Structural and biochemical characterization of two heme binding sites on α<inf>1</inf>-microglobulin using site directed mutagenesis and molecular simulation
© 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...
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th-mahidol.432182019-03-14T15:04:17Z Structural and biochemical characterization of two heme binding sites on α<inf>1</inf>-microglobulin using site directed mutagenesis and molecular simulation Sigurbjörg Rutardottir Elena Karnaukhova Chanin Nantasenamat Napat Songtawee Virapong Prachayasittikul Mohsen Rajabi Lena Wester Rosenlöf Abdu I. Alayash Bo Åkerström Lunds Universitet Center for Biologics Evaluation and Research Mahidol University Biochemistry, Genetics and Molecular Biology Chemistry © 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. 2018-12-11T02:24:37Z 2019-03-14T08:04:17Z 2018-12-11T02:24:37Z 2019-03-14T08:04:17Z 2016-01-01 Article Biochimica et Biophysica Acta - Proteins and Proteomics. Vol.1864, No.1 (2016), 29-41 10.1016/j.bbapap.2015.10.002 18781454 15709639 2-s2.0-84946606197 https://repository.li.mahidol.ac.th/handle/123456789/43218 Mahidol University SCOPUS https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84946606197&origin=inward |
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Biochemistry, Genetics and Molecular Biology Chemistry 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 |
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© 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. |
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Lunds Universitet |
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Lunds Universitet Sigurbjörg Rutardottir Elena Karnaukhova Chanin Nantasenamat Napat Songtawee Virapong Prachayasittikul Mohsen Rajabi Lena Wester Rosenlöf Abdu I. Alayash Bo Åkerström |
format |
Article |
author |
Sigurbjörg Rutardottir Elena Karnaukhova Chanin Nantasenamat Napat Songtawee Virapong Prachayasittikul Mohsen Rajabi Lena Wester Rosenlöf Abdu I. Alayash Bo Åkerström |
author_sort |
Sigurbjörg Rutardottir |
title |
Structural and biochemical characterization of two heme binding sites on α<inf>1</inf>-microglobulin using site directed mutagenesis and molecular simulation |
title_short |
Structural and biochemical characterization of two heme binding sites on α<inf>1</inf>-microglobulin using site directed mutagenesis and molecular simulation |
title_full |
Structural and biochemical characterization of two heme binding sites on α<inf>1</inf>-microglobulin using site directed mutagenesis and molecular simulation |
title_fullStr |
Structural and biochemical characterization of two heme binding sites on α<inf>1</inf>-microglobulin using site directed mutagenesis and molecular simulation |
title_full_unstemmed |
Structural and biochemical characterization of two heme binding sites on α<inf>1</inf>-microglobulin using site directed mutagenesis and molecular simulation |
title_sort |
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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2018 |
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https://repository.li.mahidol.ac.th/handle/123456789/43218 |
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1763496522936221696 |