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J. Biol. Chem., Vol. 281, Issue 7, 4364-4370, February 17, 2006

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Molecular Structure of a 9-MDa Icosahedral Pyruvate Dehydrogenase Subcomplex Containing the E2 and E3 Enzymes Using Cryoelectron Microscopy^*

Jacqueline L. S. Milne¹, Xiongwu Wu, Mario J. Borgnia, Jeffrey S. Lengyel^¶, Bernard R. Brooks, Dan Shi, Richard N. Perham^¶, and Sriram Subramaniam

From the Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, the Laboratory of Computational Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, and the ^¶Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom

The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate. We have previously reported the molecular architecture of an 11-MDa subcomplex comprising the 60-mer icosahedral dihydrolipoyl acetyltransferase (E2) decorated with 60 copies of the heterotetrameric (₂₂) 153-kDa pyruvate decarboxylase (E1) from Bacillus stearothermophilus (Milne, J. L. S., Shi, D., Rosenthal, P. B., Sunshine, J. S., Domingo, G. J., Wu, X., Brooks, B. R., Perham, R. N., Henderson, R., and Subramaniam, S. (2002) EMBO J. 21, 5587-5598). An annular gap of 90 Å separates the acetyltransferase catalytic domains of the E2 from an outer shell formed of E1 tetramers. Using cryoelectron microscopy, we present here a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100-kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of 75 Å between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 coordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations of E3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain.

Received for publication, April 21, 2005 , and in revised form, November 15, 2005.

^* This research was supported by grants from the Center for Cancer Research at the NCI, National Institutes of Health (to J. L. S. M. and S. S.). 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.

¹ To whom correspondence should be addressed: Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bldg. 50 Rm. 4306, 50 South Dr., Bethesda, MD, 20892. Tel.: 301-594-2063; Fax: 301-480-3834; E-mail: jmilne{at}nih.gov.

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