A new topology of ACBP from Moniliophthora perniciosa.
Acyl-CoA binding protein (ACBP) is a housekeeping protein and is an essential protein in human cell lines and in Trypanosoma brucei. The ACBP of Moniliophthora perniciosa is composed of 104 amino acids and is possibly a non-classic isoform exclusively from Basidiomycetes. The M. perniciosa acbp gene was cloned, and the protein was expressed and purified. Acyl-CoA ester binding was analyzed by isoelectric focusing, native gel electrophoresis and isothermal titration calorimetry. Our results suggest an increasing affinity of ACBP for longer acyl-CoA esters, such as myristoyl-CoA to arachidoyl-CoA, and best fit modeling indicates two binding sites. ACBP undergoes a shift from a monomeric to a dimeric state, as shown by dynamic light scattering, fluorescence anisotropy and native gel electrophoresis in the absence and presence of the ligand. The protein's structure was determined at 1.6 A resolution and revealed a new topology for ACBP, containing five alpha-helices instead of four. alpha-helices 1, 2, 3 and 4 adopted a bundled arrangement that is unique from the previously determined four-helix folds of ACBP, while alpha-helices 1, 2, 4 and 5 formed a classical four-helix bundle. A MES molecule was found in the CoA binding site, suggesting that the CoA site could be a target for small compound screening.
Departamento de Ciências Biológicas, Laboratório de Proteômica, Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz, Ilhéus, BA, CEP 45662-900, Brazil.
Paulo S
PS
Monzani
Humberto M
HM
Pereira
Fernando A
FA
Melo
Flávio V
FV
Meirelles
Glaucius
G
Oliva
Júlio C M
JC
Cascardo
D000214:Acyl Coenzyme A / Q000378:metabolism; D000363:Agaricales / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D003460:Crystallization; D026261:Diazepam Binding Inhibitor / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D000214:Acyl Coenzyme A; D026261:Diazepam Binding Inhibitor; C042140:arachidoyl-coenzyme A; C040349:S-tetradecanoyl-coenzyme A