Mutations in a Specific Human Serum Albumin Thyroxine Binding Site Define the Structural Basis of Familial Dysalbuminemic Hyperthyroxinemia

Chung-Eun Ha,Nadhipuram V Bhagavan,David M Jameson,Charles E Petersen

doi:10.1074/jbc.271.32.19110

Chung-Eun Ha, Nadhipuram V Bhagavan + Show 2 more

Open Access

https://doi.org/10.1074/jbc.271.32.19110

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Abstract

The familial dysalbuminemic hyperthyroxinemia (FDH) phenotype results from a natural human serum albumin (HSA) mutant with histidine instead of arginine at amino acid position 218. This mutation results in an enhanced affinity for thyroxine. Site-directed mutagenesis and a yeast protein expression system were used to synthesize wild type HSA and FDH HSA as well as several other HSA mutants. Studies on the binding of thyroxine to these HSA species using equilibrium dialysis and quenching of tryptophan 214 fluorescence suggest that the FDH mutation affects a single thyroxine binding site located in the 2A subdomain of HSA. Site-directed mutagenesis of HSA and thyroxine analogs were used to obtain information about the mechanism of thyroxine binding to both wild type and FDH HSA. These studies suggest that the guanidino group of arginine at amino acid position 218 in wild type HSA is involved in an unfavorable binding interaction with the amino group of thyroxine, whereas histidine at amino acid position 218 in FDH HSA is involved in a favorable binding interaction with thyroxine. Neither arginine at amino acid position 222 nor tryptophan at amino acid position 214 appears to favorably influence the binding of thyroxine to wild type HSA.

Highlights

As previously shown [23, 30] the emission spectrum of HSA overlaps significantly with the absorption spectrum of thyroxine, suggesting that quenching of tryptophan 214 occurs via a nonradiative energy transfer process
The original hypothesis of this study, i.e. that the enhanced affinity of R218H (FDH) HSA for thyroxine results from changes in the structure of a specific high affinity binding site located in subdomain 2A, appears to be correct
A comparison of thyroxine binding data obtained by the fluorescence quenching and equilibrium dialysis techniques shows that introduction of specific structural changes into subdomain 2A affected a single high affinity thyroxine binding site

Summary

Introduction

As previously shown [23, 30] the emission spectrum of HSA overlaps significantly with the absorption spectrum of thyroxine, suggesting that quenching of tryptophan 214 occurs via a nonradiative energy transfer process. Given the strong (sixth power) distance dependence of Forster type energy transfer [38], transfer can only occur when thyroxine is bound to HSA, i.e. free thyroxine does not contribute to the quenching. A high concentration of HSA is titrated with ligand to approximate, as closely as possible, stochiometric binding. In this case a plot of fluorescence versus the ligand/HSA ratio shows an initial monotonic decrease in fluorescence, which plateaus at a minimum value reflecting the fraction of fluorescence not quenched by bound ligand. A lower concentration of HSA is titrated with ligand, and the fraction of HSA molecules with bound ligand can be calculated knowing the quenching efficiency determined from the stochiometric binding isotherm described above. Thyroxine and thyroxine analog concentrations were determined by the dry weight method

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