Structural and biochemical basis for polyamine binding to the Tp0655 lipoprotein of Treponema pallidum: putative role for Tp0655 (TpPotD) as a polyamine receptor

Abstract

Tp0655 of Treponema pallidum, the causative agent of syphilis, is predicted to be a 40 kDa membrane lipoprotein. Previous sequence analysis of Tp0655 noted its homology to polyamine-binding proteins of the bacterial PotD family, which serve as periplasmic ligand-binding proteins of ATP-binding-cassette (ABC) transport systems. Here, the 1.8 A crystal structure of Tp0655 demonstrated structural homology to Escherichia coli PotD and PotF. The latter two proteins preferentially bind spermidine and putrescine, respectively. All of these proteins contain two domains that sandwich the ligand between them. The ligand-binding site of Tp0655 can be occupied by 2-(N-morpholino)ethanesulfanoic acid, a component of the crystallization medium. To discern the polyamine binding preferences of Tp0655, the protein was subjected to isothermal titration calorimetric experiments. The titrations established that Tp0655 binds polyamines avidly, with a marked preference for putrescine (Kd=10 nM) over spermidine (Kd=430 nM), but the related compounds cadaverine and spermine did not bind. Structural comparisons and structure-based sequence analyses provide insights into how polyamine-binding proteins recognize their ligands. In particular, these comparisons allow the derivation of rules that may be used to predict the function of other members of the PotD family. The sequential, structural, and functional homology of Tp0655 to PotD and PotF prompt the conclusion that the former likely is the polyamine-binding component of an ABC-type polyamine transport system in T. pallidum. We thus rename Tp0655 as TpPotD. The ramifications of TpPotD as a polyamine-binding protein to the parasitic strategy of T. pallidum are discussed.

Figure 1

The structure of rTp0655. (A) Ribbons-style representation, with the N-domain in blue, the C-domain in green, and the connection region in purple. A stick model of one of the bound molecules of MES is also shown; oxygen atoms are red, carbon atoms are gray, the nitrogen atom is blue, and the sulfur atom is yellow. (B) A close-up of the bound MES. The color scheme for atoms is the same as in part (A), except carbon atoms belonging to the N-domain are colored light blue, carbon atoms from the C-domain are green, and carbon atoms from the connector region are purple. Hydrogen bonds or ionic interactions are shown as dashed black lines.

Figure 2

Stereo view of the superposition of rTp0655 (blue), PotD (green), and PotF (red). The lines are smoothed traces through the C_α backbone of the respective proteins. The bound ligand found in each structure is shown as a stick model.

Figure 3

Structures and atom numbering convention for the polyamines studied in this report. The structures are aligned such that they demonstrate their respective homologies to spermidine.

Figure 4

Assessment of polyamine binding to apo-rTp0655 using ITC. The upper panel of each part is a time course of the power supplied to the reaction cell during the course of the experiment to keep the cell at a constant temperature. In the bottom parts, the circles represent the integrated heats from each injection. The solid line in these parts is the fitted binding isotherm assuming a 1:1 interaction between polyamines and apo-rTp0655. In the three titrations shown, the compounds injected into solutions of apo-rTp0655 were putrescine (A), spermidine (B), and 1,3-DAP (C).

Figure 5

Schematic of the polyamine-binding site in Tp0655, PotD, PotF with spermidine as a reference. Listed are all residues that interact with ligand in PotD and PotF as well as structurally equivalent (based on Cα positions) residues in the other proteins. Residues in lower-case letters are structurally equivalent but their side chains do not establish interactions with the ligand.

Figure 6

Sequence similarity among COG0687 PotD-like proteins. The y-axis is the sequence number in Tp0655 numbering. The horizontal bars represent the percentage of COG0687 members that have an amino acid similar to the predominant type at that position. Four types of amino acids were defined: hydrophobic, polar, acidic, and basic. The colored bars denote positions in the alignment that putatively contact bound polyamine at anchor positions N1 (red), N6 (green), or N10 (blue).