Factors Governing the Substitution of La³⁺ for Ca²⁺ and Mg²⁺ in Metalloproteins:  A DFT/CDM Study

Abstract

Trivalent lanthanide cations are extensively being used in biochemical experiments to probe various dication-binding sites in proteins; however, the factors governing the binding specificity of lanthanide cations for these binding sites remain unclear. Hence, we have performed systematic studies to evaluate the interactions between La3+ and model Ca2+ - and Mg2+ -binding sites using density functional theory combined with continuum dielectric methods. The calculations reveal the key factors and corresponding physical bases favoring the substitution of trivalent lanthanides for divalent Ca2+ and Mg2+ in holoproteins. Replacing Ca2+ or Mg2+ with La3+ is facilitated by (1) minimizing the solvent exposure and the flexibility of the metal-binding cavity, (2) freeing both carboxylate oxygen atoms of Asp/Glu side chains in the metal-binding site so that they could bind bidentately to La3+, (3) maximizing the number of metal-bound carboxylate groups in buried sites, but minimizing the number of metal-bound carboxylate groups in solvent-exposed sites, and (4) including an Asn/Gln side chain for sites lined with four Asp/Glu side chains. In proteins bound to both Mg2+ and Ca2+, La3+ would prefer to replace Ca2+, as compared to Mg2+. A second Mg2+-binding site with a net positive charge would hamper the Mg2+ --> La3+ exchange, as compared to the respective mononuclear site, although the La3+ substitution of the first native metal is more favorable than the second one. The findings of this work are in accord with available experimental data.

Identification and Dissection of Ca2+-binding Sites in the Extracellular Domain of Ca2+-sensing Receptor

“…The geometrically predicted locations with 2-4 negatively charged ligands, strong negative surface potential, and functionally necessary residues were more likely to bind Ca 2ϩ . The model structures of CD2 variants with grafted potential Ca 2ϩ -binding sites of the CaSR were generated by SWISSMODEL (33,34 52 and Gly 53 of CD2 in the plasmid pGEX-2T (denoted as CD2-CaSR1 and CD2-CaSR2, respectively) by PCR using an established protocol (63). Two mutants, CD2-CaSR1-E228A/E229A and CD2-CaSR2-E378A/E379A, were also made by site-directed mutagenesis.…”

Engineering aluminum binding affinity in an isolated EF-hand from troponin C: A computational site-directed mutagenesis study

“…The ''EEEEE'' mutant appears that it will bind the 3+ metals tightly, although maybe not with much specificity. This site does satisfy recommendations made by Dudev et al [21] for reengineering sites to bind another lanthanide, La 3+ , by minimizing the solvent exposure and flexibility of the metal-binding cavity, freeing both carboxylate oxygen atoms of aspartate/glutamate side chains so they can bind bidentately in the metal-ion binding site, and maximizing the number of metal-bound carboxylate groups in buried sites. However, we do not retain an asparagine/glutamine side chain in our proposed Al 3+ -binding peptide since Al 3+ is much smaller and has a higher charge density than the lanthanides.…”

“…Soft sulfur ligands are uncommon. In systematic studies in which La 3+ replaced Ca 2+ and Mg 2+ in metalion binding sites, it was found that the La 3+ cation was favored and facilitated by minimizing the solvent exposure and flexibility of the metal-binding cavity, freeing both carboxylate oxygen atoms of aspartate/glutamate side chains so they could bind bidentately in the metal-ion binding site, maximizing the number of metal-bound carboxylate groups in buried sites, and including an asparagine/glutamine side chain in sites with four aspartate/ glutamate side chains [21]. Holding the ligand constant, the coordination is most strongly affected by the metal's size rather than its charge, especially if the ligand is neutral.…”

A theoretical study of the principles regulating the specificity for Al(III) against Mg(II) in protein cavities

“…Charge transfer is highest in these conformation, which favours the attachment of trivalent cations [3]. In the most solvent exposed areas, the preferred conformation for exchange is 3M because of the polarization effect.…”

“…Providing accurate metal-site structures in proteins is crucial for several studies regarding the design of drugs [1,2], metal toxicity, specificity of proteins or development of new techniques [3]. Lately, the importance of the combination of experimental and computational works has been addressed [4,5], specially when studying the structure and thermodynamics of the metal binding to a protein.…”