Title

Protein-ligand interactions: (I) The study of an ankyrin repeat-mediated protein-protein interaction. (II) Metal specificities of the Tro and Znu transporters of Treponema pallidum, the syphilis spirochete

Date of Completion

January 2008

Keywords

Biology, Microbiology|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Part(I) focuses on the frequently observed ankyrin repeat (ANK) motif, a structural scaffold evolved for mediating protein-protein interactions. Often, protein-protein interactions are driven by an energetic hot spot, which consists of a few interface residues contributing the majority of binding energy. To investigate ANK-mediated molecular recognition, the GA-binding protein (GABP) αβ complex served as a model, particularly focusing on the ANK domain of the GABPβ. Analysis of the interaction between the GABP subunits by isothermal titration calorimetry (ITC) measured a binding constant of 6.0×108 M−1. Of the 16 GABPβ interface residues chosen for mutagenesis, three spatially contiguous hydrophobic and aromatic residues were shown to form a binding free energy hot spot (ΔΔG>2.0 kcal/mol). Three non-hot spot residues are intermediate contributors (ΔΔG∼1.0 kcal/mol), which create a canopy-like structure over the hot spot residues to possibly occlude solvent and orientate the subunits. Part(II) centers on one of the tenets of microbial pathogenesis—the struggle for transition metals between a bacterial pathogen and its mammalian host. Previous studies demonstrated that Treponema pallidum (Tp) encodes a cluster-9 (C9) transporter (troABCD) whose solute-binding protein component (TroA) ligands Zn2+ and Mn2+ with essentially equal affinities. Bioinformatic analysis revealed that Tp encodes an additional C9 transporter (Tp0034-36) orthologous to Zn2+-uptake (Znu) systems in other bacteria; the binding protein component, ZnuA, contains a His-rich tract characteristic of C9 Zn2+-binding proteins. Metal analysis and metal-reconstitution studies demonstrated that ZnuA is a Zn2+-binding protein and confirmed that TroA binds Zn 2+, Mn2+, and Fe. Circular dichroism showed that ZnuA, but not TroA, undergoes conformational changes in the presence of Zn 2+. ITC analysis demonstrated that TroA binds Zn2+ and Mn2+ with affinities ∼100-fold greater than those previously reported; parallel studies revealed that ZnuA contains multiple Zn 2+-binding sites, two of which are presumed to be located within the binding pocket and His-rich tract. Expression analysis confirmed that both transporters are simultaneously expressed in Tp. Collectively, these findings indicate that Tp procures metals via the concerted utilization of its general metal (Tro) and Zn2+ (Znu) transporters. Sequestration of periplasmic Zn2+ by ZnuA may free up TroA binding capacity for Fe and Mn2+. ^