Increased backbone flexibility in threonine45-phosphorylated hirudin upon pH change

Area of Science:

• Biochemistry
• Structural Biology
• Posttranslational Modifications

Background:

• Protein phosphorylation is a key regulatory mechanism controlling protein function through conformational changes.
• The precise molecular details of how phosphorylation-induced structural alterations occur, including the phosphate group's ionic state and range of influence, remain incompletely understood.

Purpose of the Study:

• To investigate the short-range structural effects of threonine phosphorylation on the protein hirudin.
• To elucidate the relationship between the phosphate ester's ionic state and observed conformational changes.

Main Methods:

• Structural characterization of phosphorylated and unphosphorylated hirudin using Circular Dichroism (CD) and 1H Nuclear Magnetic Resonance (NMR) spectroscopy.
• Hydrogen exchange monitored by MALDI-TOF mass spectrometry to assess structural dynamics.

Main Results:

• Threonine phosphorylation at Thr(45) induced significant structural changes and increased flexibility in a segment of at least seven amino acid residues.
• These conformational alterations were dependent on the phosphate ester group being in its dianionic state, observed within a ~10 Å radius around pThr(45).
• The transition from monoanionic to dianionic phosphate occurred between pH 5.2 and 8.5, indicating a pH-dependent conformational switch.

Conclusions:

• Phosphorylation-induced conformational changes in hirudin are critically dependent on the ionic state of the phosphate group.
• The findings reveal a general mechanism for phosphorylation-dependent conformational switching at physiological pH.
• This study provides molecular insights into the short-range effects of threonine phosphorylation on protein structure and dynamics.