Figure 5. Structure and regulation of XPD.

(A) Structure of XPD colored by domain. N- and C-terminal segments (blue and purple, respectively) of XPD are indicated. An ADP molecule superposed from the structure of DinG (Cheng and Wigley, 2018) denotes the nucleotide-binding pocket in XPD RecA1, which is empty in our structure. (B) Structure of the FeS domain. Residues critical for XPD enzymatic activity (blue) and damage verification (teal) are indicated. The R112H mutation causes TTD in human patients. ARCH domain insertion brown. DNA superposed from (Cheng and Wigley, 2018). The region corresponding to the view in this panel (but viewed from the back side) is indicated in (A). (C) Interaction network of XPD with surrounding TFIIH subunits (interacting regions colored, remainder grey). (D) Cartoon model for repression and de-repression of XPD by MAT1, XPB, and p62. (E) XPD-p44 interacting regions (defined as residues within <4 Å of the neighboring protein) are colored in dark green (XPD) and dark red (p44). Residues discussed in the text are shown as sticks; those with mutation data (natural variants or experimental constructs) are colored yellow on XPD, teal on p44. The remainder of the β4-α5 loop harboring the synthetic p44 mutations is colored teal as well.

Figure 5—figure supplement 1. Structure of XPD.

(A) Close-up view of the XPD N- and C-terminal segments near the nucleotide binding pocket of the enzyme. Y14 and Y18 may stabilize nucleotides when bound (not present in our structure). (B) View of the nucleotide binding site of XPD with superposed ADP from nucleotide-bound DinG (PDB ID 6FWS (Cheng and Wigley, 2018)). Residues mutated in human disease are shown as sticks and colored (purple: XP, orange: XP/CS, dark green, TTD). These include Y18, which we propose could form a stacking interaction with the base of a bound nucleotide, as well as residues in conserved helicase motifs (see C). (C) Same view as (B), but with conserved helicase motifs color-coded. Motifs Q, I, II, and IV are involved in nucleotide binding and hydrolysis (Fairman-Williams et al., 2010). (D) View of the back side of XPD, with the N- and C-terminal segments indicated. The C-terminal segment interacts with the linker between XPD RecA1 and RecA2 (residues 476–486). CX-MS crosslinks support the assignment of the C-terminal segment. The K751Q polymorphism has been associated with cancer therapy outcomes (Mlak et al., 2018; Peters et al., 2014), though functional differences between the variants has been debated (Clarkson and Wood, 2005). (E, F) Electrostatic surface (E) and cartoon (F) representations of the XPD structure, highlighting the pore between the XPD ARCH an FeS domains through which DNA (superposed from PDB ID 6FWR (Cheng and Wigley, 2018)) passes while being translocated by XPD. (G, H) Comparison of the structures of the human and archaeal FeS domains (F), Thermoplasma, PDB ID 4A15 (Kuper et al., 2012), (G), Sulfolobus, PDB ID 3CRV (Fan et al., 2008)). There are differences at the secondary structure level, and functionally important residues are only partially conserved. (I) Sequence alignment of FeS domains from human, Sulfolobus, and Thermoplasma XPD. With the exception of the conserved cysteine residues that bind the FeS-cluster, sequence conservation in this domain is relatively low. Residues discussed in the text are indicated. (J–M) Charge complementary of regions of negative and positive electrostatic potential near the MAT1 3-helix bundle and the XPD ARCH domain interface (circled).

Figure 5—figure supplement 2. XPD-p44 interaction.

(A) Top: XPD-p44 (XPD green, p44 red) interaction surfaces are shown in darker shade (interacting residues in p44 β4-α5 loop are shown in teal). Bottom: Charge complementarity of the interface (two major complementary regions numbered 1 and 2). (B) Detailed view of the β4-α5 loop (teal) with mutations discussed in the text. XPD-p44 interacting regions (defined as residues within <4 Å of neighboring protein) are colored in dark green (XPD) and dark red (p44). Residues discussed in the text are shown as sticks; those with mutation data (natural variants or experimental constructs) are colored yellow on XPD, teal on p44. (C) Detailed view of the interactions formed by XPD R722, which is mutated in TTD. (D) View of XPD residues R616, D673, and G675 in the context of p62 and p44. (E) XPD-p44-p62 interaction region. p62 segments interacting with both XPD and p44 are colored dark blue and yellow.