Structure/function analysis of the ubiquitous SLC26A/SulP transporter/channel family

Lead Research Organisation: University of Dundee
Department Name: Molecular Medicine

Abstract

Membrane transporters play crucial roles in fundamental cellular functions and normal physiological processes such as nervous influx conduction, nutrient uptake, removal of waste or oxygen transport during respiration. Therefore studies on solute carrier proteins have had great impact on our understanding of human disease and the design of effective drugs. About 30% of current clinically-marked drugs have as their targets membrane transporters or channels. The solute carrier from the SLC26A family of proteins was defined 15 years ago and it has been shown that these proteins belong to the ubiquitous SulP/SLC26A family of anion channels/transporters conserved from bacteria to man. The human genome encodes at least 10 proteins of this family, amongst which four have been identified as disease genes. Moreover the bacterial members of this protein family may play a role in bacterial virulence. Many important questions remain unanswered about this poorly-characterised protein family, in particular regarding their structure and mode of action. Unfortunately studies on these proteins in humans are technically very challenging. Therefore, the philosophy of my project, in agreement with the famous citation of the French biologist Jacques Monod ?What is true for Escherichia coli is also true for the elephant? is to use simple and well studied bacterial model organisms, such as Escherichia coli, which possess the SulP/SLC26A proteins. In these model organisms, powerful genetic and biochemical tools will be used to shed more light on this important class of transporter. Although my project is fundamental, any advance in our understanding of the mode of action of these transport systems will have a potential impact on society, given the importance of these proteins in different biological processes, including human disease.

Technical Summary

Studies on solute carrier proteins have had great impact on our understanding of human disease: about 30% of clinically used drugs have as their targets membrane transporters/channels.
The solute carrier 26 (SLC26) protein family was defined 15 years ago. Since then, it has been shown that these transporters belong to the SLC26A/SulP family of anion transporters conserved from bacteria to man. The functional importance of the family is highlighted by several inherited human diseases caused by mutations in SLC26A genes and the putative role of the SulP protein in bacterial pathogenicity.
Although our knowledge of this protein family has increased over the past few years, limited structural information is available and several important questions still remain to be answered. These include the identification of the conformational changes that occur during the transport cycle and the structural features important for mediating ion movement through this remarkable family of transporters. Answering this may lead to an understanding of the relationship between SLC26A mutations and inherited diseases, which remains almost completely unknown. Tackling this problem would be extremely difficult using human SLC26A proteins, as they are very large compared to the bacterial homologues, and large membrane proteins are notoriously very challenging to handle for biochemical characterisation. Studies on bacterial membrane proteins have frequently proven crucial for catalyzing progress in the structure/function studies of human membrane proteins. Therefore, this research proposal aims to develop structure/function analysis to shed more light on this important family of membrane proteins, using bacterial members as model systems. The two major objectives of this project are:
1- To obtain essential information on the structure of the SLC26A/SulP proteins, more specifically:
1.1.-to define the topology, the quaternary structure, and obtain a low resolution structure,
1.2-to attempt to solve the 3D structure of one (or more) of SLC26/SulP proteins as well as their isolated cytoplasmic regulatory domain.
2- To define how the protein functions, more specifically
2.1-to determine the substrate(s) transported by developing transport assays
2.2-to define the mechanism of transport as well as identify important residues for the process through a detailed structure/function analysis.

Publications

10 25 50
 
Description MD simulation Ulrich Zachariae 
Organisation University of Dundee
Department School of Dentistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide molecular model of proten that Ulrich#s team test by molecular dynamic simulation
Collaborator Contribution Ulrich uses molecullar dynamic simulation to eveluate in silico the stability of model protein
Impact an article: Conserved structure and domain organization among bacterial Slc26 transporters. Compton EL, Page K, Findlay HE, Haertlein M, Moulin M, Zachariae U, Norman DG, Gabel F, Javelle A. Biochem J. 2014 Oct 15;463(2):297-307. doi: 10.1042/BJ20130619.
Start Year 2013
 
Description Membrane protein Laboratory 
Organisation Diamond Light Source
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise of Prof. So Iwata and Isabel Moraes is very helpful for the biochemical characterisation, crystallization and the resolution of the X-ray structure of bacterial SulP proteins
Collaborator Contribution Through this collaboration we have acess to all the facilities of the MPL and expertise of So Iwata on Membrane proteins.
Impact Through this collaboration we have acess to all the facilities of the Membrane Protein Laboratory (Diamond Light Source Ltd).
Start Year 2011
 
Description PELDOR analysis of SulP/SLC26A protein 
Organisation University of Dundee
Department College of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide purified proteins
Collaborator Contribution They use peldor to measure distance inside proteins
Impact Manuscript in preparation
Start Year 2011
 
Description SANS analysis 
Organisation Alternative Energies and Atomic Energy Commission (CEA)
Country France 
Sector Public 
PI Contribution Dr. Frank Gabel is helping on solving the low resolution structure of the bacterial SulP proteins by Small Angle Neutron Scattering
Collaborator Contribution Acess to the neutron beamline D22 at the Institute Laue-Langevin (Grenoble, France).
Impact - Article PMID: 21659513 - Acess to the neutron beamline D22 at the Institute Laue-Langevin (Grenoble, France).
Start Year 2011
 
Description Solid Supported Membrane electrophysiology 
Organisation Julich Research Centre
Country Germany 
Sector Public 
PI Contribution Purification and Biochemical characterisation of bacterial Slc26A transporter
Collaborator Contribution Use SSM technology to measure Slc26A transport activity
Impact not applicable
Start Year 2013
 
Description X Ray Crystallography 
Organisation University of St Andrews
Department Centre for Biomolecular Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Prof. James Naismith's expertise is helpful for the crystallization and the resolution of the X-ray structure of bacterial SulP proteins.
Collaborator Contribution James Naismith's expertise is helpful for the crystallization and the resolution of the X-ray structure of bacterial SulP proteins.
Impact Article PMID: 21659513
Start Year 2011