Project

amino acid transport protein; crystallization; electron crystallography; membrane protein; peptide transport protein; structure; three-dimensional crystal; transmission electron microscopy; two-dimensional crystal; X-ray crystallography

Lead
Lay summary
Membrane proteins (MPs) reside in the cell membrane where they fulfill innumerous key functions. More than 30 percent of the eukaryotic genome accounts for MPs. Two thirds of all known drugs on the market target MPs, thus highlighting their critical importance in human health. While the unique structures of more than 15’000 soluble proteins are solved, the number of unique MP structures is only 190. Clearly, there is an urgent need for structural information on MPs in order to understand their function at the molecular level and to advance the structure-based design of therapeutic drugs. About 20 percent of the MPs are membrane transporters, which play a critical role in a variety of physiological processes and are implicated in numerous diseases. Membrane transporters represent important drug targets and can furthermore be exploited for drug delivery strategies, for example for the transport of prodrugs.Our project focuses on the high-resolution structure determination of selected amino acid and peptide transporters. Both are important for nutrient uptake, but are also involved in several human diseases such as the primary inherited aminoacidurias, cystinuria and lysinuric protein intolerance, cancer, etc. Our target transporters comprise prokaryotic, mammalian and human members. For structure determination, we overexpress, purify and crystallize selected transporters and analyze the crystals by cryo-transmission electron microscopy/electron crystallography and X-ray crystallography. The elucidation of structures from these two important classes of transport proteins, i.e. amino acid and peptide transporters, will contribute to our understanding of their function and transport mechanism. This information should also facilitate the development of novel pharmaceutical drugs using a structure-based drug design strategy.

Direct link to Lay Summary

Last update: 21.02.2013

Number	Title	Start	Funding scheme

Membrane proteins (MPs) fulfill innumerous key functions in all living cells and account for more than 30 percent of the eukaryotic proteomes. Two thirds of all known drugs on the market target MPs, thus highlighting their critical importance in human health. While the unique structures of more than 15’000 soluble proteins are solved, the number of unique MP structures is only 171. Clearly, there is an urgent need for structural information on MPs in order to understand their function at the molecular level and to advance the structure-based design of therapeutic drugs. About 20 percent of the MPs are membrane transporters, which play a critical role in a variety of physiological processes and are implicated in numerous diseases. Membrane transporters represent important drug targets and can furthermore be exploited for drug delivery strategies, for example for the transport of prodrugs.In the present application, we propose to focus on the high-resolution structure determination of selected amino acid transporters from the amino acid/polyamine/organocation (APC) superfamily (SLC7*) and of peptide transporters from the proton dependent oligopeptide transporter (POT)/peptide transporter (PTR) families (SLC15). Target transporters comprise prokaryotic, mammalian and human members from the APC superfamily (including a heteromeric amino acid transporter (HAT)) and from the POT/PTR family. In this application, the bacterium Escherichia coli and the yeast Pichia pastoris are the expression systems of choice for MP overexpression. We propose to determine the structure of members from these transporter families using 2D and 3D crystals, and cryo-transmission electron microscopy (TEM)/electron crystallography and X-ray crystallography. Besides wild-type transporters, we will attempt to crystallize point mutants that are more stable. As an alternative to the mentioned crystallographic approaches, TEM and single particle analysis will be used to determine the low-resolution 3D structure of a HAT thus unveiling the supramolecular organization of the light (SLC7) and heavy (SLC3) HAT subunits. Low- and medium-resolution protein structure determination by TEM and single particle analysis requires small amounts of pure protein (micrograms) compared to 2D and 3D crystallization (miligrams). For this purpose, we want to establish expression of membrane transporters and their complexes in the promising Xenopus oocyte system.The elucidation of structures from these two important classes of transport proteins, i.e. amino acid and peptide transporters, will contribute to our understanding of their function and transport mechanism. This information should also facilitate the development of novel pharmaceutical drugs using a structure-based drug design strategy.* The solute carrier (SLC) gene series provided by the Human Genome Organisation (HUGO) Nomenclature Committee refer to solute carriers of higher organisms