SERCA mutant E309Q binds two Ca(2+) ions but adopts a catalytically incompetent conformation

Area of Science:

• Biochemistry and Molecular Biology
• Structural Biology
• Membrane Protein Function

Background:

• The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is a vital ion pump that couples ATP hydrolysis to Ca(2+) transport across membranes.
• Efficient Ca(2+) transport relies on intricate communication between Ca(2+) binding sites and the phosphorylation site over long distances within the protein.
• Understanding the mechano-structural basis of this intramolecular signaling is key to elucidating SERCA's function.

Purpose of the Study:

• To investigate the structural and functional consequences of the SERCA E309Q mutation on Ca(2+) binding and ATP hydrolysis.
• To elucidate the role of Glu(309) in Ca(2+) coordination and its impact on intramolecular communication within SERCA.

Main Methods:

• X-ray crystallography was employed to determine the structure of the SERCA E309Q mutant in the presence of Ca(2+) and an ATP analogue.
• Functional assays were performed to assess Ca(2+) binding affinity, cooperativity, and phosphorylation rates of the mutant.
• Analysis of structural changes, including the positioning of transmembrane segment M1 and the A-domain, was conducted.

Main Results:

• The crystal structure revealed that the E309Q mutant binds Ca(2+) at both site I and site II, forming a non-catalytic Ca2E1 state.
• Ca(2+) binding to both sites in E309Q occurs with micromolar affinity but lacks cooperativity.
• While the mutant can be phosphorylated by ATP, its maximal rate is significantly reduced, linked to impaired A-domain positioning due to altered charge and hydrogen bonding at site II.

Conclusions:

• The E309Q mutation disrupts the normal Ca(2+) binding cooperativity and impairs the mechano-structural rearrangements necessary for efficient SERCA function.
• Glu(309) is critical for proper Ca(2+) coordination at site II, influencing the positioning of key domains and segments involved in the catalytic cycle.
• This study provides insights into the long-range communication mechanisms within SERCA, highlighting the importance of specific residues for pump activity.