Using structural information to change the phosphotransfer specificity of a two-component chemotaxis signalling...

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Two-component signal transduction pathways, involving histidine protein kinases (HPKs) and response regulators (RRs), are vital for bacterial environmental responses.
• Specificity in phosphotransfer reactions is critical to prevent crosstalk in bacteria with numerous pathways (e.g., over 150).
• The bacterial chemotaxis pathway is a well-characterized example of two-component signaling.

Purpose of the Study:

• To determine the crystal structure of the CheA(3) histidine-containing phosphotransfer domain complexed with its cognate response regulator, CheY(6).
• To identify the structural determinants responsible for the specificity of the phosphotransfer interaction between CheA(3) and CheY(6).
• To investigate the potential for re-engineering phosphotransfer signaling specificity.

Main Methods:

• X-ray crystallography was used to obtain the 1.40 Å crystal structure of the CheA(3)-CheY(6) complex.
• Surface plasmon resonance (SPR) was employed to assess protein binding and interaction specificity.
• Site-directed mutagenesis was performed to investigate the role of specific amino acid residues in binding and phosphotransfer.

Main Results:

• The crystal structure revealed a critical methionine finger on CheY(6) that interacts with a hydrophobic pocket in CheA(3).
• Mutagenesis of this methionine and adjacent residues abolished CheA(3)-P binding and phosphotransfer to CheY(6).
• Introducing the identified residues into noncognate CheYs altered their specificity, enabling interaction and phosphotransfer with CheA(3)-P.

Conclusions:

• Specific amino acid residues, particularly a methionine finger, are key determinants for CheA-CheY interaction specificity.
• The study successfully re-engineered phosphotransfer signaling specificity by modifying these residues.
• These findings provide crucial insights into how specificity is achieved in abundant two-component signal transduction pathways.