The histidine phosphocarrier protein, HPr, binds to the highly thermostable regulator of sigma D protein, Rsd, and its isolated helical fragments.

The phosphotransferase system (PTS) controls the preferential use of sugars in bacteria and it is also involved in other processes, such as chemotaxis. It is formed by a protein cascade in which the first two proteins are general (namely, EI and HPr) and the others are sugar-specific permeases. The Rsd protein binds specifically to the RNA polymerase (RNAP) σ⁷⁰ factor. We first characterized the conformational stability of Escherichia coli Rsd. And second, we delineated the binding regions of Streptomyces coelicolor, HPr^sc, and E. coli Rsd, by using fragments derived from each protein. To that end, we used several biophysical probes, namely, fluorescence, CD, NMR, ITC and BLI. Rsd had a free energy of unfolding of 15 kcal mol^-1 at 25 °C, and a thermal denaturation midpoint of 103 °C at pH 6.5. The affinity between Rsd and HPr^sc was 2 μM. Interestingly enough, the isolated helical-peptides, comprising the third (RsdH3) and fourth (RsdH4) Rsd helices, also interacted with HPr^sc in a specific manner, and with affinities similar to that of the whole Rsd. Moreover, the isolated peptide of HPr^sc, HPr^9-30, comprising the active site, His15, also was bound to intact Rsd with similar affinity. Therefore, binding between Rsd and HPr^sc was modulated by the two helices H3 and H4 of Rsd, and the regions around the active site of HPr^sc. This implies that specific fragments of Rsd and HPr^sc can be used to interfere with other protein-protein interactions (PPIs) of each other protein.

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