Anion Exchange in Bacteria

Johns Hopkins University, Baltimore MD

Investigators

Abstract

This project concerns OxlT, an unusual transport protein found in the Gram-negative anaerobe, Oxalobacter formigenes. OxlT catalyzes the exchange of external divalent oxalate for internal monovalent formate. This electrogenic exchange, along with proton consumption during intracellular decarboxylation, constitutes a virtual proton pump that generates a proton-motive force allowing O. formigenes to extract energy from oxalate. Such proton-motive metabolic cycles are widely spread in microbiology, and to understand these events at a molecular level this project will focus on OxlT, the antiport protein at the center of this process in O. formigenes. Antibody against the OxlT N-terminus is available, the gene is cloned, and fully functional protein is purified after heterologous expression in Escherichia coli. Therefore, tools are available to study structure-function relationships for OxlT in two important areas: (1) A major experimental goal will be the determination of helix-helix proximity, using single-cysteine and double-cysteine variants as reagents in disufide trapping and cysteine-based cross-linking. In parallel, infrared spectroscopy will be used to ask whether there are changes in helix orientation accompanying the binding of substrate. (2) The more ambitious work will focus on 2-dimensional crystallography of OxlT. In this case, the goal is to capitalize on the remarkable stability of the OxlT-substrate complex, as well as the genetic tractability of OxlT, to enable formation of 2D crystals for analysis by electron microscopy. Preliminary trials show formation of fragile tubes of molecular dimension, and further effort over the next few years should be informative. The way in which small molecules move across cell membranes is central to many aspects of biology, including biotechnology and plant biology. As examples, consider the acquisition of sugar by the red blood cell, or the origin of drug resistance in microorganisms, or the adaptation to high salinity by plants. All these events are mediated by proteins within the Major Facilitator Superfamily, a vast collection of evolutionarily related transporters that includes OxlT as one of its better-characterized members. Study of OxlT may therefore help characterize the fundamental properties of this important group. This is especially true of the projected crystallographic work, since no member of the Major Facilitator Superfamily has been visualized at this level of resolution before. Understanding how such transporters are arranged within the membrane would greatly accelerate future studies that can serve the public interest by the design of novel inhibitors, activators, and substrates of these important proteins.