Structural basis for a hand-like site in the calcium sensor CatchER with fast kinetics

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Calcium ions are crucial intracellular signaling molecules.
• Green fluorescent protein (GFP) variants are used as biological sensors.
• Engineered GFP, CatchER, detects endoplasmic reticulum calcium with a fast off-rate.

Purpose of the Study:

• To determine the high-resolution crystal structures of CatchER.
• To investigate the metal ion binding site and stoichiometry of CatchER.
• To understand the structural basis for CatchER's fast kinetic properties and optical characteristics.

Main Methods:

• X-ray crystallography at 1.78-1.20 Å resolution.
• Analysis of CatchER in apo form and complexed with calcium or gadolinium.
• Structural comparison with wild-type GFP and enhanced GFP.

Main Results:

• CatchER exhibits 1:1 binding stoichiometry in solution.
• Two distinct metal ion binding positions were observed within the CatchER binding site.
• Structural differences in Thr203 and Glu222 side chains correlate with chromophore conformations and optical properties.
• Calcium binding may alter the conformational equilibrium of Glu222, affecting optical properties.

Conclusions:

• The unique structure of CatchER's binding site explains its fast kinetic properties.
• Calcium binding influences CatchER's conformation and optical characteristics.
• CatchER serves as a valuable tool for studying calcium signaling in the endoplasmic reticulum.