PHOSPHORYLATION-INDUCED TORSION-ANGLE STRAIN IN THE ACTIVE-CENTER OF HPR, DETECTED BY NMR AND RESTRAINED MOLECULAR-DYNAMICS REFINEMENT

The structure of the phosphorylated form of the histidine-containing p hosphocarrier protein HPr from Escherichia coli has been solved by NMR and compared with that of unphosphorylated HPr. The structural change s that occur upon phosphorylation of His 15, monitored by changes in N OE patterns, (3)J(NHH alpha)-coupling constants, and chemical shifts, are limited to the region around the phosphorylation site. The His 15 backbone torsion angles become strained upon phosphorylation. The rele ase of this strain during the phosphoryl-transfer to Enzyme II facilit ates the transport of carbohydrates across the membrane. From an X-ray study of Streptococcus faecalis HPr (Jia Z, Vandonselaar M, Quail JW, Delbaere LTJ, 1993, Nature 361:94-97), it was proposed that the obser ved torsion-angle strain at residue 16 in unphosphorylated S. faecalis HPr has a role to play in the protein's phosphocarrier function. The model predicts that this strain is released upon phosphorylation. Our observations on E. coli HPr in solution, which shows strain only after phosphorylation, and the fact that all other HPrs studied thus far in their unphosphorylated forms show no strain either, led us to investi gate the possibility that the crystal environment causes the strain in S. faecalis HPr. A l-ns molecular dynamics simulation of S. faecalis HPr, under conditions that mimic the crystal environment, confirms the observations from the X-ray study, including the torsion-angle strain at residue 16. The strain disappeared, however, when S. faecalis HPr was simulated in a water environment, resulting in an active site conf iguration virtually the same as that observed in all other unphosphory lated HPrs. This indicates that the torsion-angle strain at Ala 16 in S. faecalis HPr is a result of crystal contacts or conditions and does not play a role in the phosphorylation dephosphorylation cycle.