Adenovirus mediated expression "in vivo" of the chemokine receptor CXCR1.

A major hurdle in the structural analysis of membrane proteins is the expression of a functional and homogeneous form of the protein. Except for rhodopsin, most G protein-coupled receptors (GPCRs) are endogenously expressed at very low levels. Heterologous expression of GPCRs in bacteria, yeast, insect cells or mammalian cell lines often yields proteins with large amounts of misfolded proteins and heterogeneous posttranslational modifications. Here, we report a novel mammalian "in vivo" system for the expression of the chemokine receptor CXCR1. This receptor was expressed in liver of mice infected with adenovirus encoding CXCR1. Liver plasma membranes from infected mice displayed high-levels of (125)I-labeled human interleukin-8 (IL-8) binding. The pharmacological profile of the recombinant CXCR1 expressed "in vivo" was similar to those expressed in neutrophils. We found that the incorporation of the detergent solubilized CXCR1 into phospholipid vesicles in the presence of Gi/Go proteins is required for the reconstitution of (125)I-IL-8 binding. On the basis of the presence of the several endogenous His residues and glycosylation moieties in CXCR1 we fractionated the detergent-solubilized plasma membranes by employing Ni- and Concanavalin A-based chromatography. Fractions enriched with CXCR1 were monitored by (125)I-IL-8-bound to the receptor and Western blots with anti-CXCR1 antibodies. This robust expression system could be readily applied for the expression of GPCRs and other eukaryotic membrane proteins.

Solution structure and dynamics of myeloid progenitor inhibitory factor-1 (MPIF-1), a novel monomeric CC chemokine.

MPIF-1, a CC chemokine, is a specific inhibitor of myeloid progenitor cells and is the most potent activator of monocytes. The solution structure of myeloid progenitor inhibitor factor-1 (MPIF-1) has been determined by NMR spectroscopy. The structure reveals that MPIF-1 is a monomer with a well defined core except for termini residues and adopts the chemokine fold of three beta-strands and an overlying alpha-helix. In addition to the four cysteines that characterize most chemokines, MPIF-1 has two additional cysteines that form a disulfide bond. The backbone dynamics indicate that the disulfide bonds and the adjacent residues that include the functionally important N-terminal and N-terminal loop residues show significant dynamics. MPIF-1 is a highly basic protein (pI >9), and the structure reveals distinct positively charged pockets that could be correlated to proteoglycan binding. MPIF-1 is processed from a longer proprotein at the N terminus and the latter is also functional though with reduced potency, and both proteins exist as monomers under a variety of solution conditions. MPIF-1 is therefore unique because longer proproteins of all other chemokines oligomerize in solution. The MPIF-1 structure should serve as a template for future functional studies that could lead to therapeutics for preventing chemotherapy-associated myelotoxicity.

13C NMR chemical shifts can predict disulfide bond formation.

The presence of disulfide bonds can be detected unambiguously only by X-ray crystallography, and otherwise must be inferred by chemical methods. In this study we demonstrate that 13C NMR chemical shifts are diagnostic of disulfide bond formation, and can discriminate between cysteine in the reduced (free) and oxidized (disulfide bonded) state. A database of cysteine 13C C(alpha) and C(beta) chemical shifts was constructed from the BMRB and Sheffield databases, and published journals. Statistical analysis indicated that the C(beta) shift is extremely sensitive to the redox state, and can predict the disulfide-bonded state. Further, chemical shifts in both states occupy distinct clusters as a function of secondary structure in the C(alpha)/C(beta) chemical shift map. On the basis of these results, we provide simple ground rules for predicting the redox state of cysteines; these rules could be used effectively in NMR structure determination, predicting new folds, and in protein folding studies.

Spectroscopic characterization of chemokines: relevance for quality control and standardization.

Chemokines are mediators of inflammation and trafficking of cells of the immune system including a pivotal role in the recruitment and activation of leukocytes. Due to their involvement in a variety of disease processes, chemokines are potential therapeutic targets. The use of chemokines as pharmaceuticals will require that the folded state and the association properties of the protein are well characterized. In this report, we describe the utility of nuclear magnetic resonance spectroscopy as a tool to study these aspects of chemokine structural properties.

Disulfide bridges in interleukin-8 probed using non-natural disulfide analogues: dissociation of roles in structure from function.

The structural and functional roles of the two disulfide bridges in interleukin-8 (IL-8) were addressed using IL-8 analogues with covalently modified disulfide bridges. The analogues were prepared using chemical synthesis by replacement of a cysteine for either homocysteine, penicillamine, or selenocysteine and on folding resulted in a covalently modified disulfide. Deletion of either of the two disulfide bridges by replacement of either cysteine pair with alanine resulted in loss of both structure and function. In contrast, all of the analogues with modified disulfide bridges had native tertiary fold as determined by nuclear magnetic resonance spectroscopic methods. Their structural similarity provided a rational basis for assessing the functional effects of the changes to the disulfide. Modification to the disulfide bridge between cysteines 9 and 50 had only a modest effect on IL-8 function. In contrast, alterations to the 7-34 disulfide bridge resulted in a dramatic reduction in biological potency. Thus, although both disulfide bridges are required for maintenance of the native tertiary fold, their role in determining IL-8 activity is distinct. We propose that 7-34 disulfide has a direct role in determining receptor binding and activation, whereas the 9-50 was not directly involved. The synthesis of non-natural disulfide analogues is a novel general approach to structure-activity relationships of disulfide bridges. The demonstration that the participation of disulfide bridges in function can be dissociated from their effects on the stability of the tertiary structure suggests that this method will lead to increased understanding of the roles of disulfide bridges in proteins.

CAMRA: chemical shift based computer aided protein NMR assignments.

A suite of programs called CAMRA (Computer Aided Magnetic Resonance Assignment) has been developed for computer assisted residue-specific assignments of proteins. CAMRA consists of three units: ORB, CAPTURE and PROCESS. ORB predicts NMR chemical shifts for unassigned proteins using a chemical shift database of previously assigned homologous proteins supplemented by a statistically derived chemical shift database in which the shifts are categorized according to their residue, atom and secondary structure type. CAPTURE generates a list of valid peaks from NMR spectra by filtering out noise peaks and other artifacts and then separating the derived peak list into distinct spin systems. PROCESS combines the chemical shift predictions from ORB with the spin systems identified by CAPTURE to obtain residue specific assignments. PROCESS ranks the top choices for an assignment along with scores and confidence values. In contrast to other auto-assignment programs, CAMRA does not use any connectivity information but instead is based solely on matching predicted shifts with observed spin systems. As such, CAMRA represents a new and unique approach for the assignment of protein NMR spectra. CAMRA will be particularly useful in conjunction with other assignment methods and under special circumstances, such as the assignment of flexible regions in proteins where sufficient NOE information is generally not available. CAMRA was tested on two medium-sized proteins belonging to the chemokine family. It was found to be effective in predicting the assignment providing a database of previously assigned proteins with at least 30% sequence identity is available. CAMRA is versatile and can be used to include and evaluate heteronuclear and three-dimensional experiments.

Solution structure of eotaxin, a chemokine that selectively recruits eosinophils in allergic inflammation.

The solution structure of the CCR3-specific chemokine, eotaxin, has been determined by NMR spectroscopy. The quaternary structure of eotaxin was investigated by ultracentrifugation and NMR, and it was found to be in equilibrium between monomer and dimer under a wide range of conditions. At pH

Expression, purification, and characterization of the carboxyl-terminal region of the Na+/H+ exchanger.

The Na+/H+ exchanger is a pH regulatory protein that is responsible for removal of excess intracellular protons in exchange for extracellular Na+. It is a plasma membrane protein with a large cytoplasmic carboxyl terminal domain that regulates activity of the membrane domain. We overexpressed and purified the cytoplasmic domain that was produced in Escherichia coli. This region (516-815 amino acids) was under control of the tac promoter from the plasmid pGEX-KG and was fused with glutathione S-transferase. Upon induction, the fusion protein was principally found in inclusion bodies. Purified inclusion bodies were solubilized and fractionated using preparative SDS polyacrylamide gel electrophoresis. To obtain free Na+/H+ exchanger protein the fusion protein was dialyzed against cleavage buffer and cleaved at the thrombin cleavage site between glutathione S-transferase and the Na+/H+ exchanger domain. Free Na+/H+ exchanger protein was obtained by rerunning the sample on preparative gel electrophoresis. The final yield of the purified protein was 2.15 mg protein/L of cell culture. After exhaustive dialysis the secondary structure of the purified protein was assessed using circular dichroism spectroscopy. The results indicated that the protein was 35% alpha-helix, 17% beta-turn, and 48% random coil. They suggest that the cytoplasmic domain is structured and some regions may be compact in nature.

The purified myxoma virus gamma interferon receptor homolog M-T7 interacts with the heparin-binding domains of chemokines.

The myxoma virus T7 protein M-T7 is a functional soluble gamma interferon receptor homolog that has previously been shown to bind gamma interferon and inhibit its antiviral activities in a species-specific manner, but gene knockout analysis has suggested a further role for M-T7 in blocking leukocyte influx into infected lesions. We purified M-T7 to apparent homogeneity and showed that M-T7 is an N-linked glycoprotein that appears to be a stable homotrimer with a molecular mass of approximately 113 kDa in solution. M-T7, in addition to forming inhibitory complexes with rabbit gamma interferon, was also shown to bind to human interleukin-8, a prototypic member of the chemokine superfamily. Moreover, M-T7 was able to interact promiscuously with all members of the CXC, CC, and C chemokine subfamilies tested. Binding of human RANTES to M-T7 can be competed by rabbit gamma interferon and also by cold RANTES competitor with a 50% inhibitory concentration of 900 nM. Although M-T7 retains binding to a number of interleukin-8 N-terminal (ELR) deletion mutants, binding to mutants containing deletions in the C-terminal heparin-binding domain of interleukin-8 is abrogated. Furthermore, heparin effectively competes the interaction of M-T7 with the chemokine RANTES but not with rabbit gamma interferon. We propose that this novel M-T7 interaction with members of the chemokine superfamily may be facilitated through the conserved heparin-binding domains found in a wide spectrum of chemokines and that M-T7 may function by modulating chemokine-glycosaminoglycan interactions in virus-infected tissues.

Neutrophil-activating peptide-2 and melanoma growth-stimulatory activity are functional as monomers for neutrophil activation.

Neutrophil-activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA) are members of the chemokine family of inflammatory proteins. The structures of NAP-2, determined by x-ray crystallography, and MGSA, elucidated by NMR spectroscopy, revealed a tetramer and dimer, respectively. In order to address the relevance of multimeric species to their activities on neutrophils, analogs of NAP-2 and MGSA were synthesized in which the backbone amide proton of Leu-22 in NAP-2, and Val-26 in MGSA, was substituted with the bulky methyl group (NH --> NCH3). These analogs were shown to be monomeric by sedimentation equilibrium ultracentrifugation studies and were similar to the corresponding native protein in assays for neutrophil elastase release and Ca2+ mobilization from IL-8R1 and IL-8R2 transformed cells. Sedimentation equilibrium studies of the native NAP-2 and MGSA were also carried out to address the association behavior. For NAP-2, there was no evidence for the tetramer, but an equilibrium between monomers and dimers and the dissociation constant was calculated to be 50-100 microM. Similarly, MGSA showed a monomer-dimer equilibrium with a Kd of approximately 5 microM. The data from the monomeric analogs and also the calculation of dissociation constants indicate that NAP-2 and MGSA have a tendency to associate above the concentrations required for maximal activity or for receptor activation, but at functional concentrations they are predominantly monomers.

Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1.

The three-dimensional structure of stromal cell-derived factor-1 (SDF-1) was determined by NMR spectroscopy. SDF-1 is a monomer with a disordered N-terminal region (residues 1-8), and differs from other chemokines in the packing of the hydrophobic core and surface charge distribution. Results with analogs showed that the N-terminal eight residues formed an important receptor binding site; however, only Lys-1 and Pro-2 were directly involved in receptor activation. Modification to Lys-1 and/or Pro-2 resulted in loss of activity, but generated potent SDF-1 antagonists. Residues 12-17 of the loop region, which we term the RFFESH motif, unlike the N-terminal region, were well defined in the SDF-1 structure. The RFFESH formed a receptor binding site, which we propose to be an important initial docking site of SDF-1 with its receptor. The ability of the SDF-1 analogs to block HIV-1 entry via CXCR4, which is a HIV-1 coreceptor for the virus in addition to being the receptor for SDF-1, correlated with their affinity for CXCR4. Activation of the receptor is not required for HIV-1 inhibition.

1H NMR evidence that Glu-38 interacts with the N-terminal functional domain in interleukin-8.

In order to assess the importance of the buried Glu-38 observed in the structure of interleukin-8, an analog in which Glu-38 was replaced with Ala (E38A analog) was investigated by 1H NMR spectroscopy and neutrophil activation. Detailed analysis of the NMR NOESY data showed that the solution structure of the E38A analog is essentially the same as that for the native protein. Also, the neutrophil elastase activity of the E38A analog was similar to that of the native protein. However, the Gln-8 and Cys-9 amide proton chemical shifts, which are significantly downfield-shifted in the native protein, exhibit more 'normal' values. This observation indicates that in the native protein, Glu-38 side-chain carboxylate interacts with Gln-8 and Cys-9 amide protons. Although the N-terminal residues are critical for function, this interaction is not essential for neutrophil activation.

Structural characterization of a monomeric chemokine: monocyte chemoattractant protein-3.

1H-NMR spectroscopy and analytical ultracentrifugation studies reveal that monocyte chemoattractant protein-3 (MCP-3) is a monomer. NMR solution structure shows that MCP-3 adopts an alphabeta fold similar to what is observed in structures of other known chemokines. However, MCP-3 is unique in that it does not show a propensity to form dimers. The closely related chemokines MCP-1 and MCP-2 show a monomer-dimer equilibrium in sedimentation equilibrium studies (approximately 0.2-2 mg/ml). As these proteins are present at nanomolar concentrations in vivo, the results suggest that they are monomeric at functional concentrations and that the monomer is the functionally significant form of MCP-1, MCP-2 and MCP-3.

Myxoma virus T2 protein, a tumor necrosis factor (TNF) receptor homolog, is secreted as a monomer and dimer that each bind rabbit TNFalpha, but the dimer is a more potent TNF inhibitor.

The myxoma virus T2 (M-T2) gene expresses a secreted protein that contains significant sequence similarity to the ligand binding domains of the cellular tumor necrosis factor (TNF) receptors, specifically inhibits the cytolytic activity of rabbit TNFalpha and is an important virulence factor for myxoma virus infection in rabbits. M-T2 protein was overexpressed from vaccinia virus vectors, purified to apparent homogeneity, and found to specifically protect mouse and rabbit cells from lysis by rabbit TNFalpha at molar ratios comparable with the soluble versions of the host tumor necrosis factor receptors. M-T2 secreted from virus-infected cells is detected as both a monomer and a disulfide-linked dimer, both of which were shown by Scatchard analysis to bind rabbit TNFalpha (Kd values of 170 pM and 195 pM, respectively), values that are comparable with the affinities of mammalian TNFs with their receptors. In contrast to the rabbit ligand, M-T2 interacts with mouse TNFalpha with a much lower affinity, Kd of 1.7 nM, and was unable to inhibit the cytolytic activity of this ligand on mouse cells. Although both monomeric and dimeric forms bound rabbit TNFalpha with comparable affinity, the dimeric M-T2 protein was a far more potent inhibitor of rabbit TNFalpha, presumably because it can more effectively prevent dimerization of TNF receptors than can the M-T2 monomer.

1H NMR solution structure of an active monomeric interleukin-8.

The solution structure of a monomeric form of interleukin-8 (IL-8) has been solved using 1H NMR spectroscopy. The chemically synthesized nonnatural analog [IL-8 (4-72) L25 NH-->NCH3] has the same activity as that of native IL-8. Thirty structures were generated using the hybrid distance geometry and simulated annealing protocol using the program X-PLOR. The structure is well-defined except for N-terminal residues 4-6 and C-terminal residues 67-72. The rms distribution about the average structure for residues 7-66 is 0.38 A for the backbone atoms and 0.87 A for all heavy atoms. The structure consists of a series of turns and loops followed by a triple-stranded beta sheet and a C-terminal alpha helix. The structure of the monomer is largely similar to the native dimeric IL-8 structures previously determined by both NMR and X-ray methods. The major difference is that, in the monomeric analog, the C-terminal residues 67-72 are disordered whereas they are helical in the two dimeric structures. The best fit superposition of the backbone atoms of residues 7-66 of the monomer structure on the dimeric IL-8 structures showed rms differences of 1.5 and 1.2 A respectively. The turn (residues 31-35), which is disulfide linked to the N-terminal region, adopts a conformation in the monomer similar to that seen in the dimeric X-ray structure (rms difference 1.4 A) and different from that seen in the dimeric NMR structure (rms difference 2.7 A).(ABSTRACT TRUNCATED AT 250 WORDS)

A double mutant of sperm whale myoglobin mimics the structure and function of elephant myoglobin.

The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, 1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K. (1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln64(E7) substitution lowers oxygen affinity approximately 5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe29(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature.

Structure-activity relationships of chemokines.

Structural analysis of chemokines has revealed that the alpha/beta structural-fold is highly conserved among both the CXC and CC chemokine classes. Although dimerization and aggregation is often observed, the chemokines function as monomers. The critical receptor binding regions are in the NH2-terminal 20 residues of the protein and are the least ordered in solution. The flexible NH2-terminal region is the most critical receptor binding site and a second site also exists in the loop that follows the two disulfides. The well-ordered regions are not directly involved in receptor binding but, along with the disulfides, they provide a scaffold that determines the conformation of the sites that are critical for receptor binding. These general requirements for function are common to all the chemokines. For the CC chemokines, receptor activation and receptor binding regions are separate within the 10 residue NH2-terminal region. This has allowed identification of high affinity analogs that do not activate the receptor and are potent antagonists.

1H NMR studies of interleukin 8 analogs: characterization of the domains essential for function.

1H NMR studies were carried out on interleukin 8 (IL-8) analogs in order to probe the structural features that are essential for receptor binding and function. The analogs studied were the chemically synthesized IL-8 (1-72), a series of N-terminally truncated derivatives (4-72, 5-72, and 6-72), and derivatives with single amino acid substitutions (I10A, R6K, and H33A). Previous functional studies have shown that the N-terminal residues, especially the residues at positions 4-6, and the beta turn containing Cys-34, which is disulfide linked to Cys-7, are important for receptor affinity and functional activity [Clark-Lewis, I., Schumacher, C., Baggiolini, M., & Moser, B. (1991) J. Biol. Chem. 266, 23128-23134; Clark-Lewis, I., Dewald, B., Loetscher, M., Moser, B., & Baggiolini, M. (1994) J. Biol. Chem. (in press)]. The 6-72 and R6K analogs also showed properties of an antagonist. Analysis of the 1H NMR parameters such as chemical shifts, amide proton chemical shift temperature coefficients, and NOESY data indicates that the core structure is the same for all these proteins. Small differences were observed in some of the NMR properties for some of the residues in the N-terminal region and the turn containing Cys-34. Detailed analysis suggests that there is no correlation between these differences and observed function. Thus functional differences between the N-terminal analogs are a direct consequence of changes in receptor binding due to substitutions/deletions in the N-terminal sequence and not due to structural changes elsewhere.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between the steric bulk of the distal E7 and E11 residues and the tilt of the FeCN unit in cyanometmyoglobin as determined by NMR from the orientation of the magnetic axes in single and double point mutants.

The amino acids in the heme pocket of sperm whale myoglobin single E11 and double E7 and E11 point mutants in the metcyano form have been assigned by NMR methods to assess the role of steric bulk in modulating ligand tilt. The five mutants investigated are the single mutants His64(E7)-->Gly (H[E7]G), Val68(E11)-->Ile (V[E11]I), and Val68(E11)-->Ala (V[E11]A) and the double mutants His64-(E7)-->Gly:Val68(E11)-->Ile (H,V[E7,E11]G,I) and His64(E7)-->Gly:Val68(E11)-->Ala (H,V[E7,E11]G,A). The dipolar (NOESY) contacts on the proximal side of the heme confirm a conserved molecular structure for all of the mutants. The proximal residue coordinates, together with the dipolar shifts for proximal side residues, quantitatively yield the orientations of the magnetic susceptibility tensors, whose major axis corresponds to the orientation of the ligand. It is observed that upon reduction of the steric bulk in the V[E11]A mutant, the tilt of the ligand is significantly reduced (approximately 8 degrees) from that in the wild type (WT) (approximately 16 degrees), with little change in the direction of tilt. In the case of increased steric bulk at position 68 in the V[E11]I mutant, it is observed that the extent and direction of the tilt are essentially the same as in WT, and it is shown that this is due to the fact that Ile68 is oriented in the pocket with its C delta H3 directed away from the iron.(ABSTRACT TRUNCATED AT 250 WORDS)