Model for the CylR2/DNA complex

Chris M. Pillar has shown that CylR2 binds specifically to the 25-bp IR1 inverted repeat that exists within the cytolysin promoter [108]. The NMR shift perturbation results of CylR2 with a 22-bp fragment of the IR1 repeat sequence (Figures4.11,4.12) further demonstrated that similar regions in dimeric CylR2 are interacting with DNA as compared to the N-terminal domain of 434 repressor. Furthermore, HN-RDCs for CylR2 when bound to DNA showed that there are no major changes in the backbone structure of CylR2 upon binding to DNA (Figure 4.9).

Based on the combined information from crystallographic (see 4.3.1) and solution NMR studies (see 4.3.3), a model of the CylR2/DNA complex was constructed. In agreement with the absence of major conformational changes in CylR2 upon binding to DNA, a starting structure for the complex was built by superposition of the struc-ture of the unbound CylR2 dimer onto the N-terminal domain of 434 repressor and replacement of the 434 operator by standard linear B-DNA. This initial model was refined using the protein-DNA docking program Monty [90, 91] guided by information obtained from the NMR chemical shift perturbation studies: Side chains of residues 10, 16-20, 24-32 and 34-45 were allowed to rotate freely in accordance with the chem-ical shift differences (Figure 4.12). In addition, energy bonuses were given during the simulation to complex structures where the backbone amides of Tyr39, Asn40, Gln44, Leu45 and Ala46 are in close contact to the DNA and when the NH₂ of Gln29 contacts the DNA reflecting the strong chemical shift changes upon addition of DNA.

Figure 4.14 shows the model for the CylR2/DNA complex obtained from this dock-ing approach. It agrees with established dimeric HTH/DNA interactions [112, 121];

binding occurs with dyad symmetry on two adjacent major grooves through their recognition helix. In a similar manner as observed for the 434 repressor/O_R1 com-plex structure, side chains of Ser27 and Gln29 form a van der Waals pocket to receive the methyl group of thymine 4’ (Figures4.12 and 4.14B). Moreover, complex models indicate a bidentate hydrogen bonding for the NH₂ of Arg28 to either G8, T7 or C6 and a hydrogen bond between the side chain of Gln29 and bp 4. The hydrogen bond of Gln29 to bp 4 is likely to be formed with O of Gln29 as the hydrogen bond acceptor, because the C^δ resonance showed a strong downfield shift of ∼0.9 ppm while N and H remained nearly unchanged (Figures4.12 and4.14B). For the side chain of Arg28 only C^α and C^β could be assigned, whereas both backbone and side chain resonances were missing for Ser27 in the complex. The disappearance or strong perturbation of these resonances upon binding to DNA supports their importance for complex for-mation. At the N-terminus of helix 2, Gln17 forms van der Waals contacts and a hydrogen bond with the phosphate of T9, in agreement with the disappearance of the side chain resonances of the Gln17 carbonyl group upon DNA-binding. Docking

re-A

G8

R28 R28

T7 G8 T7

C6 C6

Q29 Q29

S27 S27

T4' T4'

B

Figure 4.14: (A) Overall model of the CylR2/DNA complex structure. (B) Detailed stereo view indicating important protein-DNA interactions in the major groove. The DNA is shown with the sense strand in yellow and the antisense strand in cyan. Side chains of CylR2 and nucleobases interacting by van der Waals contacts and/or hydrogen bonds are indicated.

sults und chemical shift changes also suggest that bp 6 accepts a hydrogen bond from Asn32. NMR resonances from the carbonyl side chain of Asn32 were neither found for free nor complexed CylR2, but the C^β and C^α of Asn32 were shifted by 2.8 ppm and 1.1 ppm downfield, respectively (Figure 4.14B). Very important for stabilizing the CylR2/DNA complex is the (α3-α4) loop: for both CylR2 molecules a hydrogen bond is formed from the main chain amide group of Asn40 to the phosphate of C1 in more than 30 % of docking solutions, in agreement with a downfield shift of 1.1 ppm of its amide proton resonance.

The best-fit of DNA to the dimeric structure of CylR2 was obtained for a DNA

bending parameter of 0.4. For a more extended DNA, formation of protein/DNA in-teractions, which were expected on the basis of the NMR chemical shift perturbation studies, was not possible for both molecules of the CylR2 dimer simultaneously. When the DNA was more strongly bent, on the other hand, no docking solutions could be obtained that provided the necessary space to accommodate both CylR2 molecules in the orientation and position that were observed for the uncomplexed CylR2 structure.

This orientation and position were experimentally indicated by the very good agree-ment of the experiagree-mental HN-RDCs for DNA-bound CylR2 with the X-ray structure of free CylR2 (Figure4.9B). Similar to the 434 repressor/DNA complex, the DNA is relatively straight in the middle of the operator and bends towards the ends to enable interactions with the side chains of Arg28 and Gln29 (Figure4.14B). The helical axes of individual bp, when projected onto the mean plane of bending, lie on a circle with a radius of 65 ˚A (Figure 4.14A). A specific feature of the 434/DNA complex is a strong compression of the minor groove due to insertion of the side chains of Arg43 of both molecules of the dimeric 434 repressor: the two positively charged side chains allow a closer approach of the negatively charged phosphates [118]. In CylR2, on the other hand, Arg43 is replaced by a serine; hence, the positive side chains are absent within the minor groove and no compression of the minor groove is required to dock DNA onto CylR2.