Analysis of Ca2+/Mg2+ selectivity in α-lactalbumin and Ca2+-binding lysozyme reveals a distinct Mg2+-specific site in lysozyme

• Published in: Proteins, structure, function, and bioinformatics Vol. 78; no. 12; pp. 2609 - 2624
• Main Authors: Permyakov, Sergei E., Khokhlova, Tatyana I., Uversky, Vladimir N., Permyakov, Eugene A.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-09-2010
• Subjects: ligand binding; lysozyme; metal binding; metal selectivity; phase diagram; phase transition; state diagram; thermal stability; thermodynamics; α-lactalbumin
• ISSN: 0887-3585; 1097-0134
• DOI: 10.1002/prot.22776

The triggering of Ca2+ signaling pathways relies on Ca2+/Mg2+ specificity of proteins mediating these pathways. Two homologous milk Ca2+‐binding proteins, bovine α‐lactalbumin (bLA) and equine lysozyme (EQL), were analyzed using the simplest “four‐state” scheme of metal‐ and temperature‐induced structural changes in a protein. The association of Ca2+/Mg2+ by native proteins is entropy‐driven. Both proteins exhibit strong temperature dependences of apparent affinities to Ca2+ and Mg2+, due to low thermal stabilities of their apo‐forms and relatively high unfavorable enthalpies of Mg2+ association. The ratios of their apparent affinities to Ca2+ and Mg2+, being unusually high at low temperatures (5.3–6.5 orders of magnitude), reach the values inherent to classical EF‐hand motifs at physiological temperatures. The comparison of phase diagrams predicted within the model of competitive Ca2+ and Mg2+ binding with experimental data strongly suggests that the association of Ca2+ and Mg2+ ions with bLA is a competitive process, whereas the primary Mg2+ site of EQL is different from its Ca2+‐binding site. The later conclusion is corroborated by qualitatively different molar ellipticity changes in near‐UV region accompanying Mg2+ and Ca2+ association. The Ca2+/Mg2+ selectivity of Mg2+‐site of EQL is below an order of magnitude. EQL exhibits a distinct Mg2+‐specific site, probably arising as an adaptation to the extracellular environment. Proteins 2010. © 2010 Wiley‐Liss, Inc.