Klaus Maier

Structure of the chemokine receptor CXCR1 in phospholipid bilayers

CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation. CXCR1 is a class A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors. Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences, as well as addition of stabilizing antibodies and small molecules that facilitate crystallization in cubic phase monoolein mixtures. The intracellular loops of GPCRs are crucial for G-protein interactions, and activation of CXCR1 involves both amino-terminal residues and extracellular loops. Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer.

Optimization of purification and refolding of the human chemokine receptor CXCR1 improves the stability of proteoliposomes for structure determination.

The human chemokine receptor CXCR1 is a G-protein coupled receptor that has been successfully expressed in E. coli as inclusion bodies, and purified and refolded in multi-milligram quantities required for structural studies. Expression in E. coli enables selective and uniform isotopic labeling with (13)C and (15)N for NMR studies. Long-term chemical and conformational stability and oligomeric homogeneity of CXCR1 in phospholipid bilayers are crucial for structural studies under physiological conditions. Here we describe substantial refinements in our previously described purification and reconstitution procedures for CXCR1 in phospholipid bilayers. These refinements have led to the preparation of highly purified, completely monomeric, proteoliposome samples that are stable for months at 35°C while subject to the high power radiofrequency irradiations of solid-state NMR experiments. The principal changes from the previously described methods include: 1) ensure that CXCR1 is pure and homogeneously monomeric within the limits of detection (>98%); 2) monitor and control the pH at all times especially following the addition of TCEP, which serves as a reducing agent but also changes the pH; 3) slowly refold CXCR1 with the complete removal of all traces of SDS using a KCl precipitation/dialysis method; and 4) ensure that the molar ratio between the CXCR1 and the phospholipids does not change during refolding and detergent removal. NMR samples prepared with these protocols yield reproducible results over a period of many months at 35°C. This purification and refolding protocol is likely to be applicable with minimal changes to other GPCRs as well as other membrane proteins.

Electrophoretic differentiation of multiple forms of phosphofructokinase in the tissue fluid of the rat skeletal muscle

Abstract 1. 1. Phosphofructokinase (ATP: d -fructose-6-phosphate phosphotransferase, EC 2.7.1.11) from rat skeletal muscle was prepared using a squeezing technique by which 40% of the muscle was obtained as a clear tissue fluid containing 60% of the total enzyme activity. Phosphofructokinase thus obtained was characterized by means of analytical zone electrophoresis and preparative disc electrophoresis in 3% polyacrylamide gels. 2. 2. Only a small homogeneous fraction of the enzyme (containing 10% of phosphofructokinase activity) showed an electrophoretic migration to the anode, whereas the major part (90%) of the enzyme remained at the starting point as an aggregated form, with a molecular weight exceeding 1.5·10 6 . This electrophoretic behaviour was found to be independent of the buffer system used, the reduction of the SH groups of the enzyme, or the protein concentration of the sample. 3. 3. Homogenization of the muscle in buffers of different ionic strength during the fractional tissue extraction and homogenization of the muscle after squeezing caused a significant alteration of the electrophoretic behaviour of phosphofructokinase. In these cases only one enzymatically active fraction could be detected. This form of the enzyme did not differ electrophoretically from the low molecular form of phosphofructokinase obtained physically by the squeezing technique. 4. 4. The multiple forms of phosphofructokinase from rat muscle, as demonstrated electrophoretically, may be explained by different aggregated forms of one and the same enzyme. 5. 5. The physiological importance of these findings is discussed.

Refolded G protein-coupled receptors from E. coli inclusion bodies

G protein-coupled receptors (GPCRs) are the largest receptor family in eukaryotes with about 2000 representatives in the human genome. Of these, about 1500 GPCRs transmit signals in intracellular communication, while the remaining part are involved in the recognition of sensory signals such as odorants, gustatory substances and, through the visual pigment rhodopsin, light [1, 2].