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J. Biol. Chem., Vol. 282, Issue 8, 5944-5958, February 23, 2007

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Crystal Structure of the Tp34 (TP0971) Lipoprotein of Treponema pallidum

IMPLICATIONS OF ITS METAL-BOUND STATE AND AFFINITY FOR HUMAN LACTOFERRIN^*

Ranjit K. Deka¹, Chad A. Brautigam¹, Farol L. Tomson, Sarah B. Lumpkins^¶, Diana R. Tomchick, Mischa Machius, and Michael V. Norgard²

From the Departments of Microbiology and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390 and the ^¶University of Oklahoma, Norman, Oklahoma 73019

The Tp34 (TP0971) membrane lipoprotein of Treponema pallidum, an obligate human pathogen and the agent of syphilis, was previously reported to have lactoferrin binding properties. Given the non-cultivatable nature of T. pallidum, a structure-to-function approach was pursued to clarify further potential relationships between the Tp34 structural and biochemical properties and its propensity to bind human lactoferrin. The crystal structure of a nonacylated, recombinant form of Tp34 (rTp34), solved to a resolution of 1.9Å_, revealed two metaloccupied binding sites within a dimer; the identity of the ion most likely was zinc. Residues from both of the monomers contributed to the interfacial metal-binding sites; a novel feature was that the -sulfur of methionine coordinated the zinc ion. Analytical ultracentrifugation showed that, in solution, rTp34 formed a metal-stabilized dimer and that rTp34 bound human lactoferrin with a stoichiometry of 2:1. Isothermal titration calorimetry further revealed that rTp34 bound human lactoferrin at high (submicromolar) affinity. Finally, membrane topology studies revealed that native Tp34 is not located on the outer surface (outer membrane) of T. pallidum but, rather, is periplasmic. How propensity of Tp34 to bind zinc and the iron-sequestering lactoferrin may relate overall to the biology of T. pallidum infection in humans is discussed.

Received for publication, November 1, 2006 , and in revised form, December 7, 2006.

The atomic coordinates and structure factors (code 2O6C, 2O6D, 2O6E, and 2O6F) 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 NIAID, National Institutes of Health Grant AI-56305 and by Welch Foundation Grant I-0940. Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by the United States Dept. of Energy, Office of Energy Research under Contract W-31-109-ENG-38. 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 Fig. 1.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Microbiology, UT Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75390. Tel.: 214-648-5900; Fax: 214-648-5905; E-mail: michael.norgard{at}utsouthwestern.edu.

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