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

Abstract

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 structureto-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 1.9 Å , 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.

Fitting two- and three-site binding models to isothermal titration calorimetric data

“…As a result, the method has been extended to study more complex interactions, featuring two [23–26] and three [20,27–29] distinct binding sites. While the cited efforts examined strongly featured isotherms or properly constrained analyses, other, more ambitious studies must contend with some countervailing facts.…”

Characterization of a Dipartite Iron Uptake System from Uropathogenic Escherichia coli Strain F11

“…In the copper-loaded FetP crystal structure, copper was coordinated by residues His 44 , Met 90 , His 97 , and His 127 * from the other respective subunit as well as forming a weaker interaction with Glu 46 . These five aminoacyl residues are conserved in the 100 closest homologs of FetP in a BLAST search (data not shown), including the three previously characterized homologs, ChpA from the marine magnetotactic Vibrio strain MV-1 (10), P19 from the pathogenic Campylobacter jejuni (11), and Tp34 (TP0971) from Treponema pallidum (12).…”

“…Recombinant Tp34 from T. pallidum, soaked in 10 mM Zn(II), bound a Zn(II) atom in a degenerated octahedral geometry that is coordinated by 3 His, 1 Glu, and 1 Met residue, corresponding to the copper-coordinating residues from FetP (12). The authors discuss that this geometry may represent a novel zinc-binding site or that zinc could have bound adventitiously to a site that would bind a different metal in vivo.…”

“…Because metal binding was required for dimerization of Tp34, a FetP homolog from T. pallidum (12), dimerization of FetP was studied using analytical ultracentrifugation (Table S2). The experimentally determined mass of FetP was 35,283 Da, which was independent of variations in sodium chloride concentration or the presence or absence of transition metals, indicating a stable dimer.…”

“…A general relationship between FetP homologues and iron metabolism has been reported previously. For example, ChpA from the marine magnetotactic Vibrio strain MV-1 is required for magnetosome production (10); P19 from pathogenic Campylobacter jejuni is required for growth under iron limitation (11); Tp34 (TP0971) from Treponema pallidum is a lactoferrinbinding protein (12). All three homologs can also form homodimers and bind either copper or zinc.…”