Abstract
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.
Highlights
The mammalian branched-chain ␣-ketoacid dehydrogenase complex (BCKDC)2 is a member of the highly conserved ␣-ketoacid dehydrogenase complex family comprising pyruvate dehydrogenase complex (PDC), ␣-ketoglutarate dehydrogenase complex (KGDC), and the BCKDC with similar structure and function [1]
In patients with inherited maple syrup urine disease (MSUD), the activity of the BCKDC is deficient, which results in the accumulation of branched-chain ␣-ketoacids and amino acids [3]
E1 and E2 components are specific for the BCKDC, whereas the E3 component is common among the three ␣-ketoacid dehydrogenase complexes [1]
Summary
Sample Preparation—Three recombinant proteins containing various E2 domains were studied in this article, the di-domain protein (hbDD, residues 1–166), the lipoyl-bearing domain protein (hbLBD, residues 1– 84), and the subunit binding domain protein (hbSBD, residues 104 –152). To produce the hbSBD protein, a tobacco-etch virus (TEV) protease cleavage site was cloned in the linker region of hbDD between positions 103 and 104. The target protein was eluted with the same buffer containing 100 mM imidazole. To produce isotopically labeled samples for NMR structural determination, the cell was cultured in M9 minimal medium, supplemented with 15NH4Cl (1g/liter), 13C-labeled glucose (2g/liter), and 13C/ 5N-labeled Celtone base powder (0.5 g/liter, Spectra Stable Isotopes). The purified hbSBD contains an extra glycine in front of Glu104, which was left behind after the TEV cleavage. The NMR samples contained ϳ1–2 mM protein in 50 mM phosphate buffer at pH 7.5, 100 mM NaCl, 0.02% (w/v) NaN3, and 10% (v/v) D2O. 15N T1, 15N T2, and steady state heteronuclear 1H-15N NOE data were obtained at 295 K using standard pulse sequences [25]. Two sets of T1 and T2 experiments, and three pairs of the steady-state heteronuclear
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