Structure of the NPr:EIN^Ntr Complex: Mechanism for Specificity in Paralogous Phosphotransferase Systems.

Paralogous enzymes arise from gene duplication events that confer a novel function, although it is unclear how cross-reaction between the original and duplicate protein interaction network is minimized. We investigated HPr:EI^sugar and NPr:EI^Ntr, the initial complexes of paralogous phosphorylation cascades involved in sugar import and nitrogen regulation in bacteria, respectively. Although the HPr:EI^sugar interaction has been well characterized, involving multiple complexes and transient interactions, the exact nature of the NPr:EI^Ntr complex was unknown. We set out to identify the key features of the interaction by performing binding assays and elucidating the structure of NPr in complex with the phosphorylation domain of EI^Ntr (EIN^Ntr), using a hybrid approach involving X-ray, homology, and sparse nuclear magnetic resonance. We found that the overall fold and active-site structure of the two complexes are conserved in order to maintain productive phosphorylation, however, the interface surface potential differs between the two complexes, which prevents cross-reaction.

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