Structure of the subunit binding domain and dynamics of the di-domain region from the core of human branched chain alpha-ketoacid dehydrogenase complex.
The homo-24-meric dihydrolipoyl transacylase (E2) scaffold of the human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) contains the lipoyl-bearing domain (hbLBD), the subunit-binding domain (hbSBD) and the inner core domain that are linked to carry out E2 functions in substrate channeling and recognition. In this study, we employed NMR techniques to determine the structure of hbSBD and dynamics of several truncated constructs from the E2 component of the human BCKDC, including hbLBD (residues 1-84), hbSBD (residues 111-149), and a di-domain (hbDD) (residues 1-166) comprising hbLBD, hbSBD and the interdomain linker. The solution structure of hbSBD consists of two nearly parallel helices separated by a long loop, similar to the structures of the SBD isolated from other species, but it lacks the short 3(10) helix. The NMR results show that the structures of hbLBD and hbSBD in isolated forms are not altered by the presence of the interdomain linker in hbDD. The linker region is not entirely exposed to solvent, where amide resonances associated with approximately 50% of the residues are observable. However, the tethering of these two domains in hbDD significantly retards the overall rotational correlation times of hbLBD and hbSBD, changing from 5.54 ns and 5.73 ns in isolated forms to 8.37 ns and 8.85 ns in the linked hbDD, respectively. We conclude that the presence of the interdomain linker restricts the motional freedom of the hbSBD more significantly than hbLBD, and that the linker region likely exists as a soft rod rather than a flexible string in solution.
Chang
Chi-Fon
CF
Genomics Research Center and Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115, Republic of China.
Chou
Hui-Ting
HT
Lin
Yi-Jan
YJ
Lee
Shin-Jye
SJ
Chuang
Jacinta L
JL
Chuang
David T
DT
Huang
Tai-Huang
TH
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) / Q000737:chemistry*; D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits
D021122:Protein Subunits; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)