John S. Mills

IDENTIFICATION OF A LIGAND BINDING SITE IN THE HUMAN NEUTROPHIL FORMYL PEPTIDE RECEPTOR USING A SITE-SPECIFIC FLUORESCENT PHOTOAFFINITY LABEL AND MASS SPECTROMETRY

A novel fluorescent photoaffinity cross-linking probe, formyl-Met-p-benzoyl-l-phenylalanine-Phe-Tyr-Lys-ε-N-fluorescein (fMBpaFYK-fl), was synthesized and used to identify binding site residues in recombinant human phagocyte chemoattractant formyl peptide receptor (FPR). After photoactivation, fluorescein-labeled membranes from Chinese hamster ovary cells were solubilized in octylglucoside and separated by tandem anion exchange and gel filtration chromatography. A single peak of fluorescence was observed in extracts of FPR-expressing cells that was absent in extracts from wild type controls. Photolabeled Chinese hamster ovary membranes were cleaved with CNBr, and the fluorescent fragments were isolated on an antifluorescein immunoaffinity matrix. Matrix-assisted laser desorption ionization mass spectrometry identified a major species with mass = 1754, consistent with the CNBr fragment of fMBpaFYK-fl cross-linked to Val-Arg-Lys-Ala-Hse (an expected CNBr fragment of FPR, residues 83–87). This peptide was further cleaved with trypsin, repurified by antifluorescein immunoaffinity, and subjected to matrix-assisted laser desorption ionization mass spectrometry. A tryptic fragment with mass = 1582 was observed, which is the mass of fMBpaFYK-fl cross-linked to Val-Arg-Lys (FPR residues 83–85), an expected trypsin cleavage product of Val-Arg-Lys-Ala-Hse. Residues 83–85 lie within the putative second transmembrane-spanning region of FPR near the extracellular surface. A 3D model of FPR is presented, which accounts for intramembrane, site-directed mutagenesis results (Miettinen, H. M., Mills, J., Gripentrog, J., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol.159, 4045–4054) and the photochemical cross-linking data.

Characterization of the Binding Site on the Formyl Peptide Receptor Using Three Receptor Mutants and Analogs of Met-Leu-Phe and Met-Met-Trp-Leu-Leu

The formyl peptide receptor (FPR) is a chemotactic G protein-coupled receptor found on the surface of phagocytes. We have previously shown that the formyl peptide binding site maps to the membrane-spanning region (Miettinen, H. M., Mills, J. S., Gripentrog, J. M., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol. 159, 4045–4054). Recent reports have indicated that non-formylated peptides, such as MMWLL can also activate this receptor (Chen, J., Bernstein, H. S., Chen, M., Wang, L., Ishi, M., Turck, C. W., and Coughlin, S. R. (1995) J. Biol. Chem. 270, 23398–23401.) Here we show that the selectivity for the binding of different NH2-terminal analogs of MMWLL or MLF can be markedly altered by mutating Asp-106 to asparagine or Arg-201 to alanine. Both D106N and R201A produced a similar change in ligand specificity, including an enhanced ability to bind the HIV-1 peptide DP178. In contrast, the mutation R205A exhibited altered specificity at the COOH terminus of fMLF, with R205A binding fMLF-O-butyl >fMLF-O-methyl > fMLF, whereas wt FPR bound fMLF >fMLF-O-methyl ∼fMLF-O-butyl. These data, taken together with our previous finding that the leucine side chain of fMLF is probably bound to FPR near FPR 93VRK95 (Mills, J. S., Miettinen, H. M., Barnidge, D., Vlases, M. J., Wimer-Mackin, S., Dratz, E. A., and Jesaitis, A. J. (1998)J. Biol. Chem. 273, 10428–10435.), indicate that the most likely positioning of fMLF in the binding pocket of FPR is approximately parallel to the fifth transmembrane helix with the formamide group of fMLF hydrogen-bonded to both Asp-106 and Arg-201, the leucine side chain pointing toward the second transmembrane region, and the COOH-terminal carboxyl group offMLF ion-paired with Arg-205.

Functional Epitope on Human Neutrophil Flavocytochrome b558

mAb NL7 was raised against purified flavocytochrome b558, important in host defense and inflammation. NL7 recognized the gp91phox flavocytochrome b558 subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the 498EKDVITGLK506 region of gp91phox. In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (Km). mAb NL7 did not inhibit translocation of p47phox, p67phox, or Rac to the plasma membrane, and bound its epitope on gp91phox independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91phox on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47phox binding sites. We conclude that the 498EKDVITGLK506 segment resides on the cytosolic surface of gp91phox and represents a region important for oxidase function, but not substrate or cytosolic component binding.

The N-Formyl Peptide Receptor

The chemotaxis of phagocytic leukocytes from the blood to tissues identifies the acute (neutrophil) and chronic (macrophages) inflammatory condition (1). This hallmark process is central to host defense against microorganisms, to mediation of the immune response, and to repair of injured tissues. When unregulated, however, it is also the key vehicle of tissue damage and injury. Consequently, leukocyte chemotaxis, its ligands, and its receptors continue to be the focus of intense study. With a comprehensive molecular understanding of the structure-function relationships of chemotactic activation, intervention that mitigates injury but enhances microbial killing may become possible.

Variable responses of formyl peptide receptor haplotypes toward bacterial peptides.

The chemoattractant neutrophil formyl peptide receptor (FPR) binds bacterial and mitochondrial N-formylated peptides, which allows the neutrophils to find the bacterial source and/or site of tissue damage. Certain inflammatory disorders may be due in part to an impaired innate immune system that does not respond to acute bacterial damage in a timely fashion. Because the human FPR is encoded by a large number of different haplotypes arising from ten single-nucleotide polymorphisms, we examined the possibility that some of these haplotypes are functionally distinct. We analyzed the response of three common FPR haplotypes to peptides from Escherichia coli, Mycobacterium avium ssp. paratuberculosis, and human mitochondria. All three haplotypes responded similarly to the E. coli and mitochondrial peptides, whereas one required a higher concentration of the M. avium peptide fMFEDAVAWF for receptor downregulation, receptor signaling, and chemotaxis. This raises the possibility of additional bacterial species differences in functional responses among FPR variants and establishes a precedent with potentially important implications for our innate immune response against bacterial infections. We also investigated whether certain FPR haplotypes are associated with rheumatoid arthritis (RA) by sequencing FPR1 from 148 Caucasian individuals. The results suggested that FPR haplotypes do not significantly contribute toward RA.

CCR5 and CXCR4 antagonist and agonist binding sites

Several small molecule antagonists of the HIV co-receptors CCR5 and CXCR4 are being developed as HIV entry inhibitors, but side effects have been observed in clinical trials that are likely due to agonist activity and/or cross-reactivity with closely related receptors. In order to develop high resolution maps of the binding sites for these antagonists that can be exploited to improve their activity and specificity, we are synthesizing derivatives of CCR5 and CXCR4 antagonists which contain photo-crosslinking groups at a variety positions in the molecules and that retain high affinity and activity against the receptors. Derivatives of two CCR5 antagonists and one CXCR4 antagonist have been crosslinked to their receptors on cells, and the interaction sites are being mapped by mass spectrometry. Techniques for purification, crosslinking, CNBr and/or trypsin digestion, and LC/MS/MS mass spectrometry of highly hydrophobic peptides initially developed using the rhodopsin GPCR system have been applied with some success to CCR5 and CXCR4, although these represent particular challenges due to the relative paucity of Met residue cleavage sites compared to rhodopsin and resistance to tryptic digestion. Nevertheless, peptides covering most of the extracellular and transmembrane regions of CCR5 that could interact with an antagonist can be identified, and affinity purification methods are being developed to isolate CXCR4 peptides crosslinked to derivatives of the antagonist T-140 that should facilitate the identification and localization of the crosslinking sites within the proteins.

CCR5 Antagonist and Agonist Binding Site Structures

Several small molecule antagonists of the HIV co-receptors CCR5 and CXCR4 are being developed as HIV entry inhibitors, but side effects have been observed in clinical trials that are likely due to agonist activity and/or crossreactivity with closely related receptors. In order to develop high resolution maps of the binding sites for these antagonists that can be exploited to improve the activity and specificity, we are synthesizing derivatives of CCR5 and CXCR4 antagonists which contain photocrosslinking groups at a variety positions in the molecules and that retain high affinity and activity against the receptors. Derivatives of two CCR5 antagonists have been crosslinked to affinity-purified CCR5 or CCR5 expressed on cells, and the interaction sites are being mapped by mass spectrometry. Techniques for purification, crosslinking, CNBr and/or trypsin digestion, and LC/MS/MS and MALDI-TOF mass spectrometry of highly hydrophobic peptides initially developed using the rhodopsin GPCR system have been applied with some success to CCR5. The peptide photo-crosslinked to a derivative of the antagonist TAK-779 has been identified, and modeling of the interaction with CCR5 suggests that it binds within the transmembrane region of the receptor and is oriented parallel to the transmembrane helices – in striking contrast to the perpendicular orientation expected for GPCR agonists. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005