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Originally published In Press as doi:10.1074/jbc.M003386200 on June 8, 2000

J. Biol. Chem., Vol. 275, Issue 35, 26799-26805, September 1, 2000
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NMR Studies of Active N-terminal Peptides of Stromal Cell-derived Factor-1

STRUCTURAL BASIS FOR RECEPTOR BINDING*

Elena L. ElisseevaDagger, Carolyn M. SlupskyDagger, Matthew P. Crump§, Ian Clark-Lewis, and Brian D. Sykes||

From the Protein Engineering Network of Centers of Excellence and the Department of Biochemistry, 713 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

Received for publication, April 20, 2000, and in revised form, June 7, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stromal cell-derived factor 1 (SDF-1), a member of the CXC chemokine family, is the only chemokine to bind to the receptor CXCR4. This receptor is also a co-receptor for syncytia-inducing forms of HIV in CD4+ cells. In addition, SDF-1 is responsible for attracting mature lymphocytes to the bone marrow and can therefore contribute to host versus graft rejection in bone marrow transplantation. Clearly, by manipulating SDF-1 activity, we could find a possible anti-viral AIDS treatment and aid in bone marrow transplantation. SDF-1 binds to CXCR4 primarily via the N terminus, which appears flexible in the recently determined three-dimensional structure of SDF-1. Strikingly, short N-terminal SDF-1 peptides have been shown to have significant SDF-1 activity. By using NMR, we have determined the major conformation of the N terminus of SDF-1 in a 17-mer (residues 1-17 of SDF-1) and a 9-mer dimer (residues 1-9 of SDF-1 linked by a disulfide bond at residue 9). Residues 5-8 and 11-14 form similar structures that can be characterized as a beta -turn of the beta -alpha R type. These structural motifs are likely to be interconverting with other states, but the major conformation may be important for recognition in receptor binding. These results suggest for the first time that there may be a link between structuring of short N-terminal chemokine peptides and their ability to activate their receptor. These studies will act as a starting point for synthesizing non-peptide analogs that act as CXCR4 antagonists.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemokines are an important class of proteins in the immune system that act to recruit leukocytes to sites of inflammation and infection by interacting with specific receptors on the cell surface of their target cells (for reviews see Refs. 1 and 2). In addition, chemokine receptors are coupled to several heterotrimeric G-proteins in natural killer cells that can recognize and kill transformed or infected cells (3). Chemokines have been implicated in auto-immune diseases and allergic disorders (reviewed in Refs. 4 and 5), and several chemokine receptors are necessary cofactors that permit HIV-11 cell entry (6-13). Human chemokines are approximately 70-80 residues in length and share substantial sequence and structural similarity (14). There are two major classes of chemokines, the CC chemokines (RANTES, MCP-1, MIP-1alpha , and MIP-1beta ) and the CXC chemokines (IL-8, NAP-2, MGSA, and SDF-1), so named because of the spacing between the cysteine residues near the N terminus of these proteins.

Stromal cell-derived factor-1 (SDF-1) is a member of the CXC chemokine family and is expressed constitutively in a broad range of tissues. SDF-1 has a fundamental role in trafficking, export, and homing of bone marrow cells (15). Tissue distribution of SDF-1 suggests that it may have a role in immune surveillance rather than inflammation (16). SDF-1 has exceptionally strong sequence conservation between species (17) and is the only known natural ligand for the CXC chemokine receptor 4 (CXCR4) (8). The recently solved structure of SDF-1 (18) reveals that it has a global fold similar to other chemokines with a flexible N-terminal region followed by a loop, three antiparallel beta -strands, and one C-terminal alpha -helix. Interest in SDF-1 has grown since CXCR4 was identified as a co-receptor for syncytia-inducing forms of HIV in CD4+ T-cells. Through interaction with CXCR4, SDF-1 inhibits replication of the syncytia-inducing form (T-tropic) of HIV-1 (7, 8). SDF-1 also appears to be important for attracting mature lymphocytes to the bone marrow. Antagonism of this function before the harvest of the bone marrow for transplantation could be clinically beneficial (16). In addition, SDF-1 receptors are coupled to multiple G-proteins that may be important for initiating motility of natural killer cells (3). Thus, SDF-1 plays an important role in mobilizing the immune system and may be important for the treatment of AIDS patients and in bone marrow transplantation. A low molecular weight antagonist for SDF-1 could provide a possible therapeutic in these areas.

It has recently been observed in the CXC class of chemokines that important residues for receptor binding are at the N terminus and the loop region (RFFESH) following the two disulfide bridges (14, 15, 18-22), with the N terminus being the most critical receptor binding site (14). It is therefore tempting to suggest that the N terminus alone could be sufficient for binding activity. These two sites appear unstructured in the solution structure of SDF-1 (18). However, short N-terminal peptides of SDF-1 were found to have SDF-1 activity (15). This is a very striking and important observation. Several sequences corresponding to residues 1-8, 1-9 monomer, 1-9 dimer, and 1-17 all bind to CXCR4 and induce intracellular calcium release and chemotaxis in T lymphocytes. It was determined that the 1-17 and 1-9 dimer peptides were similar in terms of receptor binding, whereas the 1-8 and 1-9 monomer peptides had significantly lower affinity. The 1-9 dimer had the greatest activity of all the peptides tested when compared with native SDF-1 with the 1-17, 1-9 monomer, and 1-8 peptides showing decreasing activity. The basis for the enhanced activity of the 1-9 dimer remains uncertain. Finally, P2G, an SDF 1-9 analog, attained binding similar to that of the 1-9 dimer yet acted as a receptor antagonist (15).

Although in the solution structure of SDF-1, the N-terminal region has significant flexibility (18), it is of interest to see if there is a significantly populated conformation of these peptides that might mimic the receptor bound conformation. We present here structural data on the 1-9 dimer as well as the 1-17-mer obtained using NMR spectroscopy at 8 °C. Although the 1-9 dimer and 1-17-mer peptides are conformationally flexible, analysis of the ensemble of structures calculated from the NMR data revealed a major family that consists of a beta -turn structural motif. This motif was not detected in the structure of native SDF-1. These data support the structuring of the peptides into turns that may be important for recognition in receptor binding. By understanding the structural elements necessary for receptor binding, we hope to be able to develop therapeutics that are more cost-effective mimics of the peptide itself.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peptide Synthesis-- The N-terminal fragments of SDF-1 1-9, KPVSLSYRC, and 1-17, KPVSLSYRCPCRFFESH, were synthesized by solid phase peptide synthesis and purified by reverse phase HPLC. The 1-9 dimer was prepared by oxidizing the 1-9 monomer peptide under dilute conditions in 100 mM ammonium bicarbonate buffer at pH 8.5. The solution was magnetically stirred for 24 h and then lyophilized. Verification of complete oxidation was indicated by reverse phase HPLC, electrospray mass spectroscopy, and NMR spectroscopy.

NMR Sample Preparation-- The samples were prepared by dissolving each peptide in 500 µl of 90% H2O/10% D2O or 99.9% D2O, containing 20 mM CD3COO-Na+ and 1 mM NaN3, to a concentration of approximately 5 mM. 2,2-Dimethyl-2-sila-pentane sulfonate was added to a concentration of 1 mM as an internal chemical shift reference. The pH was subsequently adjusted to 5.0 using NaOH and HCl solutions (or NaOD for D2O samples, pH adjusted to 5.0 with no correction for isotope effects).

NMR Spectroscopy-- 1H NMR spectra for the 1-9 monomer, 1-9 dimer, and 1-17 monomer peptides were acquired at 600 MHz using a Varian Unity 600 spectrometer. TOCSY, NOESY, and double quantum filtered COSY spectra acquired at 8 °C were used for 1H resonance assignments. The WATERGATE pulse sequence (23) was used for solvent suppression for spectra in H2O. Mixing times for NOESY experiments were set at 200, 300, 400, 500, and 600 ms to determine NOE build-up rates, which were found to be linear up to 500 ms. ROE spectroscopy data were collected for the 9-mer monomer with mixing times of 60, 120, and 150 ms.

Structure Calculations-- NOESY experiments with 500-ms mixing times were used for the integration of the NOE cross-peaks because build up rates were approximately linear up to this mixing time, and this spectrum gave best signal to noise ratio for our measurements. The integral volumes were converted into distance restraints using a reference distance of 2.5 Å between the ortho (delta ) and meta (epsilon ) protons of the tyrosine ring. The NOE connectivities were classified as strong, medium, weak, and very weak, corresponding to upper distance restraints of 2.8, 3.5, 4.5, and 5.5 Å, respectively. Upper limits for nonstereospecifically assigned protons were corrected appropriately with center averaging.

Structure calculations were performed on the 9-mer dimer and 17-mer using the simulated annealing method employing the SHAKE algorithm implemented in X-PLOR (24) at an initial simulated annealing temperature of 800 K with 8000 high temperature and 6000 cooling steps. The initial structure was an extended chain, and the target function contained only potential terms for covalent geometry, experimental distance restraints, and a van der Waals' repulsion term for nonbonded contacts. The final structures, generated using the simulated annealing method, had no NOE violations >0.25 Å nor dihedral violations >5°. The 9-mer dimer was also subjected to the time-averaged distance restraint method as described in Ref. 25. Families of structures were extracted by superimposing the backbone of residues 5-8 within one monomeric unit and utilizing the program NMRCLUST (26).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sequential Assignment and 3JNH-Calpha H Coupling Constants-- Three N-terminal fragments of SDF-1, which were found to have SDF-1 activity (15), were studied. The sequence of the 9-mer monomer (to distinguish from the 9-mer dimer) corresponds to residues 1-9 of SDF-1. The 9-mer dimer represents two 9-mer monomers connected by a disulfide bond through Cys9. The sequence of the 17-mer corresponds to residues 1-17 of SDF-1. Two-dimensional homonuclear proton spectra, TOCSY, NOESY, and double quantum filtered COSY spectra were collected at 8 °C for all three peptides in aqueous solution. In addition, ROESY spectra were collected for the 9-mer monomer because the peptide was too small for suitable build-up of NOEs. Resonance assignments were made using standard two-dimensional methods (27).

Chemical shift data were obtained for the 9-mer monomer, 9-mer dimer, and 17-mer of SDF-1. The 9-mer monomer and 9-mer dimer have identical chemical shifts (except for Cys9, which is in its reduced (delta Halpha 4.40) and oxidized (delta Halpha 4.66) forms, respectively), suggesting that both strands of the dimer have the same conformation but do not interact with each other. Chemical shifts of residues 1-7 of the 17-mer are identical to those of the 9-mer monomer and 9-mer dimer (data not shown).

Vicinal proton coupling constants, 3JNH-Calpha H, were measured from well digitized one-dimensional proton spectra and, where measurements were not possible because of the resonance overlap, from double quantum filtered COSY spectra. For all three peptides, the 9-mer monomer, 9-mer dimer, and 17-mer, the coupling constants were all greater than 6 Hz and less than 8 Hz, indicating that these residues could potentially adopt any of a number of different phi  angles. These data could therefore not be included in the structure calculations.

Short and Medium Range NOESY Connectivities and Secondary Structure-- The region of the NOESY spectrum of the 9-mer dimer containing dNN(i,i+1) NOEs and NOEs from amide protons to ring protons of Tyr7 is shown in Fig. 1A. Regions of the 9-mer dimer NOESY spectrum including medium range NOEs dalpha N(i,i+2), dbeta N(i,i+2), and NOEs from alpha  and side chain protons to the ring protons of Tyr7 are shown in Fig. 1B. NOEs detected for the 9-mer dimer were assumed to arise from intra-monomeric contacts because there was no evidence of inter-monomer interactions. Moreover, this distinction was confirmed when the same NOEs were observed for residues 1-9 of the 17-mer and similar ROEs were observed for the 9-mer monomer (data not shown). Summaries of sequential and medium range NOE connectivities for the 9-mer dimer and 17-mer are illustrated in Fig. 2.


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  		Fig. 1.   Regions of a 500-ms NOESY spectrum of the 1-9 dimer. A, the dNN region indicating the dNN(i,i+1) NOE connectivities and NOE connectivities between amide protons and Tyr7 aromatic ring protons. B, the dalpha N(i,i+2) and dbeta N(i,i+2) connectivities characterizing the beta -turn. C, the NOE connectivities between alpha  and side chain protons and the Tyr7 aromatic ring protons indicating that the Tyr7 aromatic ring is involved in the local structure.


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  		Fig. 2.   Summary of sequential and medium range NOE connectivities, and amide proton temperature coefficients for the SDF-1 1-9 dimer (A) and the SDF-1 17-mer (B) as observed by NMR spectroscopy. Backbone NOE connectivities are indicated by horizontal lines between residues, with the line width indicating the relative magnitude for NOEs observed in the 500-ms NOESY spectrum.

Observed NOEs suggest the presence of a beta -turn conformation for the 9-mer dimer and 17-mer comprising residues Leu5, Ser6, Tyr7 and Arg8. The presence of a beta -turn is usually indicated by dalpha N(2,3), dalpha N(3,4), dalpha N(2,4), dNN(2,3), and dNN(3,4) NOESY connectivities, where the numbering refers to the residue position in the turn. NOESY spectra for the 9-mer dimer and 17-mer peptide show a dalpha N (2, 4) cross-peak between Ser6 and Arg8 and a dNN (2, 3) cross-peak between Ser6 and Tyr7. In addition, a dNN (1, 2) is observed between Leu5 and Ser6 as well as Arg8 and Cys9, and a dalpha N connectivity is observed between Tyr7 and Cys9 (Figs. 1 and 2), suggesting that Cys9 is also involved in the local structure. Apart from the backbone proton NOEs, NOEs to the Tyr7 ring protons were observed from the amide and alpha -protons of Cys9, Ser6, and Arg8, beta  and delta  protons of Leu5, and gamma  protons of Val3 (Fig. 1, A and C). These connectivities show that the Tyr7 side chain is stabilized in the structure most likely by hydrophobic interactions with surrounding residues.

There is evidence of a second local structure in the 17-mer involving residues Cys11, Arg12, and Phe13 (data not shown). This is particularly interesting because the CRF portion of the CRFFESH sequence is a partial palindrome of the tail of the 9-mer sequence (KPASLSYRC) involved in the formation of the first beta -turn. A dNN(i,i+1) NOESY connectivity was observed for residues Arg12 and Phe13 as well as a dalpha N(i,i+2) cross-peak between Cys11 and Phe13. In addition, NOEs were observed between Phe13 aromatic ring protons and the amide protons of residues Cys11, Arg12, and Phe14 as well as the Calpha protons of Cys11 and Arg12. These data suggest that the Phe13 aromatic ring is stabilized in the structure, although it is less well defined than the Tyr7 aromatic ring.

Temperature Coefficients of Amide Protons-- To further characterize the structuring of the 9-mer dimer and the 17-mer, we measured amide temperature coefficients (Delta delta /Delta T). Although for a rigid structure, exposed NHs typically have gradients in the range of -6.0 to -8.5 ppb/°C, hydrogen-bonded or protected NHs apparently have Delta delta /Delta T of -2.0 ± 1.4 ppb/°C (28). For peptide fragments, however, Delta delta /Delta T values may lie anywhere between -28 to +12 ppb/°C, resulting in a correlation between the gradient and structure that lies outside the rules mentioned above. Conformational averaging in peptides appears to be the major source of deviant values of Delta delta /Delta T whereby temperature-induced changes in the population of the folded state are the major contributor to the observed NH shift temperature gradient for partially structured peptides (28). A plot of Delta delta /Delta T versus the chemical shift deviation (CSD) of the amide proton provides a better correlation with partial structuring of a peptide at lower temperatures.

To measure Delta delta /Delta T values for the 9-mer and 17-mer, TOCSY spectra were acquired at 5, 10, 15, 20, and 25 °C. Chemical shift deviations were derived from the lowest temperature set included (5 °C). Random coil chemical shifts (29) were corrected to 5 °C according to Refs. 30 and 28. Fig. 3 shows a plot of the CSD versus Delta delta /Delta T for the 9-mer dimer and 17-mer peptides. The dashed line represents the cutoff of Delta delta /Delta T between exposed and sequestered NHs of proteins. Gradients above the dashed line indicate exposed NHs, whereas those below indicate sequestered NHs. All of the residues in the 9-mer dimer and 17-mer are above the dashed line, indicating that these amides are somewhat exposed. However, according to Andersen et al. (28), peptides that are structured at lower temperatures and become unstructured upon warming have a slope of the Delta delta /Delta T versus NH-CSD graph in the -8 to -20 ppt/°C range. In addition, the gradient/CSD plot must display a correlation coefficient greater than 0.7 and significant NH and alpha H CSD values for reasonable assessment of NH sequestration. For the 9-mer dimer, the slope of the graph for residues 5-8 is -8 ppt/°C with an R2 of 0.7 (for the unstructured residues, the slope was -8 ppt/°C with an R2 of 0.3). For the 17-mer, residues 5-8 had a slope of -10 ppt/°C with an R2 of 0.8; residues 11-14 had a slope of -4 ppt/°C with an R2 of 0.8; for the unstructured residues, the slope was -5 ppt/°C with an R2 of 0.4. The NH and alpha H CSD values are shown in Table I. The greatest chemical shift deviations occur for residues 5-8 in the 9-mer dimer and 17-mer. In addition, residues 11-15 in the 17-mer show larger chemical shift deviations.


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  		Fig. 3.   NH Delta delta /Delta T versus CSD for the 9-mer dimer and 17-mer. Circles represent residues in the 17-mer, while squares represent residues in the 9-mer dimer. Filled circles and squares correspond to potentially structured residues (residues 5-8 for the 9-mer dimer and 17-mer and residues 12-14 for the 17-mer). The dashed line corresponds to Delta delta /Delta T = -7.8(CSD) -4.4, which provides the best differentiation of sequestered NHs in the protein data base (28).

                              
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Table I
NH and Halpha 1H NMR chemical shift deviations
CSD for NHs were derived at 5 °C with appropriate random coil chemical shift correction as described in Refs. 28 and 29. CSDs for Halpha chemical shifts were calculated from peptide data at 8 °C, and random coil chemical shift data were calculated at 25 °C from Ref. 29.

Structure Calculations and Analysis-- 49 inter-residue and 54 intra-residue NOEs were used to construct distance restraints for each monomeric unit of the 9-mer dimer. For structure calculations of the 17-mer, 96 inter-residue and 80 intra-residue distance restraints were used. No explicit dihedral or hydrogen bonding restraints were applied. Structure calculations were performed using a simulated annealing protocol (24) for both the 9-mer dimer and 17-mer peptides. For both peptides, a family of 80 structures was calculated. Structures with the lowest energy and NOE violations of no more than 0.25 Å were selected from each group. Conformationally related subfamilies of structures were then extracted using the program NMRCLUST (26) (Table II).

                              
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Table II
Structural statistics and atomic root mean square differences
ENOE was calculated with a square well potential with a force constant of 50 kcal/mol · Å2. EVDW was calculated with a force constant of 4 kcal/mol · Å-4 where final van der Waals' radii were set to 0.75 times their value in the CHARMM forcefield.

For the 9-mer dimer, 47 structures were selected out of 80. A major family of 28 structures, which corresponds to 60% of the selected structures, was extracted by superimposing the backbone of residues 5-8 within one monomeric unit. For the 9-mer dimer, the spread was found to be 0.41 Å. NMRCLUST detected four other minor subfamilies, each of them consisting of less than 10% of the selected structures. PROCHECK_ NMR (31) was used to determine that residues in the major subfamily adopted allowed conformations in (phi ,psi ) space. The precision of the torsion angles of the residues involved in the local structure, within the major subfamily, was also assessed in terms of the order parameter S (32). The angular order parameter is a statistical parameter that assumes a value of 1 if a given torsion angle is identical in every member of the structure family and equals 0 if the angle is completely undefined (Table III). Although the coupling constants for this dynamic peptide are in the 6-8 Hz range, the major conformational family of structures at 8 °C has phi ,psi dihedral angles defining the turn as (-95 ± 8, 73 ± 19) for Ser6 and (-115 ± 19, 56 ± 14) for Tyr7 with corresponding (phi ,psi ) order parameters of (0.99, 0.95) for Ser6 and (0.95, 0.97) for Tyr7. This turn is classified as a beta -alpha R type by the nomenclature of Wilmot and Thornton (33), with no hydrogen bond present in the turn. Backbone superposition of residues 5-8 of the subfamily members onto residues 5-8 of the subfamily representative structure (residues 4-9 are shown) is illustrated in Fig. 4A.

                              
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Table III
phi , psi , chi 1 and S values for the 9-mer dimer
Angles and order parameters for residues 3-9 were computed from the structures from the major family determined by clustering residues 5-8.


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  		Fig. 4.   A, major subfamily of 28 SDF-1 1-9 dimer peptide simulated annealing structures superimposed on the subfamily representative structure. B, major subfamily of 17 SDF-1 1-17 peptide simulated annealing structures illustrating residues 11-15 superimposed on the subfamily representative structure (shown as a thick line).

Our goal was to find the conformation of the peptides that may be recognized by CXCR4. Because it is likely that the peptides undergo rapid interconversion between several substates, it is plausible that the measured NOEs reflect an average of these states. Traditional simulated annealing calculations will then, not surprisingly, produce a single dominant "average" structure. To further explore the behavior of the calculated families, time-averaged distance restraints (25) were employed on the 9-mer dimer peptide. The conformationally related subfamilies of structures produced using time-averaged distance restraints were extracted using NMRCLUST. 35 structures were calculated starting with the structures generated by conventional simulated annealing methods. These structures were then grouped into subfamilies as described above. Out of 35 structures, five families were found. The most populated of the families contained 15 structures with a backbone root mean square deviation of 0.84 ± 0.12 Å for residues 5-8 (data not shown). The fold is very similar to what was obtained using conventional analysis alone, although with a higher root mean square deviation, indicating greater flexibility in the case of simulations with time-averaged restraints.

For calculation of the 17-mer structure, 47 of the lowest energy structures were selected (out of 80) with NOE violations of no more than 0.25 Å. Cluster analysis was performed with the use of NMRCLUST (26) by superimposing residues 5-8. A major family of 26 structures with cluster spread of 0.51 Å was found. Within the subfamily, the phi ,psi angles defining the turn at residues 5-8 are similar to that found for the 9-mer dimer. In addition, the order parameters are similar, indicating the same beta -alpha R type turn as detected in the 9-mer dimer.

To investigate the possibility of a second local structure in the 17-mer, cluster analysis was performed by superimposing residues 11-14. This yielded a major family of the 17 conformers with a higher cluster spread of 1.1 Å. The phi ,psi angular order parameters of residues 11-14 in the major conformational family are (0.14, 0.80), (0.94, 0.99), (0.90, 0.47), and (0.51, 0.59) respectively, indicating that Arg12 and Phe13 assume an ordered conformation in all members of the subfamily (Table IV). The phi ,psi angles of Arg12 and Phe13 are (-128 ± 20, 41 ± 7) and (-149 ± 26, 117 ± 86) within the subfamily. Backbone superposition of residues 11-14 of subfamily members onto residues 11-14 of the representative structure is presented in Fig. 4B (thick line). Although the structure is less well defined compared with the first beta  turn, the "bent" conformation of Phe13 side chain resembles the conformation of the Tyr7 aromatic ring. The conformation of the Phe14 side chain, however, appears to be completely disordered.

                              
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Table IV
phi , psi , chi 1 and S values for the 17-mer
Angles and order parameters for residues 3-9 were computed from the structures from the major family determined by clustering residues 5-8. Angles and order parameters for residues 10-17 were computed from the structures in the major family determined by clustering residues 11-14.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

For most chemokines in the CC and CXC families studied to date, it has been shown that the important residues for receptor binding and activation lie in the N-terminal region and the loop immediately following the first two cysteines in the sequence (34-37). SDF-1 is no exception, and there have been studies showing activities of SDF-1 N-terminal peptides (15). Although the N terminus appears disordered in the NMR structure (18), we wanted to determine whether some structure does, in fact, exist that would account for the high affinity and biological activity of these N-terminal peptides. We surmise that if such a conformation exists, it could provide a starting point for synthesizing non-peptide analogs to act as antagonists or amplifiers of SDF-1 activity.

We studied three peptides: a 9-mer monomer, a 9-mer dimer, and a 17-mer. Similar medium-range NOEs were observed for the 9-mer dimer and 17-mer as well as ROEs for the 9-mer monomer, respectively. The activity of the 1-9 dimer is 10 fold more potent than the 1-9 monomer. Because there are similar ROEs in the 1-9 monomer upon comparison with the NOEs in the 1-9 dimer, a similar structure should be present in the 1-9 monomer. The lower activity of the 1-9 monomer could be simply due to the fact that there are two sites on the receptor that need to be bound, and the 1-9 monomer cannot fill those sites alone. It is probable that there is two-site binding to the receptor, where binding occurs tightly to the first site, and subsequently because of the proximity of the second binding portion of the peptide, finds its way to bind to the second site. Alternatively, the increased potency of the dimer could be purely statistical because there is twice the concentration of the 1-9 peptide that can exchange in and out of the active site. Because the 17-mer binds tighter than the 9-mer monomer, the former explanation is likely correct and strengthens the recently proposed two-step mechanism for SDF-1 receptor binding and activation (18, 21). According to this mechanism, there are two chemokine receptor-binding sites. An initial interaction occurs with the RFFESH loop region (12-17) that follows the CXC motif. The disordered part of the N-terminal region is subsequently proposed to become structured during binding, establishing contacts within the receptor groove. It has been proposed that SDF-1 N-terminal peptides do not require the presence of a structured loop region for their activity (15). The present study shows in fact that these peptides are able to adopt specific conformations.

The preferred three-dimensional conformations of the 9-mer dimer and 17-mer were determined by NMR spectroscopy at 8 °C. Fig. 4 illustrates the turn structure generated using conventional simulated annealing techniques. There appears to be a beta -turn motif formed by residues 5-8 of the 1-9 dimer and 17-mer peptides with the aromatic ring of Tyr7 situated within a hydrophobic pocket involving Leu5 and Cys9. In addition, the 17-mer contains a second beta -turn structural motif comprising residues 11-14. That this beta -turn structural motif is real in the 9-mer dimer and not an average of several conformations was confirmed by using the time-averaged distance restraint method (data not shown). Further confirmation that one major folded conformation was achieved at 8 °C was illustrated by the plot of the NH temperature gradient versus chemical shift deviation (Fig. 3). The structured regions of peptides in conformational equilibrium should have correlation coefficients greater than 0.7 and slopes in the -8 to -20 ppt/°C range. The slope and correlation coefficient for residues 5-8 of the 9-mer dimer and the 17-mer meet these criteria. Although residues 11-14 of the 17-mer appear not to meet all of the criteria (slope = -4 ppt/°C), they have significant chemical shift deviations, suggesting that they have a preferred conformation at 8 °C. In contrast, the unstructured residues had low correlation coefficients for the 9-mer dimer the 17-mer. Although there was no evidence for NH sequestration at 5 °C (no Delta delta /Delta T versus CSD values below the dashed line), it is most likely due to the fact that the beta -turn, which is of the beta -alpha R type, does not normally have a hydrogen bond stabilizing its structure.

These structural motifs may not have been detected in the native SDF-1 simply because they are not present in the SDF-1 structure. The other, more likely possibility is that the same structural motif is present in native SDF-1, but essential NOEs were not detected because much shorter mixing times for the NOESY experiments were used for the protein as compared with the peptide. The rotational correlation time, tau c, is proportional to the cube of molecular radius. The NOE intensity is a function of the correlation time, which is shorter for more flexible parts of the protein and tends to build up faster in the well structured core of the protein. Longer mixing times were not employed in the original SDF-1 study, and generally they tend not to be used for solving protein structure because of the effects of spin diffusion. However, it would seem that these experiments could be employed for detecting structuring in the more flexible regions of a protein.

The SDF-1 1-17 peptide has a similar binding constant to the 9-mer dimer (15). We found that it contains the same beta -alpha R turn formed by residues 5-8 in the 1-9 dimer as well as a second beta -turn involving residues 11-14 within the 12RFFESH17 sequence. This beta -turn is structurally similar to that found in the native SDF-1 structure, and superposition of residues 11-14 in the native structure with 11-14 of the peptide results in a backbone root mean square deviation of 0.78. Given the complementarity of residues 7-9 and 11-13 in the sequence, chemical shift deviations and NOEs should lead to similar beta -turn patterns in both peptides, suggesting that two turns are required for optimal binding to the receptor.

It has been shown that this beta -alpha R turn motif is present in the conformation of the peptides in solution and, in addition, represents the major conformational family. Peptide antagonists for SDF-1 have already been synthesized (38); however, clinical use of such peptides would be of limited use because of the high cost. High hydrophobicity and the presence of aromatic rings in more or less stabilized conformations relates this motif to the recently found highly potent nonpeptide antagonists of a CC chemokine receptor (39). We believe that the structural studies of peptides with SDF-1 activity that are presented in this paper may lead to the development of new low molecular weight non-peptide compounds that will mimic this structure and bind to CXCR4 with agonist and antagonist properties.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors contributed equally to this work.

§ Present address: Dept. of Biochemistry and Molecular Biology, University of Southampton, Bassett Crescent East, Southampton, SO16 7PX, UK.

Present address: Biomedical Research Centre, University of British Columbia, 2222 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.

|| To whom correspondence should be addressed. Tel.: 780-492-6540; Fax: 780-492-1473; E-mail: brian.sykes@ualberta.ca.

Published, JBC Papers in Press, June 8, 2000, DOI 10.1074/jbc.M003386200

    ABBREVIATIONS

The abbreviations used are: HIV, human immunodeficiency virus; SDF-1, stromal cell derived factor 1; NOE, Nuclear Overhauser enhancement; NOESY, NOE spectroscopy; ROE, rotating frame effect Overhauser enhancement; ppt, parts per thousand; HPLC, high pressure liquid chromatography; CSD, chemical shift deviation; TOCSY, total correlation spectroscopy.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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