1. Volume 23, Issue 6, Pages 1066-1077 (June 2015)
Interactions of the Chemokine CCL5/RANTES with Medium-Sized Chondroitin Sulfate Ligands 
Courtney Deshauer, Ashli M. Morgan, Eathen O. Ryan, Tracy M. Handel, James H. Prestegard, Xu Wang 
Structure 
Volume 23, Issue 6, Pages (June 2015)
DOI: /j.str
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2. Structure 2015 23, 1066-1077DOI: (10.1016/j.str.2015.03.024)
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3. Figure 1 Structure of CS444 and CS644
The difference in sulfation position at the non-reducing end GalNAc is highlighted in bold.
Structure  , DOI: ( /j.str )
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4. Figure 2 CS444-Induced Chemical Shift Changes in E66S CCL5
(A) 15N-HSQCs of E66S-CCL5 in the presence of different concentrations of CS444. The concentration of protein is 40 μM and the concentrations of CS444 are 0 (dark green), 20 (brown), 40 (yellow), 80 (pink), 120 (light green), and 160 (light blue) μM. Detailed sections showing movements of representative residues from the N loop and the 40s loop are shown on the right.
(B) Scaled and combined chemical shift changes of each residue. The combined chemical shift changes were calculated using the formula ΔδS=ΔδH,Hz2+(1.7ΔδN,Hz)2. Positions of S5 and R45, which broadened beyond detection during the titration, are indicated by asterisks. Other residues missing from the graph are prolines.
Structure  , DOI: ( /j.str )
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5. Figure 3 Sections of 15N-HSQCs of 30 μM E66S-CCL5 at 40°C
(Black) E66S-CCL5 without CS444, both monomer and dimer species are visible. (Red) E66S-CCL5 with two molar equivalents of CS444. The assignments of signals representing monomeric and dimeric species are made based on (Schnur et al., 2013) and (Duma et al., 2007).
Structure  , DOI: ( /j.str )
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6. Figure 4
Quantitative Measurement of Paramagnetic TEMPO-Induced Transverse Relaxation (R2,PRE)
(A) Graph and surface plot of amide proton R2,PRE produced by TEMPO-labeled CS444.
(B) Graph and surface plot of amide proton R2,PRE produced by TEMPO-labeled CS644. Residues Y14, A16, and R21 disappeared upon addition of CS644-TEMPO but reappeared upon its reduction. They are given the same magnitude of R2,PRE as residue Y3, which showed the biggest change.
Structure  , DOI: ( /j.str )
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7. Figure 5 Intermolecular NOEs Detected between E66S CCL5 and CS444
(A) 13C-filtered/edited NOESY of 200 μM 13C-labeled E66S-CCL5 in the presence of one molar equivalent of CS444. CCL5 proton chemical shifts correspond to the X coordinates of the cross peaks while ligand proton chemical shifts correspond to the Y coordinates of the cross peaks.
(B) HSQC-NOESYs of 200 μM deuterated E66S CCL5 with ILV methyl protonation in the presence (black) and absence (red) of one molar equivalent of CS444. Two new cross peaks can be seen in the spectrum collected in the presence of the ligand. They correspond to contacts between methyl groups on I15 and L19.
Structure  , DOI: ( /j.str )
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8. Figure 6
Cross Saturation Spectra of Deuterated E66S CCL5 Complexed to CS444
Reference 15N-HSQC (left) and STD 15N-HSQC (right) spectra of perdeuterated E66S-CCL5. Three residues showed the most prominent difference.
Structure  , DOI: ( /j.str )
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9. Figure 7
Saturation Difference Spectra of CS444 in the Presence of E66S CCL5
(A) STD 1D of 1 mM CS444 with 50 μM 13C-labeled E66S-CCL5. Possible assignments of the peaks are indicated.
(B) Sections of F3-coupled HSQC-NOESY collected on the same sample. Cross peaks between ligand protons and protein protons can be seen.
Structure  , DOI: ( /j.str )
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10. Figure 8 Model of the CCL5 Dimer Complexed to CS444
(A) A representative frame from the most energetically favored models of the CCL5-CS444 complex. The ribbon representation of the complex with side chains of selective amino acids is shown in gray. The CS444 ligand is shown in the stick representation with the non-reducing end and the reducing end sugar labeled GlcA1 and GalNAc3, respectively.
(B) Surface representation of the same structure with residues known to contact CS444 colored in blue. The protein is in the same orientation as (A).
(C) Schematic illustration of the intermolecular hydrogen-bond contacts observed between E66S CCL5 and CS444. Curved arrows indicate contacts observed between CCL5 residues and functional groups on CS444 in the simulation. The percentages indicate the fraction of 50 most energetically favored frames containing the contact.
Structure  , DOI: ( /j.str )
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11. Figure 9
Comparisons of Chemokines’ Interactions with the Receptor N Terminus and E66S CCL5’s Interaction with GAGs
(A) Structure of vMIP-II (surface) bound to N terminus of CXCR4 (ball-and-stick).
(B) Model of vMIP-II bound to the CCR5 N terminus constructed using the crystal structure of the CXCR4-vMIP-II. Basic residues involved in contacting the CCR5 N terminus are colored in blue and labeled.
(C) Model of CS444-bound CCL5 determined in this study. Basic residues involved in binding GAGs are colored in blue.
(A) and (B) are adapted from Qin et al. (2015).
Structure  , DOI: ( /j.str )
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