The AB complex adopts a highly elongated shape in solution
To obtain insights about the shape of the AB DNA damage sensor in solution, we measured SAXS from preparations of the complex that had been purified away from isolated UvrA and UvrB. The data, recorded as 15 1-s exposures at three different protein concentrations: 1, 2, and 4 mg/mL [Fig. 1(A)], were of high quality as indicated by three measures. First, comparison of the 15 individual scattering curves revealed close juxtaposition with no outliers (not shown). This indicates that our samples experienced no radiation damage during the measurement. Second, a Guinier plot (ln I(q) against q2) revealed a straight line in the low-q region [Fig. 1(A), inset] for all three protein concentrations. Third, radius of gyration (Rg), a quantity extracted from the Guinier plot, revealed consistent values (62.84 Å, 63.03 Å, and 66.06 Å for the 1, 2, and 4 mg/mL measurements, respectively, see also Table I). These last two measures indicated that our preparations showed little aggregation and/or interparticle interference, especially at low protein concentrations. The increase in Rg with the 4 mg/mL measurement suggests that the sample might have aggregated or exhibited interparticle attraction at high concentration. Therefore, we have restricted our analysis to the data collected at the lowest protein concentration.
Figure 1. X-ray scattering curves, pair distribution function, and SAXS-derived molecular envelope of the AB complex. (A) Curves representing SAXS from the AB complex measured at three different protein concentrations. Inset: the corresponding Guinier plots (ln I(q) against q2). (B) Pair distribution function, P(r), calculated from the scattering data collected from 1 mg/mL sample concentration. (C) Close correspondence between the bead model calculated from the SAXS data and the UvrA2–UvrB2 crystal structure (Model #1, Fig. 2(A), the tetramer is generated by a crystallographic two-fold axis from the PDB 3UWX). Superposition of the UvrA2–UvrB2 crystal structure (Model #1)20 onto the SAXS-derived bead model shows good agreement. The SAXS-derived bead model is shown using semitransparent gray spheres with a radius of 11 Å. The AB crystal structure is shown using ribbon representation. The two UvrA protomers are shown in different shades of green, whereas the two UvrB molecules are shown in different shades of magenta. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Figure 2. Evaluation of five potential AB models using SAXS. The left panel shows scattering curves computed from five potential models for the AB complex (red) in comparison to the experimental (gray) curve. The right panel depicts the P(r) curve calculated from the models (red) and experimental data (gray). The AB structural models are displayed in a surface representation with the two UvrA molecules colored in different shades of green, and the two UvrB molecules colored in different shades of magenta. Rg coord. and Dmax coord. are calculated directly from atomic coordinates using the program MOLEMAN2.34Rg calc. scatt. is from pair distribution function29 calculated using scattering curves derived from coordinates.33Dmax calc. scatt. is from the scattering curves calculated from coordinates using the program CRYSOL.33
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An important overall conclusion from inspection of the scattering data is that the AB complex is a highly elongated entity in solution. This is evident from the pair distribution function, P(r), calculated from the experimental data [Fig. 1(B) and Ref.29], which in addition, provided an estimate of the longest chord (Dmax) in the damage sensor: 213.7 Å.
We next used the SAXS data collected from the AB sensor, in combination with prior structural and biochemical data, to identify the conformation and composition of the sensor in solution. The issue of the precise identity of the physiological entity arose during determination of the crystal structure when we observed that the AB sensor complex crystallized with one AB heterodimer in the crystallographic asymmetric unit (PDB 3UWX).20 As it is well established that UvrA is a dimer in solution,22, 35 we concluded that the physiological entity must be composed of two copies of UvrA and two copies of UvrB. However, inspection of the packing in the crystal revealed four possible candidates for the biologically relevant entity. To identify the species in solution, we analyzed these four models, plus a fifth model that contained two copies of UvrA and one copy of UvrB (referenced by a large number of studies1, 22, 23) in the context of five parameters derived from the SAXS data (the scattering curve, the radius of gyration (Rg), the maximum particle dimension (Dmax), the pair distribution function (P(r)), and the solvent envelope). The five models are shown in Figure 2. As noted previously, Model #1 displays a dimer configuration (termed the “closed groove” conformation) not observed in any previous structure of UvrA, which all resemble the “open tray” conformation.20 In this context, none of the other possible models suggested by packing in the crystal (Models #2, #3, and #4) contain the dimer seen in the open tray conformation. Also, consideration of A2B1 as a candidate (Model #5) was restricted to a species that contained the dimer observed in the closed groove configuration of UvrA.20
Comparison of the scattering curves calculated from the five candidate models of the AB complex reveals that only Model #1 agrees with the measured SAXS data [Fig. 2(A)]. Models #2, #3, and #4 display a poor fit over the entire resolution range [Fig. 2(B–D)]. Notably, the X-ray scattering calculated from the A2B1 model (Model #5, [Fig. 2(E)]) shows a poor fit at very low resolution; this feature is especially informative as this is the range that contains size information and where one would expect close agreement if this model represented AB complex in solution.
We obtained further support for Model #1 by consideration of other SAXS-derived quantities. Analysis of the linear Guinier region (q•Rg < 1.3) of the experimental scattering curve provided an estimate of the Rg of the AB complex in solution (62.84 Å). This value compares well with the value calculated from coordinates of Model #1 (64.2 Å). In contrast, the corresponding values for Models #3, #4, and #5 diverged from that obtained from the experiment (Table I and Fig. 2). We note that the Rg calculated from Model #2 is closer to the experimental value than those of Models #3, #4, and #5, thus potentially making a case for the physiological relevance of this model. However, Model #2 can be eliminated from consideration as it is organized around UvrA dimer contacts to a domain (the “insertion” domain) that we have shown previously to be dispensible for dimerization.11 Model #1 is also favored by comparison of the SAXS-derived Dmax value against the value calculated from the coordinates (213.7 vs. 220 Å). Moreover, the large divergence of Dmax values calculated from the other models eliminates them from consideration. In addition, analysis of the pair distribution function, P(r) from the various models, argues in favor of Model #1 as the physiological entity. As illustrated in Figure 2, only the pair distribution function calculated from Model #1 agrees with the curve calculated from the experimental data.
To further explore the elongated shape suggested by the one-dimensional SAXS data, we performed an ab initio calculation of the three-dimensional shape of the AB sensor from the scattering curve. This procedure, as implemented in the computer program DAMMIN, uses simulated annealing to modify a starting volume filled with dummy atoms, so as to maximize the agreement between a scattering curve calculated from this volume and that measured from the experimental sample.36 The starting volume is chosen to be a sphere, with a radius equal to one-half of the longest chord from the P(r) curve calculated from the data. We repeated this procedure 50 times to obtain independent models; these were superimposed, averaged, and filtered to the average volume. In this context, two types of reconstructions were performed, the first without imposition of symmetry (P1) and the second with imposition of two-fold symmetry (P2, the crystal structure of the A2B2 tetramer is two-fold symmetric). The models that emerged form the P1 and P2 calculations were very similar in appearance. We decided to focus our analysis on the P1-derived reconstruction as the AB sensor may not obey two-fold symmetry in solution. This P1-derived calculation produced an average volume with dimensions of approximately 80 × 80 × 210 Å [Fig. 1(C)]; the NSD between individual models used in averaging were in the range of 0.81–0.89 as calculated by SUPCOMB.30
Comparison of the volume that emerged from the procedure above, visually and quantitatively, points to close agreement with Model #1 and rules out other models [Fig. 1(C)]. The overall dimensions of the SAXS-derived envelope (80 × 80 × 210 Å) follow those seen in the crystal structure (80 × 90 × 210 Å). Superposition of the UvrA2–UvrB2 heterotetramer (Model #1) to the bead model revealed a good fit visually; this fit was characterized by a normalized special discrepancy (NSD) of 1.3 (Ref.30). We note that the SAXS-derived volume, which represents the hydrated volume of the complex in solution, is slightly smaller than that expected for UvrA2–UvrB2. In addition, upon superposition, some parts of the crystallographic model lie outside the SAXS volume. We speculate that this could be due to a combination of factors, including intrinsic flexibility of the AB sensor, partial dissociation of the complex in solution, errors in measurements and calculation, and the low resolution of the scattering data.
Taken together, the five experimental quantities extracted from the SAXS data establish that the shape of the AB sensor and the disposition of the UvrB molecules seen in the crystal structure (Model #1) reports faithfully on the species in solution.
Nucleotide-dependent changes in the AB damage sensor or UvrA were not observed by SAXS
Both UvrA and Uvr B are ATPases, and ATP plays crucial, but incompletely understood roles, in genome scanning, lesion recognition, and UvrB loading onto the lesion DNA.1, 37–41 To obtain insights into how nucleotide affects the conformation of UvrA and the AB complex, we measured SAXS data for isolated UvrA and the AB complex in the presence of ATP and ADP. Data were collected at three different protein concentrations (1, 2, and 4 mg/mL) in buffer containing either ATP or ADP. As illustrated in Figure 3, the scattering profiles of both UvrA and the AB complex in the presence of the two nucleotides showed small differences. At this time, we have chosen not to interpret these differences as significant because of our recent discovery that UvrA might be natively interconverting in solution between open and closed forms with quite different structures11, 20; interconversion is likely to be more pronounced when ATP is present, as it is in some of our measurements. Our two crystal structures of UvrA (isolated11 and part of the UvrAB complex20) give a hint as to the nature of these large structural changes. However, to understand the effects of nucleotide on the structure of UvrA, conformationally homogeneous preparations of UvrA with defined nucleotide state are needed. We are currently developing a procedure that will provide us with such samples using a combination of biochemical trapping and site-directed mutagenesis.
Figure 3. Nucleotide-dependent conformational changes of UvrA and AB complex were not observed by SAXS. SAXS curve and pair distribution function of AB complex (A and B) and isolated UvrA (C and D) measured from 1 mg/mL sample in the presence of ADP and ATP are very similar. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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While this manuscript was under review, a new crystal structure of Bacillus subtilis UvrB was reported (Webster et al. Nucleic Acids Res. 2012. Epub 2012/07/04. doi: 10.1093/nar/gks633. PubMed PMID: 22753105). The most notable feature of this new structure is the presence of a two-fold symmetric UvrB dimer in the crystallographic asymmetric unit (PDB 3V4R); biochemical methods established the solution relevance of this arrangement. We note that the architecture and stoichiometry of the UvrAB sensor (PDB 3UWX), as described above, are consistent with the new UvrB dimer structure. In this regard, we speculate that the arrangement of UvrB in the complete UvrAB sensor (PDB 3UWX) and that found in the new UvrB dimeric structure may represent different, sequential, stages of the damage recognition pathway.