Structural studies of histidine-containing phosphocarrier protein from Enterococcus faecalis

Area of Science:

• Structural Biology
• Biochemistry
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• Histidine-containing phosphocarrier protein (HPr) is crucial in bacterial phosphotransferase systems.
• The active site loop structure of HPr is critical for its catalytic function but remains debated.
• Previous crystal structures suggested backbone strain in E. faecalis HPr's active site.

Purpose of the Study:

• To elucidate the secondary structure and local geometry of the active site of Enterococcus faecalis HPr.
• To investigate the presence of backbone torsion strain at position 16 in solution.
• To compare the active site geometry of E. faecalis HPr with other known HPr structures.

Main Methods:

• Complete sequential assignment of the 1H-NMR spectrum using multidimensional NMR techniques.
• Determination of coupling constants to calculate phi angles and assess backbone torsion strain.
• Comparative analysis of NMR and X-ray crystallographic structures of HPr from various organisms.

Main Results:

• E. faecalis HPr exhibits a stable and well-defined structure in aqueous solution, similar to other bacterial HPrs.
• No significant backbone torsion strain at position 16 was detected in the dominant solution conformation.
• The active-site loop demonstrates conformational flexibility, with phosphorylated HPr existing in two substates.

Conclusions:

• The crystal structure's observed backbone strain at position 16 is not present in the dominant solution state of E. faecalis HPr.
• The active-centre loop of HPr is conformationally adaptable in solution, particularly upon phosphorylation.
• NMR data provide detailed insights into the phosphate-binding site (His15) and its local environment.