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 tumor growth1-3. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signaling pathways result in neutrophil migration to the site of inflammation2. 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 receptors4-7. Despite its importance, its molecular mechanism is poorly understood due to the limited structural information available. Recently, structure determination of GPCRs has advanced by tailoring the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences8, as well as addition of stabilizing antibodies and small molecules9 that facilitate crystallization in cubic phase monoolein mixtures10. The intracellular loops of GPCRs are critical for G-protein interactions11 and activation of CXCR1 involves both N-terminal residues and extracellular loops2,12,13. Our previous NMR 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 activity14. 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.

In photosynthetic organisms, tetrapyrrole-mediated retrograde signals are proposed to contribute to a balanced nuclear gene expression (NGE) in response to metabolic activity in chloroplasts. We followed an experimental short-term approach that allowed the assessment of modified NGE during the first hours of specifically modified enzymatic steps of the Mg branch of tetrapyrrole biosynthesis, when pleiotropic effects of other signals can be avoided. In response to 24-h-induced silencing of CHLH, CHLM, and CHL27 encoding the CHLH subunit of Mg chelatase, the Mg protoporphyrin methyltransferase and Mg protoporphyrin monomethylester cyclase, respectively, deactivated gene expression rapidly led to reduced activity of the corresponding enzymes and altered Mg porphyrin levels. But NGE was not substantially altered. When these three genes were continuously inactivated for up to 4 d, changes of transcript levels of nuclear genes were determined. CHL27 silencing for more than 24h results in necrotic leaf lesions and modulated transcript levels of oxidative stress-responsive and photosynthesis-associated nuclear genes (PhANGs). The prolonged deactivation of CHLH and CHLM results in slightly elevated transcript levels of PhANGs and tetrapyrrole-associated genes. These time-resolved studies indicate a complex scenario for the contribution of tetrapyrrole biosynthesis on NGE mediated by (1)O2-induced signaling and feedback-regulated ALA synthesis.

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 13C and 15N 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.

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