Structural and biochemical characterization of two heme binding sites on α1-microglobulin using site directed mutagenesis and molecular simulation.
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.


CONCLUSIONS
The model provides a structural basis for the functional activities of A1M: heme binding activity of A1M.
Division of Infection Medicine, Lund University, Lund, Sweden.
Sigurbjörg
S
Rutardottir
Laboratory of Biochemistry and Vascular Biology, Division of Hematology Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, MD, USA.
Elena
E
Karnaukhova
Center of Data Mining and Biomedical Informatics, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand; Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand.
Chanin
C
Nantasenamat
Center of Data Mining and Biomedical Informatics, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand.
Napat
N
Songtawee
Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand.
Virapong
V
Prachayasittikul
Laboratory of Biochemistry and Vascular Biology, Division of Hematology Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, MD, USA.
Mohsen
M
Rajabi
Division of Infection Medicine, Lund University, Lund, Sweden.
Lena Wester
LW
Rosenlöf
Laboratory of Biochemistry and Vascular Biology, Division of Hematology Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, MD, USA.
Abdu I
AI
Alayash
Division of Infection Medicine, Lund University, Lund, Sweden. Electronic address: sigurbjorg.rutardottir@med.lu.se.
Bo
B
Åkerström
D000510:Alpha-Globulins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002942:Circular Dichroism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance
D000510:Alpha-Globulins; D011994:Recombinant Proteins; C016001:alpha-1-microglobulin; D006418:Heme
Heme; Molecular simulation; Site-directed mutagenesis; α(1)-Microglobulin