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

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.