STRUCTURAL STUDIES OF HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN FROM ENTEROCOCCUS-FAECALIS

Based on the complete sequential assignment of the H-1-NMR spectrum by multidimensional NMR techniques the secondary structure and the local geometry of the active site of histidine-containing phosphocarrier pr otein (HPr) from Enterococcus faecalis were elucidated. We present a c omparative analysis of the active site in the seven known structures o f HPr from different organisms determined by NMR or X-ray crystallogra phy. In catalysis, HPr is phosphorylated at the ring N delta 1 of His1 5. No general agreement exists in literature regarding the structure o f the active-centre loop. In the crystal structure of HPr from E. faec alis, a torsion strain of the backbone at position 16 was observed, wh ich was assumed to be important to the catalytic mechanism. Coupling c onstants were determined in order to calculate phi angles to establish whether there are strained torsion angles in HPr from E. faecalis in the solution state. The evaluation of data obtained indicate a stable and well-defined structure of HPr from E. faecalis, with an overall fo ld similar to that found in HPr from other bacteria. We find that in t he active-site region there are relatively large variations in local g eometry between the evaluated structures. In HPr from E. faecalis, a p articularly detailed view of the phosphate-binding His15 and residues in close spatial proximity was obtained by determination of coupling c onstants obtained from the double-quantum-filtered COSY spectrum. Our data indicate that in aqueous solution, in the dominant conformational state there is no torsion strain of the backbone at position 16, as o bserved in the crystal state. The maximum population of a strained con formation in solution can be estimated to be smaller than 23%. The ana lysis of the data suggests that the active-centre loop is able to adop t different conformations in solution. A similar observation was made for HPr from E. faecalis phosphorylated at its regulatory site (Ser46) . P-31-NMR shows that phosphorylated HPr exists in two conformational substates with nearly equal populations.