PDBsum entry 1vmc

Cytokine

PDB id: 1vmc

Contents

Protein chain

61 a.a. *

* Residue conservation analysis

PDB id:

1vmc

Name:

Cytokine

Title:

Stroma cell-derived factor-1alpha (sdf-1alpha)

Structure:

Stromal cell-derived factor 1. Chain: a. Fragment: sdf-1alpha (residues 22-89). Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: cxcl12, sdf1. Expressed in: escherichia coli. Expression_system_taxid: 562

NMR struc:

1 models

Authors:

E.K.Gozansky,G.M.Clore

Key ref:

E.K.Gozansky et al. (2005). Mapping the binding of the N-terminal extracellular tail of the CXCR4 receptor to stromal cell-derived factor-1alpha. J Mol Biol, 345, 651-658. PubMed id: 15588815 DOI: 10.1016/j.jmb.2004.11.003

Date:

20-Sep-04 Release date: 01-Mar-05

Protein chain

P48061  (SDF1_HUMAN) -  Stromal cell-derived factor 1 from Homo sapiens

Seq: 93 a.a.

Struc: 61 a.a.

DOI no: 10.1016/j.jmb.2004.11.003

J Mol Biol 345:651-658 (2005)

PubMed id: 15588815

Mapping the binding of the N-terminal extracellular tail of the CXCR4 receptor to stromal cell-derived factor-1alpha.

E.K.Gozansky, J.M.Louis, M.Caffrey, G.M.Clore.

ABSTRACT

The solution structure of monomeric stromal cell-derived factor-1alpha (SDF-1alpha), the natural ligand for the CXCR4 G-coupled receptor, has been solved by multidimensional heteronuclear NMR spectroscopy. The structure has a characteristic chemokine fold and is in excellent agreement with the individual subunits observed in the crystal structures of dimeric SDF-1alpha. Using various peptides derived from the N-terminal extracellular tail of the CXCR4 receptor, we show that the principal determinants of binding reside in the N-terminal 17 residues of CXCR4, with a major contribution from the first six residues. From 15N/1HN chemical shift pertubation studies we show that the interaction surface on SDF-1alpha is formed by the undersurface of the three-stranded antiparallel beta-sheet bounded by the N-terminal loop on one side and the C-terminal helix on the other. This surface overlaps with but is not identical to that mapped on several other chemokines for the binding of equivalent peptides derived from their respective receptors.

Selected figure(s)

Figure 1.
Figure 1. Structure of SDF-1a. (a) Example of a 1H(F[1])/13C(F[2]) plane from the 4D 13C/15N-separated NOE spectrum (mixing time=120 ms) taken at 15N(F[3])=130.4 ppm/1HN(F[4])=10.22 ppm, which corresponds to the Ne1H of Trp57. (Note that extensive folding was employed for 13C(F[2]) dimension which was recorded with a sweep width of 20.71 ppm; peaks folded an even number of times have positive contours, shown by continuous lines, while peaks folded an odd number of times have negative contours shown by broken lines; thus, for example, the absolute 13C shift of Leu55d1 is at 25.5 ppm). (b) Ribbon drawing of the restrained regularized mean structure of SDF-1a (green, b-sheet; cyan, helix; dark blue, 3^10 helix; and brown, loops). (c) Backbone (N, C^a, C atom) best-fit superposition of the final 100 simulated annealing structures (red) with the two disulfide bridges shown in yellow. (Residues 1-7 are not shown since they are disordered in solution.) (d) Isosurface of the re-weighted atomic density map (purple) for selected side-chains drawn at a value of 20% maximum,12 calculated from the 100 simulated annealing structures; the backbone of the restrained regularized mean structure is shown as a blue tube and side-chain coordinates within the atomic density map are shown in red. Note that the atomic density map for Arg8 and Arg12 clearly indicates that these two side-chains occupy multiple rotameric states. SDF-1a with an additional three residues (Ser-Asp-Gly) at the N terminus was cloned into the pET11a vector and expressed in Escherichia coli BL-21(DE3). Cells were grown at 37 °C in minimal medium with 15NH[4]Cl and/or 13C[6]-glucose as the sole nitrogen and carbon sources, respectively. Cells derived from 1 l of culture were suspended in 80 ml of buffer A (50 mM Tris-HCl (pH 8), 10 mM EDTA, 10 mM dithiothreitol (DTT)]), followed by the addition of lysozyme (100 µg/ml) and sonicated at 4 °C. The insoluble recombinant protein was washed by resuspension in 70 ml of buffer containing 50 mM Tris-HCl (pH 8), 10 mM EDTA, 10 mM DTT and 2 M urea and subsequently in buffer A. The insoluble fraction was pelleted by centrifugation at 20,000g for 30 minutes at 4 °C. The final pellet was solubilized in 50 mM Tris-HCl (pH 8.0), 7.5 M guanidine-HCl, 5 mM EDTA, 100 mM DTT to yield a protein concentration of vert, similar 20 mg/ml. 30 mg of protein was applied on a Superdex-75 column (HiLoad 2.6 cm×60 cm, GE Healthcare, NJ) equilibrated in 50 mM Tris-HCl (pH 8), 4 M guanidine-HCl, 5 mM EDTA, 5 mM DTT, and eluted at a flow-rate of 3 ml/minute at ambient temperature. Peak fractions were pooled and vert, similar 12 mg (0.25 mg/ml) of protein in the column buffer was folded at room temperature against 4 l of buffer in three steps: first against 1 M guanidine-HCl, 50 mM Tris-HCl (pH 8), 50 mM NaCl, 5 mM EDTA overnight and then twice against 20 mM Tris-HCl (pH 8), 0.1 M NaCl for 5-6 hours. The protein was concentrated to vert, similar 2 ml and applied on a Superdex-75 column (HiLoad 2.6 cm×60 cm) in 50 mM sodium phosphate buffer (pH 4.8). Peak fractions eluting at a retention volume between 200 ml and 225 ml corresponding to the monomeric folded SDF-1a were pooled and concentrated. Samples for NMR contained vert, similar 1 mM protein in 50 mM phosphate buffer (pH 5.5). All NMR experiments were carried out at 35 °C on Bruker DMX500 and DMX600 spectrometers. Spectra were analyzed using the programs PIPP, CAPP and STAPP.22 Assignment was carried out using 3D double and triple resonance experiments (HNCACB, HNCO, CBCA(CO)NH, C(CCO)NH, H(CCO)NH, HCCH-COSY, HCCH-TOCSY).11 NOE distance restraints (1.8-2.7 Å, 1.8-3.5 Å, 1.8-5.0 Å and 1.8-6.0 Å, corresponding to strong, medium, weak and very weak NOE cross-peak intensities) were derived from 3D 15N-separated and 13C-separated NOE experiments and 4D 13C/15N-separated and 13C/13C-separated NOE experiments.11 Three-bond J couplings (3J[HNHa], 3J[NCg], 3J[C'Cg] and 3J[CaCd] couplings) were measured using quantitative J correlation spectroscopy.23 Side-chain torsion angle restraints were derived from 3J couplings combined with information from the NOE data.11 1D[NH] residual dipolar couplings were obtained from the difference in 1J[HN] couplings measured in liquid crystalline (5% bicelles, 3 : 1 DMPC:DHPC) medium and in isotropic (water) medium.24 Backbone /q torsion angle restraints were derived from backbone chemical shifts using the program TALOS.25 The structures were calculated using well-established procedures26 from the experimental restraints by simulated annealing in torsion angle space27 using the program Xplor-NIH.28 The non-bonded contacts in the target function were represented by a quartic van der Waals repulsion term24 supplemented by torsion angle13 and hydrogen-bonding14 database potentials of mean force, and a radius of gyration restraint to ensure optimal packing.29 Structure Figures were generated with the programs VMD-XPLOR30 and RIBBONS.31 Reweighted atomic density probability maps (contoured at 20% of maximum value) were calculated from the ensemble of simulated annealing structures as described.12

Figure 2.
Figure 2. Comparison of the current NMR structure of monomeric SDF-1a (red, labeled as NMR(1VMC)), with (a) the coordinates of one subunit from two independent crystal structures of dimeric SDF-1a solved at resolutions of 2.2 Å (blue, 1A15)9 and 2.0 Å (green, 1AQG7),10 and (b) the original monomeric NMR structure (gray, 1SDF) solved by Crump et al.4 Note that the orientation of the helix with regard to the underlying b-sheet in the 1SDF structure differs by vert, similar 35° from that in the other three structures, and the conformation of the loop connecting strand b3 to the helix is significantly different as well.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2005, 345, 651-658) copyright 2005.

Figures were selected by an automated process.