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J. Biol. Chem., Vol. 281, Issue 42, 31544-31552, October 20, 2006

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Structures of R- and T-state Escherichia coli Aspartokinase III

MECHANISMS OF THE ALLOSTERIC TRANSITION AND INHIBITION BY LYSINE^*

Masayo Kotaka¹, Jingshan Ren¹, Michael Lockyer, Alastair R. Hawkins^¶, and David K. Stammers²

From the Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, Arrow Therapeutics Ltd., Trinity Street, Borough, London SE1 1DA, and ^¶Institute of Cell and Molecular Biosciences, Catherine Cookson Building, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom

Aspartokinase III (AKIII) from Escherichia coli catalyzes an initial commitment step of the aspartate pathway, giving biosynthesis of certain amino acids including lysine. We report crystal structures of AKIII in the inactive T-state with bound feedback allosteric inhibitor lysine and in the R-state with aspartate and ADP. The structures reveal an unusual configuration for the regulatory ACT domains, in which ACT2 is inserted into ACT1 rather than the expected tandem repeat. Comparison of R- and T-state AKIII indicates that binding of lysine to the regulatory ACT1 domain in R-state AKIII instigates a series of changes that release a "latch", the 15-K loop, from the catalytic domain, which in turn undergoes large rotational rearrangements, promoting tetramer formation and completion of the transition to the T-state. Lysine-induced allosteric transition in AKIII involves both destabilizing the R-state and stabilizing the T-state tetramer. Rearrangement of the catalytic domain blocks the ATP-binding site, which is therefore the structural basis for allosteric inhibition of AKIII by lysine.

Received for publication, June 20, 2006 , and in revised form, July 26, 2006.

The atomic coordinates and structure factors (code 2J0W and 2J0X) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* Financial support was received from Arrow Therapeutics and the United Kingdom Medical Research Council. 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 Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 44-1865-287-565; Fax: 44-1865-287-547; E-mail: daves{at}strubi.ox.ac.uk.

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