Human α-fetoprotein as a Zn2+-binding protein. Tight cation binding is not accompanied by global changes in protein structure and stability

Abstract

The binding of zinc to human alpha-fetoprotein (AFP) isolated from human umbilical cord serum was studied by fluorimetric Zn(2+)-titration. We found that the total number of strong binding sites for zinc on this protein was 5: AFP has one very strong (dissociation constant, K(d)<10(-8) M) and at least four lower affinity zinc binding sites (K(d)<10(-5) M). Fourier transform infrared (FTIR) analysis revealed that aspartate and histidine residues could be involved in the strong coordination of zinc. Intriguingly, binding of zinc to the protein does not induce structural changes that can be detected by circular dichroism, FTIR, intrinsic fluorescence or (1,1')-bi-(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) binding. Finally, scanning microcalorimetry measurements showed that stability of the protein is also unaffected by zinc binding in spite of the strength of the coordination. Such strong interactions without major structural consequences are highly unusual, and AFP may therefore be the first characterized representative of a new class of ligand-binding proteins.

Binding of Zn(II) to Tropomyosin Receptor Kinase A in Complex with Its Cognate Nerve Growth Factor: Insights from Molecular Simulation and in Vitro Essays

“…Potentiometric data indicate that the zinc ion coordination environment involves four imidazole nitrogen atoms. The stability constant value of such ZnL 2 complex species (log β = 6.30) is in line with those of zinc complexes with proteins or peptide fragments, 40,50 indicating an effective affinity of this TrkA domain for the metal. DFT calculations and NMR measurements confirm a four-imidazole tetrahedral arrangement with a 2Nδ2Nε coordination mode without the involvement of other peptide groups.…”

Therapeutic Strategies for Gaucher Disease: Miglustat (NB-DNJ) as a Pharmacological Chaperone for Glucocerebrosidase and the Different Thermostability of Velaglucerase Alfa and Imiglucerase

“…The discrepancy can be due to NB-DNJ binding to the unfolded thermally denatured state. A small or negligible stability increase, or even a further destabilization, in the presence of bound ligand has been reported for other proteins. − Other small molecules, like N -nonyldeoxynojirimycin (NN-DNJ) and isofagomine (IFG), show a stabilization effect on GlcCerase much larger than that observed for NB-DNJ. ,, In the case, of IFG, T m changes of ∼9 °C and ∼4 °C at pH 7.4 and 5.2, respectively, have been reported . In addition, high binding affinities of ≤12 nM and ≥50 nM at neutral and mildly acidic pH, respectively, have been reported for IFG binding to GlcCerase. , The higher affinity for IFG explains the larger stabilization effect on GlcCerase compared to NB-DNJ.…”

Mutant p53s and chromosome 19 microRNA cluster overexpression regulate cancer testis antigen expression and cellular transformation in hepatocellular carcinoma

“…Notably, many of these gene products require, or bind to zinc for their function [examples: AFP, BIRC5/survivin, OMA1 18 – 20 ], or their function is antagonized by zinc [Example: BAK1 21 ]. Of particular interest, elevated AFP expression is a clinical biomarker for poor prognosis of HCCs 22 and is a major zinc binding protein found in human plasma primarily expressed by liver 18 . AFP mRNA is significantly elevated in the p53TI cluster (Fig.…”