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Protein Science (2004), 13:2022-2028. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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NMR structure of CXCR3 binding chemokine CXCL11 (ITAC)

Valerie Booth1,3, Ian Clark-Lewis2,4 and Brian D. Sykes1

1 Protein Engineering Network of Centres of Excellence (PENCE) and University of Alberta, Edmonton, Alberta, T5G 2S2, Canada
2 University of British Columbia, Biomedical Research Centre and Department of Biochemistry and Molecular Biology, Vancouver, British Columbia, V6T 1Z3, Canada

Reprint requests to: Brian D. Sykes, University of Alberta, 713 HMRC, Edmonton, AB, T5G 2S2, Canada; e-mail: brian.sykes{at}ualberta.ca; fax: (780) 492-0886.

(RECEIVED April 5, 2004; FINAL REVISION May 13, 2004; ACCEPTED May 14, 2004)


   Abstract
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 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
CXCL11 (ITAC) is one of three chemokines known to bind the receptor CXCR3, the two others being CXCL9 (Mig) and CXCL10 (IP-10). CXCL11 differs from the other CXCR3 ligands in both the strength and the particularities of its receptor interactions: It has a higher affinity, is a stronger agonist, and behaves differently when critical N-terminal residues are deleted. The structure of CXCL11 was determined using solution NMR to allow comparison with that of CXCL10 and help elucidate the source of the differences. CXCL11 takes on the canonical chemokine fold but exhibits greater conformational flexibility than has been observed for related chemokines under the same sample conditions. Unlike related chemokines such as IP-10 and IL-8, ITAC does not appear to form dimers at millimolar concentrations. The origin for this behavior can be found in the solution structure, which indicates a {beta}-bulge in {beta}-strand 1 that distorts the dimerization interface used by other CXC chemokines.

Keywords: ITAC; CXCL11; structure; NMR; chemokine

3 Present address: Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, A1B 3X7, Canada. Back

4 Deceased. Back

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04791404.


   Introduction
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 Abstract
 Introduction
Results
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ITAC (CXCL11) is a member of the chemokine family of small secretory proteins involved in immune and inflammatory responses. Chemokines play a key role in these processes by promoting recruitment and activation of different subpopulations of leukocytes (Mackay 2001). They fall into two main subfamilies based on the arrangement of the first two of four conserved cysteines, adjacent in CC chemokines and separated by one amino acid in CXC chemokines (Fernandez and Lolis 2002). Chemokines exert their effects by interacting with an appropriate seven-transmembrane G protein-coupled receptor on the surface of a leukocyte (Rojo et al. 1999). Knowledge of the structural basis for the interaction of chemokines with their receptors is of significant importance to understanding chemokine function and to the design of therapeutic interventions for a number of pathologies that involve chemokines (Power and Proudfoot 2001).

The receptors follow a similar nomenclature to the chemokines, with CC receptors generally interacting with CC chemokines and CXC receptors generally with CXC chemokines, although there are some examples of binding across families. ITAC binds to and activates the receptor CXCR3, as do IP-10 (CXCL10) and Mig (CXCL9; Loetscher et al. 1998). While ITAC, IP-10, and Mig are agonists for CXCR3, they can also act as antagonists for CCR3 (Loetscher et al. 2001). As well as binding to the same receptors, ITAC, IP-10, and Mig also show similarities to each other in that they share an individual branch of the phylogenetic tree (O’Donovan et al. 1999), are induced primarily by IFN-{gamma} and are produced by macrophages as well as other cell types (Farber 1997; Cole et al. 1998; Laich et al. 1999; Tensen et al. 1999). However, ITAC also shows a number of functional differences from IP-10 and Mig.

ITAC has higher binding affinity for CXCR3 than either IP-10 or Mig, and is a more potent activator of CXCR3 (Meyer et al. 2001; Sauty et al. 2001; Clark-Lewis et al. 2003). ITAC is also a more potent antagonist to CCR3 than IP-10 or Mig (Loetscher et al. 2001). Structure–activity studies have shown that if its first three residues are deleted, ITAC retains significant binding affinity but loses the ability to activate CXCR3. By contrast, deletion of the N-terminal few residues of IP-10, Mig, or eotaxin (an agonist of CCR3) causes these proteins to lose binding capacity (Proost et al. 2001; Clark-Lewis et al. 2003). N-terminal deletions of ITAC are physiologically relevant, as the peptidase DPP-IV has been found to cleave the N-terminal two residues of ITAC, IP-10, and Mig in vivo, and thus regulates their effects on CXCR3 (Proost et al. 2001; Ludwig et al. 2002). Interestingly, the time scale observed for DPP-IV cleavage is significantly faster for ITAC than for IP-10 or Mig (Proost et al. 2001). We have previously determined the structure of IP-10 (Booth et al. 2002), and were interested in determining the structure of ITAC, in part to look for clues as to the origin of the observed differences in binding between the two proteins.


   Results
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 Abstract
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The majority of chemokines studied have been observed to form dimers at mM concentrations (Baggiolini et al. 1997), which is not ideal for NMR studies, especially NMR studies of a protein without 13C and/or 15N isotope labels. Previous NMR studies of chemokines have made use of an NH to N-CH3 mutation in one of the residues of each monomer,along the dimer interface. N-methyl mutations of leucine 27 of IP-10 (Booth et al. 2002), leucine 25 in IL-8 (Rajarathnam et al. 1995), and valine 26 of MGSA (Rajarathnam et al. 1997) all disrupted dimerization without affecting the structure of the protein. Hence, we first decided to make the analogous mutation in ITAC. Surprisingly, N-methyl mutation of alanine 27 lead to unfolding of the protein, as judged by the lack of dispersion observed in proton NMR spectra (data not shown). Thus, the NMR investigation presented herein proceeded with wild-type ITAC, rather than the mutant.

To provide information about the oligomerization state of ITAC, the 1H transverse relaxation rate of wild-type ITAC was measured using a jump and return spin-echo pulse sequence (Plateau and Gueron 1982; Gueron et al. 1992) and found to be T2 = 34 msec at 30°C (data not shown). The T2 value can be used to estimate the apparent molecular weight of the protein using the following relations for correlation time: {tau}c {approx} 1/5T2 (Anglister et al. 1993) and {tau}c {approx} MW/2 (Booth et al. 2002). This gives an apparent molecular weight of about 11 kDa, and indicates that the wild-type protein (MW = 8.3 kDa) is mostly monomeric.

Initial studies of ITAC proceeded with a sample of wild-type ITAC that contained no isotope labels. 2D homo-nuclear TOCSY and NOESY spectra were collected at 30°C and 40°C and pH 5. Difficulties in completing the frequency assignments from this data were encountered due to spectral overlap and apparent conformational heterogeneity. A second ITAC sample was synthesized that contained 15N isotope labeled leucine (four residues) and valine (five residues). The new sample was analyzed by 2D 15N-1H-HSQC, a spectrum that would be expected to show nine peaks (one for each 15N labeled residue) if the sample is conformationally homogeneous. At pH 5 and temperature 30°C, the peaks in the HSQC appear doubled and in some cases tripled, indicating two or three different conformations exist under these conditions (Fig. 1AGo). At pH 5 and a temperature of 40°C, there is much less conformational heterogeneity, but minor peaks are still apparent (Fig. 1BGo). 15N-1H-HSQC spectra were then acquired at a range of temperatures from 5°C to 45°C and pH from 4.5 to 6.3 in an attempt to find conditions where ITAC was structurally homogeneous. Lower temperature and higher pH caused increases in conformational heterogeneity. At a temperature of 45°C and pH 4.5, ITAC appeared to be primarily in one conformation (Fig. 1CGo) and additional NMR spectra (2D NOESY and TOCSY, 3D 15N-edited NOESY) were acquired under these conditions. At this point, it became possible to determine the frequency assignments for residues 9 to 70 of the major conformation of ITAC under all three sample conditions. However, even at a temperature of 45°C, pH 4.5, ITAC still showed evidence of conformational heterogeneity, in that residues 43, 51, and 52 possessed more than one set of resonances.



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  		Figure 1. 15N-1H-HSQC spectra of partially 15N-labeled ITAC1–73 (all L,V 15N-labeled for a total of nine labeled residues) with conditions (A) pH 5.0, 30°C; (B) pH 5.0, 40°C; and (C) pH 4.5, 45°C. Resonance assignments are indicated on C.

 
Structure calculations made use of data from all three sample conditions in the initial stages; however, only data from the 45°C, pH 4.5 spectra were used in the final refinement, as detailed in Materials and Methods. The statistics for the structure calculation are shown in Table 1Go, and a summary of the NOEs identified per residue is shown in Figure 2Go. An ensemble of calculated structures that fulfill the experimentally derived restraints is shown in Figure 3Go. The structure exhibits the canonical chemokine fold with a three-stranded antiparallel {beta}-sheet packed against an {alpha}-helix, a long N-terminal loop stabilized by two disulfide bonds, and one turn of a 310 helix just N-terminal to the first {beta}-strand. Overall, the resolution of the final ITAC structure is somewhat lower than what has been obtained for other chemokines solved by similar methods, as judged by the backbone RMSD and the number of residues found in allowed regions of the Ramachandran plot.


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  		Table 1. Structural statistics for the ensemble of 10 calculated structures
 


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  		Figure 2. Number of unambiguous (A) and ambiguous (B) interresidue NOEs per residue. NOEs are classed as long (>5 residue difference), medium, and sequential. Unambiguous NOEs are those that could be unambiguously assigned by the end of the ARIA structure determination, and ambiguous NOEs are those that could not be unambiguously assigned by the end of the structure determination but were included as ambiguous restraints. Hence, for the ambiguous NOEs, the contribution of each possible assignment is displayed, resulting in more NOEs being displayed than were necessarily used in the structure calculation.

 


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  		Figure 3. Structure of ITAC. (A) Backbone trace of the ensemble of 10 lowest energy calculated structures. (B) Representative structure in a ribbon representation with secondary structure elements and cysteine residues labeled. (C) 90° rotation of B. (D) Hydrogen bonds in the {beta}-bulge region with H-N bonds shown in blue, and C = O bonds shown in red. Residues 8–71 only are shown in figures A–C. A and D were produced using MOLMOL (Koradi et al. 1996). B and C were produced using MolScript (Kraulis 1991) and Raster3D (Merritt and Bacon 1997).

 
Although the three {beta}-strands and {alpha}-helix are reasonably well defined in the structure, the calculated structures display a higher degree of variability in the region N-terminal to the 310 helix, termed the N-loop region, and most of the loops between secondary structure elements (see ensemble in Fig. 3AGo and RMSD values in Table 1Go). The N-terminal eight residues of ITAC could not be assigned, and as in other chemokine structures, this region is assumed to be unstructured unless bound to the receptor (Booth et al. 2003). The observed disorder in the N-loop region and the loop connecting {beta}-strand 3 to the {alpha}-helix of ITAC is greater than what has been observed in previously determined chemokine structures where these structural elements have been better defined (see Fernandez and Lolis (2002) for a list of chemokine structures). For the loops of ITAC between {beta}-strands 2 and 3 and between {beta}-strand 3 and the {alpha}-helix, this variability can be attributed to the lack of observable NOEs in these regions (Fig. 2Go), which suggest these residues do not take on a defined structure. The story is somewhat different for the N-loop region in that a significant number of NOEs were observed in this region (Fig. 2Go), although fewer long-range NOEs than were observed for the {beta}-sheet. The N-loop and the loop between {beta}-strand 3 and the {alpha}-helix are proximal to {beta}-strand 3, which possess two residues for which multiple sets of resonances could be assigned, indicating that these two residues take on more than one distinct conformation. These observations are suggestive of increased mobility in this region of the protein, although inferring dynamics based solely on NOE observations and multiple chemical shift assignments is potentially misleading. However, studies have shown that NMR structure ensembles can reflect mobility of the protein in that the conformational space sampled by the ensembles overlaps well with the conformational space defined using molecular dynamics simulations (Abseher et al. 1998).

The observed NOEs and the pattern of hydrogen bonds indicated by the calculated structures suggested that the N-terminal section of the first {beta}-strand contains a {beta}-bulge with residue 44 hydrogen bonding to both residue 25 (i.e., T44 C = O•••H-N E25) and 26 (i.e., T44 N-H•••O = C K26) (Fig. 2BGo). The hydrogen bonding, side-chain orientation and dihedral angle pattern matches the C-(15) bulge with residue X = T44, residue 1 =E25, residue 2=K26 described in Chan et al. (1993). This distortion helps explain why the N-methyl mutation of alanine 27 lead to unfolding of the protein, as will be discussed further in the next section.


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Numerous structure–activity studies of chemokines have shown that they possess two main sites that interact with their receptors (Clark-Lewis et al. 1995; Loetscher and Clark-Lewis 2001; Fernandez and Lolis 2002), and have led to a two-step model for the interaction, in which receptor binding and activation are dissociated. In the initial event, the N-loop region of the chemokine, comprised of the segment between the second cysteine residue and the 310- helix, recognizes and binds the receptor ("docking"). This initial contact facilitates subsequent binding and proper positioning of the flexible N-terminal region of the chemokine to the receptor, which leads to its activation ("triggering"). The region of the receptor responsible for chemokine docking has been localized to residues 22 to 42 of CXCR3 (Rajarathnam et al. 1995; Booth et al. 2002) located on the extracellular side of the membrane, N-terminal to the first transmembrane helix. A study using CXCR1/CXCR3 chimeras identified extracellular loops 1 and 2 of CXCR3 as being essential for triggering of CXCR3 by ITAC (Xanthou et al. 2003).

Figure 4AGo presents the two-step binding model with a rudimentary molecular model of the structure of CXCR3 bound to a ITAC. This is not presented as a rigorous molecular model, but rather to portray the character and proper scaling of the interaction in a more realistic way than can be done by a simple, conceptual line drawing. In particular, it should be noted that while mutagenic studies of receptor often suggest a number of receptor "sites" involved in triggering, the spatial constraints of the receptor are such that it is probably more realistic to visualize the interaction surface, not as a set of discrete sites, but rather as one contiguous binding site formed by contributions from one or more loops and possibly some of the helical residues of the receptor.



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  		Figure 4. Binding of ITAC to CXCR3. (A) Two-step model of ITAC-receptor interaction (see text for details). The rudimentary receptor model was produced by first aligning the sequence of CXCR3 to the GPCR consensus sequence (Baldwin et al. 1997), building the transmembrane helices using the structures 1F88 [PDB] and 1L9H [PDB] as templates and then using SwissPDBViewer (Guex and Peitsch 1997) to add in the loops. ITAC was positioned with respect to the receptor using SwissPDBViewer (Guex and Peitsch 1997). The receptor is shown primarily in gray, with red indicating the position of residues 22–42, which correspond to the segment of CXCR3 known to contain the residues that bind to the N-loop region of chemokines. The chemokine is shown in green, with residues 12–17 of the N-loop region colored cyan and the N-terminal 8 residues colored in yellow. (B) Electrostatic surface representation of IP-10 with the approximate location of the CXCR3 N terminus shown as a thick, red line (Booth et al. 2002). (C) Electrostatic surface representation of ITAC. In B and C, disordered residues 1–7 are omitted and residue 8 is labeled to show where these omitted residues would be located. The N-loop residues thought to be most critical for binding to CXCR3 in step 1 (12–17) and two extra residues of IP-10 (19,38) shown in previous studies (Booth et al. 2002) to contact CXCR3 are also labeled. All panels were made using MOLMOL (Koradi et al. 1996).

 
One study has indicated that loops 1 and 2 of CXCR3 appear to be essential for activation by ITAC, while slightly different patterns were observed for the other CXCR3 binding chemokines, with IP-10 triggering requiring loops 1, 2, and 3, and Mig triggering requiring loops 2 and 3 only (Xanthou et al. 2003). There are additional indications that the nature of ITAC’s receptor interaction is somewhat different from the other CXCR3 binding chemokines. ITAC has the highest affinity for CXCR3, and is also the most potent agonist (Meyer et al. 2001; Sauty et al. 2001; Clark-Lewis et al. 2003). Deletion of the N-terminal three residues of ITAC results in a potent antagonist that competes for binding of CXCR3 with the full-length protein. In contrast, deletion of the N-terminal three residues of IP-10 or Mig leads to loss of binding capacity (Clark-Lewis et al. 2003), indicating that the N-loop region alone is not sufficient for receptor binding. IP-10/ITAC hybrids in which residues 12–17 in the N-loop of IP-10 are replaced with residues 12–17 of ITAC show comparable binding to wild-type ITAC, indicating that much of the additional binding strength of ITAC originates in this region of the chemokine (Clark-Lewis et al. 2003). This lead us to compare the binding surfaces of ITAC and IP-10 to see if the higher affinity of ITAC could be rationalized based on the experimentally determined structures.

In a previous study, we identified regions of IP-10 that interact with the N terminus of CXCR3 and defined a path on the N-loop face of IP-10 along which the receptor lies (Booth et al. 2002; Fig. 4BGo). This path includes a hydrophobic patch formed by residues 12–17 of the N-loop, as well as notches along either side, in which parts of the receptor may lie. The N-loop face of ITAC shows a similar character, with the 12–17 region forming a surface significantly more hydrophobic than that of IP-10. The increased hydrophobicity in this critical region may underlie the higher affinity of CXCR3 for ITAC than for IP-10 or Mig.

The NMR studies suggested that the N-loop, the third {beta}-strand, and the loop between this strand and the {alpha}-helix are more flexible than the opposite face of ITAC and also more flexible than has been observed in other chemokines. Because these apparently more mobile structural elements are all located on the CXCR3 binding face of ITAC, this observation brings up the possibility that increased flexibility of this region is related to ITAC’s greater binding affinity.

The structural analysis of ITAC lead to the identification of an additional quirk of ITAC—a {beta}-bulge on the first {beta}-strand (Fig. 3DGo). This bulge can only be tentatively identified on the basis of the structure alone but is supported by two additional observations: (1) HN to N-CH3 mutation of alanine 27 leads to unfolding of the protein; (2) ITAC shows less of a propensity to dimerize than other CXC chemokines. The bulge causes an exaggeration of the normal twist in strand 1. This distortion leads to a partial burying of the HN group of alanine 27, which would explain why the HN to methyl mutation of this residue led to unfolding. CXC chemokines often dimerize along this{beta}-strand by forming interchain{phi} {beta}-sheet interactions, and so distortion of this region is consistent with our observation that ITAC has less propensity to dimerize than other chemokines, such as IP-10 (Booth et al. 2002). Although most chemokines appear to dimerize at the high concentrations used in structural studies, they are monomeric at physiological concentrations (Clark-Lewis et al. 1995; Fernandez and Lolis 2002) and a monomeric mutant of IL-8 was found to be fully functional in in-vivo assays of receptor binding and activation (Rajarathnam et al. 1994). Thus, the functional significance of ITAC being monomeric at high concentrations is not obvious.


   Materials and methods
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Sample preparation
Protein with the sequence of human ITAC 1–73 was synthesized using either natural isotope abundance for all amino acids, or with 15N isotope labeled leucine and valine. ITAC was synthesized by stepwise solid-phase methods using t-butoxylcarbonyl protection chemistry. After hydrogen fluoride deprotection, the polypeptide was folded and purified from reverse-phase HPLC as described previously (Clark-Lewis et al. 1997). The mass spectroscopy results agreed with the expected MWs for all samples: observed MW for ITAC (1–73) was 8303.14 ± 1.00; observed MW for ITAC NMeAla 27 was 8316.42 ± 0.64; the observed MW for N15 Leu and Val ITAC was 8312.50. The samples were lyophilized and stored at 4°C.

NMR spectroscopy and structure calculation
For NMR data collection, the sample was dissolved in buffer containing 20 mM deuterated sodium acetate, 1 mM 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS), 1 mM sodium azide, 90% H2O/10% D2O, or 99.90% D2O and adjusted to pH 4.5, 5.0 or 6.3. The concentration of ITAC was 2.0 mM.

NMR spectra used in resonance frequency and NOE assignment were collected on the NANUC Varian Inova 800 MHz spectrometer with three different sample conditions: condition 1, pH 5.0, temperature 30°C; condition 2, pH 5.0, temperature 40°C; and condition 3, pH 4.5, temperature 45°C. NMR data were processed using NMRPipe software (Delaglio et al. 1995), and spectral analysis was assisted using the program NMRView (Johnson and Blevins 1994). Assignment of IP-10 proton resonances was achieved using homonuclear NOESY (mixing time 120 msec) and TOCSY (mixing time 44 msec) spectra. Frequency assignments for residues 9–70 were obtained for the protein under all three sample conditions.

Hydrogen bond restraints were defined for secondary structural elements where clearly indicated by NOE patterns and 3JNH-H{alpha} values as measured by DQ-COSY. Hydrogen bond restraints were added as H•••O = 2.5 Å and N•••O = 3.5 Å in an antiparallel pattern between strands 1 and 2 (26O to 30O on strand 1, 40N to44N on strand 2) and between strands 2 and 3 (41N to 43N on strand 2, 50O to 52O on strand 3), and in an {alpha}-helical pattern for residues 61 to 69. Dihedral angle restraints were set at {phi} = –120 ± 30° {varphi} = 120 ± 40° for residues 26–31, 38–48, and 51–54, and {phi} = –60 ± 30° {varphi} = 120 ± 40° for residues 61 to 70.

These structure calculations were performed using CNS version 1.1 (Brunger et al. 1998) with ambiguous restraints for iterative assignment (ARIA; Nilges et al. 1997). Initial input to ARIA included 188 manually assigned NOE derived distance restraints (112 sequential, 34 medium, 42 long), dihedral angle restraints, and hydrogen bond restraints. The structures used in the initial iteration were four different model structures of ITAC generated by Swiss Model (Guex and Peitsch 1997; Schwede et al. 2003) based on the structures of IP-10 (PDB ID: 1LV9 [PDB] ), NAP-2 (PDB ID: 1NAP [PDB] ), GRO-A (PDB ID: 1MGS [PDB] ), and MIP-2 (PDB ID: 1MI2 [PDB] ). These model structures sampled a wide variety of conformational space (backbone RMSD to mean for residues 9–70 was 1.7 Å) upon which to base the initial NOE assignments. Also entered were frequency assignments and NOESY peak lists from all three conditions. Cross-peaks in the NOESY spectra were picked using AUTOPSY (Koradi et al. 1998). The frequency window tolerance for assigning NOEs with ARIA was 0.015 ppm. To reduce the number of mis-assigned peaks in the initial stages of the structure calculation, at each ARIA step, the ARIA-generated NOE restraints were filtered to retain only those restraints observed in at least two out of the three NOESY spectra. The ARIA parameter p was varied from 0.99 in the initial iteration to 0.80 in the final iteration, and {nu}tol went from 1.0 to 0.25 Å. In the final iteration, only NOEs derived from the best data set (with conditions pH 4.5 and temperature 45°C) were used. These structures were refined using the water refinement routine in ARIA. The final structures were based on 678 unambiguous restraints, 257 ambiguous restraints, plus the hydrogen bond and dihedral angle restraints detailed above (see Table 1Go for structural statistics). Twenty structures were refined and the lowest energy ten of these were retained for analysis. The co-ordinates, restraints and resonance assignments have been deposited in the Protein Data Bank (PDB ID: 1RJT [PDB] ) and the BioMagResBank (BMRB accession no. 6038 [BMRB] ).


   Acknowledgments
 
This work was funded by a grant from the PENCE. V.B. is supported by fellowships from the Canadian Institutes of Health Research (CIHR) and the Alberta Heritage Fund for Medical Research (AHFMR). We acknowledge the Canadian National High Field NMR Centre (NANUC) for their assistance and the use of the facilities. Operation of NANUC is funded by CIHR, the Natural Science and Engineering Research Council of Canada (NSERC), and the University of Alberta. Thanks also to Jan Rainey for help in preparing the CXCR3 receptor model, and Steffen Graether for comments on the manuscript.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


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