mapping intramolecular interactions between domains in hmgb1 using a tail-truncation approach

doi:10.1016/j.jmb.2007.09.075 J. Mol. Biol. (2007) 374, 1286–1297

Available online at www.sciencedirect.com

Mapping Intramolecular Interactions between Domainsin HMGB1 using a Tail-truncation Approach

Matthew Watson†, Katherine Stott† and Jean O. Thomas⁎

Department of BiochemistryUniversity of Cambridge80 Tennis Court RoadCambridge CB2 1GA, UK

Received 2 May 2007;received in revised form26 September 2007;accepted 26 September 2007Available online2 October 2007

*Corresponding author. E-mail [email protected].† M.W. and K.S. contributed equaAbbreviations used: HMG, high-m

HSQC, heteronuclear single quantunuclear Overhauser effect; NOESY,

0022-2836/$ - see front matter © 2007 E

The mechanism underlying negative regulation of HMGB1-DNA interac-tion by the acidic C-terminal tail is ill defined. To address this issue, we havedevised a novel NMR chemical-shift perturbation mapping strategy toelucidate interactions between the tail, which consists solely of aspartic acidand glutamic acid residues, and the twowell characterizedHMG-box DNA-binding domains. A series of HMGB1 tail-truncation mutants differing fromeach other by five residues was generated. Chemical-shift perturbationmapping using these mutants shows that tails of different lengths bind withdifferent affinities. Nevertheless, the truncated tails bind along the samepath on the HMG boxes as the full-length tail, differences in length beingmanifested in differences in the “reach”. The tail makes extensive contactswith the DNA-binding surfaces of both HMG boxes, thus explaining thebasis of negative regulation of HMGB1–DNA interaction by the tail.

© 2007 Elsevier Ltd. All rights reserved.

Keywords: HMG box; NMR spectroscopy; acidic tail; chemical-shiftperturbation
Edited by P. Wright
Introduction

High-mobility group protein B1 (HMGB1) is anabundant non-histone chromosomal protein that isexpressed ubiquitously inmammalian cells. HMGB1bends DNA significantly, and it appears to actprimarily as an architectural facilitator in theassembly of nucleoprotein complexes, in which thebound DNA is often tightly bent.1–3 HMGB1 has atripartite structure consisting of two homologoustandem HMG-box DNA-binding domains of ∼80amino-acid residues (the A and B boxes) linked by a20-residue basic extension to a 30-residue C-terminalacidic tail composed entirely of aspartic acid andglutamic acid residues. The HMG box binds to linearDNA in the minor groove with little or no sequencespecificity, inducing a significant bend in the DNA,and has a preference for distorted DNA structures(e.g. four-way junctions, bulges, minicircles andcisplatin-modified DNA). Despite their sequenceand structural similarity, the A and B HMG boxes

ess:

lly to this work.obility group;

m coherence; NOE,NOE spectroscopy.

lsevier Ltd. All rights reserve

appear to have distinct functional roles, the A boxbeing the primary determinant of structure-specificDNA binding, and the B box of DNA bending.4–6

The structures of both the HMG boxes ofHMGB1 have been determined,7–9 and theirDNA-binding properties characterized,3 but thestructure and role of the acidic tail are poorlyunderstood. The tail generally down-regulatesDNA binding by the tandem HMG boxes invitro,10,11 with the exception of binding to high-affinity DNA substrates, such as minicircles.12

There is growing evidence that the acidic tail hasan essential role in several important functions ofthe protein, e.g. stimulation of transcription,13,14

and facilitation of chromatin remodelling.15,16 Thetail may also influence post-translational modifica-tion of the protein.17 A previous NMR studyidentified several residues from the body of theprotein involved in the interaction with the tail.18

However, due to the low sequence complexity andthe unstructured nature of the HMGB1 acidic tail,chemical-shift assignments are problematic toobtain, and detailed mapping of the interaction ofthe tail with the body of the protein has not hithertobeen possible. In this study, we describe a novelNMR chemical-shift perturbation mapping strategythat circumvents the need for chemical-shift assign-ments. A series of tail-truncationmutants are used tomap the interaction of distinct sections of the acidic

d.

http://dx.doi.org/10.1016/j.jmb.2007.09.075

1287Mapping Intramolecular Interactions in HMGB1

tail of HMGB1 with specific residues in the body ofthe protein.

Figure 2. CD analysis of HMGB1 and the tail-trunca-tion proteins. (a) Far-UV and (b) near-UV CD spectra ofHMGB1 (Δ0) and the tail-truncation proteins (Δ5–Δ30) atprotein concentrations of 6.5–8.6 μM and 63–85 μM,respectively.

Results

The tail-truncation mutants are shown in Figure 1.The proteins are termed Δ0 (full-length HMGB1; 30tail residues), Δ5 (25 tail residues), Δ10 (20 tailresidues), Δ15 (15 tail residues), Δ20 (ten tailresidues), Δ25 (five tail residues) and Δ30 (the tail-less protein; previously termed AB′12).

Secondary and tertiary structure are unaffectedby tail length

Far-UV circular dichroism (CD) spectra of full-length HMGB1 and the tail-truncation proteinsshow that all the proteins have a high α-helicalcontent (Figure 2(a)). Removal of the acidic tail(cf. Δ0 with Δ30, which lacks the tail) resulted in anincrease in negative mean residue ellipticity at208 nm and at 222 nm, indicating an increase inthe proportion of α-helix in the truncated protein,consistent with a predominantly random-coil con-formation for the tail. The spectra of the various tail-truncation proteins lie between those of HMGB1and Δ30 (Figure 2(a)).The near-UV CD spectra show that the proteins

are overall folded (Figure 2(b)). Interestingly,removal of the tail (Δ30) causes a change in thespectrum, suggesting that the tail interacts with theboxes at positions close to one or more of thehydrophobic cores (two per HMG box), and maycause a small conformational change in one or both

Figure 1. HMGB1 and the tail-truncation proteins. (a) Domtruncation mutants. (b) SDS/18%-PAGE (gel stained with Ctruncation proteins Δ5–Δ30 (lanes 3–8); lane 1, molecular maindicated in red, the minimal B domain in blue and the acidic tpositions of the α-helices (grey boxes) determined by NMR sp

boxes. The spectra appear to fall into two groups:those (Δ5 and Δ10) that resemble the full-lengthprotein, Δ0, and those (Δ15, Δ20, and Δ25) that

ain structure of HMGB1 and the tail sequences of the tail-oomassie brilliant blue) of HMGB1 (lane 2) and the tail-ss marker. (c) Sequence of rat HMGB1. The A domain isail in green. The diagram below the sequence indicates theectroscopy of the isolated domains.7,9

1288 Mapping Intramolecular Interactions in HMGB1

more closely resemble the tail-less protein, Δ30,suggesting that the region of the tail responsible forthese spectral changes is the five-residue segmentbetween Δ10 and Δ15 (200EEEED204).

The tail affects thermal stability and DNAbinding in a length-dependent manner

The acidic tail is known to modulate the DNA-binding properties of HMGB1,10–12,19 and its ther-mal stability,18,20 presumably through interactionswith one or both of the HMG boxes and the basiclinkers. The effect of tail truncation was assessed byCD thermal denaturation and DNA-cellulose bind-ing experiments. Comparison of the results for Δ0and Δ30 (Figure 3(a)(i)) shows clearly that the acidictail stabilizes the protein against thermal dena-turation, as shown previously.18,20 Examination ofthe truncated proteins shows that the stabilization islength-dependent for tails from 0 to 15 residues(Δ30–Δ15; Figure 3(a)(ii)). Constructs with morethan 15 tail residues (Δ10, Δ5, and Δ0 (Figure 3(a)

Figure 3. Effect of tail length on the thermal stability afollowed by measurement of ellipticity at 222 nm. The curveComparison of HMGB1 and the tail-less protein (Δ30); (ii) comthese conditions (Δ30, Δ25, Δ20, and Δ15); (iii) comparison oflength HMGB1). The addition of each five-residue tail segmen46.8 °C for Δ20, and 50.3 °C for Δ15; i.e. the proteins that couproteins was bound to DNA-cellulose and eluted with buffers cSDS/18%-PAGE (gel stained with Coomassie brilliant blue) of2–13); lane 1 in each case shows the unbound protein. Represen(Δ30) are shown. (ii) Quantification of the elution profiles by

(iii))) were prone to aggregation upon denaturationand could not be refolded. The affinity of theproteins for DNA-cellulose increased with decreas-ing tail length, as expected (Figure 3(b)), althoughΔ20 and Δ25 bound with similar affinities, suggest-ing that the five residues by which Δ20 and Δ25differ may interact with the boxes in a manner thatdoes not affect DNA binding. The alternativeexplanation, that these tail residues do not interactat all with the body of the protein, was not consistentwith NMR data (discussed below).

Interaction of 15N-labelled tail peptide withHMGB1 lacking the tail (Δ30)

An earlier study suggested that the interaction ofthe tail of HMGB1 with the body of the proteincould be studied using Δ30 (there termed ABbt) anda separate tail peptide, and chemical-shift changes in15N-labelled Δ30 that occurred upon addition of theunlabelled tail peptide were mapped.18 We havecarried out the reciprocal experiment using the 15N-

nd DNA binding of HMGB1. (a) Thermal denaturations are shown as the percentage loss of helical content. (i)parison of the proteins that could be fully refolded underthe proteins that could not be refolded (Δ10, Δ5, and full-t increases Tm, which is 43.6 °C for Δ30, 45.0 °C for Δ25,ld be refolded. (b) Binding to DNA-cellulose. Each of theontaining increasing concentrations of NaCl (as shown). (i)TCA-precipitated proteins from the eluted fractions (lanestative gels for full-length HMGB1 and the tail-less proteindensitometry, using BSA as a loading control.


labelled tail peptide in order to look at the tailcomponent of the complex.UnlabelledΔ30 was titrated into a solution of 15N-

labelled tail up to a molar ratio of 4:1 (Δ30 to tail)and 1H-15N heteronuclear single quantum coher-ence (HSQC) NMR experiments were recorded(Figure 4). The spectrum of the tail peptide alonecontains approximately the expected number ofpeaks and (as predicted for approximately neutralpH) these peaks lie within the random-coil region ofthe spectrum (∼8.1–8.6 ppm).21 Upon addition ofΔ30, the tail resonances shift and broaden, indica-tive of an interaction. No new peaks appear,indicating that the resonances are in fast exchangeon the NMR chemical-shift time-scale. Up to a Δ30to tail ratio of 1:1, the chemical shifts become morelike those of the tail in full-length HMGB1 (thespectra overlay to a large extent; Figure 4), suggest-ing that the major binding mode of the tail is native-like, and that the interaction between the tail and therest of the protein shows a considerable degree ofspecificity, as suggested previously.18 A largeproportion of the tail resonances shift, indicatingthat the tail makes extensive contacts with the bodyof the protein.As the molar ratio of Δ30 to tail peptide is in-

creased beyond 1:1, the tail resonances become lesslike those in the native protein. Evidently, saturationof the native binding mode is reached at a 1:1 molarratio; up to 4:1, additional binding modes becomeincreasingly populated, until no further change isseen. This is most clearly illustrated by inspection ofthe backbone amide resonance of the C-terminal Gluresidue (Figure 4, inlay). At a Δ30 to tail molar ratioof 1:1 (magenta), this resonance overlays well with

Figure 4. Binding of unlabelled Δ30 to 15N-labelledtail peptide. 1H-15N HSQC spectra of the free 15N-labelledtail peptide (blue) and the tail peptide in the presence ofunlabelled Δ30 at Δ30 to tail ratios of 1:1 (magenta) and4:1 (yellow) were overlaid with the corresponding regionof the 1H-15N HSQC spectra of the full-length protein(black). The inlay shows the 1H-15N resonance for theC-terminal residue (Glu214). Optimal overlap with thefull-length protein occurs at a 1:1 molar ratio.

the corresponding resonance in full-length HMGB1(black), but no longer overlays at a Δ30 to tail molarratio of 4:1 (yellow). When bound toΔ30, the amide-proton chemical shifts of the tail peptide are stillwithin the random-coil region of the spectrum andno medium-range or strong sequential nuclearOverhauser effect (NOE) (e.g. dNN(i,i+1)) indicativeof secondary structure22 are observed in NOEspectroscopy (NOESY)-HSQC spectra, even forlonger mixing times (up to 250 ms; data notshown). This indicates that the tail peptide, and byinference the tail in HMGB1, lacks a definedsecondary structure when bound to the rest of theprotein.

Chemical-shift assignments for thetail-truncation mutants

Initial 1H and 15N assignments were made usingHSQC and NOESY-HSQC spectra recorded for Δ25,which did not suffer from the spectral overcrowdingand peak broadening seen for the proteins withlonger tails. Assignments were then propagatedto the other spectra and additional NOESY ortotal correlation spectroscopy-HSQC spectra wererecorded where necessary to resolve ambiguities.The following percentages of backbone resonanceswere assigned (excluding tail resonances):Δ30, 95%;Δ25, 96%; Δ20, 94%; Δ15, 94%; Δ10, 95%; Δ5, 95%;and Δ0 (HMGB1), 95%. Unfortunately, due toextensive overlap in the central random-coil regionof the spectra and the lack of sequential NOEinformation, only the two C-terminal resonances ofthe acidic tail could be sequentially assigned. In theNOESY spectra of the full-length protein (Δ0), weakNOE crosspeaks are observed between the HN ofGlu and Asp in the acidic tail, and resonances fromresidues elsewhere in the protein (Figure 5(a)).Those at ∼3 ppm presumably correspond to theHδ and Hε protons of Arg and Lys residues, res-pectively, in the boxes or linkers. However, thesecannot be assigned sequence-specifically due to thelarge number of Arg (8) and Lys (43) residues, andthe heavy overlap of their side-chain resonances.The assignments of the NOEs observed at otherchemical shifts are similarly ambiguous.Two new intrinsic structural features were ob-

served for the B domain in the context of the AB′didomain (Δ30) that had not been seen in theoriginal study of the minimal B box (residues 88–164).7 The first is a two-residue extension (Arg-Ala)to helix III of the B box, indicated by large chemical-shift changes, increased line-widths and additionalsequential and medium-range NOEs for residuesArg162 and Ala163 compared with their counter-parts in the isolated domain. Evidently, the last turnof the helix is destabilized by truncation at residue164, possibly by the presence of the chargedcarboxylate group. Interestingly, the longer helixwas observed in a complex of the chimeric proteinSRY.B with DNA where, although the B box istruncated, the helix is stabilized by interaction withDNA.23 The second new feature observed in the B

Figure 5. NMR spectroscopy of HMGB1 and the tail-truncation proteins. (a) Strip plots from 3D NOESY-15N-HSQCspectra. Long-range NOEs observed for the tail in the context of full-length HMGB1 (gold) are indicated by asterisks. Theequivalent plane for the free tail peptide (black) is shown alongside for comparison. (b) Overlay of the 1H-15N HSQCspectra for Δ30 (red), Δ25 (beige), Δ20 (dark blue), Δ15 (magenta), Δ10 (light blue), Δ5 (green) and full-length HMGB1(yellow). Boxed sections are enlarged in (c). (i) Ala136, which does not shift; (ii) Lys111 and (iii) Lys49, which displaymoderate shifts between Δ5 and Δ15; (iv) Ile158 which displays a moderate shift between Δ10 and Δ25; (v) Thr76, whichdisplays large shifts that reverse direction after Δ10; (vi) Trp48 Hε1.


domain is a type-I β-turn formed by residues Asp90-Pro91-Asn92-Ala93. This was also seen in the SRY.B/DNA complex,23 and may be important forredirecting the inter-domain linker of HMGB1from the phosphate backbone back to the minorgroove, such that the B box is positioned correctlyfor DNA binding.

Mapping the interaction of the acidic tail with thebody of the protein using tail-truncation mutants

By superimposing HSQC spectra of HMGB1 andthe tail-truncation proteins (Figure 5(b)) it waspossible to identify the contacts made by each five-residue tail segment using chemical-shift perturba-


tions. The peaks shift to different extents withincreasing tail length; peaks representative of thedifferent kinds of behavior are indicated by boxes inFigure 5(c). Some peaks (e.g. Ala136) do not shift,and are therefore assumed not to be involved in tailbinding. Others (e.g. Lys111) show small shifts thatincrease steadily as the tail length increases, but formost peaks that shift, although the shifts are in thesame direction, the increase is uneven. This isexemplified by Lys49 and Ile158; the chemical shiftof Lys49 changes most between Δ5 and Δ15,whereas Ile158 shows the greatest change betweenΔ10 and Δ25. We deduce from this that Lys49contacts the section of the tail that is added in goingfrom Δ15 to Δ5 (i.e. residues 200–209), and thatIle158 contacts residues in the range 190–204. Thr76is an example of a small group of residues (75-78,170-172) that reverse direction with addition of thedistal ten tail residues (Δ10 to Δ0). Interestingly, alarge number of residues (including Lys49, Ile158and Trp48 Hε1) reverse direction very slightly withaddition of the last five residues (Δ5 to Δ0). The“back-tracking” of peaks is discussed below.Chemical-shift changes upon successive addition

of each five-residue tail segment to the tail-lessprotein (Δ30), abbreviated as Δδ(Δn, Δ30), areshown in Figure 6(a). Shift changes for Asn, Glnand Trp side-chains correlate well with the corre-sponding shift changes in the backbone resonancesand so are omitted. With the exception of a smallgroup of peaks in the spectrum of Δ15 (discussedbelow), all residues appear to give rise to a single,tracking peak, indicative of fast exchange on theNMR chemical-shift time-scale. The resulting che-mical shift is therefore a weighted average of theshifts of the free and tail-bound forms, and thusthere are two contributions to the chemical-shiftchange, the shift difference (δbound−δfree) and themole fraction of the tail-bound form (xbound). Sincethe affinity of the tail for the rest of the protein isvery likely to increase with tail length, a resonancethat shifts on contacting residue n of the tail can beexpected to shift further in tails that contain morethan n residues, even though it may make noadditional contacts with these residues, since xboundhas increased. Inspection of the overlaid (Δn, Δ30)chemical-shift differences in graphical form (Figure6(b)) confirms this assumption; the envelope foreach Δδ(Δn+5, Δ30) plot fits neatly under the nextΔδ(Δn, Δ30) set, but only after scaling by xbound(Δn)/xbound(Δn+5) to take into account the increasein xbound. Therefore, in order to compensate for the

Figure 6. NMR chemical-shift perturbation mapping ofChanges in backbone 1H-15N shifts (Δδ) for HMGB1 and theprotein (Δ30), indicated as Δδ(Δn, Δ30). (b) Shift differences (five-residue shorter protein (Δδ(Δn+5, Δ30), grey) scaled by(Δn+5) (see the text for details). (c) Shift differences obtained bΔδ(Δn, Δ30)–[Δδ(Δn+5, Δ30) × xbound(Δn)/xbound(Δn+5)]. Tthe schematic at the bottom shows the location of the α-helidifferences (inset in (c)) were created using MOLSCRIPT24 andunstructured linker regions in yellow. Residues that shift moreas space-fill.

effect of increasing xbound, the shift differences forthe (Δn+5, Δ30) set were subtracted from the (Δn,Δ30) set after the appropriate scaling had beenapplied. The scaling factors were approximately 1.3,1.2, 1.2, 1.0 and 1.0 for (Δ25, Δ30), (Δ20, Δ30), (Δ15,Δ30), (Δ10, Δ30) and (Δ5, Δ30), respectively,indicating that the tail affinities of Δ20 and Δ15are similar, and that the tail is essentially fully boundat lengths in excess of 20 residues. The shift differ-ences extracted in this way (Figure 6(c)) aredescribed by the expression:

DyðDn;D30Þ�½DyðDnþ 5;D30Þ � xboundðDnÞ=xboundðDnþ 5Þ�

and correspond directly to the new contacts for eachfive-residue section of the “growing” tail.Upon addition of the first five tail residues to the

tail-less protein (Figure 6(c)(i)), shifts occur pre-dominantly in the 20-residue basic extensionbetween the B box and the tail, with small shiftsin the N and C-terminal regions of the B box andthe C-terminal region of the A box. With furtherextension of the tail from five to ten residues(Figure 6(c)(ii)), large changes are seen in the N andC-terminal regions of both boxes and the inter-boxlinker, as well as several smaller shifts throughoutthe concave face of the B box. When the tail isextended to 15 residues (Δ20 to Δ15; Figure 6(c)(iii))the trend is the same, with additional shifts in theN-terminal strand of the A box and further shifts atthe N and C-terminal regions of both boxes, but thenew shift changes are small, implying that thissection of the tail does not make extensive contactswith the body of the protein. With the extension ofthe tail from 15 to 20 residues (Δ15 to Δ10; Figure 6(c)(iv)) the trend changes, with the largest newchemical-shift perturbations being predominantlyin residues from the N-terminal strand and helices Iand II of the A box, on the concave face. This trendcontinues as the tail is extended from 20 to 25residues (Figure 6(c)(v)), with additional changes inthe basic linker between the B box and the tail.Finally, the last five residues of the tail cause smallchemical-shift changes, in both the forward andreverse directions, over most of the protein, imply-ing that these residues exhibit multiple non-specificbinding modes.Peak line-widths broaden significantly for most

peaks as the tail length is increased, indicating adecrease in the overall tumbling rate. The spectrumofΔ15 contains some peaks, generally those in the N

the interaction of the tail with the body of HMGB1. (a)tail-truncation mutants (Δn) compared with the tail-lessΔδ(Δn, Δ30), black) overlaid with shift differences for thean appropriate factor corresponding to xbound(Δn)/xboundy subtraction of the grey from the black data-sets in (b); i.e.he positions of proline residues are indicated by asterisks;ces in the HMG boxes. Molecular representations of shiftRaster3D25 with the A box in red, the B box in blue, andthan 1.5 standard deviations from the mean are rendered

Figure 6 (legend on previous page)



and C-terminal regions of the A and B domains, forwhich the largest overall shift changes are seen, thatshow evidence of additional peak broadening (anexample, Ile158, is shown in Figure 5(c)(iv)), imply-ing that the exchange rate for these resonances isfast-to-intermediate on the chemical-shift time-scale.In a subset of these broadened peaks, two distinctpeaks can be resolved (an example, Thr76, is shownin Figure 5(c)(v)). This indicates the presence of twospecies, interconverting on a time-scale that is slowcompared with the NMR chemical-shift time-scale,but both species are in fast exchange between thefully bound and the unbound forms. This peakdoubling is likely to represent two subtly-distinctbinding modes of the 15-residue tail.

Discussion

While previous studies agree that the tail of HMGB1binds to the body of the protein,18–20,26 no clearpicture has emerged as to whether one or both boxesare involved, possibly as a result of differences inmethodology, and the nature of the interaction isunclear. We find that the tail interacts with bothboxes. The interaction appears to be specific, despitethe basic nature of the HMG boxes and linkers, andthe highly acidic nature and low sequence complex-ity of the tail. The tail interacts in a similar mannerwhether covalently attached or as a separate peptidein the presence of the tail-less protein (Δ30). Theresidues from the body of the protein that showsignificant chemical-shift perturbations in the pre-sence of the tail are qualitatively similar to thosereported previously;18 detailed comparison of thedata in the two studies is inappropriate due todifferences in experimental conditions.CD spectroscopy shows that the tail in full-length

HMGB1 adopts a random-coil conformation, andthat truncation of the tail does not significantly affectthe structure of the folded HMG boxes (Figure 2).This is also apparent from the 1H-15N HSQC spectraof the truncated proteins (Figure 5(b)) where,although many chemical-shift changes occur, theoverall appearance of the spectra remains verysimilar. In addition, both long-range NOEs andstrong dNN(i,i+1) NOEs between residues in theα-helical regions seen in the NOESYexperiments arepreserved for each truncation (data not shown). Thetail, although specifically bound, as shown by long-range NOEs between the tail and the boxes/linkers(Figure 5(a)), does not appear to adopt any stablesecondary structure. This is evident from the poorchemical-shift dispersion and the lack of strongsequential or medium-range NOEs, both in full-length HMGB1 and in the complex formed byaddition of Δ30 to the free tail peptide. Moreover,the tail displays only very limited dynamics onthe nano- to picosecond time-scale. {1H}15N hetero-nuclear NOE values recorded at 500 MHz are closerto those observed for residues in short loops and injunctions between structured and unstructuredregions than in fully-unstructured proteins (∼+0.2

in the heavily overlapped region corresponding toresidues 185–212, −0.2 for residue 213, and −0.6 forthe C-terminal residue 214).The use of the novel mapping method based on a

tail-truncation strategy is justified by the observa-tion that the shortened tails interact with the body ofthe protein in the same mode as the equivalentresidues in full-length HMGB1 and differ only intheir “reach” and affinity. Tails of different lengththat differ in affinity may nonetheless be comparedby the use of appropriate normalizing factors. Theproximal half of the tail (residues 185–199) primarilycontacts the N- and C-terminal regions of each box,and the concave face of the B box. The next tenresidues (200–209) contact the concave face of the Abox. Relatively large chemical-shift changes areobserved for the side-chain resonances of Trp48(Figure 5(c)(vi)), which lies in the major hydropho-bic core of the A box.9 A corresponding step-changeoccurs in the near-UV CD spectra of the proteins(Figure 2(b)), which is sensitive to the environmentof aromatic residues. The five residues that extendthe tail from Δ15 to Δ10 (residues 200–204) appearto be involved directly in recruiting the A box to theassembly. The two subtly different forms resolved inthe spectra forΔ15 may arise because, in the absenceof a sufficient number of tail residues for A-boxrecruitment, residues 200–204 (or a subset of these)make alternative non-native contacts.As mentioned above, a small number of peaks

begin to reverse direction at a tail length of 20residues (Δ10; Figure 6(c)(v) and (vi)). It is possiblethat upon recruitment of the A box by the tail, theboxes compete for tail contacts in the tail-boundstate, weakening interactions that were alreadypresent with shorter tails, or that a conformationalchange occurs. Most residues reverse direction veryslightly with the addition of the last five tail residues(Δ5 to Δ0; Figure 6(c)(vi)). Taken at face value, fromthe chemical-shift changes, these appear to contact alarge number of residues, and may be inferred tointeract non-specifically; whether the shifts are dueto a global loosening/tightening effect is unclear. Inany event, it may be assumed that this region of thetail is relatively delocalized and thus potentiallyaccessible for recruitment by other partners. Thiswould be consistent with the proposed role of the C-terminal 210DDDDE214 in binding to the N-terminaltail of histone H3 to position HMGB1 on thenucleosomal linker DNA in chromatin.14

It is striking that the tail interacts extensively withthe concave faces of the boxes, which are known tobe the DNA-binding surfaces.9,23,27 Given theinherent dynamic nature of the bound tail, it islikely that the perturbation data represent a staticpicture of the average of the tail-bound states inwhat must be a dynamic equilibrium between themore compact, tail-bound form(s) and “open”, freeforms in which the DNA-binding surfaces areaccessible. Given that the open forms representonly a small fraction of the population at any onetime, it is not surprising that only binding to veryhigh-affinity substrates (e.g. minicircles) is relatively


unaffected by the tail.12 This is also a property of thesingle HMG box protein HMG-D, which has a muchshorter acidic tail (12 residues).28

It is well established that no interaction is ob-served between the two HMG boxes in the absenceof the tail, where the domains undergo independentanisotropic diffusion.29 It is striking from themolecular representations of the tail-interactingresidues in Figure 6(c), where the boxes are shownas non-interacting entities in a linear arrangement,that some of the five-residue tail portions contactboth boxes, or regions that are distant in this veryopen representation. There are two possible expla-nations for this, one being that the tail may bepopulating more than one binding mode, and isdistributed between the two boxes. A more likelyscenario would seem to be a “closed” structure, inwhich the tail, rather than being elongated andtracking across the surface of a linear arrangementof the two HMG-box domains of the protein,draws in the basic boxes and linkers around it. Arepresentation of this is postulated in Figure 7,showing how the boxes and linkers might beorganised, such that the residues identified by themapping in Figure 6 (including residues on theDNA-binding surfaces of both boxes and in the basiclinkers) all make extensive and simultaneous contactwith the tail.In summary, we describe here a novel methodol-

ogy for the study of intramolecular interactions inHMGB1 that may prove useful in studies of othersystems comprising elements of intrinsicallyunstructured proteins that are problematic to assign(e.g. due to a low sequence complexity) in theirinteraction with structured partners. Using thisapproach, we show for the first time that the acidic

Figure 7. A representation of a “compact” tail-boundform of HMGB1. Structural representations of the A (red)and B (blue) boxes were created from the known structures(1AAB and 1HME) using MOLSCRIPT24 and Raster3D.25

The representations of the basic linkers (yellow) and theacidic tail (green) were added using a line-drawing tool.The DNA-binding faces would be rendered inaccessible inthis “closed” assembly (see the text).

tail of HMGB1 makes extensive intramolecularcontacts with the known DNA-binding surfaces ofboth HMG boxes.9,23,27 The DNA-binding surfacesare occluded and perhaps even internalized in acompact three-dimensional structure. These resultsprovide a clear mechanism for how the tail couldnegatively regulate the affinity of the HMG boxesfor most DNA substrates. When the tail is displacedby DNA it would become available for possibleinteraction with histones or other proteins. Inaddition, proteins interacting with the acidic tail infree HMGB1 may liberate the boxes and enhancetheir binding to DNA. It is worth noting that,despite its low level of sequence complexity anduniformly acidic character, the tail binds in aspecific, but nonetheless largely or wholly electro-static manner. These results add to the growing con-sensus that the tail plays a pivotal role in the functionof HMGB1.

Materials and Methods

Plasmids

Plasmid pT7.7 HMGB1-AB′ has been described.29

Plasmid pBAT4 HMGB1 contains the coding sequencefor rat HMGB1 PCR-amplified with a 5′-NcoI site and a3′-HindIII site from plasmid pT7.7cm rHMGB130 using5′-CATGCCATGGGCAAAGGAGATCC-3′ as the forwardprimer and 5′-GATGATAAGCTTATTCATCATCATCATC-3′ as the reverse primer, and inserted into plasmidpBAT4.31 Plasmids pBAT4 Δ5 to Δ25 contain the codingsequences for HMGB1 residues 1–209 (Δ5), 1–204 (Δ10),1–199 (Δ15), 1–194 (Δ20), and 1–189 (Δ25), which had beenPCR-amplified from pBAT4 HMGB1 with a 5′-NcoI siteand a 3′-blunt end using the same forward primer: 5′-CATGCCATGGGCAAAGGAGATCC-3′ and the follow-ing reverse primers: 5′-CTTATTCATCATCATCTTATTC-TTCTTCATCTTC-3′, (Δ5) 5′-CATCTTCTTCTTCATCTTA-ATCTTCCTCTTCTTC-3′, (Δ10) 5′-CTTCATCTTCCTCTT-CTTACTCCTCTTCCTCATC-3′, (Δ15) 5′-CTTCCTCC-TCTTCCTCTTACTCTTCATCCTCCTC-3′, (Δ20) 5′-TTAG-TCGTCTTCCTCTTCCTTCTTTTTCTTGCTC-3′, (Δ25).Plasmid pHAT3 HMGB1-tail, which encodes the acidic

tail of HMGB1 with an N-terminal thrombin-cleavable Histag, contains the coding sequence for the tail PCR-amplified from pBAT4 HMGB1 with a 5′-NcoI site and a3′-HindIII site using: 5′-CATGCCATGGAAGAGGAA-GACGACGAGG-3′ as the forward primer and: 5′-GAT-GATAAGCTTATTCATCATCATCATC-3′ as the reverseprimer and inserted into plasmid pHAT3 (similar topHAT).31 Plasmid construct sequences were verified byDNA sequencing.

Expression and purification of 15N-labelled HMGB1,the tail-truncation mutants (Δ5–Δ30) and unlabelledΔ30

15N-labelled proteins were expressed in Escherichia coliBL21(DE3) cells transformed with the appropriate plas-mid and grown in MOPS minimal medium32 supplemen-ted with 6 mM [15N]ammonium chloride as the solenitrogen source, and 50 μg/ml of carbenicillin. Expressionof 15N-labelled HMGB1Δ0, Δ5, Δ10 and Δ15 was carried


out as described for HMGB1,12 but with shaking at200 rpm during induction. Expression of 15N-labelledHMGB1Δ20, Δ25 and Δ30 was carried out as describedfor the individual domains;7,9 unlabelled Δ30 wasexpressed similarly but in 2×YT medium.Cell extracts containing recombinant HMGB1Δ0, Δ5

andΔ10 were prepared as described,7 and adjusted to 2 Mammonium sulfate on ice. After centrifugation to removeunwanted precipitated proteins, the supernatant wasdesalted by dialysis and the soluble proteins purifiedfurther by cation-exchange and anion-exchange chroma-tography using HiLoad SP Sepharose HP and Resource Qcolumns (GE Healthcare), respectively. Bound proteinswere eluted from each column using a 100 ml linear NaClgradient (0–1 M). HMGB1Δ15, Δ20, Δ25 and Δ30 werepurified as described for the single A and B domains.7,9

Protein samples were concentrated and buffer-exchangedinto 10 mM sodium phosphate (pH 7.0), 1 mM EDTA,1 mM dithiothreitol (DTT) using a Vivaspin 2 concentrator(Sartorius) with a 10 kDa molecular mass cut-off. Accurateprotein concentrations were determined by automatedamino acid analysis.

Expression and purification of the 15N-labelled tailpeptide

E. coli BL21(DE3) cells were transformed with plasmidpHAT3 HMGB1-tail and grown in MOPS minimalmedium as described above. Cells were grown toA600=0.7, protein expression was induced with 0.5 mMisopropyl β-D-thiogalactopyranoside and cells weregrown for a further 5 h at 37 °C. Cell extract containingrecombinant His-tagged acidic tail was prepared asdescribed,7 but in buffer containing 5 mM imidazole andno EDTA. The tail peptide was bound to nickel-NTA resin(Qiagen), which was washed with buffer containing20 mM imidazole, and the tail peptide was then elutedwith buffer containing 100 mM imidazole. The samplewas desalted by dialysis and further purified using aResource Q column (see the previous section). The His tagwas released from the tail peptide by cleavage withcyanogen bromide and removed by binding to SPSepharose HP resin (at pH 5.0 to protonate the His tag).The peptide was concentrated and buffer-exchanged asdescribed above, using a concentrator with a 3 kDa cut-off.

DNA-cellulose binding assay

HMGB1 or the tail-truncation proteins (20μg) wereallowed to bind to 10 mg of DNA-cellulose (Sigma) in10 mM sodium phosphate (pH 7.0), 1 mM DTT, 1 mMEDTA, 10 μg/ml of bovine serum albumin (BSA) for 1 h at4 °C. The DNA-cellulose was washed successively withthe same buffer containing NaCl at increasing concentra-tions (50 mM to 650 mM). Proteins were precipitated with25% (w/v) trichloroacetic acid, subjected to SDS/18%-PAGE and quantified by densitometry, normalizing forloading with respect to BSA.5

Circular dichroism (CD) spectroscopy

CD measurements were made using a JASCO J-810spectropolarimeter fitted with a Peltier temperaturecontroller. Spectra were recorded at 15 °C, with proteinconcentrations 6.5–8.6 μM in 10 mM sodium phosphate(pH 7.0) for far-UV measurements, and 63–85 μM in10 mM sodium phosphate (pH 7.0), 1 mM DTT, 1 mM

EDTA for near-UV measurements. Spectra were recordedwith a data pitch of 1 nm using a 1 mm (far UV) or 1 cm(near UV) path-length quartz cuvette (Hellma). Far-UV(250–200 nm) CD spectra are represented as mean residuemolar ellipticity, [θ]MRW, based on the appropriate meanresidue weights, and near-UV (350–250 nm) CD spectra asmolar ellipticity [θ]; protein concentrations were deter-mined by automated amino acid analysis. Spectra wereaveraged over five (far UV) or ten (near UV) accumula-tions and corrected for buffer absorbance using themanufacturer's software.For thermal denaturation experiments, samples were

prepared as for far-UV CD. Ellipticity at 222 nm wasfollowed over the temperature range 15–85 °C withheating at a rate of 1 °C/min; measurements were madeat 1 °C intervals. The temperature gradient was thenreversed to check whether the proteins refolded.

NMR spectroscopy

NMR measurements were made on samples containing0.3–1.4 mM protein, 10% (v/v) 2H2O in 10 mM sodiumphosphate (pH 7.0), 1 mM EDTA and 1 mM deuteratedDTT. Experiments were generally carried out at 25 °C,with supplementary experiments recorded at 15 °C or 5 °Cto facilitate the assignment of resonances from loops andlinkers, which were more prominent at lower tempera-tures. Data were collected on Bruker DRX500 and DRX800spectrometers equipped with triple-resonance HCN probeheads and actively-shielded z-gradients.HN and N assignments were derived from established

versions of 3D total correlation spectroscopy-15N-HSQCand NOESY-15N-HSQC experiments.33 In all experiments,water suppression was achieved using established flip-back methods to return the water magnetisation to thez-axis and WATERGATE.34 States-TPPI was used forquadrature detection in all indirect dimensions. Datawere processed using the AZARA suite of programs(v. 2.7, © 1993-2007; Wayne Boucher and Department ofBiochemistry, University of Cambridge, unpublished).Assignments were made using Analysis v. 1.0.35

The chemical-shift changes relative to Δ30 werecalculated for each spectrum according to the expression:

DyðDn;D30Þ ¼ M½ðDyHÞ2 þ ðDyN=10Þ2�Scaling factors (xbound(Δn)/xbound(Δn+5)) were estimatedby overlaying the graphs, as shown in Figure 6(b), toextract the unique shifts for each spectrum (Figure 6(c)).Shift changes were considered in order of decreasing size,and judged to be significant if they were greater than 1.5standard deviations from the mean shift change for eachspectrum.

Data Bank accession numbers

Chemical-shift assignments have been deposited withthe BioMagResBank under the accession numbers 15502and 7408-7413.

Acknowledgements

We thank Drs Keng-Boon Lee, Michelle Webb andBill Broadhurst for contributions to the initial stages


of the project, and Dr Marko Hyvönen for the giftof plasmids pBAT4 and pHAT3. This work wassupported by the Biotechnology and BiologicalSciences Research Council of the UK (grant toJ.O.T. and a studentship to M. Watson).

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mapping intramolecular interactions between domains in hmgb1 using a tail-truncation approach

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