The objective of this proposal is to understand structural details in the bilayer that relate to transport properties of membrane protein channels. Specifically, we wish to understand the structure/function relationships for the mammalian water channels AQP1 and AQP4 and the pore-forming anthrax toxin protective antigen by high-resolution electron crystallography.
Our specific aims are listed below.
AIM I. Examine at the structural level the inhibition of water transport in AQP1 by mercurial compounds. We will apply electron diffraction to quantitate in 3-dimensions structural changes in AQP1 upon mercurial binding. This will allow us to understand AQP1 function based on its modulation by the pharmacological inhibitor.
AIM II. Examine the structural/functional roles of residues involved in the selective water transport in AQP1. We will examine the functional roles of polar and charged amino acids in the putative membrane-spanning region of AQP1 using a combined functional and structural approach. Amino-acid substitutions that lead to altered function without structural perturbation will allow us to identify residues critical for water transport.
AIM III. Structural studies on the AQP4 water channel. The AQP4 water channel expressed primarily in brain elicits highest osmotic water permeability. We will crystallize AQP4 in the lipid bilayer and use it as a model to understand and identify at the structural level factors responsible for diversity in solute transport mediated by aquaporins.
AIM I V. Structural studies on anthrax toxin protective antigen. The structure of the soluble and membrane-integrated complex of protective antigen (PA63) heptamers with and without bound lethal factor (LF) will be studied using single particle image analysis and conical-tilt reconstructions. This will allow us to understand the binding of LF and test the proposed porin-like model for the membrane-embedded domain of PA63. The selective expression of AQP1 and homologous water channels believed to be involved in fluid absorption and/or secretion makes them an important pharmacological target. The pore-forming anthrax toxin has been shown to have a potential for delivery of macromolecules across the bilayer. Thus structural studies on these systems will potentially have impact on structure-based drug design.
| Ren, Gang; Quispe, Joel; Leppla, Stephen H et al. (2004) Large-scale structural changes accompany binding of lethal factor to anthrax protective antigen: a cryo-electron microscopic study. Structure 12:2059-66 |
