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J. Biol. Chem., Vol. 281, Issue 11, 7413-7420, March 17, 2006

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High Resolution Crystal Structures and Molecular Dynamics Studies Reveal Substrate Binding in the Porin Omp32^*

Ulrich Zachariae, Thomas Klühspies, Sharmila De, Harald Engelhardt¹, and Kornelius Zeth^¶

From the Departments of Molecular Structural Biology and ^¶Membrane Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany and the Physics Department, Dinabandhu Mahavidyalaya, 743235 Bongaon, West Bengal, India

The porin Omp32 is the major outer membrane protein of the bacterium Delftia acidovorans. The crystal structures of the strongly anion-selective porin alone and in complex with the substrate malate were solved at 1.5 and 1.45 Å resolution, respectively, and revealed a malate-binding motif adjacent to the channel constriction zone. Binding is mediated by interaction with a cluster of two arginine residues and two threonines. This binding site is specific for Omp32 and reflects the physiological adaptation of the organism to organic acids. Structural studies are combined with a 7-ns unbiased molecular dynamics simulation of the trimeric channel in a model membrane. Molecular dynamics trajectories show how malate ions are efficiently captured from the surrounding bulk solution by the electrostatic potential of the channel, translocated to the binding site region, and immobilized in the constriction zone. In accordance with these results, conductance measurements with Omp32 inserted in planar lipid membranes revealed binding of malate. The anion-selective channel Omp32 is the first reported example of a porin with a 16-stranded -barrel and proven substrate specificity. This finding suggests a new view on the correlation of porin structure with substrate binding in specific channels.

Received for publication, October 6, 2005 , and in revised form, January 19, 2006.

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

^* This work was supported by a grant from the DFG (En 144/3). 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 data.

¹ To whom correspondence should be addressed. Tel.: 49-89-8578-2650; Fax: 49-89-8578-2641; E-mail: engelhar{at}biochem.mpg.de.

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