Publication: Structural and biochemical characterization of two heme binding sites on α<inf>1</inf>-microglobulin using site directed mutagenesis and molecular simulation
Issued Date
2016-01-01
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18781454
15709639
15709639
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2-s2.0-84946606197
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Mahidol University
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SCOPUS
Bibliographic Citation
Biochimica et Biophysica Acta - Proteins and Proteomics. Vol.1864, No.1 (2016), 29-41
Suggested Citation
Sigurbjörg Rutardottir, Elena Karnaukhova, Chanin Nantasenamat, Napat Songtawee, Virapong Prachayasittikul, Mohsen Rajabi, Lena Wester Rosenlöf, Abdu I. Alayash, Bo Åkerström Structural and biochemical characterization of two heme binding sites on α<inf>1</inf>-microglobulin using site directed mutagenesis and molecular simulation. Biochimica et Biophysica Acta - Proteins and Proteomics. Vol.1864, No.1 (2016), 29-41. doi:10.1016/j.bbapap.2015.10.002 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/43218
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Title
Structural and biochemical characterization of two heme binding sites on α<inf>1</inf>-microglobulin using site directed mutagenesis and molecular simulation
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Abstract
© 2015 Elsevier B.V. Background α1-Microglobulin (A1M) is a reductase and radical scavenger involved in physiological protection against oxidative damage. These functions were previously shown to be dependent upon cysteinyl-, C34, and lysyl side-chains, K(92, 118,130). A1M binds heme and the crystal structure suggests that C34 and H123 participate in a heme binding site. We have investigated the involvement of these five residues in the interactions with heme. Methods Four A1M-variants were expressed: with cysteine to serine substitution in position 34, lysine to threonine substitutions in positions (92, 118, 130), histidine to serine substitution in position 123 and a wt without mutations. Heme binding was investigated by tryptophan fluorescence quenching, UV-Vis spectrophotometry, circular dichroism, SPR, electrophoretic migration shift, gel filtration, catalase-like activity and molecular simulation. Results All A1M-variants bound to heme. Mutations in C34, H123 or K(92, 118, 130) resulted in significant absorbance changes, CD spectral changes, and catalase-like activity, suggesting involvement of these side-groups in coordination of the heme-iron. Molecular simulation support a model with two heme-binding sites in A1M involving the mutated residues. Binding of the first heme induces allosteric stabilization of the structure predisposing for a better fit of the second heme. Conclusions The results suggest that one heme-binding site is located in the lipocalin pocket and a second binding site between loops 1 and 4. Reactions with the hemes involve the side-groups of C34, K(92, 118, 130) and H123. General significance The model provides a structural basis for the functional activities of A1M: heme binding activity of A1M.