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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/43218
Title: Structural and biochemical characterization of two heme binding sites on α<inf>1</inf>-microglobulin using site directed mutagenesis and molecular simulation
Authors: 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
Keywords: Biochemistry, Genetics and Molecular Biology;Chemistry
Issue Date: 1-Jan-2016
Citation: Biochimica et Biophysica Acta - Proteins and Proteomics. Vol.1864, No.1 (2016), 29-41
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.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84946606197&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/43218
ISSN: 18781454
15709639
Appears in Collections:Scopus 2016-2017

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