Similarities and differences between yeast and vertebrate calmodulin: An examination of the calcium-binding and structural properties of calmodulin from the yeast Saccharomyces cerevisiae

• Published in: Biochemistry (Easton) 1993-04, Vol.32 (13), p.3261-3270
• Main Authors: Starovasnik, Melissa A, Davis, Trisha N, Klevit, Rachel E
• Format: Article
• Language: English
• Subjects: Amides - chemistry; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Base Sequence; Binding and carrier proteins; Binding Sites; Biological and medical sciences; CALCIO; CALCIUM; Calcium - metabolism; Calmodulin - chemistry; Calmodulin - metabolism; CATION; CATIONES; DNA Mutational Analysis; Fundamental and applied biological sciences. Psychology; Fungal Proteins - chemistry; Fungal Proteins - metabolism; Hot Temperature; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Protein Binding; Protein Conformation; Protein Denaturation; PROTEINAS; PROTEINE; Proteins; Recombinant Proteins; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - chemistry; Sequence Alignment; Sequence Deletion
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00064a008

The Ca2+-binding and structural properties of calmodulin (CaM) from the yeast Saccharomyces cerevisiae (yCaM) were analyzed by flow dialysis and NMR spectroscopy. Full-length yCaM and two truncated versions of yCaM were expressed in Escherichia coli and purified. yTR1 (residues 1-76) and yTR2 (residues 75-147) are similar to the vertebrate CaM fragments TR1 and TR2, which are generated by limited proteolysis with trypsin. As was found for the fragments of vertebrate CaM, the yCaM fragments retain native conformation and are useful for examining structure and metal-binding properties by NMR. Evidence for a short beta-sheet in each domain, as well as characteristic NOES to aromatic residues, suggests that yCaM folds similarly to vertebrate CaM. Furthermore, although the previously considered "invariant" glycine at position 6 is replaced by a histidine in site II of yCaM,the far downfield chemical shift of His-61's amide proton suggests that this site adopts a conformation similar to that found in other EF-hand sites. Macroscopic Ca2+-binding constants were determined for yCaM by flow dialysis, revealing three high-affinity sites (dissociation constants were 5.2, 3.3, and 2.3 micromolar in the presence of 1 mM MgCl2 and 100 mM KCl). Positive cooperativity was observed among all sites. Ca2+ binding was also monitored indirectly by one-dimensional NMR. Titrations of the fragment molecules reveal that two binding sites reside in the N-terminal domain (sites I and II) and one in the C-terminal domain (site III). All three sites exhibit slow-exchange behavior in the intact protein, but site III exhibits fast-exchange behavior in the isolated C-terminal domain fragment (yTR2). Thus, an interaction between the two domains of intact yCaM affects the behavior of site III. These results with yCaM differ from those of vertebrate CaM in terms of Ca2+-binding stoichiometry, affinity of sites I and II, relative affinity of sites in the N- and C-terminal domains, and the exchange behaviors observed