENP-A–H4)2tetramer,making the modelled (Scm3–Cse4–H4)2hexamer incompatible with DNA binding (Supplementary Fig.20).Moreover,the Scm3loop in the scSCH structure blocks loop 2of H4,which makes contacts with DNA in the canonical nucleosome (Fig.4a,b).

Thus,the structure of scSCH indicates that retention of Scm3in association with centromere DNA is unlikely to occur via binding of Scm3(CBD)to Cse4/H4,as binding of DNA and Scm3(CBD)to Cse4/H4is mutually incompatible.Instead,Scm3(CBD)behaves as a specific histone chaperone,and the retention of Scm3with Cse4/H4on centro-meric DNA requires its distinct DNA-binding domain (H.Xiao and C.Wu,manuscript in preparation).

The structure of scSCH reveals that a subregion within the CATD,including four Cse4-specific residues in the N-terminal region of the a 2helix of Cse4,is necessary and sufficient for specific recognition by Scm3.Thus,the remainder of the CATD of Cse4should be important for association with other proteins for Cse4functions.The CBD of Scm3uses both induced histone conformation changes 26,27and direct steric occlusion 28to prevent Cse4–H4in the Scm3–Cse4–H4complex from DNA binding (Supplementary Fig.21).Conversely,Cse4–H4,with a conformation similar to that of CENP-A–H4in the (CENP-A–H4)2tetramer,is unfavourable for Scm3(CBD)binding but favours DNA binding,indicating a competition mechanism for Scm3and HJURP as CenH3-specific chaperones 22.

METHODS SUMMARY

All the proteins used in the present study were overexpressed in Escherichia coli and purified using Ni-NTA column (Qiagen),ion exchange,gel filtration and reverse-phase HPLC (Waters).Uniformly isotope-labelled proteins were produced using M9medium with 15NH 4Cl,13C-D -glucose and D 2O as the sole source of the isotopes.The molecular weight and stoichiometry of the complex were determined by velocity and equilibrium sedimentation experiments on a Beckman Coulter Proteome XL-I analytical ultracentrifuge at 20.0u C.The multi-dimensional NMR spectra were collected on Bruker 500,600,800and 900MHz and Varian 600and 800MHz instruments.The structure was calculated using the distance constraints measured by NMR and the program Xplor-NIH.Mutations were made using a quick-change kit.The binding constants were measured on the MicroCal VP-ITC instrument.

Full Methods and any associated references are available in the online version of the paper at https://www.sodocs.net/doc/2817480805.html,/nature.

Received 21October 2010;accepted 17January 2011.Published online 16March 2011.1.

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M103

223

332

2′3′

L98

Y99

L98G196

Cse4

CENP-A

Scm3DNA

DNA

Figure 4|Scm3induces large conformational changes in Cse4and H4and prevents loop 2of H4from binding to DNA.a ,Cse4–H4in scSCH.The extended a 3helix in H4is shown in light green.The loop of Scm3pushes loop 1of Cse4away from loop 2of H4and prevents loop 2of H4from binding to DNA.DNA is modelled to bind the loop 2region of H4based on the canonical nucleosome structure 29.b ,CENP-A–H4in the (CENP-A–H4)2tetramer.DNA is modelled to bind to the loop 2region of H4based on the canonical nucleosome struture 29

Figure 3|Altered interactions in the CATD region in scSCH.a ,The region of the a 2helices of Cse4and H4in scSCH,showing that there is little side-chain interaction between the two helices.b ,The corresponding region of the a 2helices of Cse4and H4in the Cse4–H4dimer structure obtained by homology modelling based on the structures of H3and H4in the nucleosome,showing that there are many hydrophobic interactions.c ,The region of the a 2helices of H3and H4in the nucleosome.d ,Region of loop 1of Cse4and the loop 2of H4.The side chains of the hydrophobic residues are shown in stick representation and orange.The extra three residues in loop 1of Cse4are shown in stick representation.e ,The corresponding loop 1of H3and loop 2of H4in the nucleosome structure.The hydrophobic residues are shown in stick representation and orange.

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Supplementary Information is linked to the online version of the paper at

https://www.sodocs.net/doc/2817480805.html,/nature.

Acknowledgements We thank J.Ying,K.Varney,J.F.Ellena and J.Gruschus for help collecting NMR spectra,A.Bax for discussion,C.Klee and M.Lichten for comments on the manuscript,and D.Cleveland for plasmids of human CENP-A and H4histones.This work is supported by the intramural research programs of NCI,NIDDK and NHLBI. Author Contributions Z.Z.and H.F.contributed equally to this work.Z.Z.performed protein engineering,biochemical and ITC studies.B.-R.Z.contributed to protein sample preparation.B.-R.Z.and L.M.M.J.contributed to the analysis of ITC data.H.F.,K.H.,A.Z. and N.T.collected the NMR spectra.H.F.and Z.Z.analysed the NMR data and H.F. solved the structure.R.G.performed the sedimentation experiments.H.X.provided initial plasmids and guidance in cloning.C.W.proposed the project and participated in manuscript writing.Y.B.contributed to the overall strategy,project management and writing of the manuscript.All authors read and commented on the manuscript. Author Information The atomic coordinates have been deposited in the Protein Data Bank under accession code2L5A.Reprints and permissions information is available at https://www.sodocs.net/doc/2817480805.html,/reprints.The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at

https://www.sodocs.net/doc/2817480805.html,/nature.Correspondence and requests for materials should be addressed to Y.B.(yawen@https://www.sodocs.net/doc/2817480805.html,).

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METHODS

Protein sample preparation.All proteins were expressed in E.coli(BL21-codonPlus(DE3)-RIL)with pET vectors(Stratagene).N-terminal His6-tagged fragments of Cse4and Scm3and their mutants were first purified via Ni-NTA (Qiagen)whereas H4and non-His-tagged fragments of Cse4and Scm3were first purified via SP sepharose(GE Healthcare).They were next subjected to reverse-phase HPLC purification using acetonitrile and water as solvents.Purified proteins were lyophilized.Isotope-labelled proteins for NMR studies were produced by growing E.coli cells in M9media with15NH4Cl,U-13C6-glucose,and D2O as the sole source for nitrogen,carbon and deuterium,respectively.For the measurement of side-chain NOEs,specific methyl labelling(-13CH3)for Ile,Leu and Val residues was also made following the protocol of ref.30.

To prepare the Cse4,H4and Scm3complexes,lyophilized proteins were first dissolvedinH2O.Theirconcentrationsweredeterminedbymeasuringtheabsorbance at280nm.Equal amounts of each species were mixed together and dialysed against 10mM Tris-HCl and2M NaCl at pH7.4and4u C.After centrifugation,the soluble fractions were subjected to gel filtration on Superdex7510/300GL column(GE Healthcare).The eluted complexes were concentrated with an Amicon with Ultra Ultracel-10membrane(Millipore)and exchanged to a final buffer of50mM MES at pH5.6.The Cse4–H4complexes were made in the same way.Scm3(80–211)samples for NMR study were prepared by dissolving them in8M urea and dialysis against correspondingbuffer.scSCHandallothersingle-chainmoleculesderivedfromscSCH are purified with Ni-NTA(Qiagen)under native conditions(20mM Tris-HCl and 0.5M NaCl at pH8.0),followed by gel filtration with Superdex20010/60column at 4u C(GE healthcare).The fractions containing the target protein were combined and concentrated and exchanged with the final buffer(50mM MES at pH5.4). Analytical ultracentrifugation.Sedimentation velocity experiments were con-ducted in duplicate at20.0u C on a Beckman Coulter Proteome XL-I analytical ultracentrifuge.400m l of the sample of35m M in50mM MES(pH5.6)was loaded in a double sector centrepiece cell and analysed at a rotor speed of50,000r.p.m.One-hundred scans were acquired as single absorbance measurements(l5280nm)at 7.1-min intervals using a radial spacing of0.003cm.Data were analysed in SEDFIT 11.71in terms of a continuous c(s)distribution to obtain a sedimentation coefficient, s,and molecular mass M(ref.31).Solution densities r were measured at20.0u C on a Mettler Toledo DE51density meter and solution viscosities g were measured using a Cannon–Ubbelohde viscometer and Cannon-CT500constant temperature bath set at20.00u C.The partial specific volume v of the complex was calculated in SEDNTERP1.09(ref.32).c(s)analyses were carried out using an s-value range of 0.5to6.0with a linear resolution of100and a confidence level(F-ratio)of0.68.The analyses,implemented using time-independent noise corrections,returned root mean square deviation(r.m.s.d.)values for the best fits of0.0040absorbance units. Sedimentation equilibrium experiments were conducted at20.00u C on a Beckman Optima XL-A.135m l volumes of the complex were studied at loading concentra-tions of20,39and78m M,along with the sample recovered from the sedimentation velocity experiments.Experiments were carried out using six-channel centrepiece cells at rotor speeds ranging from18,000to34,000r.p.m.In all cases data were acquired as an average of four absorbance measurements at wavelengths of280and 250nm using a radial spacing of0.001cm.Sedimentation equilibrium at each speed was achieved within40h.Data were analysed globally in terms of a single ideal species using SEDPHAT6.21(refs32,33).

NMR experiments.NMR experiments were performed on Bruker500,600,800 and900MHz and Varian600and800MHz spectrometers at35u C.The following experiments were recorded.2D:[1H,1H]-NOESY,[1H,15N]-TROSY,[1H,13C]-HMQC,15N-{1H}NOE;TROSY version3D:HNCACB,HNCOCACB,HNCA, HNCOCA,HNCO,HNCACO;3D HBHACONH,HCCH-TOCSY,CCH-TOCSY,CCC(CO)NH,[1H,15N]-NOESY-HSQC,[1H,15N]-NOESY-HSQC ([13C]methyl-labelled sample),[1H,13C]-NOESY-HSQC,[1H,13C]-NOESY-HSQC([13C]methyl-labelled sample).The spectra were processed using NMRPipe34and analysed with NMRView35.

Structure calculation.Structure calculation was done using the program Xplor-NIH36.The NOE-derived restraints were subdivided into four classes,strong,med-ium,weak and very weak,by comparison with NOEs of protons separated by known distances as described previously37.Backbone dihedral angle restraints(w and y angles)were obtained from analysis of1H a,HN,13C a,13C b,13CO and15N chemical shifts by using the program TALOS38.Two constraints per hydrogen bond(dNH-O#2.2A?and dN-O#3.2A?)were added in the final structure calculation after initial NOE-derived structures were obtained.The program PROCHECK_NMR39 was used to evaluate the quality of the calculated structures.

Isothermal titration calorimetric experiments.The ITC experiments were per-formed on a MicroCal VP-ITC by injecting Scm3(83–169)solution(250m M)to a solution of single-chain Cse4–H4or their mutants(His6-KK-Cse4(151–207)-LVPRGS-H4(45–103))(20m M)in a chamber of1.4ml at25u C in50mM MES (pH5.4)and0.1M NaCl.Twenty-nine injections(each of10m l)were made and the heat released was analysed.The data were analysed as described previously40. Pull-down experiments.Pull-down experiments were carried out in50mM sodium phosphate,25mM imidazole,2M NaCl,pH8.0at room temperature. Ni-NTA(Qiagen)beads were mixed with His6–Scm3(66–169)with a final con-centration of6m M.Approximately10-fold excess of(Cse4–H4)2or(H3–H4)2or their mutants was mixed with beads and incubated.The incubation was at25u C for30min.The beads were washed with the same buffer three times.The complex formed on the beads was eluted with250mM imidazole and analysed by SDS–PAGE.Beads without His6–Scm3were also incubated with corresponding(Cse4–H4)2under identical conditions to assess background binding and the integrity of the tetramer.No nonspecific binding was identified in2M NaCl.For molecules derived from thrombin-digested single-chain proteins,the complex was incubated with Ni-NTA(Qiagen)beads at25u C for30min and then washed three times.The final complex formed on the beads was eluted with either8M urea or250mM imidazole.The eluted molecules were analysed by SDS–PAGE.

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molecular-weight proteins by solution NMR spectroscopy.Nature Protocols1, 749–754(2006).

31.Schuck,P.Size-distribution analysis of macromolecules by sedimentation velocity

untracentrifugation and Lamm equation modeling.Biophys.J.78,1606–1619 (2000).

32.Cole,J.L.,Lary,J.W.,Moody,T.P.&Laue,T.M.Analytical ultracentrifugation:

sedimentation velocity and sedimentation equilibrium.Methods Cell Biol.84,

143–179(2008).

33.Schuck,P.On the analysis of protein self-association by sedimentation velocity

analytical ultracentrifugation.Anal.Biochem.320,104–124(2003).

34.Delaglio,F.et al.NMRPipe:a multidimensional spectral processing system based

on UNIX pipes.J.Biomol.NMR6,277–293(1995).

35.Johnson,B.A.&Blevins,R.A.NMRView:a computer program for the visualization

and analysis of NMR data.J.Biomol.NMR4,603–614(1994).

36.Schwieters,C.D.,Kuszewski,J.,Tjandra,N.&Clore,G.M.The Xplor-NIH NMR

molecular structure determination package.J.Magn.Reson.160,65–73(2003).

37.Zhou,Z.et al.NMR structure of chaperone Chz1complexed with histones

H2A.Z-H2B.Nature Struct.Mol.Biol.15,868–869(2008).

38.Cornilescu,G.,Delaglio,F.&Bax,A.Protein backbone angle restraints from

searching a database for chemical shift and sequence homology.J.Biomol.NMR 13,289–302(1999).

https://www.sodocs.net/doc/2817480805.html,skowski,R.A.et al.AQUA and PROCHECK-NMR:programs for checking the

quality of protein structures solved by NMR.J.Biomol.NMR8,477–486(1996).

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Biochemistry43,4170–4178(2004).

LETTER RESEARCH

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