A citrate-binding site in calmodulin

Abstract

Calmodulin (CaM) is a major Ca2+ messenger which, upon Ca2+ activation, binds and activates a number of target enzymes involved in crucial cellular processes. The dependence on Ca2+ ion concentration suggests that CaM activation may be modulated by low-affinity Ca2+ chelators. The effect on CaM structure and function of citrate ion, a Ca2+ chelator commonly found in the cytosol and the mitochondria, was therefore investigated. A series of structural and biochemical methods, including tryptic mapping, immunological recognition by specific monoclonal antibodies, CIDNP-NMR, binding to specific ligands and association with radiolabeled citrate, showed that citrate induces conformational modifications in CaM which affect the shape and activity of the protein. These changes were shown to be associated with the C-terminal lobe of the molecule and involve actual binding of citrate to CaM. Analyzing X-ray structures of several citrate-binding proteins by computerized molecular graphics enabled us to identify a putative citrate-binding site (CBS) on the CaM molecule around residues Arg106-His107. Owing to the tight proximity of this site to the third Ca(2+)-binding loop of CaM, binding of citrate is presumably translated into changes in Ca2+ binding to site III (and indirectly to site IV). These changes apparently affect the structural and biochemical properties of the conformation-sensitive protein.

A mutually induced conformational fit underlies Ca2+-directed interactions between calmodulin and the proximal C terminus of KCNQ4 K+ channels

“…The crystallization buffer contained citrate, which also binds Ca 2ϩ but with much lower affinity than CaM (K a ϭ 10 3.5 M Ϫ1 ) (55). Citrate has also been shown to directly interact with the C-lobe of CaM, but we do not observe such an interaction in this structure (56). We also point out that the Ca 2ϩ /CaM:Q1AB structure was not obtained in a high-citrate buffer (48) but is very similar to this structure (RMSD ϭ 1.68 Å) and was also found to lack Ca 2ϩ ion density in A, representative schematic of a KCNQ protein subunit depicting the S1-S6 transmembrane helices, the intracellular N and C termini, and the A domain (teal) and B domain (gold) within the proximal half of the C terminus, referred to as the RD.…”

Injectable Self-Setting Ternary Calcium-Based Bone Cement Promotes Bone Repair

“…Owing to the rapid degradation of the bone cement in the early stages, large amounts of Ca 2+ and citric acid were released, which contributed to early bone regeneration. Studies have found that citric acid can mediate growth factors involved in angiogenesis and promote bone regeneration. , Many animal models have demonstrated that the incorporation of citric acid into biomaterials can promote bone formation; however, the role of extracellular citric acid in bone differentiation remains elusive. ,− In addition, citric acid acts as a Ca 2+ chelator and is involved in many cellular processes with Ca 2+ by regulating calmodulin, a major Ca 2+ messenger . However, the synergistic effect of citric acid and Ca 2+ on bone repair requires further investigation.…”

“… 22 , 41 − 43 In addition, citric acid acts as a Ca 2+ chelator and is involved in many cellular processes with Ca 2+ by regulating calmodulin, a major Ca 2+ messenger. 44 However, the synergistic effect of citric acid and Ca 2+ on bone repair requires further investigation.…”

Stability of buffer-free freeze-dried formulations: A feasibility study of a monoclonal antibody at high protein concentrations

“…79 generally limited to 1-2 mL. High protein concentrations 80 ()100 mg/mL) are needed to ensure sufficient therapeutic effect 81 in that small volume [5,6]. 82 There is consequently a certain need for understanding 83 development of highly concentrated freeze-dried formulations 84 (HC-FDF).…”