Nucleic Acids Research, 2009, Vol. 37, No. 14 4553 Figure 6. DNA binding and recognition in CrgA. (A) Sequence alignment of the DBD of CrgA with two further LTTR family members [E. coli, CysB and OxyR) and the three HTH proteins (DtxR from Corynebacterium diphtheriae (56), OhrR from Bacillus subtilis (55) and NarL from E. coli (54)] used for modeling. Residues boxed in yellow, contact the DNA in the co-structures of DtxR (PDB code 1F5T), OhrR (PDB code 1Z9C) and NarL (PDB code 1ZG5) or are predicted to contact DNA in CrgA. Residues important for DNA binding identiﬁed by mutational analysis in CysB, OxyR and CrgA are shown in red (11,57). Residues underlined form the activating region identiﬁed in CysB (64). The residue conservation scores of CrgA obtained with Consurf, based on 70 LTTRs from the Swissprot database, are shown above the alignment. Only residues of the structurally equivalent a2 and a3 are shown for DtxR, OhrR and NarL. (B) EMSA on CrgA wild type (2.5 pmol) and CrgA R55Q mutant (1.5 pmol) mixed in a 1:1 molar ratio with the reported 63-bp CrgA DNA footprint (18,48). (C) One DBD of CrgA with modeled DNA showing selected residues discussed in this report. (D) Structural comparison of a DBD and LH pair from CrgA and CbnR (Ca RMSD 1.1 A˚2 for 86 equivalent residues). R55 of CrgA is labeled for orientation purposes in panel E. (E) Electrostatic surface potential of the CrgA octamer showing one DNA-binding pair with modeled DNA (DxtR, PDB code 1F5T). activation of a ring-like structure in a mass spectrometer typically results in structural collapse of the quaternary topology. This is then followed by an expansion of the protein complex. Minimal activation however allows the transmission of a ring-like protein complex with little compromise to the presence of the cavity (52). In the case of the apo CrgA octamer, a reduction and then increase drift time was observed for the lowest charge state ion (+28) of the protein complex (Supplementary Figure S4). This is consistent with a small collapse, assigned to the loss of the cavity, followed by much greater expansion as the complex is activated and unfolded, The holo CrgA octamer with two DNA strands exhibits a similar collapse for its lowest charge state (+30) but to a smaller degree than the +28 charge state of the apo CrgA octamer. The diﬀerence between the initial drift time and minimum drift time for the most collapsed struc- ture, normalized for the charge of the ion, is smaller for the holo (22.80 ms) than for the apo (28.84 ms) form of the CrgA octamer. Moreover, the energy required to retain the most compact state for the DNA-bound CrgA octa- mer is greater for the holo form compared to the apo CrgA. Both the higher activation required to collapse the structure and lower level of collapse observed for the holo CrgA suggest that the DNA contributes to stabiliza- tion of the CrgA octamer. Moreover, these results suggest that the ring-like conformation of CrgA is maintained upon DNA binding. Comparison of CrgA and CrgA-DNA complexes by analytical centrifugation To complement the analysis by MS, sedimentation veloc- ity experiments were carried out on CrgA either alone or in the presence of the same DNA probe used in MS. In the absence of DNA (Figure 5A), the sedimentation proﬁle was dominated by a species of 9.4 S, accounting for 90% of the observed molecules, followed by a small tail at higher s values. This tail did not consist of well-deﬁned peaks and indicated a range of interactions, which were either non-speciﬁc or very transitory. In the presence of DNA, a major peak of 9.6 S was observed together with a series of species, which were well resolved and extended to 25 S. The proportion of observed molecules in the 9.6 S peak was 74%. Calculation of apparent weight c(s,M) distributions for both samples (Supplementary data Figure S6) showed that the 9.4 S CrgA species observed in the absence of DNA corresponded to a diﬀused peak between 200 and 350 kDa whereas the major 9.6 S species in the sample with DNA gave a well-deﬁned peak at 170 kDa (Figure 5). Peaks at higher values were also observed including a clear non-trivial peak at 2 MDa (Supplementary data ﬁgure S6) corresponding to the