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Crystal structure of transhydrogenase domain III at 1.2 Å resolution

Nature Structural Biology volume 6, pages 1126–1131 (1999)Cite this article

Abstract

The nicotinamide nucleotide transhydrogenases (TH) of mitochondria and bacteria are membrane-intercalated proton pumps that transduce substrate binding energy and protonmotive force via protein conformational changes. In mitochondria, TH utilizes protonmotive force to promote direct hydride ion transfer from NADH to NADP, which are bound at the distinct extramembranous domains I and III, respectively. Domain II is the membrane-intercalated domain and contains the enzyme's proton channel. This paper describes the crystal structure of the NADP(H) binding domain III of bovine TH at 1.2 Å resolution. The structure reveals that NADP is bound in a manner inverted from that previously observed for nucleotide binding folds. The non-classical binding mode exposes the NADP(H) nicotinamide ring for direct contact with NAD(H) in domain I, in accord with biochemical data. The surface of domain III surrounding the exposed nicotinamide is comprised of conserved residues presumed to form the interface with domain I during hydride ion transfer. Further, an adjacent region contains a number of acidic residues, forming a surface with negative electrostatic potential which may interact with extramembranous loops of domain II. Together, the distinctive surface features allow mechanistic considerations regarding the NADP(H)-promoted conformation changes that are involved in the interactions of domain III with domains I and II for hydride ion transfer and proton translocation.

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Figure 1: a,Stereo figure of the electron density for NADP in domain III of bovine TH at 1.2 Å resolution.
Figure 2: Alignment of sequences of TH domains homologous with domain III of bovine TH.
Figure 3: Stereo view showing the details of the NADP binding site.
Figure 4: a, The distribution of conserved (yellow) and semi-conserved (green) residues on the surface of domain III.

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Acknowledgements

The authors thank N. Kresge for preparing several of the figures and the staff of the Stanford Synchrotron Radiation Laboratory for their generous assistance. The authors are indebted to U. Genick and E. Getzoff for their generous assistance in the use of their single crystal microspectrophotometer. This work was supported by a United States Public Health Service Grant to Y. Hatefi.

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Authors and Affiliations

  1. Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, 92037, California, USA

    G. Sridhar Prasad, Vandana Sridhar & C. David Stout

  2. Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, 92037, California, USA

    Mutsuo Yamaguchi & Youssef Hatefi

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  1. G. Sridhar Prasad
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  2. Vandana Sridhar
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Correspondence to C. David Stout.

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Prasad, G., Sridhar, V., Yamaguchi, M. et al. Crystal structure of transhydrogenase domain III at 1.2 Å resolution . Nat Struct Mol Biol 6, 1126–1131 (1999). https://doi.org/10.1038/70067

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