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Figure 8
Estimation of the arrangement of the two PDZ domains in full-length AaRseP. (a, b) Structural alignment of the Fab–PDZ models onto the 3D reconstruction. (a) The 605 ns snapshot in the MD trajectory of the NZ-1 Fab–PDZ-N (181-PA14-184) pair, whose conformation was most frequently sampled during the MD simulation, was fitted into the 3D reconstruction model of class 1 shown in Fig. 7[link](a) based on the position of the Fv region. (b) The 347 ns snapshot, which showed a similar conformation to that of the complex 2 structure, in the MD trajectory calculated from the complex 1 structure of the NZ-1 Fab–PDZ-C (235-PA14-236) pair was fitted into the 3D reconstruction model shown in Fig. 7[link](c) based on the position of the Fv region. (c) Structural model of size exclusion. The approximate arrangement of the two PDZ domains from AaRseP is shown together with the model of RseA from E. coli to help understand the size-exclusion mechanism. The crystal structure of the periplasmic domain of RseA in complex with RseB (PDB entry 3m4w) is drawn in the RseA model. RseB is shown as a blue ribbon model with a partial transparent surface. The RseA periplasmic domain is shown as magenta ribbon model, and the side chain of Val148 is highlighted with an orange sphere model. The atomic model of the PDZ tandem from AaRseP was built based on the structural alignments in (a) and (b). Firstly, the two 3D reconstruction models were aligned based on the AaRseP molecule composed of a sphere and two protrusions. Next, the position of the PA14-inserted PDZ-N and PDZ-C domains were aligned onto the respective 3D models as described in (a) and (b). Finally, the crystal structures of wild-type PDZ-N and PDZ-C domains were independently aligned onto the models of the PA14-inserted mutants. The aligned models of PDZ-­N and PDZ-C were merged to build the entire PDZ tandem model. The PA14-insertion sites and the carboxylate-binding loops within the ligand-binding grooves are highlighted with magenta and red sphere models, respectively. Glu158 and Asp162, shown as blue sphere models, were modeled to be close to the transmembrane region based on the structural alignment. The corresponding residues in EcRseP were estimated to be membrane-proximal residues based on protection from chemical modification. In both 3D reconstruction models the PDZ-C domain was separated from the TM domain by a gap, which might suppress the entry of the RseB-bound full-length RseA to the active center as the size-exclusion filter.

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ISSN: 2059-7983
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