4552 Nucleic Acids Research, 2009, Vol. 37, No. 14 Figure 5. Plots of sedimentation coeﬃcient population distribution (c(s)) against s. Each clearly deﬁned peak represents a single species, the main peak in both cases being ﬁt with a Gaussian to allow for precise calculation of the s value (red dashed line). (A) CrgA. (B) CrgA with DNA. The 2D cluster map contains four major groups with a large main cluster of 2928 sequences containing the tetra- meric LTTR CbnR in addition to other characterized LTTRs including MetR and OxyR. CrgA forms part of a smaller cluster of 921 (19%) protein sequences, which includes the close E. coli homolog YafC (59% sequence identity) as well as proteins annotated as AaeR and PtxR (Supplementary Figure S3). This partitioning of LTTR sequences indicates that CrgA is not unique and we suggest that the octameric assembly of CrgA may be shared with other LTTRs within this cluster. Although the overall oligomeric states of CrgA and CbnR are diﬀerent, there are parallels in the way that the CrgA octamer and CbnR tetramer are held together. The DBD–LH module of these two LTTRs is particularly well conserved (Ca RMSD of 1.1 A˚ for 86 equivalent resi- dues) and probably within the wider family (Figure 6D). Dimerization of the coiled coil controls the overall spacing and position of the two wHTH motif within an LTTR DNA-binding pair and will thus impart some of the gen- eral characteristics of an LTTR DNA-binding site, for example the length of the palindromic bases recognized. The RD interface does not show the same level of struc- tural conservation between the two structures. Although 25/35 of the equivalent residues involved in formation of the CrgA RD interface are also found at the CbnR RD interface, in CbnR the two RD-IIs also interface with each other through residues 248–253 whereas in CrgA they are clearly separate. MS reveals a double octamer CrgA–DNA complex Previously, DNase I protection assays of the intergenic region between crgA and the divergently transcribed gene, mdaB, identiﬁed two 30-bp CrgA DNA-binding sites (crgA sites 1 and 2) separated by 3 bp, each contain- ing the characteristic LTTR-binding motif, T-N11-A (48). We have conﬁrmed the speciﬁc interaction between puri- ﬁed CrgA and this 63-bp CrgA footprint in a gel-shift experiment. As observed by Deghmane et al. (16), there is not a complete shift of the probe by CrgA indicating that binding under the conditions of electrophoresis is rel- atively weak (Figure 6B). In order to determine the precise stoichiometry of the interaction between CrgA and the target DNA, MS was carried out on the isolated CrgA– DNA complex. A mixed population of CrgA–DNA com- plexes was detected. It was found that a single CrgA octamer could bind one, two or three lengths of DNA (Figure 3C). Furthermore, in the presence of DNA a CrgA 16mer was detected bound to a maximum of four DNA strands (Figure 3C). This double octamer species was not observed in the apo form suggesting that the pres- ence of the DNA is required to stabilize an interaction between the two CrgA octameric rings. The peak intensi- ties give an indication of the relative populations of the diﬀerent protein–DNA species in solution; however, par- ticularly for complexes with a similar composition, abso- lute solution populations maybe aﬀected by subtle diﬀerences in the ionization properties as a result of each CrgA–DNA complexes’ unique stoichiometry (49,50). It is interesting that in the 16mer arrangement, all four DNA- binding sites for each octamer are occupied suggesting some co-operativity of DNA binding. In fact, as indicated by modeling, once one DNA binds orthogonally to two CrgA octamer rings the DNA-binding sites at the remain- ing three corners of each octamer are aligned such that it is more favorable for three additional strands to bind (see below). A nano-electrospray mass spectrum of the DNA strand alone is shown as an insert to Figure 3C. The pre- dicted mass of the DNA is 38 788 Da and is in close agree- ment to the measured mass of 38 885 1 Da (series D) calculated using the +14 to +10 charge states. The three additional charge states series (A–C) are likely to be the result of nucleic acid truncations. In order to investigate further the eﬀect of DNA bind- ing on the CrgA protein ion mobility mass spectrometry (IM-MS) was used to compare the apo CrgA octamer with a holo CrgA octamer binding two DNA stands. IMMS separates ions not only by their m/z mass and charge but also by their drift time mobility through a nitrogen ﬁlled T-wave drift cell which is inﬂuenced by the species shape and size (51). Additionally, suﬃcient gas phase