to 25 S. The calculated sedimentation coeﬃcient for the CrgA hexadecamer is 17.75 S though the experimental S value may well be smaller as a result of extra hydrody- namic drag referred to above for the octamer. Therefore it is not possible to unambiguously designate one of the observed peaks as the hexadecamer. The failure of MS to detect any oligomers larger than the hexadecamer may be due to a combination of their low abundance and/or ineﬃcient ionization compared to the octamer and hexadecamer. Diﬀerences in the size range of oligo- mers detected by MS compared to AUC have been reported in a study of amyloid ﬁbril formation (53). Interaction of CrgA with its DNA target site The CrgA residues likely to contact the DNA were identi- ﬁed using modeling with the DNA co-structures of DtxR, OhrR and NarL (Figure 6A) (54–56). M29, A30, S32, A33, S35, R36, I37, K39 and R40 in the turn preceding or within the recognition helix (a3) are predicted to con- tact the major groove and residues of the wing are pre- dicted to contact the minor groove of the target DNA. The conserved arginine residues present in the wing appear to have an important role in DNA binding in LTTRs. An R50 (equivalent to CrgA R52) mutant in OxyR and additionally an R55Q mutation in CrgA abo- lishe all DNA binding on electrophoretic mobility shift assay (EMSA) (Figure 6B and C) (57). In addition resi- dues N4-E7 may also make contact with the DNA, though the nature of these interactions are diﬃcult to predict as the orientation of the a1 helix is not well conserved within the wHTH family. The DBD pairs of CbnR and CrgA are highly similar suggesting that individual DBD pairs of LTTRs bind to DNA in a similar way (Figure 6D). The overall length of DNA bound by a single LTTR DBD pair would be in the region of 70–80 A˚ (20–24 bp) with a distance of 33 A˚ (equivalent to 1 helical turn of DNA) between the two recognition helices (Ca of S32) and 63 A˚ between the two wings (Ca of R55). Almost all LTTRs bind to the T-N11-A motif and the more highly conserved residues of the wHTH are likely to contact these T/A bases (57). It follows that the 11-bp spacing of these bases indicates that the molecular determinant for LTTR consensus selec- tivity is likely to lie within the a-helical core of the DBD pair and not in the outer wing regions. A possible candi- date for conferring this selectivity is the serine/threonine at residue 35, one of the most highly conserved residues in the recognition helix of the LTTR family (Figure 6A) and required for DNA binding in OxyR, CysB and GcvA (57–59). Furthermore, modeling suggests that in a DBD pair of CrgA, the two S35 residues would be capable of making base contacts to the major groove of DNA on opposite strands with 11 bp in between (Figure 6C). Perhaps, further indication of a role for this Ser/Thr in LTTR motif recognition is MauR, an LTTR that unusu- ally contains an alanine rather than a Ser/Thr at this posi- tion binds to DNA lacking the classical LTTR motif (60). The calculated electrostatic potential of the outer sur- face of the octamer showed localized regions of both pos- itive and negative charge. Taken together, with the Nucleic Acids Research, 2009, Vol. 37, No. 14 4555 orientation of the wHTH motif within CrgA suggests that, unless there is a signiﬁcant structural rearrangement upon DNA binding, the DNA would bind across the octa- meric disc at an angle of 608 (axis between adjacent corners) and not directly around the periphery of the disc (Figure 6E). The distance between DBD pairs (mea- sured between S32) at adjacent corners is 91 A˚ . Therefore 26 bp or more of bridging DNA would be required to link these adjacent DBD pairs together, if they were to engage DNA sequence elements in the same stretch of DNA. However, the spacing of the two LTTR consensus sites within the 63-bp CrgA sequence footprint is just 15 bp, equivalent to 50 A˚ of DNA and would not be long enough to bridge two DBD pairs of a single CrgA octamer (18,48). We conclude from these observations that the two binding sites, which make up the 63-bp sequence footprint of CrgA cannot be bound by the same octamer. Model of the CrgA–DNA complex As mentioned above, the published DNA footprinting data for CrgA show that the protein interacts with two adjacent LTTR-binding motifs in the CrgA/MdaB pro- moter region (48). We have shown that CrgA assembles as an octamer and have argued from an examination of the crystal structure that geometric constraints preclude the simultaneous binding of both sites by the same CrgA octamer. The MS of CrgA–DNA complexes showed the binding of single octamers to DNA and sedimentation velocity experiments conﬁrmed that single octamers are the predominant species observed in the presence of DNA. We conclude that in this case, only one of the two LTTR-binding motifs in the DNA are involved in CrgA binding. Consistent with this, EMSA assays showed that in addition to binding to the 63-bp CrgA DNA footprint, CrgA binds to the individual CrgA site 1 and site 2 sequences (Figure 7B). The mobilities of these 30-bp DNA fragments were clearly shifted by CrgA. No shift was observed for a randomized control oligonucleo- tide of similar GC base content, indicating that the inter- action with the CrgA sites was sequence speciﬁc (Figure 7B). In contrast to the EMSA with the 63-bp sequence, no deﬁned band corresponding to a CrgA– DNA complex for these shorter DNA fragments was observed. This possibly indicates that the presence and occupancy of both binding sites is required for the forma- tion of a stable complex in the EMSA. In these experi- ments, we did observe some material that did not enter the gel, which may correspond to high-molecular-weight com- plexes. However, given the propensity of the protein to aggregate it could also include non-speciﬁcally aggregated material. The MS of CrgA–DNA complexes showed in addition to the binding of single octamers to DNA, the unexpected stabilization of a double octameric species by DNA bind- ing. Sedimentation velocity experiments also indicated that higher order oligomers of CrgA are induced by the addition of DNA. Intriguingly, in crystal form B of CrgA, two octamers were observed stacked together providing a view of how CrgA could be self-associated (Figure 7C).