Specific conformation and Ca(2+)-binding mode of yeast calmodulin: insight into evolutionary development

Area of Science:

• Molecular Biology
• Biochemistry
• Protein Structure and Function

Background:

• Vertebrate calmodulin has independent N- and C-terminal lobes connected by a flexible linker.
• Saccharomyces cerevisiae calmodulin (yCaM) displays unique inter-lobe interactions affecting calcium (Ca2+) binding.
• Understanding these interactions is crucial for elucidating yCaM's regulatory mechanisms.

Purpose of the Study:

• To investigate the functional consequences of inter-lobe interactions in yCaM.
• To determine the role of specific Ca2+ binding sites and C-terminal residues in yCaM's affinity and target recognition.
• To propose a model for Ca2+ and target binding in yCaM.

Main Methods:

• Site-directed mutagenesis to create yCaM mutants with impaired Ca2+ binding in one lobe.
• Analysis of C-terminal residue truncation mutants.
• Polyacrylamide gel electrophoresis to assess Ca2+-induced mobility shifts.
• Enzyme activity assays to measure Cmk1p activation.

Main Results:

• Ca2+-bound N-lobe associates with Ca2+-free C-lobe, restoring wild-type Ca2+ affinity.
• Truncation of the C-terminal three residues resulted in hyper Ca2+ affinity.
• These C-terminal residues are essential for Ca2+-induced structural changes and Cmk1p activation.

Conclusions:

• A stable intermediate formed by N-lobe Ca2+ binding and inter-lobe interaction ensures proper Ca2+ affinity.
• The C-terminal three residues prevent aberrant stabilization of this intermediate, maintaining normal Ca2+ affinity and target enzyme recognition.
• A model for Ca2+ and target binding in yCaM is proposed, with evolutionary implications discussed.