GST pulldowns with CheD’s two binding partners (CheC and McpAc) show reduced binding by GST-CheD-F102E.

<p>Lanes 1 (GST-CheD only control), 2 (+CheC), 3 (+McpAc) and 4 (+McpCc) contain GST-CheD bound to glutathione beads. Lanes 5 (GST-CheD-F102E only control), 6 (+CheC), 7 (+McpAc) and 8 (+McpCc) contains GST-CheD-F102E bound to glutathione beads. Multiple bands of the Mcps correspond to receptor deamidation.</p

Sequence alignment of various CheDs shows that residues M101 and F102 (boxed) are conserved in species that also contain CheC.

<p>Abbreviations for species that contain CheC: Bsub, <i>Bacillus subtilis</i>; Bhal, <i>Bacillus halodurans</i>; Cace, <i>Clostridium acetobutylicum</i>; Cthe, <i>Clostridium thermocellum</i>; Dhaf, Desulfitobacterium hafniense; Oihe, Oceanobacillus iheyensis; Phor, <i>Pyrococcus horikoshii</i>; Tmar, <i>Thermotoga maritima</i>; Tten, <i>Thermoanaerobacter tengcongensis</i>. Abbreviations for species that do not contain CheC: Atum, <i>Agrobacterium tumefaciens</i>; Bbur, Borrelia burgdorferi; Ccre, Caulobacter crescentus; Neur, <i>Nitrosomonas europaea</i>; Paer, <i>Pseudomonas aeruginosa</i>; Rsph, <i>Rhodobacter sphaeroides</i>; Smel, <i>Sinorhizobium meliloti</i>; Xcam, <i>Xanthomonas campestris</i>.</p

A CheC/CheD structural model based on the <i>T. maritima</i> crystal structure (PDB ID 2F9Z) shows residues at the binding interface [<b>21</b>].

<p>Residues M101 and F102 on CheD are highlighted in cyan, and residue D149 on CheC is highlighted in yellow.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Western blots of <i>in vitro</i> deamidation assays of McpAc show CheD-mediated receptor modifications.

<p>Deamidation of the receptor fragment results in higher-migrating bands on a polyacrylamide gel. CheC is added, as indicated, and inhibits deamidation. Lanes 2 and 3 show McpAc incubated with wild-type GST-CheD, and lanes 4–11 show McpAc incubated with various GST-CheD mutants.</p

Kinetic parameters of CheC-CheD binding from SPR experiments.

<p>Kinetic parameters of CheC-CheD binding from SPR experiments.</p