Structure of the DNA repair helicase XPD

Abstract

The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalyzing DNA duplex opening localized to the transcription start site or site of DNA damage, respectively. XPD has a 5' to 3' polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigmentosum, trichothiodystrophy, and Cockayne's syndrome, characterized by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 A crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans.

Figure 1. Structure of XPD

A. Comparison of the domain organisation of human and S. tokodaii XPD. Motor domain 1 is shown in cyan, motor domain 2 in salmon, the Arch domain in wheat and the FeS domain in green. The positions of the canonical motifs are shown by coloured bars, and the conserved sequences of these motifs are indicated. The C-terminal region of human XPD that interacts with the p44 protein is shown as a grey box, and is not present in the archaeal XPD. B. Overview of the structure of XPD. Domains are coloured as in 1A. The Arch domain (top of structure) arises as an insert into motor domain 1. The boundaries of the FeS cluster binding domain, which is not visible in the electron density map, are indicated by green spheres. The canonical motifs defined in 1A are indicated with the same color scheme and labelled. The phosphate ion in the ATP binding site is shown space-filled. C. S. tokodaii XPD is shown in the same orientation and color scheme as in 1B. The DNA species shown as a red cartoon is taken from the co-crystal structure of the 3′ to 5′ helicase Hel308 (PDB code 2p6r). The position of the DNA arises as a consequence from an overlay of the motor domains of the two enzymes without any further modelling. D. GRASP representation of the electrostatic surface of XPD shown in the same orientation as 1B (left) and with a rotation of 90 ° (right). The narrow channel formed between the Arch domain and motor domain 1 is lined with basic residues, and could accommodate ssDNA but not duplex DNA. E. Cartoon showing the likely path of DNA through the XPD helicase. Duplex DNA entering from the right is broken on the FeS domain, threading through the cleft between the Arch domain and motor domain 1, engaging with the conserved helicase motifs Ia, Ib, IV, V, Va and VII. The orphaned (3′) strand of the duplex may form interactions with the basic surface on the outside of the motor domain, shown in the right panel of 1D.

Figure 2. Mutations of XPD giving rise to XP and XP/CS

A. Schematic showing the domain structure and canonical motifs of S. tokodaii XPD. Domain boundaries are colored as in Figure 1A, and canonical motifs indicated with black bars. The positions of residues targeted by mutation in XP and XP/CS are indicated, with the S. tokodaii residue and numbering in black and the equivalent human residue numbering indicated immediately below in blue (XP) or orange (XP/CS). B. Structure of S. tokodaii XPD, colored as in Figure 1B, with the 8mer dU oligonucleotide derived from the co-crystal structure of the NS3 helicase shown in red (see also Figure S2). Residues mutated in XP and present in the S. tokodaii crystal structure are shown as space-filled sphere models, colored by element (carbon, yellow; nitrogen, blue; oxygen, red). Residues R437 and A438 constitute part of the loop formed by motif V that is not observed in the crystal structure. Residue D182, in motif II (the Walker B box) is omitted for clarity. C. Details as for B, with the structure rotated vertically forward by 90 °. The Arch domain is omitted for clarity. The cluster of residues at the top of domain 2 close to the expected position of the ssDNA is apparent.

Figure 3. Mutations of XPD giving rise to TTD

A. Schematic showing the domain structure and canonical motifs of S. tokodaii XPD. The positions of residues targeted by mutations causing TTD in human are indicated. S. tokodaii residues and numbering are in black and the equivalent human residue numbering indicated immediately below in red. B. Structure of S. tokodaii XPD colored as in Figure 1. The residues targeted by TTD-causing mutations are represented in space-filled and labelled sphere models as in Figure 2B. Residue K84 is on the boundary of the FeS domain and is not visible in the crystal structure. The position of the C-terminus in the archaeal XPD protein is also indicated. C. XPD structure with surface representation. The model is colored as in 3B and rotated 90 ° with respect to 3B to emphasise the bottom of domain 2. TTD mutations mapping to the bottom surface of motor domain 2 are colored blue and labelled. In eukaryotic XPD, these residues together with the C-terminal extension probably form an interaction surface with the p44 protein. D. Zoomed-in view of the boxed region of the Arch domain in Figure 3B. Residue A206 and surrounding residues are shown as space-filled molecular representations, with A206 in purple and other residues coloured according to their atom type. The TTD mutation introducing a tyrosine at this position (C259Y) is likely to cause significant disruption to the core of the Arch domain. E. Plot showing the temperature stability of the wild-type and A204Y mutant XPD enzymes from S. acidocaldarius (equivalent to S. tokodaii A206Y and human C259Y). The helicase activity of the A204Y mutant is similar to that of the wild-type protein, but the mutant enzyme was inactivated on heating for 20 min at the temperatures indicated before assaying for helicase activity. This confirms the predicted destabilising effect of the C259Y mutation.

Figure 4. Biochemical characterisation of selected mutations of XPD

A. Plot of the ssDNA-stimulated ATPase activities of the indicated wild-type and mutant proteins of S. acidocaldarius XPD. Relative ATPase activities are shown in 4D. Experiments were carried out in triplicate and means with standard errors are shown. Data were fitted by a linear fit. Rates expressed as a percentage of the wild-type rate with standard errors are shown in 4D. B. Plot of the anisotropy changes resulting from binding of the indicated wild-type and mutant XPD proteins from S. acidocaldarius to a 15 nucleotide oligonucleotide tagged with a 5′ fluorescein reporter molecule. Apparent dissociation constants are shown in 4D. Experiments were carried out in triplicate and means with standard errors are shown. Data were fitted as described in the methods. C. Helicase activities of wild-type and mutant proteins of S. acidocaldarius XPD showing the time course of unwinding of a partial DNA duplex with a 25 nt 5′ overhang. Parental and product species were quantified by phosphoimaging and helicase activity is summarised in 4D. Control lanes are: c1, boiled DNA; c2, no ATP control; c3, no XPD control. D. Summary of the biochemical properties of the wild-type and mutant proteins of S. acidocaldarius XPD. Equivalent numbering for the human and S. tokodaii XPD homologs are also indicated. Helicase activity is scored as “++” for near-wild-type levels and “−“ for completely inactive mutants. The A204Y mutant was active but temperature-sensitive, as described in figure 3E. nd – not determined.