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J. Biol. Chem., Vol. 281, Issue 38, 28345-28353, September 22, 2006

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Structure of the Subunit Binding Domain and Dynamics of the Di-domain Region from the Core of Human Branched Chain -Ketoacid Dehydrogenase Complex^*

Chi-Fon Chang, Hui-Ting Chou, Yi-Jan Lin, Shin-Jye Lee, Jacinta L. Chuang^¶, David T. Chuang^¶, and Tai-huang Huang^||1

From the Genomics Research Center and Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115, Republic of China, the ^¶Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, and the ^||Department of Physics, National Taiwan Normal University, Taipei, Taiwan 106, Republic of China

The homo-24-meric dihydrolipoyl transacylase (E2) scaffold of the human branched-chain -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 3₁₀ 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 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.

Received for publication, May 24, 2006 , and in revised form, July 14, 2006.

The atomic coordinates and structure factors (code 1ZWV) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The NMR assignment data have been deposited in the Biological Magnetic Resonance Data Bank, BMRB, accession number 7057.

^* This work was supported in part by the Academia Sinica and by Grants from the National Science Council of the Republic of China (NSC-94-2113-M-001-012) (to T.-H. H.), from the National Institutes of Health (DK-26578) (to D. T. C.), and from the Welch Foundation (I-1286) (to D. T. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.

¹ To whom correspondence should be addressed: Inst. of Biomedical Sciences, Academia Sinica, Nankang, Taipei 115, Taiwan, The Republic of China. Tel.: 886-2-2652-3036; Fax: 886-2-2788-7641; E-mail: bmthh{at}ibms.sinica.edu.tw.

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