Mutagenesis Studies of Thyroxine Binding to Human Serum Albumin Define an Important Structural Characteristic of Subdomain 2A

Petersen, Charles E.; Ha, Chung-Eun; Harohalli, Krishna; Park, David; Bhagavan, N.V.

doi:10.1021/bi970225v

Cited by 46 publications

(33 citation statements)

References 11 publications

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“…Purified recombinant wild-type HSA (Recombumin) was obtained from Delta Biotechnology (Nottingham, U.K.). HSA mutants were expressed in Pichia pastoris and purified as described (6). Before crystallization in the absence of fatty acid, the protein was defatted and purified by gel filtration using established protocols (14,25,26).…”

Section: Methodsmentioning

confidence: 99%

“…Quenching of the fluorescent emission of W214 was used to determine K d value for T 4 binding to recombinant HSA essentially as described (6,30). In experiments to assay the effect of myristate on T 4 binding to HSA, 30 M myristate was used with 1 M HSA to ensure that all myristate sites on HSA were occupied by the ligand.…”

Section: Methodsmentioning

confidence: 99%

“…It is a monomeric protein containing three homologous domains (I-III), each divided into two subdomains (A and B). The number and locations of binding sites for T 4 are not well defined (5,6), although a consensus of most recent studies (6)(7)(8)(9)(10)(11) indicates that there are likely to be high-affinity T 4 -binding sites in subdomains IIA and IIIA. These subdomains contain the two primary drug-binding sites on the protein (12,13) and also bind fatty acids (refs.…”

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confidence: 99%

“…Individuals with FDH have normal serum levels of free T 4 , but their total serum T 4 is significantly elevated, a characteristic that can lead to misdiagnosis of hyperthyroidism and inappropriate treatment (22)(23)(24). Mutational analysis of R218 and neighboring residues led to a hypothesis that the enhanced binding affinity of the HSA mutants is due to relief of steric forces (6,10), but this has not yet been tested by direct observation.…”

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confidence: 99%

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Structural basis of albumin–thyroxine interactions and familial dysalbuminemic hyperthyroxinemia

Petitpas

Petersen²,

Ha³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Human serum albumin (HSA) is the major protein component of blood plasma and serves as a transporter for thyroxine and other hydrophobic compounds such as fatty acids and bilirubin. We report here a structural characterization of HSA-thyroxine interactions. Using crystallographic analyses we have identified four binding sites for thyroxine on HSA distributed in subdomains IIA, IIIA, and IIIB. Mutation of residue R218 within subdomain IIA greatly enhances the affinity for thyroxine and causes the elevated serum thyroxine levels associated with familial dysalbuminemic hyperthyroxinemia (FDH). Structural analysis of two FDH mutants of HSA (R218H and R218P) shows that this effect arises because substitution of R218, which contacts the hormone bound in subdomain IIA, produces localized conformational changes to relax steric restrictions on thyroxine binding at this site. We have also found that, although fatty acid binding competes with thyroxine at all four sites, it induces conformational changes that create a fifth hormone-binding site in the cleft between domains I and III, at least 9 Å from R218. These structural observations are consistent with binding data showing that HSA retains a high-affinity site for thyroxine in the presence of excess fatty acid that is insensitive to FDH mutations.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Structural basis of albumin–thyroxine interactions and familial dysalbuminemic hyperthyroxinemia

Petitpas

Petersen²,

Ha³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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238

204

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“…The techniques used in this study to mutate the HSA coding region and to synthesize and purify the mutated protein have been previously described (32)(33)(34)(35).…”

Section: Synthesis and Purification Of Recombinant Hsamentioning

confidence: 99%

A Dynamic Model for Bilirubin Binding to Human Serum Albumin

Petersen¹,

Ha²,

Harohalli³

et al. 2000

Journal of Biological Chemistry

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Site-directed mutagenesis of human serum albumin was used to study the role of various amino acid residues in bilirubin binding. A comparison of thermodynamic, proteolytic, and x-ray crystallographic data from previous studies allowed a small number of amino acid residues in subdomain 2A to be selected as targets for substitution. The following recombinant human serum albumin species were synthesized in the yeast species Pichia pastoris: K195M, K199M, F211V, W214L, R218M, R222M, H242V, R257M, and wild type human serum albumin. The affinity of bilirubin was measured by two independent methods and found to be similar for all human serum albumin species. Examination of the absorption and circular dichroism spectra of bilirubin bound to its high affinity site revealed dramatic differences between the conformations of bilirubin bound to the above human serum albumin species. The absorption and circular dichroism spectra of bilirubin bound to the above human serum albumin species in aqueous solutions saturated with chloroform were also examined. The effect of certain amino acid substitutions on the conformation of bound bilirubin was altered by the addition of chloroform. In total, the present study suggests a dynamic, unusually flexible high affinity binding site for bilirubin on human serum albumin.The binding of bilirubin, a toxic metabolite of heme, to human serum albumin (HSA) 1 has been studied extensively for many years. Early medical interest in the bilirubin-HSA interaction arose when it became clear to physicians that prolonged high blood concentrations of bilirubin, which often occur in premature infants, could result in bilirubin encephalopathy (1-5). In this condition, significant amounts of bilirubin, which is toxic to all tissues, partitions from the blood to neuronal tissue causing irreversible brain damage. The prolonged high blood concentrations of bilirubin in premature infants results from underdevelopment of the liver, the organ responsible for conversion of bilirubin to a soluble form and its excretion into the bile. HSA binds bilirubin (K d ϭ 10 Ϫ7 -10 Ϫ8 M) at a high affinity site and acts as a buffer preventing the transfer of bilirubin from blood to the tissues, thus playing a critical role in impairing the development of bilirubin encephalopathy. In total, other studies on the pathology of bilirubin encephalopathy in premature infants have highlighted the importance of HSA as a bilirubin transport molecule in normal neonates (who experience a transient hyperbilirubinemia after birth) and in normal adults. These findings provided the motivation for many years of study on the bilirubin-HSA binding mechanism, making it one of the most studied of the HSA-ligand interactions (6 -11).Although some studies have suggested that lower affinity binding components (K d ϭ 10 Ϫ6 -10 Ϫ3 M) contribute to HSAbilirubin binding, most studies have primarily attempted to locate the high affinity binding site and to identify amino acid residues involved in the high affinity binding process. A number of studies th...

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Competition of cytarabine and aspirin in binding to serum albumin in multidrug therapy

et al. 2006

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The aim of this study was to describe a competition between cytarabine (araC) and aspirin (ASA) in binding with bovine serum albumin (BSA). High-affinity binding sites for both drugs were determined using a spectrofluorimetric method. Cytarabine as well as aspirin binds in the IIA hydrophobic subdomain of the transporting protein. Binding constants for araC-BSA and ASA-BSA were calculated by the Scatchard method. Analysis of ultraviolet (UV) difference spectra showed that araC, which has a higher affinity to BSA in comparison to ASA [Ka(araC) > Ka(ASA)] displaces ASA in high-affinity binding sites. The competition between drugs in low-affinity binding sites was investigated using (1)H nuclear magnetic resonance (NMR) and 13C-NMR spectra. We concluded that in the low-affinity binding sites cytarabine decreases the affinity of albumin toward aspirin. However, the interaction between araC and BSA becomes more difficult in the presence of aspirin.

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Mutagenesis Studies of Thyroxine Binding to Human Serum Albumin Define an Important Structural Characteristic of Subdomain 2A

Cited by 46 publications

References 11 publications

Structural basis of albumin–thyroxine interactions and familial dysalbuminemic hyperthyroxinemia

Structural basis of albumin–thyroxine interactions and familial dysalbuminemic hyperthyroxinemia

A Dynamic Model for Bilirubin Binding to Human Serum Albumin

Competition of cytarabine and aspirin in binding to serum albumin in multidrug therapy

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