proteins
STRUCTURE O FUNCTION O BIOINFORMATICS
The HhH domain of the human DNA repair
protein XPF forms stable homodimers
Devashish Das,1 Konstantinos Tripsianes,1 Nicolaas G. J. Jaspers,2 Jan H. J. Hoeijmakers,2
Robert Kaptein,1 Rolf Boelens,1* and Gert E. Folkers1
1 Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands
2 Department of Cell Biology and Genetics, Erasmus MC, Rotterdam, The Netherlands
ABSTRACT
The human XPF-ERCC1 protein complex
plays an essential role in nucleotide exci-
sion repair by catalysing positioned nick-
ing of a DNA strand at the 50 side of the
damage. We have recently solved the
structure of the heterodimeric complex of
the C-terminal domains of XPF and
ERCC1 (Tripsianes et al., Structure
2005;13:1849–1858). We found that this
complex comprises a pseudo twofold sym-
metry axis and that the helix–hairpin–
helix motif of ERCC1 is required for
DNA binding, whereas the corresponding
domain of XPF is functioning as a scaf-
fold for complex formation with ERCC1.
Despite the functional importance of het-
erodimerization, the C-terminal domain
of XPF can also form homodimers in
vitro. We here compare the stabilities of
homodimeric and heterodimeric com-
plexes of the C-terminal domains of XPF
and ERCC1. The higher stability of the
XPF HhH complexes under various exper-
imental conditions, determined using CD
and NMR spectroscopy and mass spec-
trometry, is well explained by the struc-
tural differences that exist between the
HhH domains of the two complexes. The
XPF HhH homodimer has a larger inter-
action interface, aromatic stacking inter-
actions, and additional hydrogen bond
contacts as compared to the XPF/ERCC1
HhH complex, which accounts for its
higher stability.
Proteins 2008; 70:1551–1563.
C 2007 Wiley-Liss, Inc.
V The Supplementary Material referred to in this article can be found online at http://www.interscience.wiley.
Key words: NMR; structure determination;
protein stability; nucleotide excision repair
(NER); DNA binding domain; helix–hair-
pin–helix-motif.
C 2007 WILEY-LISS, INC.
V PROTEINS
INTRODUCTION
Damage to the cellular DNA can lead to cancer and aging, a subject of
major inquiry.1 Thus, uncovering how repair systems target and correct
damage2 will provide a deeper understanding of genetic recombination,
chromosomal replication, and genome maintenance.3 Throughout the course
of evolution living organisms acquired several repair systems, thereby pro-
viding the cells with a broad arsenal to defy the threats of DNA lesions
arising due to exogenous and endogenous factors. The nucleotide excision
repair (NER) is a DNA repair pathway widely conserved among eukaryotic
species.4,5 The NER machinery is designed to cope with a wide variety of
bulky, helix-distorting and transcription-interfering lesions. Therefore, de-
fective NER elements result in serious prototype repair disorders in human,
such as xeroderma pigmentosum (XP), Cockayne syndrome, trichothiodys-
trophy,6,7 associated with an extreme sensitivity to UV-induced skin cancer
or many features of accelerated aging.8,9
A collection of over 25 purified proteins is sufficient to carry out in vitro
NER of damaged DNA substrates,10 many of these acting within the con-
text of stable complexes. The in vitro as well as in vivo studies agree on a
role of the XPC-HR23B-cen2 complex to bind the DNA lesion and subse-
quently recruit the multiprotein basal transcription factor TFIIH and the
XPG protein, followed by association of XPA and RPA to form a substrate
amenable to strand-specific incision.11–15 This step is performed by the
two structure-specific endonucleases, XPF-ERCC116 and XPG.17 The inci-
sions are positioned on either side of the DNA lesion, allowing removal of
a 24-32nt stretch with subsequent refilling of the resulting ss gap. The coor-
dinated action of the six repair factors XPC, RPA, XPA, TIIFH, XPG, and
XPF-ERCC1 are necessary and sufficient to carry out the dual incision
in vitro and release the excised DNA lesion.17,18
The XPF protein, in complex with ERCC1, is responsible for the incision
on the 50 side of the lesion.19 Its structure-specificity and defined polarity
is believed to participate in other genome maintenance modes as well, such
Abbreviations: HhH, helix–hairpin–helix; HSQC, heteronuclear single quantum coherence; XP, xeroderma
pigmentosum.
com/jpages/0887-3585/suppmat/
Grant sponsors: Research Council for the Chemical Sciences of the Netherlands Organization for Scientific
Research (NWO-CW); Center for Biomedical Genetics.
*Correspondence to: Rolf Boelens, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8,
3584 CH Utrecht, The Netherlands. E-mail: r.boelens@chem.uu.nl
Received 2 March 2007; Revised 11 May 2007; Accepted 19 May 2007
Published online 2 October 2007 in Wiley InterScience (www.interscience.wiley.com).
DOI: 10.1002/prot.21635
1551
 D. Das et al.
as interstrand-crosslink-repair,20–22 recombination,23,24
and telomere protection.25,26 Thus, mice deficient for
ERCC1 or XPF exhibit complex phenotypes27–29 with
severe growth defects multiple organ failure and early
death, contrasting with the UV-specific phenotype of
other NER-mutant mice such as XPA and XPC. The phe-
notypic similarity of ERCC1 and XPF knockout mice is
consistent with the functional association of the two pro-
tein partners. However, slight differences in the two mu-
tant mice may indicate an additional role for either XPF
or ERCC1. For instance, ERCC1-deficient splenic B cells
show moderately reduced class switch recombination in
vitro,30 in contrast to XPF-deficient splenocytes.31 Inter-
estingly, mutations in the Drosophila XPF-homologue
mei9 affect both mitotic and meiotic recombination,32–34
whereas no deficit in homologous recombination was
reported for the ERCC1-deficient cells from mice and
Chinese hamster.29
At present no direct evidence has been presented to
support an independent role for XPF in DNA repair in
eukaryotes. The archaeal counterpart that performs 50
incision in NER is homodimeric. The X-ray structure of
the archaeal XPF proteins35,36 describes the C-terminal
HhH motif, which mediate DNA binding and dimeriza-
tion.2,37 The presence of HhH motifs38,39 has been
reported for many other important proteins such as
RuvA,40 Uvrc,41 where a role in DNA binding has been
confirmed. Recent structural data and biochemical obser-
vations for the eukaryotic XPF-ERCC1 complex revealed
that the ERCC1 subunit of the heterodimer is required
for DNA binding activity,42,43 whereas the full-length
XPF subunit is likely to modulate the cleavage.44 These
structures indicated that XPF acts as a scaffold for
ERCC1. Since archaeal XPF family members possess the
ability to form homodimers, this raises the question
whether eukaryotic XPF can also form homodimers.
It has been shown that C-terminal HhH domains of
the XPF and ERCC1 proteins are crucial for the heterodi-
meric complex formation.45 Independent studies carried
out by us43 and Choi et al.46 confirmed that the C-ter-
minal HhH domain of ERCC1 is stable only in the pres-
ence of the XPF HhH domain. The Choi group reported
the existence of homodimers of the human XPF HhH
domain in vitro.46 This is in agreement with our present
observation, showing that the XPF HhH domain can
form homodimers both in in vitro experiments and in
E. coli strains overexpressing ERCC1-XPF. The formation
of stable XPF HhH homodimers contrasts to the C-
terminal part of ERCC1 that is unable to form a soluble
homodimer in vitro and in E. coli.43 Also there is a huge
body of evidence indicating that ERCC1-XPF hetero-
dimers are a biologically important entity.47,48 This
raises the question whether human XPF can also form a
stable homodimer as observed in archaeal repair proteins
in vivo.49,50 Here we compared the stability of the homo
and heterodimeric complexes of the XPF HhH domain
1552 PROTEINS
by CD spectroscopy and amide proton exchange studies.
Surprisingly we find that the XPF HhH homodimer is
much more stable than the biologically functional hetero-
dimeric complex of the XPF and ERCC1 HhH domains.
We also determined the solution structure of this homo-
dimeric complex and compared it to the heterodimeric
HhH complex. The high stability of the homodimeric XPF
HhH complex as compared to the heterodimeric XPF-
ERCC1 HhH complex can be well explained by the pres-
ence of a larger interaction interface, additional hydrogen
bonds, and additional aromatic ring stacking of the phe-
nylalanine residues at the interface.
RESULTS
Stability of the (XPF)2 homodimer
While overexpressing ERCC1-XPF HhH domains we
noticed the presence of an excess of XPF which could be
purified to homogeneity for biophysical and structural
studies.43 Gel filtration experiments revealed that this
XPF [823–905] domain is present as a stable homodimer
(data not shown).
The stability of the XPF homodimeric and heterodi-
meric HhH complexes was studied using CD spectros-
copy. Surprisingly, a higher stability is detected for the
XPF HhH homodimer under increasing temperature and
increasing denaturant concentration of urea and GuHCl
(Fig. 1).
The thermostability data of the XPF HhH homodimer
(20–25 lM protein in 50 mM NaPi, pH 7 and 3–5 mM
NaCl) reveals the presence of a single transition at 75–
778C [Fig. 1(a)]. Similar experiments, carried out on the
XPF-ERCC1 HhH heterodimer under identical condition,
show that the heterodimer is much less stable as judged
by the profile of the CD melting curve and is highly
dynamic based on 15N NMR relaxation data. Most im-
portantly, the melting profile of the heterodimer shows a
biphasic unfolding. The first transition is observed at 37–
388C, accompanied with an extensive protein precipita-
tion. The second transition occurs at 75–778C, the tem-
perature where XPF homodimer also undergoes unfold-
ing. We found similar results under higher ionic
strengths (100 mM NaCl and 50 mM NaPi, pH 7). We
suggest that in the first transition (378C) the XPF-
ERCC1 complex dissociates with the precipitation of
unprotected ERCC1 protein, while the stable (XPF)2
homodimer is formed. In the second transition (768C)
the (XPF)2 homodimer subsequently unfolds.
The stability of the homo and heterodimeric XPF
complexes was also assessed in experiments with urea
and GuHCl at low salt conditions (50 mM NaPi, pH 7
and 3–5 mM NaCl). The data on the XPF homodimer in
the presence of increasing urea concentration shows that
the homodimer starts to unfold around 6.0M–6.7M urea.
This is in agreement with work the carried out by Choi
DOI 10.1002/prot
 Structure and Stability of XPF HhH Dimers

Figure 1
Stability of XPF-XPF and XPF-ERCC1 complexes. (a) Plot of weight fraction of the thermal denaturation profiles of XPF-XPF and XPF-ERCC1 complex with increasing
temperature. The temperature is in 8C. (b) Denaturant-induced unfolding curves of the XPF-ERCC1 and XPF-XPF complex. The plots show weight fraction of the
unfolded proteins with increasing concentration of the denaturant GuHCL. (c,d) Denaturation plots of the XPF-ERCC1 heterodimer and XPF homodimer as a function of
urea concentration, respectively. The denaturation in (a–d) was followed by the CD ellipticity measured at 222 nm. Protein concentrations were
20–25 mM, in 50 mM
NaPi, pH 7. (e) NMR H-D exchange rate analyses of the homodimeric XPF protein complex. Protection factors of the homodimeric XPF (HhH) domain plotted as a
function of residue number. Protection factors were calculated from the rate ratio kint/kobs and are displayed as a logarithmic scale. (f) 15N-1H HSQC spectra of the XPF-
ERCC1 (black) and XPF-XPF (red). Superimposed spectra of XPF-ERCC1 (colored black) at 608C and XPF-XPF complex (red). The similarity in the two spectra shows
that in XPF-ERCC1 above 458C only homodimeric XPF is present.

DOI 10.1002/prot PROTEINS 1553

 D. Das et al.

et al. on XPF (HhH)2 homodimers.46 The XPF-ERCC1 factors for XPF-ERCC1 are at least 100 times smaller
heterodimer is much less stable in urea, showing dissoci- than for XPF homodimers at 22.38C. H/D exchange
ation already at 2.4M–2.7M urea [Fig. 1(c,d)]. These study of XPF-ERCC1 at pH 4.5 shows rates in a range of
results agree well with the lower thermostability discussed 1024–1023 s21. These results show that the H/D ex-
earlier. change for XPF-ERCC1 is at least 100 times faster than
Similarly, the data collected on the XPF homodimer in for the XPF homodimer. This agrees well with the thermal-
increasing concentration of GuHCl reflects that the and denaturant-induced unfolding studies, where we
homodimer is stable until 2.9M –3.0M GuHCl concentra- also observed a large difference in the stability of both
tion, while the data on XPF-ERCC1 indicates that the complexes.
heterodimer seems to fall apart already at 2.4M GuHCl
[Fig. 1(b)]. The denaturation profiles of ERCC1-XPF do Temperature dependence studied by NMR
not show a clear biphasic unfolding pattern as in temper-
The temperature unfolding of the homo- and hetero-
ature unfolding. The shallowness of the slope of the
dimers was also monitored by following the amide cross
denaturation curves of ERCC1-XPF as compared to the
peaks in a series of 15N-1H HSQC experiments [Fig.
(XPF)2 homodimer suggests, however, an additional tran-
1(f)]. We observe that the heterodimer amide peaks start
sition after initial disruption of the ERCC1-XPF com-
plex. to disappear and the peaks corresponding to the XPF
homodimer start to appear at 45–508C. Thus our NMR
We measured the 15N NMR R1 and R2 relaxation rates
data provide direct evidence for homodimer formation
and the 1H-15N heteronuclear NOE data for the XPF
and aggregation of ERCC1 as explanation for the first
homodimer and the XPF-ERCC1 heterodimer (supple-
transition in the CD measurements [cf. Fig.1(a)]. Apart
mentary material). For the XPF homodimer, the highest
from those of folded XPF HhH homodimer no addi-
R2 rates are observed for residues at or near the interface
tional new signals appeared, indicating that ERCC1 forms
of the complexes (D839, F840, N861, E864, and Y887)
large aggregates that are not visible in the NMR spectra.
and in the beginning of helices H2 (A849, N851, and
R853) and H4 (Q870, D871) and in the nonconserved By comparing the amide resonances for heat-treated
ERCC1-XPF and native (XPF)2, it is clear that the overall
hairpin h2 (G878, N879), indicating possible microsec-
folds for the two species after aggregation of ERCC1 are
ond–millisecond time-scale motions in these regions. The
essentially the same. We have also noticed that in addi-
R2 rates for residues in hairpin h1 (K843, G846, V847)
tion to heating under NMR conditions in the absence of
were smaller than the average, indicating a sub-nanosec-
denaturants the ERCC1-XPF heterodimer tends to slowly
ond time-scale flexibility in this part. On an average, the
change to the XPF homodimer in the course of time
R2 rates at the interface of the XPF-ERCC1 heterodimer
(months). Spectra acquired at various time-points clearly
were higher, in agreement with its reduced stability.
Unfortunately, a full relaxation analysis for both proteins supports this observation (data not shown). In this
experiment the newly formed XPF homodimer from the
was not possible due to sample instabilities over pro-
heterodimer has an identical spectrum as the native
longed relaxation measurements.
homodimer. This agrees with the high stability of the
XPF HhH homodimer, not only at high temperature and
NMR H/D exchange studies denaturant concentrations, but also at room temperature.
The stability and molecular mass of the XPF homodimer
To understand the stability of the XPF homo- and het-
was further confirmed by MALDI-TOF-MS, which showed
erodimeric complexes in atomic detail, we next per-
next to the monomeric XPF an intense peak for homodi-
formed H-D exchange experiments. Hydrogen deuterium
meric XPF (data not shown). This is a strong evidence
exchange rates were measured on the XPF homodimer
for the tight packing of the homodimer, as it survives
complex (
0.4 mM XPF2, 100 mM NaCl, 50 mM NaPi,
even under these harsh MALDI conditions.51 Our bio-
pH 5 7.06, 22.38C), and the kinetics of the exchange
physical studies show that the homodimer of the C-ter-
was monitored by acquiring a series of 15N-1H HSQC
minal domain of XPF is significantly more stable than
spectra. The amide protons at this pH exchange slowly
the biologically functional XPF-ERCC1 heterodimer. To
with an average rate of 1023 s21. We noted that most of
provide a molecular explanation, we have determined the
the amide protons of the XPF homodimer form very sta-
solution structure of the homodimeric C-terminal do-
ble hydrogen bonds with a very high protection factor
main of the XPF protein.
[Fig. 1(e)]. This agrees well with the high stability of the
(XPF)2 homodimer. The experiments performed on the
Three-dimensional structure of the (HhH)2
XPF-ERCC1 heterodimer under similar conditions indi-
domain of XPF
cated very fast amide proton exchange, both for the
ERCC1 and the XPF domains (with rates faster than The structure of the C-terminal domain of XPF (823–

0.1 s21). Since this exchange kinetics was even too fast 905) was solved by combined heteronuclear double and
to follow, we could only conclude that, the protection triple resonance NMR spectroscopy using uniformly

1554 PROTEINS DOI 10.1002/prot

 Structure and Stability of XPF HhH Dimers

ence in the fold of the hairpins that have been implicated

Table I in DNA binding in most other HhH domains. Although
Structural Statistics of the Structure Ensemble of the C-Terminal Domain
(823–905) of Homodimeric Human XPF the first hairpin (h1 and h10 ) consisting of residues (843–
847) is structurally conserved, the second hairpin is
RMSD (
) with respect to mean (backbone/heavy)a absent in our XPF homodimer because of the shorter
XPF monomer 0.31  0.08/0.57  0.15
XPF dimer 0.39  0.12/0.64  0.16 intervening sequence between helix-4 and 5 [Fig. 2(b)].
No of experimental restraints The structure of the second hairpin of the human XPF
Homodimeric complex HhH homodimer is very similar to that of XPF in the
Intra residue NOEs 750 heterodimeric complex of ERCC1/XPF.
Sequential NOEs (|i – j | 5 1) 1054
Medium range NOEs (1 < |i – j |<4) 927
Long-range NOEs (|i – j | > 4) 645
Interprotein 319 Structural comparison with other
Total NOEs 3695 HhH domains
Dihedral angle restraints 194
Restraint violations The human XPF HhH domain shares a high degree of
NOE distances with violations >0.4
0.00  0.00 sequence conservation with the ERCC1 HhH domain,
Dihedrals with violations >58 0.00  0.00 primarily in hydrophobic residues. These residues are
RMSD experimental restraints
All distance restraints (3695) (
) 0.019  0.001 part of the hydrophobic interface between the two mono-
Torsion angles (194) (8) 0.38  0.06 mers, both in the homo- and heterodimeric complexes
RMSD from idealized covalent geometry indicated in Figure 2(a,b).
Bonds (
) 0.011  0.00 The helical assembly of the XPF HhH homodimer is
Angles (8) 1.26  0.05
Impropers (8) 1.38  0.10 similar to the XPF-ERCC1 heterodimer HhH com-
CNS energies after water refinement plex.42,43 We performed a fitting of the structure ele-
Evdw (kcal mol21) 2677  25 ments of the XPF homodimer and the XPF monomer
Eelec (kcal mol21) 26513  100
within the ERCC1-XPF heterodimeric complex (1z00),
Ramachandran analysis
Residues in the favored regions (%) 94.4  1.5 and the homodimeric archaeal XPF structures (1x2i,
Residues in additional allowed regions (%) 5.6  1.5 2bgw) using Profit (Table II). The fitting of the XPF
Residues in generously allowed regions (%) 0.0  0.0 homodimer indicates the very high overall structural
Residues in disallowed regions (%) 0.0  0.0
similarity within the XPF protein family, suggesting that
a
RMSD values (in Å) were calculated for 835–895 of Prot A and 835–895 Prot B XPF family of protein is structurally conserved.35,36
XPF. Small RMSD differences exist between all XPF family
members that might arise from amino acid sequence dif-
ferences. However, differences within the XPF molecule
15
N/13C-labeled protein (10 mM NaPi, pH 5, 400 mM when present as a homodimer or as a heterodimer in
NaCl) at 20.68C. The 15N-1H HSQC spectra showed a complex with ERCC1 indicate that the protein adapts its
single set of resonance, demonstrating that XPF forms a structure for an optimal interaction with the partner pro-
symmetric dimer. The structure of the dimer was deter- tein. The most notable difference is the orientation of the
mined based on 3965 experimental NMR restraints and helix-1 which together with helix-3 contributes most to
194 TALOS-based dihedral angle restraints. A significant the interaction surface. For instance, the reorientation of
number of intersubunit NOEs (394) permitted the rela- helix-1 away from helix-2 makes space to accommodate
tive positioning of the subunits. No distance violations the bulky side chains of residues F840, L841, and L842. At
larger than 0.4 Å or dihedral angle violations larger than the same time the orientation of the residues in helix-3
58 were found after structure refinement. A summary of gets altered, which interlocks the helix-5 forming a closed
the structural and restraint statistics is given in Table I. tight hydrophobic cage.
Sequence alignment of the XPF protein and an overlay of
the final 20 lowest energy conformers obtained after the
structure calculation and water refinement is shown in Larger protein interaction area and aromatic
ring stacking in XPF homodimers by
Figure 2(a–d). The lowest energy structure of the XPF
adaptation of helix-1
homodimer is showing the overall helical arrangement
[Fig. 2(e)]. A detailed structural comparison suggests that the
The present structure of the XPF homodimer shows a backbone folds of XPF in the homo- and heterodimeric
twofold symmetry. The arrangement of the double helix– complexes are very similar, but there are substantial
hairpin–helix motifs in each monomer is shown in Fig- differences in the positioning of the monomers with
ure 2(e). In archaeal XPFs this domain is essential for respect to each other, as shown in the fitted structures
both dimerization and DNA binding activity.35,36 Here [Fig. 3(a,b)]. Moreover, at the interface of the complexes,
we also observe many interactions between the two there are additional ring-stacking interactions as com-
monomers of the XPF dimer. However, we note a differ- pared to the XPF-ERCC1 heterodimer [Fig. 3(c–e)].

DOI 10.1002/prot PROTEINS 1555

 D. Das et al.

Figure 2
Sequence alignment of the C-terminal HhH motif of the XPF protein homologues. (a) Alignment of the human XPF protein with most closely matched species. The
residue part of the conserved first hairpin is indicated in green. Conserved Gly878 present in the nonconserved second hairpin is coloured blue. Conserved Asn861 which
mediates H-bond interaction via side chain amide (red circle with a black dot) with the carbonyl Phe889 (red asterisk) is coloured blue, and this interaction is absent in
XPF-ERCC1 heterodimer. Ala863 forms hydrogen (red circle with a black dot) with Ile890 (black circle). Residue Phe894 which is locked in the XPF cavity is indicated by
(red oval). (b) Sequence alignment of the XPF homologue proteins from all the three kingdoms. Residue involved in H-bonding in Archaeal XPF is indicated by (blue
circle) and (black circle). Conserved residues are highlighted in green (green square), and those in (HhH) in blue. Most important nonconserved residues are shown in
pink. Residues absent from the second hairpin of XPF are indicated as (–), if present highlighted in yellow. (c) 3D Structure of the C-terminal HhH motif of the human
XPF homodimeric complex (2aq0). Ensemble backbone view of the final 20 structures of the XPF homodimer. The protein subunits are coloured green and blue; the
hairpins in both subunits are red. (d) Superimposed representation of the XPF subunits. Color conventions are as in (c). (e) Cartoon representation of the lowest-energy
model. The helices are denoted with number 1–4 for the green colored subunit and 10 –40 for the blue subunit. The hairpins are indicated as h1 and h10 .

A clear difference of the human XPF homodimer ver- ent side chain conformations at the interface allow a sub-
sus the XPF-ERCC1 heterodimer and archaeal XPF com- stantial stacking of the aromatic groups of Phe840 and
plex is the positioning of its helices, resulting in a larger the conserved Phe889 of helix 5 [Fig. 3(c,d)]. The result-
interface of the XPF homodimer compared to XPF-
ERCC1 and archaeal XPF (AP-XPF) (1760 Å2 vs. 1534 Å2
Table II
and
1235 Å2 ), as pointed out in Figure 3(a,b). Most RMSD (Å) and Z-Score Values of the XPF Complexes
importantly, helix-1 (residues 836–841) tilts and slightly
moves away, offering additional space required for ac- Complex Z-score (XPF)2 ApXPF Pf HEF XPF ERCC1
commodating the side chains of Phe840 and Phe889. This (XPF)2 2aq0.pdb 15.1 0.3 1.5 1.7 1.3 1.8
rearrangement is accomplished with a slightly different ApXPF 2bgw.pdb 8.1 1.7 1.8 1.2 1.8 1.8
orientation of some key side chain residues of helix-1 in Pf-HEF 1x2i.pdb 8.8 1.5 1.3 0.5 1.8 1.5
XPF 1z00.pdb 10.5 1.3 1.6 1.7 2.5
the homodimer interface as compared to the XPF- ERCC1 1z00.pdb 7.7 1.9 1.7 1.7 2.5
ERCC1 interface, as is shown in Figure 4(a,b). The differ-

1556 PROTEINS DOI 10.1002/prot

 Structure and Stability of XPF HhH Dimers

Figure 3
Structures of XPF complexes. (a) Overlay of XPF of human XPF-ERCC1 heterodimer (orange) and of homodimeric human XPF (green). (b) Overlay of archaeal XPF
(magenta) and of homodimeric human XPF (green). (c) Stacking of aromatic side chains in the interface of homodimeric human XPF. (d) Ribbon view of the human
XPF-ERCC1 heterodimer. The figure shows the lowest-energy model with the aromatic phenylalanine (Phe840, Phe889, Phe894) of XPF colored in yellow and the aromatic
phenylalanines (Phe231, Phe257, Phe286 and Phe293) of ERCC1 colored in cyan. (e) Ribbon view of archaeal XPF. The aromatic phenylalanine residues are coloured in
green and cyan. The coordinates used for (a–e) are from the XPF complexes from Archaea (2bgw), human XPF-ERCC1 (1z00), and human XPF-XPF homodimer
(2aq0).

ing p–p interactions52,53 may contribute considerably to
the observed high stability of the homodimeric complex.
Figure 4(c,d) shows the contacting residues at the surface
of XPF from two different angles. Importantly, residue
Phe894 is buried in hydrophobic cavities both in the XPF
homodimer and the XPF-ERCC1 heterodimer [Fig. 4(e,f)].
A comparison of the contribution of homologous resi-
dues Phe293 of ERCC1 and Phe894 of XPF in the homodi-
meric and heterodimeric XPF complexes reveals that
these anchoring Phe894 and Phe293 residues occupy inter-
action areas of 538 Å2 and 500 Å2 in the homodimeric
and heterodimeric complexes, respectively, contributing
to a large amount to the interaction surface. In fact, the
corresponding tyrosine residue in the archaeal XPF35
contributes only
348 Å2 to the interaction surface (Ta-
ble III). WHATCHECK54 and PROCHECK55,56 analysis
of the residues surrounding the hydrophobic cavities in
the XPF-ERCC1 heterodimer and in the XPF homodimer
(in XPF residues Pro837, Leu841, Leu842, Met844, Asn834,
Asn861, Phe840, and Phe889, plus in the homodimer
Phe894, and in ERCC1 Phe231, Val232, Val235, and Leu254),
show that the homodimer is well structured.
Hydrogen bond network at the interface
A comparison of the hydrogen bonds formed in the
ERCC1-XPF heterodimer and the XPF homodimer sug-
gests that the higher stability of the latter is in part also
due to additional hydrogen bonds, as shown in Figure
4(e,f). In each of the monomer subunits the side chain
NH2 of Asn861 makes intermonomer hydrogen bonds to
the backbone carbonyl of Phe889. This hydrogen bond
serves as an important link between helix-5 and the loop
connecting helix-20 to helix-30 . This hydrogen bond,

DOI 10.1002/prot PROTEINS 1557

 D. Das et al.

Figure 4
Structures of the interaction interface of XPF complexes. The figure shows the C-terminal domains of homodimeric human XPF and heterodimeric human XPF-ERCC1
(colored orange and red). (a) Stick representation of the backbone and side chains of the helices 1 (green) and 10 (blue) of homodimeric XPF. (b) View of the equivalent
region in the heterodimeric XPF (orange)-ERCC1 (red). (c,d) Surface representation of the aromatic side chains in the interface of the XPF homodimer from two angles.
(e,f) Network of hydrogen bonds at the interface of homodimeric XPF and of the XPF-ERCC1 heterodimer, respectively.

which is absent in the XPF-ERCC1 heterodimer, engages the core of the complex. Furthermore, the backbone am-
the side chain NH2 in anchoring helix-5, thereby ena- ide proton of Ala863 at the end of helix-3 is hydrogen-
bling the proper positioning of the helix with respect to bonded to the carbonyl of Ile890 in helix-5, which fixes

1558 PROTEINS DOI 10.1002/prot

 Structure and Stability of XPF HhH Dimers
Table III
Accessible Surface Area of the Aromatic Phe or Tyr in the XPF Complexes
Cavity (
)2
Complex Monomer A
XPF homodimer 270
XPF-ERCC1 heterodimer 280ERCC1
XPF-archaea homodimer 173
the position of the two helices. This arrangement of
helix-3 and 5 is stabilized further a number of hydropho-
bic contacts. Also the side chain NH2 of Lys860, the car-
bonyl oxygen of His891, the side chain oxygen of Glu895,
and the side chain hydroxyl of Ser893 are forming hydro-
gen bonds. In the XPF-ERCC1 heterodimer the equiva-
lent hydrogen bond, His891-Glu261, participates in the
side chain-to-side chain connection,43 whereas the hydro-
gen bond between Lys860 and Glu895 is absent. In total,
18 intermolecular hydrogen bonds exists in the XPF
homodimer as compared to 13 in the XPF-ERCC1 hetero-
dimer and 9 in archaea (Ap-XPF), which is likely to be
an important factor of the (XPF)2 stability as observed in
the (H-D) exchange, temperature, and denaturant-
induced unfolding studies.
DISCUSSION
A large body of evidence indicates that XPF-ERCC1 is
an obligate heterodimer in vivo.19,47 It was noted, how-
ever, that the C-terminal HhH region of XPF that was
shown to be required and sufficient for heterodimer for-
mation in vitro45 can also form a homodimer in vitro.46
Our biophysical characterization of the C-terminal HhH
domain of XPF shows a high stability of the C-terminal
homodimeric XPF HhH complex as compared to the
heterodimeric XPF-ERCC1 HhH complex. The structural
data of the XPF homodimer can well explain this
increased stability. We find a larger number of hydrogen
bonds, a larger interaction surface area, and additional
ring-stacking for Phe840 and Phe889 of the XPF HhH
homodimer in comparison with the heterodimeric
ERCC1-XPF HhH complex. These differences account for
the increased thermostability, increased denaturant stabil-
ity, and the greater conformational stability as probed in
H/D exchange. The residues that contribute significantly
to the increased stability of the homodimer are well con-
served, such as Asn861, suggesting that the evolutionary
ancient ability to form homodimers might be of yet
unknown functional relevance.
Can human XPF form a homodimer in vivo?
In vivo, the human XPF and ERCC1 proteins are
unstable in mammalian cells in the absence of their part-
DOI 10.1002/prot
ners.57,58 In XP group F cells, that lack functional XPF
protein, a low content of ERCC1 protein is found despite
normal levels of ERCC1 mRNA.59–61 The levels of the
ERCC1 protein can be increased in the XPF-defective
cells by transfecting an expression vector encoding wild-
Monomer B
type XPF.59 This all fits well with the observations that
268 ERCC1 only folds in the presence of XPF protein. In
220XPF
175
ERCC1-defective cells, XPF protein can still be found
albeit only at a tiny fraction of the normal amount, indi-
cating that, like the HhH domain, the full-length XPF
can be present in the absence of ERCC1.62,63 Recombi-
nantly produced XPF-ERCC1 heterodimer can comple-
ment in an in vitro NER reaction while recombinant
ERCC1 can only marginally complement ERCC1-defec-
tive cell extract. Further addition of recombinant XPF
did not significantly increase the repair activity.62 It is
tempting to speculate that this recombinantly produced
XPF also forms stable dimer that fails to dissociate to
form a functional ERCC1-XPF heterodimer in vitro.
These data indicate that functional XPF-ERCC1 can
only be produced when the two proteins are present to-
gether and cotranslationally fold together. Also in vitro
folding experiments by us and others using the ERCC1
and XPF HhH domains strongly argue that XPF-ERCC1
heterodimer can only be formed when the two proteins
are present together. The interactions between XPF and
ERCC1 have been mapped to the C-terminal HhH
domains of XPF and ERCC1,45 whereas the flanking
XPF nuclease and ERCC1 central domains do not seem
to interact.42 We here find that the C-terminal HhH
domains of XPF can also form a stable homodimeric
complex. In vivo, however, no or only a marginal amount
of intact XPF is detected in the absence of ERCC1. We
cannot explain this observation. We cannot exclude that
the homodimers, as formed here using an isolated do-
main, are in vivo prevented by the presence of additional
flanking sequences in intact XPF. But given the presence
of small amounts of XPF in ERCC1-defective cells62 it
appears more probable that in vivo homodimeric XPF is
actively degraded.
Biological significance of the observations
Most of what we know about the XPF-ERCC1 protein
is based on the biochemical and structural data points to
the notion that a heterodimer is obligatory for a func-
tional complex.42,43,45 However, the ability to form
homodimer among the XPF family of endonucleases is
evolutionary conserved and apparently is not lost even
after gene duplication of the ancestral XPF leading to the
appearance of ERCC1. The structure of the XPF protein
in homodimeric and heterodimer context is so similar
that alterations that would prevent homodimer forma-
tion would also block heterodimer formation.
It is possible that the homodimer might act as a stor-
age complex, whereby the presence of excess amount of
PROTEINS 1559
 D. Das et al.
ERCC1 drives the equilibrium back to the heterodimer.
Whether an XPF homodimer has an additional role
remains to be seen, but the high stability observed here
and before for the human XPF protein fragments46,64
and in full-length Hef homodimer49,65 argues that a
function for XPF homodimer in vivo would be possible
and is worth investigating. Further studies are needed to
clarify the apparent contradiction between the much
higher stability of the XPF HhH domain in vitro and the
apparent absence of full length (XPF)2 in vivo, and to
investigate the putative role for XPF homodimers.
MATERIAL AND METHODS
Sample preparation
ERCC1-XPF was expressed and purified as described
before.43 The excess of XPF homodimers from the
ERCC1-XPF heterodimer expression was purified by col-
lecting the unbound fraction and dialysing against
50 mM Tris/bis-Tris buffer at pH 7. This fraction was
loaded on to an anion exchange column, under the same
buffer condition; the unbound fractions contained the
XPF dimer with minor impurities. Subsequently, the
unbound fraction was concentrated and dialysed against
the NMR buffer (10 mM NaPi and 400 mM NaCl, pH 5)
and the fraction was loaded on a Superdex 75 column
equilibrated to the same buffer conditions. The complex
eluted from the column as a single species, with an elu-
tion time in agreement with the XPF dimer of molecular
weight of
19 kDa. The fraction was concentrated to
nearly 450 lL with a final protein monomer concentra-
tion of 1–1.2 mM using Amicon filters with a 3-kDa cut-
off. Using this protocol, both 15N-labeled and 13C/15N-
labeled (XPF)2 samples were prepared.
NMR spectroscopy
NMR data were collected at 20.68C on a Bruker
DRX600 spectrometer equipped with a z-gradient triple-
resonance cryoprobe and a Bruker Avance 700 spectrom-
eter equipped with a 5-mm z-gradient triple-resonance
probe. The NMR samples were
1 mM (monomer con-
centration) in a buffer containing 10 mM NaPi, pH 5,
400 mM NaCl, 95%/5% H2O/D2O, and a small amount
of complex protease inhibitor (Roche).
A set of triple resonance experiments, HNCACB,
CBCACONH, HNCO, HN(CA)CO, HNCA, HN(CO)CA,
HBHA(CBCACONH), HBHANH, HN(CA)HA, (H)
CC(CO)NH-TOCSY, H(CCCO)NH-TOCSY, (H)CCH-
TOCSY, and H(C)CH-TOCSY, as described in Refs. 66
and 67, was acquired to assign the backbone and side
chain 1H, 13C, and 15N resonances at 600 MHz. A set of
NOE spectra, 3D-NOESY-(13C,1H)-HSQC, 3D-(13C)-
HMQC-NOESY-(15N,1H)-HSQC, 2D 15N-filtered/13C
edited-(1H,1H)-NOESY (tmix 5 70 ms) was acquired at
1560 PROTEINS
600 MHz using a cryogenically cooled probe (Bruker
TXI), and 3D-NOESY-(15N,1H)-HSQC (tmix 5 70 ms)
was acquired at an AVANCE 700 MHz spectrometer as
described previously.67 All the spectra were processed
using XWINNMR (Bruker, DRX) and the NMRPipe soft-
ware package68 and analyzed with Sparky69 and
NMRview.70
H/D exchange
The samples for the H/D exchange studies contained
15
N-labeled XPF homodimer or XPF-ERCC1 heterodimer
in 50 mM NaPi, pH 7.06, 100 mM NaCl, were dissolved
in 95% H2O/5% D2O and checked by a 1H-15N HSQC
spectrum and were subsequently lyophilized. Protein
monomer concentrations were
0.4 mM for (XPF)2 and
ERCC1-XPF. The spectrometer was setup using another
sample under identical conditions in water, in order to
reduce the acquisition of the first spectrum. The lyophi-
lized sample was dissolved in D2O to start the H/D
exchange. The dead time delay between dissolving the
sample in D2O and acquiring the first experiment was

12 min. A series of 1H-15N fast HSQC71 experiments
were acquired at 600 MHz at 22.38C. The cross peak vol-
umes in the 1H-15N HSQC spectra were extracted and
the decay was fitted to a single exponential using three
parameters (corresponding to the intensity at time zero,
the decay rate, and an offset), using the Levenberg–Mar-
quardt algorithm implemented in the Gnuplot 3.7
(www.gnuplot.info). Estimates for the decay rate errors
were taken from the standard errors given by Gnuplot.
The intrinsic rates kint were predicted using the program
SPHERE (www.fccc.edu/research/labs/roder/sphere.html).
Structure calculations
Initial distance constraints were generated from inte-
grated crosspeak volumes in 3D NOESY-(15N-1H)-HSQC
(smix 5 70 ms), 3D NOESY-(13C-1H)-HSQC, 3D (13C)-
HMQC-NOESY-(15N,1H)-HSQC, and 2D homonuclear
13
C-edited (1H-1H)-NOESY (smix 5 70 ms) experiments,
torsion angle restraints were derived from TALOS,72 and
hydrogen-bond restraints were derived in accordance to
the secondary shift data and NOE73 data.
A strategy combining manual assignment and auto-
matic NOE assignment using CANDID module of the
program CYANA were performed as described.74,75
Monomer and dimer protocols were performed and the
structures calculated were analyzed to distinguish inter
and intramonomer NOE constraints as described76,77;
intermonomer NOE was inconsistent with the structure
of the monomer. Initially any potential NOE that could
arise from both inter and intrasubunit contacts were
treated
P 26as21=6 ambiguous NOE restraints in the form of a
ð r Þ sum, as described.78 Subsequently, the
intersubunit NOEs were resolved during the course of
DOI 10.1002/prot
 Structure and Stability of XPF HhH Dimers
iterative refinement, and were verified by manual inspec-
tion of the calculated structures. The total number of
NOEs used in the structure calculation was cross validated
as described,79,80 which includes the manual inspection
of the total number of input peaks from the number
peaks rejected during the automatic assignment steps.
Finally the NOE restraints, dihedral, and hydrogen
bond restraints derived from the structures calculated by
CYANA were converted to CNS81 formats, and these
restraints were used in the standard RECOORD proto-
col82 with the symmetry and experimentally derived set
of intermonomer hydrogen bond restraints, and NCS
restraints were switched off for water refinement. The
structures calculated with NCS restraint force constants
(kncs 5 1.0, ksym 5 10.0 kcal mol21 Å22) during water re-
finement did not change significantly.
CD spectroscopy and thermal and
denaturant unfolding
CD measurements were recorded with a JASCO J-810
spectropolarimeter fitted with a thermostatically con-
trolled cell holder and interfaced with a water bath. The
CD melting experiments were performed in 50 mM
NaPi, pH 7, 5 mM NaCl and in 50 mM NaPi, pH 7,
100 mM NaCl, at a scan rate of 18C min21 at 222 nm
on the spectropolarimeter, by controlling the sample tem-
perature with a Peltier-type temperature controller. The
experiments were recorded in a time-course manner,
such that for each data point 10 scans were acquired
each lasting for 10 s at 222 nm. The first derivative was
calculated from the CD melting profile. The maximum
values in the derivative curves were designated as the
melting temperature, Tm. The cell length was 1 mm and
the protein concentration 20–25 lM.
CD spectra, at a constant temperature of 278C, were
collected under the same buffer and protein conditions.
For the GuHCL denaturation, the concentration of
GuHCl was increased in steps to 5M. For the urea dena-
turation, the concentrations were stepwise increased to
8M. The CD data were collected and analyzed as
described before.83
ACKNOWLEDGMENTS
The authors are grateful to Dr. A. B. Eiso, Dr. R. K.
Salinas, Dr. H. Wienk, and Dr. Aalt-JanVan Dijk for
the assistance and discussions during the project. The
authors thank Prof. B. de Kruijff for use of the CD spec-
tropolarimeter.
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PROTEINS 1563