Identification of intrinsic order and disorder in the DNA repair protein XPA

Abstract

The DNA-repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full-length protein, the minimal DNA-binding domain, revealed that one-third of this molecule was disordered. To better characterize structural features of full-length XPA, we performed time-resolved trypsin proteolysis on active recombinant Xenopus XPA (xXPA). The resulting proteolytic fragments were analyzed by electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance mass spectrometry and SDS-PAGE. The molecular weight of the full-length xXPA determined by mass spectrometry (30922.02 daltons) was consistent with that calculated from the sequence (30922.45 daltons). Moreover, the mass spectrometric data allowed the assignment of multiple xXPA fragments not resolvable by SDS-PAGE. The neural network program Predictor of Natural Disordered Regions (PONDR) applied to xXPA predicted extended disordered N- and C-terminal regions with an ordered internal core. This prediction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well-characterized intrinsically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q85-I179) despite 14 possible cut sites. For the full-length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.

Fig. 1.

(A) Full-length Xenopus XPA sequence aligned with hMBD. The Zn-finger (shaded) and poly-Glu domain (box) are labeled. The human MBD sequence begins on the second line. (B) ESI-FTICR mass spectrum of the full-length xXPA showing a large distribution of charge states from 20+ to 34+. The measured molecular weight of the pure protein (most abundant isotopic peak =30922.02 daltons) was in good agreement with the mass predicted from the sequence (calculated most abundant isotopic peak = 30922.45 daltons), and the observed isotopic distribution was consistent with the calculated one (inset).

Fig. 1.

(A) Full-length Xenopus XPA sequence aligned with hMBD. The Zn-finger (shaded) and poly-Glu domain (box) are labeled. The human MBD sequence begins on the second line. (B) ESI-FTICR mass spectrum of the full-length xXPA showing a large distribution of charge states from 20+ to 34+. The measured molecular weight of the pure protein (most abundant isotopic peak =30922.02 daltons) was in good agreement with the mass predicted from the sequence (calculated most abundant isotopic peak = 30922.45 daltons), and the observed isotopic distribution was consistent with the calculated one (inset).

Fig. 2.

(A) SDS-PAGE of xXPA partial tryptic digestion. Thirty μg of purified xXPA was digested at 1:2000 (w/w) trypsin:XPA (lanes 1–5) and at 1:200 (lanes 6–10). Aliquots were removed at 5 min (lanes 1,6), 15 min (lanes 2,7), 30 min (lanes 3,8), 60 min (lanes 4,9), and 120 min (lanes 5,10) and resolved by 4%–20% gradient SDS-PAGE. (MWM) Molecular weight marker (lane 12), Broad Range Protein Markers, New England BioLabs. Mobility of intact xXPA (lane 11) corresponds to ∼40 kD. The three dominant bands are indicated with a filled diamond, open square, and filled triangle. (B) Quantitation of the three dominant XPA fragments resulting from partial proteolysis. The Coomassie-stained gel was scanned, analyzed with NIH Image (v1.6.1), and quantities of each band indicated by the filled diamond, open square, and filled triangle in Fig. 2A ▶ were plotted vs. gel lane number.

Fig. 3.

Summary of all xXPA partial tryptic fragments identified by ESI-FTICR mass spectrometry. All potential cleavage sites are indicated as white lines in the black bar representing full-length xXPA. Amino acid positions for each fragment's N and C termini are indicated.

Fig. 4.

ESI-FTICR mass spectrometry of fragments separated by reverse-phase HPLC. ESI-FTICR mass spectra for one recovered fraction from reverse-phase chromatography after 45-min digestion with 1:200 (w/w) of trypsin:xXPA. The full spectrum was highly convoluted because of the presence of several charge states of the same fragments (insets). Charge state deconvolutions of regions containing isotopic distributions detected by the Horn Mass Transform algorithm (Horn et al. 2000) are shown in insets.

Fig. 5.

Summary of cleavage site frequency. All detected unique fragments for trypsin:xXPA 1:200 and 1:2000 (w/w) at 5, 15, 30, 45, and 60 min were analyzed. Each of the 48 possible cleavage positions is indicated on the X-axis beginning with K11 and ending with K264; the Y-axis shows the number of unique detected fragments resulting from cleavage at each possible position. The cleavage positions in the xXPA sequence for the major sites in the N-terminal region are indicated above the peaks (filled diamond, open square, and filled triangle, as in Fig. 2 ▶).

Fig. 6.

Comparison of proteolysis data with PONDR predictions and NMR structure. (Top) Full-length Xenopus XPA is depicted as a line, interspersed with all possible trypsin sites as white vertical lines (Xenopus numbering). The line below represents hMBD in the same format. Based on the refined NMR structure of hMBD (Buchko et al. 1999a), four regions with low certainty of assignment or high flexibility are indicated in gray. Assigned structural regions (α, α-helix; β, β-sheet; t, turn) are depicted below hMBD with black vertical lines separating each region. Each of the unique, experimentally observed, 1:200 and 1:2000 (w/w) trypsin:XPA proteolysis fragments corresponding to the three dominant bands on the SDS–polyacrylamide gel in Fig. 2 ▶ (filled diamond, open square, and filled triangle) are drawn as horizontal lines below. (Bottom) PONDR prediction of order/disorder in xXPA. Each residue (X-axis) is assigned a disorder score (Y-axis) by the predictor based on the attributes of amino acids surrounding the residue. Predicted scores for residues ≥0.5 signify disorder.

Fig. 7.

Comparison of PONDR attributes for the XPA N-terminal region to disordered protein families. VL1 (white bars) is one of the databases of disordered sequences from 15 different proteins used to train PONDR. Disordered sequences from three representative protein families (calcineurin [dense diagonal pattern], histone H5 [square pattern], and prions [wide diagonal pattern]) were compared with the first 97 residues of 10 XPAs (seven species; black bars). The calcineurin data set contained 21 sequences (14 species) for the amino acids that align with the known disordered region (374–468) of human calcineurin. The histone H5 data set consisted of nine sequences (seven species) for the amino acids that align with the known disordered region (101–185) of chicken histone H5. The known disordered region (23–120) of mouse prion and the corresponding regions of 70 other prions were analyzed. Coordination number reflects how frequently a given amino acid is found internally vs. externally in a protein. Net charge is the absolute value in a window of 21 amino acids. The Y-axis is the difference between the indicated protein family composition and database of all known protein structures (NRL 3D) divided by the composition of the structure database (Δ/NRL 3D). When an amino acid attribute is below zero, the disordered family has less of that attribute than ordered proteins; above zero indicates the reverse.

Fig. 8.

Comparison of PONDR's order/disorder predictions for xXPA to three programs that predict secondary structure. xXPA was analyzed by the programs PHD (Rost and Sander 1994), available at http://cubic.bioc.columbia.edu/predictprotein/; SSP-Baylor (Solovyev and Salamov 1994), available at http://dot.imgen.bcm.tmc.edu:9331/pssprediction/pssp.html; and Chou-Fasman (Chou and Fasman 1978) (Wisconsin Package Version 10.0, Genetics Computer Group). (Heavy stippled pattern) Coils and turns, (diagonal stripes) helices, (horizontal stripes) sheets, (light stippled pattern) disorder, (dark stippled pattern) order, (white) no prediction.