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J. Biol. Chem., Vol. 280, Issue 26, 24839-24848, July 1, 2005

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The ATPase Activity of BfpD Is Greatly Enhanced by Zinc and Allosteric Interactions with Other Bfp Proteins^*

Lynette J. Crowther, Atsushi Yamagata, Lisa Craig, John A. Tainer, and Michael S. Donnenberg¶

From the Division of Infectious Diseases, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201 and the Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037

Type IV pilus biogenesis, protein secretion, DNA transfer, and filamentous phage morphogenesis systems are thought to possess similar architectures and mechanisms. These multiprotein complexes include members of the PulE superfamily of putative NTPases that have extensive sequence similarity and probably similar functions as the energizers of macromolecular transport. We purified the PulE homologue BfpD of the enteropathogenic Escherichia coli bundle-forming pilus (BFP) biogenesis machine and characterized its ATPase activity, providing new insights into its mode of action. Numerous techniques revealed that BfpD forms hexamers in the presence of nucleotide. Hexameric BfpD displayed weak ATPase activity. We previously demonstrated that the N termini of membrane proteins BfpC and BfpE recruit BfpD to the cytoplasmic membrane. Here, we identified two BfpD-binding sites, BfpE_39-76 and BfpE_77-114, in the N terminus of BfpE using a yeast two-hybrid system. Isothermal titration calorimetry and protease sensitivity assays showed that hexameric BfpD-ATPS binds to BfpE_77-114, whereas hexameric BfpD-ADP binds to BfpE_39-76. Interestingly, the N terminus of BfpC and BfpE_77-114 together increased the ATPase activity of hexameric BfpD over 1200-fold to a V_max of 75.3 µmol of P_i min^-1 mg^-1, which exceeds by over 1200-fold the activity of other PulE family members. This augmented activity occurred only in the presence of Zn²⁺. We conclude that allosteric interactions between BfpD and BfpC and BfpE dramatically stimulate its ATPase activity. The differential nucleotide-dependent binding of hexameric BfpD to BfpE_39-76 and BfpE_77-114 suggests a model for the mechanism by which BfpD transduces mechanical energy to the biogenesis machine.

Received for publication, January 7, 2005 , and in revised form, April 18, 2005.

^* This work was supported by National Institutes of Health Grants R01 AI-37606 (to M. S. D.) and AI22160 (to J. A. T.), and a fellowship from The Canadian Institutes of Health Research (to L. 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.

^¶ To whom correspondence should be addressed: Division of Infectious Diseases, Dept. of Medicine, University of Maryland School of Medicine, HSF II, 20 Penn St., Baltimore, MD 21201. Tel.: 410-706-7560; Fax: 410-706-8700; E-mail: mdonnenb{at}umaryland.edu.

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