NMR solution structure and backbone dynamics of the CC chemokine eotaxin-3.

Eotaxin-3 is one of three related chemokines that specifically activate chemokine receptor CCR3. We report the 3D structure and backbone dynamics of eotaxin-3 determined by NMR spectroscopy. Eotaxin-3 is monomeric under the conditions in this study and consists of an unstructured N-terminus before the first two conserved cysteine residues, an irregularly structured N-loop following the second conserved cysteine, a single turn of 3(10)-helix, a three-stranded antiparallel beta-sheet, an alpha-helix, and an unstructured C-terminal tail. As in other chemokines, the alpha-helix packs against one face of the beta-sheet. The average backbone and heavy atom rmsd values of the 20 structures (residues 9-65) are 0.44 and 1.01 A, respectively. A comparison between the structures of eotaxin-3 and related chemokines suggests that the electrostatic potential in the vicinity of a surface groove and the structure of the beta2-beta3 turn may be important for maintaining receptor specificity. The backbone dynamics of eotaxin-3 were determined from 15N NMR relaxation data using the extended model free dynamics formalism. Large amplitude motions on the picosecond to nanosecond time scale were observed in both termini and in some residues in the N-loop, the beta1-beta2 turn, and the beta3 strand; the location of these residues suggests a possible role for dynamics in receptor binding and activation. In contrast to eotaxin, eotaxin-3 exhibits no substantial mobility on the microsecond to millisecond time scale.     

Backbone dynamics of the CC-chemokine eotaxin-2 and comparison among the eotaxin group chemokines

The eotaxin group chemokines (eotaxin, eotaxin-2, and eotaxin-3) share only 35-41% sequence identity but are all agonists for the receptor CCR3. Here we present a detailed comparison between the backbone dynamics of these three chemokines. The dynamics of eotaxin-2 were determined from 15N NMR relaxation data and compared to those obtained previously for eotaxin and eotaxin-3. For all three chemokines, the majority of residues in the first two beta-strands and the alpha-helix show highly restricted motions on the subnanosecond time scale but there is dramatically higher flexibility in the N- and C-terminal regions and also substantial mobility for residues in the N-loop region and the third beta-strand. The latter two regions form a groove on the chemokine surface that is the likely binding site for the N-terminal region of the receptor. Taken together, the available data suggest a model in which conformational rearrangements of both the chemokine and the receptor are likely to occur during binding and receptor activation.     

Eotaxin and monocyte chemotactic protein-3 use different modes of action

Eotaxin selectively binds CC chemokine receptor (CCR) 3, whereas monocyte chemotactic protein (MCP)-3 binds CCR1, CCR2, and CCR3. To identify the functional determinants of the chemokines, we generated four reciprocal chimeric chemokines-M10E9, M22E21, E8M11, and E20M23-by shuffling the N-terminus and N-loop of eotaxin and MCP-3. M22E21 and E8M11, which shared the N-loop from MCP-3, bound to monocytes with high affinity, and activated monocytes. In contrast, M10E9 and E20M23, which lacked the N-loop, failed to bind and transduce monocyte responses, identifying the N-loop of MCP-3 as the selectivity determinant for CCR1/CCR2. A BIAcore assay with an N-terminal peptide of CCR3 (residues 1-35) revealed that all chimeras except E20M23 exhibited varying degrees of binding affinity with commensurate chemotaxis activity of eosinophils. Surprisingly, E20M23 could neither bind the CCR3 peptide nor activate eosinophils, despite having both N-terminal motifs from eotaxin. These results suggest that the two N-terminal motifs of eotaxin must cooperate with other regions to successfully bind and activate CCR3.     

Tyrosine sulfation influences the chemokine binding selectivity of peptides derived from chemokine receptor CCR3

The interactions of chemokines with their G protein-coupled receptors play critical roles in the control of leukocyte trafficking in normal homeostasis and in inflammatory responses. Tyrosine sulfation is a common post-translational modification in the amino-terminal regions of chemokine receptors. However, tyrosine sulfation of chemokine receptors is commonly incomplete or heterogeneous. To investigate the possibility that differential sulfation of two adjacent tyrosine residues could bias the responses of chemokine receptor CCR3 to different chemokines, we have studied the binding of three chemokines (eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26) to an N-terminal CCR3-derived peptide in each of its four possible sulfation states. Whereas the nonsulfated peptide binds to the three chemokines with approximately equal affinity, sulfation of Tyr-16 gives rise to 9-16-fold selectivity for eotaxin-1 over the other two chemokines. Subsequent sulfation of Tyr-17 contributes additively to the affinity for eotaxin-1 and eotaxin-2 but cooperatively to the affinity for eotaxin-3. The doubly sulfated peptide selectively binds to both eotaxin-1 and eotaxin-3 approximately 10-fold more tightly than to eotaxin-2. Nuclear magnetic resonance chemical shift mapping indicates that these variations in affinity probably result from only subtle differences in the chemokine surfaces interacting with these receptor peptides. These data support the proposal that variations in sulfation states or levels may regulate the responsiveness of chemokine receptors to their cognate chemokines.     

NMR solution structure of murine CCL20/MIP-3alpha, a chemokine that specifically chemoattracts immature dendritic cells and lymphocytes through its highly specific interaction with the beta-chemokine receptor CCR6

CCL20/MIP-3alpha is a beta-chemokine expressed in the thymus, skin, and intestinal epithelial cells that exclusively binds and activates the CCR6 receptor in both mice and humans. The strict receptor binding specificity of CCL20 is exceptional; other chemokines and their receptors bind promiscuously with multiple partners. Toward determining the structural basis for the selective receptor specificity of CCL20, we have determined its three-dimensional structure by 1H NMR spectroscopy. CCL20 exhibits the same monomeric structure previously described for other chemokines: a three-stranded beta-sheet and an overlying alpha-helix. The CCL20 receptor selectivity could arise from the rigid conformation of the N-terminal DCCL motif as well as the groove between the N-loop and the beta2-beta3 hairpin, which is significantly narrower in CCL20 than in other chemokines. Similar structural features are seen in human beta-defensin 2, a small nonchemokine polypeptide reported to selectively bind and activate CCR6, which stresses their importance for the specific binding of both CCL20 and beta-defensin 2 to CCR6. CCL20's structure will be useful to design tools aimed to modulate its important biological functions.     

N-terminal domain of eotaxin-3 is important for activation of CC chemokine receptor 3

Eotaxin-3 belongs to the CC chemokine family, and specifically recognizes CC chemokine receptor (CCR) 3 that is expressed on eosinophils, basophils and helper T type 2 cells. The three-dimensional structure of eotaxin-3 determined by nuclear magnetic resonance has revealed that the N-terminal nine residues preceding the first cysteine comprise an unstructured domain, which is also observed in other chemokine molecules. In order to determine the function of the N-terminal domain of eotaxin-3, we constructed various N-terminal-deletion mutants, and then examined their binding and chemotactic activities toward eosinophils in vitro. Competitive binding studies showed that the binding affinity of truncated mutant toward CCR3 was almost the same as that of wild-type eotaxin-3 even though the N-terminal truncation involved the first through to the ninth residues. In contrast, the chemotactic activity gradually decreased with extension of the N-terminal deletion, and when the deletion extended to the eighth residue, the activity was not detected at all. Thus, the N-terminal nine residues are not critical for binding but the N-terminal eight residues are essential for activation of CCR3. The truncated eotaxin-3 proteins lacking the N-terminal eight or nine residues inhibited the chemotactic activity of chemokines that recognize CCR3. The truncated mutants can possibly be used for anti-allergic and anti-HIV-1 therapy.     

Identification of receptor binding and activation determinants in the N-terminal and N-loop regions of the CC chemokine eotaxin

Eotaxin is a CC chemokine that specifically activates the receptor CCR3 causing accumulation of eosinophils in allergic diseases and parasitic infections. Twelve amino acid residues in the N-terminal (residues 1-8) and N-loop (residues 11-20) regions of eotaxin have been individually mutated to alanine, and the ability of the mutants to bind and activate CCR3 has been determined in cell-based assays. The alanine mutants at positions Thr(7), Asn(12), Leu(13), and Leu(20) show near wild type binding affinity and activity. The mutants T8A, N15A, and K17A have near wild type binding affinity for CCR3 but reduced receptor activation. A third class of mutants, S4A, V5A, R16A, and I18A, display significantly perturbed binding affinity for CCR3 while retaining the ability to activate or partially activate the receptor. Finally, the mutant Phe(11) has little detectable activity and 20-fold reduced binding affinity relative to wild type eotaxin, the most dramatic effect observed in both assays but less dramatic than the effect of mutating the corresponding residue in some other chemokines. Taken together, the results indicate that residues contributing to receptor binding affinity and those required for triggering receptor activation are distributed throughout the N-terminal and N-loop regions. This conclusion is in contrast to the separation of binding and activation functions between N-loop and N-terminal regions, respectively, that has been observed previously for some other chemokines.     

Characterization of binding between the chemokine eotaxin and peptides derived from the chemokine receptor CCR3

The CC chemokine eotaxin plays a predominant role in eosinophil trafficking in vivo by specifically activating the chemokine receptor CCR3. We have screened a series of synthetic peptides corresponding to extracellular regions of CCR3 for their ability to bind eotaxin. A peptide corresponding to the N terminus of CCR3 (CCR3-(1-35)) bound to eotaxin with a dissociation constant of 80 +/- 38 micrometer. However, linear or cyclic peptides derived from the first and third extracellular loops of CCR3 did not bind to eotaxin. Linear and cyclic peptides derived from the second extracellular loop precipitated upon addition of eotaxin. (1)H-(15)N correlation NMR spectroscopy indicated that an extended groove in the eotaxin surface, whose edges are defined by the N-loop, 3(10)-helical turn, and beta(2)-beta(3) hairpin, is the most likely binding surface for CCR3-(1-35). NMR assignments for CCR3-(1-35) were obtained using two-dimensional and three-dimensional homonuclear NMR experiments. (15)N-Filtered TOCSY spectra indicated that the central region of CCR3-(1-35), surrounding the DDYY sequence, is involved in the interaction with eotaxin. This was supported by the observation that a truncated N-terminal peptide (CCR3-(8-23)) binds to eotaxin with a dissociation constant of 136 +/- 23 micrometer, only slightly weaker than the full-length N-terminal peptide. Taken together with previous studies, these results suggest that interactions between the N-loop/beta(3) regions of chemokines and the N-terminal regions of their receptors may be a conserved feature of chemokine-receptor complexes across the CC, CXC, and C chemokine subfamilies. However, the low affinity of the interactions observed in these studies suggests the existence of additional binding regions in both the chemokines and the receptors.     

Determination of the spatial structure of insectotoxin 15A from Buthus erpeus by (1)H-NMR spectroscopy data]

The solution structure of insectotoxin 15A (35 residues) from scorpion Buthus eupeus was determined on the basis of 386 interproton distance restraints 12 hydrogen-bonding restraints and 113 dihedral angle restraints derived from 1H NMR experiments. A group of 20 structures was calculated with the distance geometry program DIANA followed by the restrained energy minimization with the program CHARMM. The atomic RMS distribution about the mean coordinate position is 0.64 +/- 0.11 A for the backbone atoms and 1.35 +/- 0.20 A for all atoms. The structure contains an alpha-helix (residues 10-20) and a three-stranded antiparallel beta-sheet (residues 2-5, 24-28 and 29-33). A pairing of the eight cysteine residues of insectotoxin 15A was established basing on NMR data. Three disulfide bridges (residues 2-19, 16-31 and 20-33) connect the alpha-helix with the beta-sheet, and the fourth one (5-26) joins beta-strands together. The spatial fold of secondary structure elements (the alpha-helix and the beta-sheet) of the insectotoxin 15A is very similar to those of the other short and long scorpion toxins in spite of a low (about 20%) sequence homology.     

Tyrosine Sulfation of Chemokine Receptor CCR2 Enhances Interactions with Both Monomeric and Dimeric Forms of the Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)

Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1(T10C) was less effective at inducing leukocyte recruitment in vivo. In two-dimensional NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favor the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and β3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N terminus of CCR2 destabilizes the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.     

Human CC chemokine I-309, structural consequences of the additional disulfide bond

I-309 is a member of the CC subclass of chemokines and is one of only three human chemokines known to contain an additional, third disulfide bond. The three-dimensional solution structure of I-309 was determined by (1)H nuclear magnetic resonance spectroscopy and dynamic simulated annealing. The structure of I-309, which remains monomeric at high concentrations, was determined on the basis of 978 experimental restraints. The N-terminal region of I-309 was disordered, as has been previously observed for the CC chemokine eotaxin but not others such as MCP-1 and RANTES. This was followed in I-309 by a well-ordered region between residues 13 and 69 that consisted of a 3(10)-helix, a triple-stranded antiparallel beta-sheet, and finally a C-terminal alpha-helix. Root-mean-square deviations of 0.61 and 1.16 were observed for the backbone and heavy atoms, respectively. A comparison of I-309 to eotaxin and HCC-2 revealed a significant structural change in the C-terminal region of the protein. The alpha-helix normally present in chemokines was terminated early and was followed by a short section of extended strand. These changes were a direct result of the additional disulfide bond present in this protein. An examination of the I-309 structure will aid in an understanding of the specificity of this protein with its receptor, CCR8.     

Chemokine-binding specificity of soluble chemokine-receptor analogues: identification of interacting elements by chimera complementation

The specificity of chemokine-receptor interactions plays a central role in the regulation of leukocyte migration in inflammatory responses. Herein, we describe a soluble mimic of CC chemokine receptor 2 (CCR2), dubbed CROSS-N(2)E3(2), which incorporates the N-terminal region (N) and third extracellular loop (E3) elements of CCR2 displayed on the surface of a soluble protein scaffold. CROSS-N(2)E3(2) binds to the CCR2 ligand monocyte chemoattractant protein-1 (MCP-1) with a dissociation equilibrium constant of 1.1 +/- 0.1 microM but does not bind to the cognate chemokines of the receptor CCR3 (eotaxin-1, -2, and -3). Similarly, a soluble analogue of CCR3 (CROSS(5)-N(3)E3(3)) binds to eotaxin-1, -2, and -3 but not to MCP-1. Thus, these receptor analogues have the same specificity as the natural receptors. Using soluble proteins containing N and E3 elements from different receptors (CROSS-N(2)E3(3) and CROSS-N(3)E3(2)), we demonstrate that both receptor elements are required for optimal binding to the cognate chemokines. In addition, we report the binding affinities of all four CROSS proteins to a panel of two wild-type and six chimeric chemokines. These complementation studies indicate the regions of the chemokines that interact with each element of the receptors, allowing us to deduce the orientations of the receptor extracellular elements relative to the bound chemokines.     

Pi7, an orphan peptide from the scorpion Pandinus imperator: a 1H-NMR analysis using a nano-NMR Probe

The three-dimensional solution structure of a novel peptide, Pi7, purified from the venom of the scorpion Pandinus imperator, and for which no specific receptor has been found yet, was determined by two-dimensional homonuclear proton NMR methods from a nanomole amount of compound using a nano-nmr probe. Pandinus imperator peptide 7 does not block voltage-dependent K(+)-channels and does not displace labeled noxiustoxin from rat brain synaptosomal membranes. The toxin has 38 amino acid residues and, similarly to Pi1, is stabilized by four disulfide bridges (Cys6-Cys27, Cys12-Cys32, Cys16-Cys34, and Cys22-Cys37). In addition, the lysine at position 26 crucial for potassium-channel blocking is replaced in Pi7 by an arginine. Tyrosine 34, equivalent to Tyr36 of ChTX is present, but the N-terminal positions 1 and 2 are occupied by two acidic residues Asp and Glu, respectively. The dihedral angles and distance restraints obtained from measured NMR parameters were used in structural calculations in order to determine the conformation of the peptide. The disulfide-bridge topology was established using distance restraints allowing ambiguous partners between S atoms combined with NMR-derived structural information. The structure is organized around a short alpha-helix spanning residues Thr9 to Thr20/Gly21 and a beta-sheet. These two elements of secondary structure are stabilized by two disulfide bridges, Cys12-Cys32 and Cys16-Cys34. The antiparallel beta-sheet is composed of two strands extending from Asn22 to Cys34 with a tight turn at Ile28-Asn29 in contact with the N-terminal fragment Ile4 to Cys6.     

Comparison of the structure of vMIP-II with eotaxin-1, RANTES, and MCP-3 suggests a unique mechanism for CCR3 activation

Herpesvirus-8 macrophage inflammatory protein-II (vMIP-II) binds a uniquely wide spectrum of chemokine receptors. We report the X-ray structure of vMIP-II determined to 2.1 A resolution. Like RANTES, vMIP-II crystallizes as a dimer and displays the conventional chemokine tertiary fold. We have compared the surface topology and electrostatic potential of vMIP-II to those of eotaxin-1, RANTES, and MCP-3, three CCR3 physiological agonists with known three-dimensional structures. Surface epitopes identified on RANTES to be involved in binding to CCR3 are mimicked on the eotaxin-1 and MCP-3 surface. However, the surface topology of vMIP-II in these regions is markedly different. The results presented here indicate that the structural basis for interaction with the chemokine receptor CCR3 by vMIP-II is different from that for the physiological agonists eotaxin-1, RANTES, and MCP-3. These differences on vMIP-II may be a consequence of its broad-range receptor recognition capabilities.     

High level expression, activation, and antagonism of CC chemokine receptors CCR2 and CCR3 in Chinese hamster ovary cells

The specificity of leukocyte trafficking in inflammation is controlled by the interactions of chemokines with chemokine receptors. Reliable structure-function studies of chemokine-receptor interactions would benefit from cell lines that express consistent high levels of chemokine receptors. We describe herein two new Chinese hamster ovary (CHO) cell lines in which the genes for chemokine receptors CCR2 and CCR3 have been incorporated into identical positions in the host genome. CCR2 is the primary receptor for the chemokine monocyte chemoattractant protein-1 (MCP-1) whereas CCR3 is the primary receptor for the chemokines eotaxin-1, eotaxin-2 and eotaxin-3. Both receptors are expressed at >5,000,000 copies per cell, substantially higher levels than in previous cell lines, and both are competent for binding and activation by the cognate chemokines for these receptors. Using these cell lines we confirm that eotaxin-1 and eotaxin-3 can act as an agonist and an antagonist, respectively, of CCR2. In addition, we show that eotaxin-2 is an antagonist of CCR2 and MCP-1 is an agonist of CCR3. Comparison of the chemokine sequences reveals several positions that are identical in MCP-1 and eotaxin-1 but different in eotaxin-2 and eotaxin-3, suggesting that these amino acids play a role in CCR2 activation.     

Solution structure of the human CC chemokine 2: A monomeric representative of the CC chemokine subtype.

HCC-2, a 66-amino acid residue human CC chemokine, was reported to induce chemotaxis on monocytes, T-lymphocytes, and eosinophils. The three-dimensional structure of HCC-2 has been determined by 1H nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics calculations on the basis of 871 experimental restraints. The structure is well-defined, exhibiting average root-mean-square deviations of 0.58 and 0.96 A for the backbone heavy atoms and all heavy atoms of residues 5-63, respectively. In contrast to most other chemokines, subtle structural differences impede dimer formation of HCC-2 in a concentration range of 0.1 microM to 2 mM. HCC-2, however, exhibits the same structural elements as the other chemokines, i.e., a triple-stranded antiparallel beta-sheet covered by an alpha-helix, showing that the chemokine fold is not influenced by quaternary interactions. Structural investigations with a HCC-2 mutant prove that a third additional disulfide bond present in wild-type HCC-2 is not necessary for maintaining the relative orientation of the helix and the beta-sheet.     

Thermodynamic characterization of interleukin-8 monomer binding to CXCR1 receptor N-terminal domain

Chemokines elicit their function by binding receptors of the G-protein-coupled receptor class, and the N-terminal domain (N-domain) of the receptor is one of the two critical ligand-binding sites. In this study, the thermodynamic basis for binding of the chemokine interleukin-8 (IL-8) to the N-domain of its receptor CXCR1 was characterized using isothermal titration calorimetry. We have shown previously that only the monomer of IL-8, and not the dimer, functions as a high-affinity ligand, so in this study we used the IL-8(1-66) deletion mutant which exists as a monomer. Calorimetry data indicate that the binding is enthalpically favored and entropically disfavored, and a negative heat capacity change indicates burial of hydrophobic residues in the complex. A characteristic feature of chemokine receptor N-domains is the large number of acidic residues, and experiments using different buffers show no net proton transfer, indicating that the CXCR1 N-domain acidic residues are not protonated in the binding process. CXCR1 N-domain peptide is unstructured in the free form but adopts a more defined structure in the bound form, and so binding is coupled to induction of the structure of the N-domain. Measurements in the presence of the osmolyte, trimethylamine N-oxide, which induces the structure of unfolded proteins, show that formation of the coupled N-domain structure involves only small DeltaH and DeltaS changes. These results together indicate that the binding is driven by packing interactions in the complex that are enthalpically favored, and are consistent with the observation that the N-domain binds in an extended form and interacts with multiple IL-8 N-loop residues over a large surface area.     

Recognition of RANTES by extracellular parts of the CCR5 receptor

The chemokine RANTES (regulated upon activation, normal T-cell expressed and secreted) is a natural ligand of CCR5, one of the major HIV-1 coreceptors. It is secreted as part of the immune response to human immunodeficiency virus 1 (HIV-1) and inhibits infection by CCR5-dependent (R5) HIV-1 isolates. We have investigated the interaction of RANTES with several peptides derived from the extracellular domains of CCR5 by heteronuclear NMR spectroscopy in aqueous solution. We show that a peptide comprising the first 25 amino acid residues of the CCR5 N-terminal domain and sulfated at the Y10 and Y14 side-chains binds with micromolar affinity exclusively to the monomeric form of RANTES. In contrast to the tight binding of the sulfated peptide, the affinity of the same peptide in non-sulfated form was reduced by more than two orders of magnitude. Peptides derived from the CCR5 extracellular loops ECL1, ECL2 and ECL3 showed only very moderate and mostly non-specific binding. Chemical shift mapping of the interaction of the sulfated N-terminal peptide reveals a contiguous binding surface on RANTES, which comprises amino acid residues of the first beta-strand, the N-loop, the fourth beta-strand and the turns around residues 30 and 40. This binding surface largely overlaps with the dimer interface and is strongly positively charged, providing a rationale for the exclusive binding of the monomer to the peptide and the requirement of the negative sulfate groups at the Y10 and Y14 side-chains. The binding surface also largely overlaps with the segments that were identified previously as crucial for HIV blockade by peptide scanning and mutagenesis studies. These data offer new insights into the structure-function relation of the RANTES-CCR5 interaction and may be helpful for the design of novel HIV-1 inhibitors.     

Solution structure of Eucommia antifungal peptide: a novel structural model distinct with a five-disulfide motif

The three-dimensional structure in aqueous solution of Eucommia antifungal peptide 2 (EAFP2) from Eucommia ulmoides Oliv was determined using (1)H NMR spectroscopy. EAFP2 is a newly discovered 41-residue peptide distinct with a five-disulfide cross-linked motif. This peptide exhibits chitin-binding activity and inhibitory effects on the growth of cell wall chitin-containing fungi and chitin-free fungi. The structure was calculated by using torsion angle dynamic simulated annealing with a total of 614 distance restraints and 16 dihedral restraints derived from NOESY and DQF-COSY spectra, respectively. The five disulfide bonds were assigned from preliminary structures using a statistical analysis of intercystinyl distances. The solution structure of EAFP2 is presented as an ensemble of 20 conformers with a backbone RMS deviation of 0.65 (+/-0.13) A for the well-defined Cys3-Cys39 segment. The tertiary structure of EAFP2 represents the first five-disulfide cross-linked structural model of the plant antifungal peptide. EAFP2 adopts a compact global fold composed of a 3(10) helix (Cys3-Arg6), an alpha-helix (Gly26-Cys30), and a three-strand antiparallel beta-sheet (Cys16-Ser18, Tyr22-Gly24, and Arg36-Cys37). The tertiary structure of EAFP2 shows a chitin-binding domain (residues 11-30) with a hydrophobic face and a characteristic sector formed by the N-terminal 10 residues and the C-terminal segment cross-linked through the unique disulfide bond Cys7-Cys37, which brings all four positively charged residues (Arg6, Arg9, Arg36, and Arg40) onto a cationic face. On the basis of such a structural feature, the possible structural basis for the functional properties of EAFP2 is discussed.     

Recognition of a CXCR4 sulfotyrosine by the chemokine stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12)

Tyrosine sulfation of the chemokine receptor CXCR4 enhances its interaction with the chemokine SDF-1alpha. Given similar post-translational modification of other receptors, including CCR5, CX3CR1 and CCR2b, tyrosine sulfation may be of universal importance in chemokine signaling. N-terminal domains from seven transmembrane chemokine receptors have been employed for structural studies of chemokine-receptor interactions, but never in the context of proper post-translational modifications known to affect function. A CXCR4 peptide modified at position 21 by expressed tyrosylprotein sulfotransferase-1 and unmodified peptide are both disordered in solution, but bind SDF-1alpha with low micromolar affinities. NMR and fluorescence polarization measurements showed that the CXCR4 peptide stabilizes dimeric SDF-1alpha, and that sulfotyrosine 21 binds a specific site on the chemokine that includes arginine 47. We conclude that the SDF-1alpha dimer preferentially interacts with receptor peptide, and residues beyond the extreme N-terminal region of CXCR4, including sulfotyrosine 21, make specific contacts with the chemokine ligand.