solution structure and backbone dynamics of the xpc-binding domain of the human dna repair protein...

Solution structure and backbone dynamics of theXPC-binding domain of the human DNA repair proteinhHR23BByoungkook Kim1,*, Kyoung-Seok Ryu2,*, Hyun-Jin Kim1, Sung-Jae Cho1 and Byong-Seok Choi1

1 Department of Chemistry, and National Creative Research Initiative Center for the Repair System of Damaged DNA, Korea Advanced

Institute of Science and Technology, South Korea

2 Korea Basic Science Institute, Daejon, South Korea

Nucleotide excision repair (NER) is an important

pathway for the removal of DNA lesions caused by

diverse environmental factors, such as UV irradiation

and chemical modifications [1,2]. There are two human

homologs (A and B) of the yeast Rad23 protein

(hHR23A and hHR23B), both of which can form a

complex with the xeroderma pigmentosum group C

protein (XPC) [1,3]. Recent in vitro and in vivo studies

point to a role for the XPC–hHR23B complex as the

initiator of global genomic NER [1,4]. Although the

precise functions performed by hHR23A and hHR23B

alone in human NER have not yet been determined,

Keywords

hHR23B; nucleotide excision repair; stress-

inducible; structure; xeroderma

pigmentosum group C protein

Correspondence

B.-S. Choi, Department of Chemistry and

National Creative Research Initiative Center

for Repair System of Damaged DNA, Korea

Advanced Institute of Science and

Technology, Yusong-Gu, Gusong-Dong 373-1,

Daejon 305-701, South Korea

Fax: +82 42 869 2810

Tel: +82 42 869 2868

E-mail: [email protected]

*Byoungkook Kim and Kyoung-Seok Ryu

contributed equally to this work

Note

The atomic coordinates of the bundle of 20

conformers have been deposited in the

RCSB Protein Data Bank with entry code

1PVE

(Received 9 November 2004, revised

4 March 2005, accepted 17 March 2005)

doi:10.1111/j.1742-4658.2005.04667.x

Human cells contain two homologs of the yeast RAD23 protein, hHR23A

and hHR23B, which participate in the DNA repair process. hHR23B hou-

ses a domain (residues 277–332, called XPCB) that binds specifically and

directly to the xeroderma pigmentosum group C protein (XPC) to initiate

nucleotide excision repair (NER). This domain shares sequence homology

with a heat shock chaperonin-binding motif that is also found in the stress-

inducible yeast phosphoprotein STI1. We determined the solution structure

of a protein fragment containing amino acids 275–342 of hHR23B (termed

XPCB–hHR23B) and compared it with the previously reported solution

structures of the corresponding domain of hHR23A. The periodic position-

ing of proline residues in XPCB–hHR23B produced kinked ahelices and

assisted in the formation of a compact domain. Although the overall struc-

ture of the XPCB domain was similar in both XPCB–hHR23B and

XPCB–hHR23A, the N-terminal part (residues 275–283) of XPCB–

hHR23B was more flexible than the corresponding part of hHR23A. We

tried to infer the characteristics of this flexibility through 15N-relaxation

studies. The hydrophobic surface of XPCB–hHR23B, which results from

the diverse distribution of N-terminal region, might give rise to the func-

tional pleiotropy observed in vivo for hHR23B, but not for hHR23A.

Abbreviations

hHR23B, human homolog B of yeast Rad23; NER, nucleotide excision repair; RMSD, root mean square deviation; STI1, stress-inducible,

heat shock chaperonin-binding motif; UBA, ubiquitin-associated domains; UbL, ubiquitin-like domain; XPC, xeroderma pigmentosum group C

protein; XPCB, XPC binding.

FEBS Journal 272 (2005) 2467–2476 ª 2005 FEBS 2467

many reports suggest that these proteins stabilize the

XPC protein by protecting it from 26S proteasome-

dependent protein degradation [5–7]. Another bio-

chemical analysis of the damage-recognition process in

NER revealed that hHR23B is necessary for XPA ⁄replication protein A-mediated displacement of the

XPC–hHR23B complex from damaged DNA [8].

Both hHR23A and hHR23B have four well-defined

functional domains (Fig. 1A). These include an N-ter-

minal ubiquitin-like (UbL) domain, the XPC-binding

domain, and two ubiquitin-associated domains (UBA1

and UBA2) [9,10]. The UbL domain has a high bind-

ing affinity for polyubiquitin binding site 2 of the

human S5a protein, which is supposed to serve as a

shuttle delivering polyubiquitinated, degradable protein

substrates to the proteasome [10]. The UBA domains

occur in many enzymes involved in the ubiquitination

pathway and in cell-cycle check points [5,11]. It also

has been shown recently that the intramolecular inter-

action between the UbL and UBA domains of

hHR23B may regulate NER by modulating the pro-

teolysis of XPC [12,13].

The XPC-binding domain of hHR23B (residues

277–332, referred to herein as XPCB–hHR23B) houses

an XPC-stimulation activity that functions in a manner

similar to that of full-length hHR23B; this activity par-

ticipates in DNA damage discrimination in vitro and

in the enhancement of cell survival in vivo [9]. From

the amino acid sequence, Masutani et al. [9] showed

that the XPC-binding domain of hHR23B has a partly

repetitive character [that is, it contains various versions

of the sequence (P)QLLQQ(I)] and a highly amphi-

pathic nature, which is evident when the domain is

represented as a helical wheel [9]. XPC-binding

domain-like sequences are also found in protein

linking IAP with cytoskeleton (PLIC), which also has

both UbL and UBA domains [14]. Consensus motifs

from proteins with XPC-binding domain-like sequen-

ces are shown in Fig. 1B. It is interesting that the

XPC-binding domain has been classified as a heat

shock chaperonin-binding motif, which is also found in

the stress-inducible phosphoprotein STI1 [15,16]. The

presence of sequence similarity between the XPCB

domain and STI1 is not surprising, because XPC is also

induced by a kind of cellular stress (i.e. DNA damage).

Studies have also suggested that hHR23B has more

diverse in vivo functions than hHR23A. For example,

only hHR23B was codetected with XPC protein during

the affinity fractionation of mammalian crude extract,

using an immobilized glutathione-S-transferase (GST)–

S5a fusion protein [17]. S5a can bind to both polyubi-

quitinated proteins and the N-terminal ubqiuitin-like

domain of hHR23A ⁄B [18]. This might occur because

only a small fraction of cellular hHR23A exists in a

complex with XPC [7]. Experiments with knockout

mice that carry a homozygous deficiency in either the

mHR23A or mHR23B gene showed that these two pro-

teins are functionally redundant in terms of response

to DNA damage by UV light. The XPC protein was

not detected in the double knockout cell line, but

could be detected after treatment with a proteasome

inhibitor. It is interesting that only the mHR23B

knockout mouse showed defects in postnatal growth,

suggesting that mHR23B may have functions beyond

those related to XPC and DNA repair [19].

Here, we report the three-dimensional solution struc-

ture of the XPCB–hHR23B (275–342) fragment

(XPCB–hHR23B), which contains the XPC interaction

domain, and compare this with the recently reported

structure of XPCB–hHR23A [13,20]. Both overall

A

B

Fig. 1. Proteins with STI1-homologous domain and the sequence alignment of the XPCB domain with its homologs. (A) Domain presenta-

tions of the STI1-homologous proteins, showing the UbL (ubiquitin-like), UBA (ubiquitin associated), STI1 (stress inducible) and TPR (tetratrico

peptide repeat) domains. (B) Multiple sequence alignments of the XPCB domains of hHR23B and hHR23A with other STI1-homologous

domains from yeast STI1 and DSK2, and from human PLIC2 were obtained from a simple modular architecture research tool (SMART)

[16]. The Pro residues are indicated in bold.

Dynamic structure of XPC-binding domain of hHR23B B. Kim et al.

2468 FEBS Journal 272 (2005) 2467–2476 ª 2005 FEBS

structures are similar, but the N-terminal part (275–

283) of XPCB–hHR23B is more flexible than that of

hHR23A. 15N-Heteronuclear relaxation analyses were

performed with XPCB–hHR23B to gain precise infor-

mation concerning the flexibility of the NH vectors

along the peptide chain. We analyzed the diverse

hydrophobic surfaces of XPCB–hHR23B, which result

from the flexible N-terminal region, and attempt to

determine the structure of the XPC-binding surface.

Results and Discussion

Although the minimal domain of hHR23B (277–332)

binding to the XPC protein (XPCB–hHR23B) has

been reported previously [9], the solubility of this frag-

ment was too low for NMR experiments (< 0.2 mm,

data not shown). To increase the solubility of XPCB–

hHR23B for NMR analysis, we added two more

N-terminal amino acids, P275 and L276, which were

selected according to the sequence alignment of STI1

homologs (Fig. 1); as such, XPCB–hHR23B could be

concentrated up to 1 mm. Still, the peak of the15N-HSQC spectrum was broad, and its intensity was

not uniform with increasing concentrations of protein

(data not shown). The line-broadening observed at

higher protein concentrations seemed to result from

nonspecific hydrophobic interactions between XPCB–

hHR23B subunits, which were due to the higher con-

tent of hydrophobic residues in the XPCB. To reduce

these intermolecular interactions, we either included

additional amino acids from the C-terminal part of

XPCB–hHR23B (residues 333–342, QEAGGQGG

GGG) or solubilized the protein fragment in 10 mm

CHAPS buffer. The combination of these two

procedures markedly improved the quality of the 15N-

HSQC spectra for XPCB–hHR23B (Supplementary

material, Fig. S1). The presence of CHAPS had a

negligible effect on the chemical shift values in the15N-HSQC spectra (data not shown).

Although the peak regions of Hb and Hc in the15N-HSQC spectra were complicated because of the

high content of Gln, Leu and Glu, we were able to

accomplish complete side-chain assignment with the

aid of additional HCCH-COSY spectrum. After the

automatic NOE assignment and structure calculation

using cyana [21], we obtained the 1242 assigned dis-

tance restraints from the 1948 NOE cross-peaks. For

the energy-minimized final structure, we used the

amber7 program after pseudo-atom correction for the

obtained distance restraints. A stereoview of the calcu-

lated XPCB–hHR23B structures is shown in Fig. 2A,

and the statistics of structure calculation are summar-

ized in Table 1. XPCB forms a very compact, roughly

five-helix bundle: (a) helix 1 consists of residues E277

to L279 or R280 and assumes the geometry of a

310 helix; (b) helix 2 consists of residues F285 to I292,

of which the front boundary was slightly variable;

(c) helix 3 spans residues P296 to E309, which has a

310 helix that contains residues P296 to L298;

(d) helix 4 is formed by residues P311 to S318; and

(e) helix 5 consists of residues Q321 to L328. The

C-terminal Gly-rich region is a flexible random coil, as

was predicted by the chemical shift index (Fig. 3A). The

Pro residues are likely to be the cornerstones for the

boundaries of the helices, and their periodical presence

made the XPCB domain fold in a compact manner by

introducing helical breaks and turns or kinks (Figs 1

and 2A). The N-terminal part of XPCB–hHR23B

A B C

Fig. 2. NMR structure of the XPCB domain of hHR23B. (A) Stereoview of the 12 superimposed structures of XPCB–hHR23B. All Pro resi-

dues conserved among the STI1 homologs are marked in blue, and the Pro residues not conserved among the STI1 homologs are marked

in cyan. (B) Ribbon presentation of the XPCB–hHR23B three-dimensional structure. (C) Seven structures of XPCB–hHR23B (yellow) and of

XPCB–hHR23A (green, from Walters et al. [13]) are superimposed. The red and blue residues are amino acids that differ between XPCB–

hHR23B and XPCB–hHR23A. The side chains of residues with orientations that differ between hHR23B and hHR23A are shown. Also, b-N

and b-C are the N- and C-termini of XPCB–hHR23B; a-N and a-C are the N- and C-termini of hHR23A.

B. Kim et al. Dynamic structure of XPC-binding domain of hHR23B


(amino acids 275–283) was not well converged, sug-

gesting that this part of the molecule was more flexible

than the rest of the polypeptide. Indeed, the relatively

negligible long-range NOE cross-peaks in this segment

fit well with this hypothesis (data not shown). For the

rest of XPCB–hHR23B (amino acids 284–332), the

backbone regions were well converged, and the root

mean square deviation (RMSD) was 0.63 A (Table 1).

Although, the overall three-dimensional structure of

XPCB–hHR23B was very similar to that of XPCB–

hHR23A (Fig. 2C), it was difficult to identify the flexi-

bility in the N-terminal region of the two previously

reported NMR structures of XPCB–hHR23A [13,20].

The sequence homology between the XPCB regions of

hHR23A and hHR23B is very high (88%), and the

nine amino acids that differ [namely, N281(A) to D237

(B), Q287 (A) to N243 (B), I291 (A) to V247 (B), S297

(A) to A253 (B), I306 (A) to L262 (B), R308(A) to

Q264 (B), Q319 (A) to R275 (B), H323 (A) to Q279

(B) and V332(A) to P288 (B)], simply increased the

flexibility of the backbone segment, including helix 1

(Fig. 2C). Although our overall structure for

XPCB–hHR23B was more similar to the structure of

XPCB–hHR23A determined by Waters et al. (RMSD,

� 2.12 A) [13] than that by Kamionka and Feigon

(RMSD, � 3.43 A) [20], this trend is reversed when

Table 1. Statistics of structure calculation. RMSD, root mean

square deviation.

Parameter Value

Total NOE distance restraints (#) 717

i, i 241

i, i + 1 33

i, i + 2 121

i, i + 3 41

i, i + 4 89

Long-range NOE (|i – j| > 4) 1242

Total angle restraints (#) 70

/ (TALOS + experimental 3JHNHa) 42

w (TALOS) 28

20-structures from AMBER (kCalÆmol)1)

Total energy )3087.9 ± 12.3

E (NOE violation) 15.1 ± 1.6

E (Angle violation) 0.0 ± 0.0

AMBER FF99 force field

E (Non-restraint)a )3103.1 ± 11.7

20-structures PROCHECK analysis (%)

Most favored regions 78.5

Additionally allowed regions 20.2

Generously allowed regions 1.3

Disallowed regions 0.0

RMSD (A) Backbone 0.83 ± 0.306

Residues 275–330 All atoms 2.00 ± 0.350





a Summation of energies defined by AMBER force field.

A

B

C

D

E

F

G

H

Fig. 3. Relaxation studies of XPCB–hHR23B at 500 MHz field. (A)

The chemical shift index (CSI) clearly shows the well-defined five

helical regions. R1 (B), R2 (C), and 15N-1H heteronuclear NOE (D)

values were used to obtain the ordered parameters (E), the internal

correlation times (F), the exchange rates (G), and the model types

(H) from the TENSOR2 analysis. The values marked by asterisks (*)

in (C) and (G) are 24.0 ± 0.94 (s)1) and 14.8 ± 1.62 (s)1), respect-

ively.



considering the local structure. For example, the struc-

ture of hHR23B I306 differed significant from that of

the corresponding residue (L262) of hHR23A. I306

was well ordered and was part of a hydrophobic core,

whereas L262 is somewhat flexible and extrudes out

from the molecule core (Fig. 2C). However, the confi-

guration of L262 from the XPCB–hHR23A structure

of Kamionka and Feigon shows solvent exposure sim-

ilar to that of I306 in XPCB–hHR23B (Fig. 5). The

C-terminus of XPCB–hHR23B extends in a different

direction than those of both XPCB–hHR23As. This

may result from the altered amino acid sequence

(P331–V332 in hHR23B vs. P287–P288 in hHR23A),

because the consecutive prolines of XPCB–hHR23A

restrict the direction of the C-terminus in a different

way to the Pro–Val sequence in hHR23B.

We next performed 15N-relaxation experiments and

tensor2 analysis [22] to examine in more detail the

flexible characteristics of the 275–284 segment of

hHR23B. Because of the high quality of our15N-HSQC spectra, 54 of the 58 protonated backbone

nitrogen atoms were available for relaxation measure-

ments. The values of 15N R1, R2 and 15N-1H hetero-

nuclear NOEs are shown in Fig. 3. The results

obtained for residues 336–342 (GGQGGGG) were

omitted from Fig. 3, as the NOE values of these resi-

dues were in the far negative and their chemical shift

index values were assigned to 0, indicating that this

region is very flexible (Fig. 3A). Excepting the C-ter-

minal segment from E330 to G342, the heteronuclear

NOE and R2 measurements showed a uniform distri-

bution over most of the amino acid sequence, demon-

strating values typical for a globular protein. The

molecular size of XPCB–hHR23B (275–342) is

8.14 kDa, and the initial R2 ⁄R1 ratio values obtained

from 600 and 500 MHz NMR machines corresponded

to an overall correlation times (sinitc ) of 5.7 and 6.9 ns,

respectively. The higher correlation time at 500 MHz

may be caused by slight experimental differences,

including a slight difference in buffer conditions and

the lower temperature used for the experiment at

500 MHz (25 instead of 27 �C), which gives better

HSQC spectra. Because of the known dependence of

the overall correlation time sc on the molecular size of

various proteins [23], both sc values of the XPCB are

well matched to those of spherical molecules of similar

size with a smooth surface moving in an ideal liquid.

By increasing the error ranges from the fitting to sin-

gle exponential decay (1.75 and 2.0 times for the relax-

ation data at 500 and 600 MHz, respectively), it was

possible to find a proper diffusion tensor model. Fol-

lowing the determination and assignment of appropri-

ate spectral density function models for each residue,

the overall correlation time was again optimized using

tensor2. Residue-specific models were selected to

minimize the overall v2 with respect to sc. The analyzedresults of both relaxation data at 600 and 500 MHz

were quite similar (Supplementary Fig. S2; and

Fig. 3), but the values of the order parameters at

600 MHz were low at the residues 278, 281, 322 and

323. Inspection of (S2, si) parametric space for these

residues (assigned to model type 2, 4, and 5) showed a

very diverse distribution in the Monte Carlo simula-

tion using tensor2. Similar inspection of the residues

278 and 281 in the 500 MHz data showed less diverse

distribution in the (S2, si) parametric space and the

residues, 322 and 323 were assigned to model type 1.

It is possible that the quality of relaxation data at

500 MHz is better than at 600 MHz or that the field-

dependent motion results in a different model type for

the specific residues. With respect to the regions of the

XPC-binding domain that have well-defined secondary

structure (Fig. 3A), the order parameter (S2) was

higher than 0.85, whereas smaller values were usually

obtained for the less ordered C-terminal part of

XPCB–hHR23B (Fig. 3E). Although a model-free ana-

lysis of is not based on the exact physical motions of

the molecule, it can provide important information

regarding the dimensionless characteristics of the mole-

cule’s backbone. Most residues are well fitted to model

type 1–4, which can be described by the combination

of three terms; an order parameter (S2), an internal

correlation time (si), and a conformational exchange

term (Rex). From the relaxation studies, we identified

that N-terminal segment of XPCB–hHR23B (275–283)

has a distinctive exchange process (Fig. 3). Interest-

ingly, the heteronuclear NOE values of this segment

were almost similar to other well-refined regions, in

spite of the presence of internal motion (si) and a

remarkable exchange process (Rex). The NOE values

combined with the presence of internal motion and the

exchange terms in N-terminal segment show that the

N-H vectors of each residue of this region are correla-

ted in motion on the axis of helix 1 (i.e. this segment

is not independently flexible).

The XPC–hHR23B complex was reported to be sta-

ble even in 0.3 m salt, and the binding mode is driven

mainly by hydrophobic interactions [9]. Our results

show that XPCB–hHR23B has a more diverse hydro-

phobic surface than the XPCB–hHR23A, because of

the heterogeneous distribution of N-terminal segment

(Fig. 4). Two major hydrophobic trails (HTs) (HT1

and HT2) were identified in XPCB–hHR23B, one

between helix 2 and helix 3, and one between helix 3

and helix 4–5. These two trails were linked by a hydro-

phobic linker patch between helix 2 and helix 3 (P296,



L298, L299, and P300) and thus form a U-shaped

hydrophobic surface (Fig. 4A,B). The boundary and

size of the hydrophobic linker patch and HT2 were

well conserved in all calculated structures, because

these hydrophobic surfaces were formed by the well-

converged parts of XPCB–hHR23B. However, because

of secondary effects from the divergent positioning of

the flexible N-terminal region (275–283), the boundary

and size of HT1 were variable; in contrast, this vari-

ability of the HT1 region was not detected for XPCB–

hHR23A in the Kamionka and Feigon study [20].

Moreover, the HT1 region was not observed in an ear-

lier structure of XPCB–hHR23A reported by Waters

et al. [13]. The effect of the motion of the N-terminal

region on the hydrophobic surface area is more obvi-

ous in Fig. 4C. The hydrophobic interior appeared to

be relatively well covered by helix 1 in Structures 1

and 2, but its larger part was exposed to the outside in

Structure 3, because of reduced shielding by helix 1.

We calculated the solvent-accessible areas of XPCB–

hHR23A and XPCB–hHR23B for each residue. It is

clearly shown that the variation in the solvent-access-

ible area of the N-terminal part of XPCB–hHR23B is

markedly higher than that of XPCB–hHR23A (Fig. 5).

The total surface area of all these structures is very

similar; XPCB–hHR23B, � 41 nm2, XPCB–hHR23As

from Waters et al. and Kamionka and Feigon, � 41

and � 43 nm2, respectively. The diverse hydrophobic

surface, which resulted from the heterogeneous distri-

bution of the N-terminal segment of XPCB–hHR23B,

was not observed in XPCB–hHR23A and could

explain the inferior solubility of XPCB–hHR23B com-

pared with that of XPCB–hHR23A [20].

We tried to express the entire hHR23B-binding

domain of human XPC (amino acids, 496–734) in

Escherichia coli so as to identify the precise XPC con-

tact sites in the XPCB–hHR23B. However, this

domain was expressed in an insoluble form with var-

ious expression vectors (N- and C-terminal His-tag,

GST-tag, and thioredoxin-tag) and in a number of

E. coli strains. It is possible that, if we expressed por-

tions of the hHR23B-binding domain of human XPC,

the peptide segments we selected would be more amen-

able to purification in a soluble form. Therefore, we

Fig. 4. Surface presentations of the three representative structures

of XPCB–hHR23B. The hydrophobic surface is presented in yellow,

and the polar and charged surfaces are shown in white. HT1 and

HT2 are hydrophobic trail 1 and 2, respectively. HL denotes the

hydrophobic linker patch. Some structures from the AMBER7 calcula-

tion showed a short distance between the side chains of Q284 and

E309 (marked by the asterisk). This is an artifact from the electro-

static force field of AMBER7, because no NOE cross-peak between

these side chains was observed.

Fig. 5. Solvent-accessible surface areas of XPCB–hHR23A and B.

The solvent-accessible surface areas and their deviations for each

residue are shown for two XPCB–hHR23A structures; Waters et al.

[13] (A1), Kamionka and Feigon [20] (A2), and XPCB–hHR23B (B).



sought to determine which XPC peptide segments

within the hHR23B-binding domain were relevant to

the domain’s function. It was reported that a segment

of the human Stch (hStch) protein can bind to the

STI1-homologous domain (Fig. 1B) of the Chap1

(hPLIC-1) protein [24]. Therefore, we performed

sequence alignment with the hHR23B-binding domain

of XPC and hStch (because the XPCB domain is sus-

pected to shares sequence similarity with STI1), and

selected two segments with the highest degree of simi-

larity, although their values are low. We then construc-

ted two GST-tagged expression vectors that contained

DNA sequences that corresponded to the two selected

segments (which encoded amino acids 566–608 and

613–661 of the hHR23B-binding domain of XPC).

However, we not able to detect binding of either

XPCB–hHR23B or the complete hHR23B protein in

GST pull-down assays using these two constructs (data

not shown). This inability to detect XPCB–hHR23B

binding in these pull-down assays suggests that either

XPCB–hHR23B recognizes XPC regions not present in

the two selected segments or binding of the two pro-

teins requires specifically folded motifs not present in

the protein fragments. The latter hypothesis is more

likely, because the hydrophobic surface of XPCB is

delocalized in two ways, and hHR23B has been repor-

ted to stabilize XPC from heat denaturation [25]. The

slight variability of the hydrophobic surface of XPCB–

hHR23B resulting from its innate flexibility could be

another advantage of adapting to the larger binding

counter part.

The difference in flexibility of the N-terminal regions

of XPCB–hHR23B and XPCB–hHR23A may be one

way to explain why these domains, which share high

sequence homology, have such different solubilities,

even though they have similarly folded structures. The

presence of the divergent hydrophobic surface, which

results from the more flexible N-terminal part of

XPCB–hHR23B compared with hHR23A, may explain

why the former has more diverse in vivo functions

[7,18,19]. In order to further our knowledge with

respect to the mechanisms of action of these proteins,

it is crucial that we define the precise boundaries of

the STI1-homologous domain and compare the struc-

tures of these domains from various proteins. Such

analysis should help to determine the minimal unit for

proper folding to yield a functional domain. A number

of other cellular proteins exist that, like hHR23B, con-

tain the STI1, UBL and UBA domains connected by

relatively flexible linkers. It is likely that the STI1

domains of these proteins have evolved to specify and

modulate target proteins through a common mechan-

ism related to proteolysis.

Experimental procedures

Cloning and purification of the XPCB–hHR23B

domain

The cDNAs encoding the hHR23B were generously provi-

ded by F. Hanaoka (Osaka, Japan) [9]. A cDNA fragment

containing the XPC-binding motif of hHR23B (277–332),

plus two extra N-terminal residues (Pro and Leu) and 10

more C-terminal residues (333–342), was subcloned into the

pET15b vector at the NdeI and BamHI sites (Novagen,

Madison, WI, USA). The protein was expressed in the

E. coli BL21 (DE3) pLysS strain by using isopropyl thio-

b-d-galactoside induction at 37 �C. The N-terminal His-

tagged form of the XPCB–hHR23B protein was purified by

using a Ni-NTA column (Qiagen, Valencia, CA, USA), and

the terminal His-tag was removed by the thrombin diges-

tion. An additional purification step of gel permeation

chromatography was performed on a Superdex 75 column

(Amersham Biosciences). Uniformly 15N-labeled and13C, 15N-labeled XPCB–hHR23B (275–342) were obtained

by growing the bacteria in M9 minimal medium supplemen-

ted by [15N]ammonium chloride and [13C]glucose.

Acquisition and processing of NMR data

NMR samples were prepared in buffers (pH 7.0, 40 mm

sodium phosphate and 160 mm sodium chloride) with or

without 10 mm Chaps. All NMR spectra were recorded

at 27 �C using a 600 MHz, Varian INOVA spectrometer

(Varian Associates Inc., Palo Alto, CA, USA). For the

backbone and side-chain assignments of XPCB–hHR23B,

we used the following general triple-resonance experiments:

HNCACB [26], CBCA(CO)NH [27], HNCO [28], C(CC-

TOCSY-CO)N-NH [29], H(CC-TOCSY-CO)N-NH [29],

HCCH-TOCSY [30], HCCH-COSY [30], and TOCSY-

N15-HSQC (mixing time, 100 ms). For structure determin-

ation, we extracted the NOE-distance restraints from

NOESY-N15-HSQC (mixing times, 80 ms and 150 ms) and

NOESY-C13-HSQC (mixing time, 100 ms).

R1 (1 ⁄T1) values of 15N were measured from spectra

recorded with nine different delays of T ¼ 10, 50, 100, 200,

400, 600, 800, 1000, and 1200 ms with relaxation delays of

1.5 s. R2 (1 ⁄T2) values were determined from spectra recor-

ded with duration delays of 10, 30, 50, 70, 130, 190, and

250 ms with relaxation delays of 1 s. Steady-state 15N-1H

NOEs were measured following the method described in

Farrow et al. [31] using proton saturation periods of 3 and

5 s, and then the average 15N-1H NOE was obtained. Addi-

tional R1 values (20, 40, 80, 140, 240, 400, 800, and

1200 ms with a delay of 3 s) and R2 values (16.8, 33.5,

50.3, 67.0, 100.5, 134.0, 184.3, and 234.6 ms with a delay of

1 s) and two independent set heteronuclear NOEs (satura-

tion period of 3 s) were obtained using a cryoprobe-

installed 500 MHz, Bruker Avance at the Korea Basic



Science Institute (Daejon, South Korea). The buffer condi-

tion was slightly different to that used in the 600 MHz

NMR machine (pH 7.2, 50 mm Hepes and 200 mm NaCl),

because the presence of highly charged and small ion, such

as phosphate, increases the 90 degree pulse length in the

cryoprobe. All NMR data were processed using nmrpipe

[32] and analyzed using sparky [33]. The errors of R1 and

R2 were estimated from the errors in the single exponential

decay fitting, and those of the 15N-1H NOEs were obtained

from the difference of two independent experiments. The

values of error were adjusted for the proper tensor2 analy-

sis, by increasing of the errors from the fitting (1.75 and 2.0

times for the relaxation data of 500 and 600 MHz, respect-

ively), in which the maximum and minimum errors were

fixed to 7.5 and 3.0%, respectively. Spectral density func-

tions assuming an isotropic rotational diffusion tensor were

calculated in 1000-step Monte Carlo simulations using the

tensor2 program [22].

Structure calculation and analysis

In total, 28 sets of dihedral angle restraints (/, u) with

good prediction scores were gathered from TALOS chem-

ical shift analysis [34], and an additional 14 angles

restraints (/) were obtained from the intensity-modulated15N-HSQC experiment [35]. The automatic NOE assign-

ment and structure calculations were performed using the

cyana program [21] and the 1948 NOE cross-peaks (1165

from 13C-NOESY-HSQC and 783 from 15N-NOESY-

HSQC). The 50 conformers with the lowest final target

function values with pseudo-atom correction were the input

for structure refinement with the amber7 program using an

Amber FF99 force field [36]. The 50 conformers were simu-

lated, annealed, and energy-minimized for 15 ps. During

this calculation, the generalized Born model was applied

for a better simulation of an electrostatic interaction in a

vacuum. We deleted a few distance restraints that were con-

sistently violated during the structure calculation and

increased the upper boundary of some distance restraints

slightly by comparing with the NOESY spectra. However,

we tried to retain the original values obtained from the

auto-assignment and the structure calculation using cyana

when the distance violations were relatively small. Among

the 30 structures with the lowest total energies, we selected

20 structures with the lowest NMR restraint violations for

further analysis. There were no angle restraint violations

for the final 20 structures and there were 33–50 distance

restraint violations (0.11 ± 0.04 A) among 1242 total dis-

tance restraints for each structure. The quality of the struc-

tures was assessed using the refined energy terms and the

procheck program [37]. The surface electrostatic potential

distribution of the best overall model of XPCB–hHR23B

was calculated with delphi [38]. The solvent-accessible

surface area for XPCB domains were calculated using

gromacs (v3.2.1) [39] and chimera [40] was used to

analyze the structures and to prepare drawings of the struc-

tures.

Acknowledgements

Financial support was obtained from the National

Creative Research Initiatives of the Korean Ministry

of Science and Technology. Molecular graphics images

were produced using the Chimera package from the

Computer Graphics Laboratory of the University of

California, San Francisco (supported by NIH P41 RR-

01081).

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Supplementary material

The following material is available from http://www.

blackwellpublishing.com/products/journals/suppmat/EJB/

EJB4667/EJB4667sm.htm.

Fig. S1. 15N-HSQC spectrum of XPCB–hHR23B.

Fig. S2. Relaxation studies of XPCB–hHR23B at 600

MHz field.



solution structure and backbone dynamics of the xpc-binding domain of the human dna repair protein...

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