Mutagenesis studies of thyroxine binding to human serum albumin define an important structural characteristic of subdomain 2A.
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. In our earlier study, site-directed mutagenesis and a yeast protein expression system were used to synthesize FDH HSA and several other HSA mutants. Measurement of the binding of these HSA mutants to thyroxine and several thyroxine analogs using equilibrium dialysis and quenching of tryptophan 214 fluorescence allowed us to propose a preliminary model of thyroxine binding to the 2A subdomain of wild type and FDH HSA. In this study, we have produced several other HSA mutants. By comparing the binding affinity of these mutants for thyroxine and tetraiodothyroacetic acid to the binding affinity of other mutants, we were able to suggest a new model for thyroxine binding to the 2A subdomain of HSA. We found that the substitution of arginine at position 218 with alanine increased the binding affinity for thyroxine by 2 orders of magnitude relative to the binding affinity of wild type HSA for thyroxine. A more accurate understanding of the mechanism of thyroxine binding to HSA has allowed us to define an important structural characteristic of subdomain 2A, one of the two principal binding sites on HSA for small hydrophobic ligands.
Petersen
C E
CE
Department of Biochemistry and Biophysics, University of Hawaii, Honolulu 96822, USA.
Ha
C E
CE
Harohalli
K
K
Park
D
D
Bhagavan
N V
NV
D001120:Arginine / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006981:Hyperthyroxinemia / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012709:Serum Albumin / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013974:Thyroxine / Q000031:analogs & derivatives / Q000378:metabolism*
D012709:Serum Albumin; D006639:Histidine; D001120:Arginine; C011126:tetraiodothyroacetic acid; D013974:Thyroxine