Histidine-containing phosphocarrier protein, HPr, was one of the early prot
ein tertiary structures determined by two-dimensional H-1-NMR. Tertiary str
uctures for HPrs from Escherichia coli, Bacillus subtilis, and Staphylococc
us aureus have been obtained by H-1 NMR and the overall folding pattern of
HPr is highly conserved, a beta alpha beta beta alpha beta alpha arrangemen
t of three alpha-helices overlaying a four-stranded beta-sheet. High-resolu
tion structures for HPrs from E. coli and B. subtilis have been obtained us
ing N-15- and C-13-labeled proteins. The first application of NMR to the un
derstanding of the structure and function of HPr was to describe the phosph
ohistidine isomer, N-delta 1-P-histidine in S. aureus phospho-HPr, and the
unusual pK(a)s of the His-15 side chain. The pK(a) values for the His-15 im
idazole from more recent studies are 5.4 for HPr and 7.8 for phospho-HPr fr
om E. coli, for example. A consensus description of the active site is prop
osed for HPr and phospho-HPr. In HPr, His-15 has a defined conformation and
N-caps helix A, and is thus affected by the helix dipole. His-15 undergoes
a small conformational change upon phosphorylation, a movement to allow th
e phosphoryl group to be positioned such that it forms hydrogen bonds with
the main chain amide nitrogens of residue 16 (not conserved) and Arg-17. In
teractions between residue I:! side chain (not conserved: asparagine, serin
e, and threonine) and His-15, and between the Arg-17 guanidinium group and
the phosphoryl group, are either weak or transitory.