 | Acta Crystallographica Section D Acta Crystallographica Section D BIOLOGICAL CRYSTALLOGRAPHY |
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![[Figure 6]](kw5082fig6mag.jpg)
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Figure 6
Nucleotide-state sensing in ClpB NBD2. (a) Sensor 1 motif: structure of ClpB NBD2 R621Q in complex with AMPPCP showing alternative conformations for the P-loop residue Thr597 and the sensor 1 residue Asn709, which are involved in a hydrogen-bonding network around the nucleotide. Depending on the nucleotide state, Thr597 can form a hydrogen bond to either the γ-phosphate of ATP (1) or, in the absence of the γ-phosphate, to sensor 1 residue Asn709 (2). Currently, it is unclear how such a switch is further communicated through the molecule. However, it has been proposed previously that the sensor 1 motif is involved in hydrolysis, coordinating the attacking water together with the Walker B glutamate Glu668 (Gai et al., 2004  ). This might only be possible when Asn709 is not captured in the interaction with Thr597; thus, in the ATP-bound state. Distances are given in Å. (b) Sensor 2 motif: ClpB NBD2 in the ADP and ATP state, respectively, showing different positions of the sensor 2 residue Arg806, which is not influenced by crystal contacts in the structures shown in this figure. In the ADP state (left panel; ClpB NBD2 R621Q + ADP), Arg806 interacts with the β-phosphate of the nucleotide (d = 3.0 Å), which is also the case in the MANT-dADP-bound structure (d = 2.8 Å) shown in Fig. 3(b) and in another recently published ADP-bound NBD2 structure (d = 2.8 Å; Biter et al., 2012). This direct interaction between Arg806 and the nucleotide in the ADP state contributes to the nucleotide-binding energy, which is in agreement with the observation that the R806A mutation causes impaired ADP binding (Table 2). In the ATP state (right panel; ClpB NBD2 R621Q + AMPPCP + GdmCl), Arg806 must change its position because it would otherwise clash with the γ-phosphate. Indeed, it bends away from the phosphates (d = 3.8 Å), thereby no longer contributing to the nucleotide binding energy significantly, which is supported by our nucleotide-binding kinetics measurements showing that the R806A mutation has no significant effect on ATP binding (Table 2). The clearly different behaviour of Arg806 in the presence of ADP compared with ATP demonstrates that this residue functions as a bona fide nucleotide sensor. |
![[Figure 6]](kw5082fig6.jpg)
| BIOLOGICAL CRYSTALLOGRAPHY |
ISSN: 1399-0047
