1. Volume 23, Issue 7, Pages 1199-1213 (July 2015)
Structures of Orf Virus Chemokine Binding Protein in Complex with Host Chemokines Reveal Clues to Broad Binding Specificity 
Rafael M. Couñago, Karen M. Knapp, Yoshio Nakatani, Stephen B. Fleming, Michael Corbett, Lyn M. Wise, Andrew A. Mercer, Kurt L. Krause 
Structure 
Volume 23, Issue 7, Pages (July 2015)
DOI: /j.str
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2. Structure 2015 23, 1199-1213DOI: (10.1016/j.str.2015.04.023)
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3. Figure 1 Crystal Structure of the ORFV CKBP Monomer
(A) Cartoon representation of the ORFV CKBP monomer showing β sheets I (left) and II (right). Secondary structural elements are labeled, as are the N and C termini. A flexible loop (β7-β8 loop) between residues 164 and 191 (shown as a dashed line) could not be built into density. The β2-β3 loop is indicated by a red ellipse.
(B) Electrostatic surface potential representations of β sheets I (left) and II (right). The molecular surfaces are colored by potential (kT/e) as indicated by the level bar.
Structure  , DOI: ( /j.str )
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4. Figure 2
Comparison of the ORFV CKBP Structure with Other Type-II CKBPs
(A and B) Cartoon representations of the monomeric subunit of the ORFV CKBP dimer, the CPV vCCI, and the VACV A41 protein (from left to right). (A) Monomers are arranged with β sheet I forward and (B) β sheet II forward. Secondary structural elements are labeled and primary structure is colored in a gradient from the N terminus (blue) to the C terminus (red). Secondary structure elements exclusive to ORFV CKBP (α helices 1 and 3, and β strand 11) are emphasized with a red arrow. The β7-β8 loop of CPV vCCI and VACV A41 protein is indicated by black arrows.
(C) Structure-based sequence alignment of ORFV CKBP and other type-II CKBPs. CPV, cowpox virus; EV, ectromelia virus; RPV, rabbitpox virus; VACV, vaccinia virus. Secondary structure elements and residue numbers are shown for the mature ORFV CKBP structure above the alignment. α Helices are shown as yellow cylinders; β strands of β sheet I are shown as blue arrows and those of β sheet II as red arrows. The light-gray box above the alignment represents the β7-β8 loop, and the dashed lines within this box indicate the missing residues from the ORFV CKBP structure. Highly conserved residues are highlighted in black and gray. Black triangles above the alignment represent conserved cysteine residues involved in disulfide bonds; red triangles with a black outline represent conserved cysteine residues present in other type-II CKBPs but not present in ORFV CKBP; blue triangles represent basic residues co-located on β sheet I of ORFV CKBP; red triangles represent acidic residues on β sheet II involved in ORFV CKBP-chemokine binding; open circles represent hydrophobic residues on β sheet II involved in ORFV CKBP-chemokine binding; stars highlight the ORFV CKBP glycosylation sites; the letter d represents residues at the ORFV CKBP dimer interface.
Structure  , DOI: ( /j.str )
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5. Figure 3 ORFV CKBP with Bound Chemokines
(A) Electrostatic surface potential representation of the ORFV CKBP monomer and cartoon representation of bound CCL2 (green). CCL2 secondary structure elements are labeled. Key CCL2 residues that interact with ORFV CKBP are shown as ball-and-stick representations. The ORFV CKBP chemokine binding site regions are outlined with a dashed line. The molecular surfaces are colored by electrostatic potential (kT/e) as indicated.
(B) Crystal structures of CCL2, CCL3, and CCL7 in complex with ORFV CKBP. Electrostatic surface potential representation of the ORFV CKBP monomer showing β sheet II, and main-chain representations of bound CCL2 (green), CCL3 (yellow), and CCL7 (blue). For clarity, only the ORFV CKBP protein from the CCL2 co-crystal structure is shown. CCL3 and CCL7 are shown superimposed on the binding surface following superposition of CKBP from the three co-crystal structures. For CCL3 the complete tracing is shown, although parts are missing in the electron density. Glycosylation at the ORFV CKBP N80 glycosylation site is shown as a purple ball-and-stick representation. Molecular surfaces are colored by potential (kT/e) as indicated.
Structure  , DOI: ( /j.str )
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6. Figure 4 Molecular Interactions at the ORFV CKBP-CCL2 Interface
(A) Atomic spheres representations for the ORFV CKBP monomer (left) and bound CCL2 (right). CCL2 has been rotated 180° about the axis to show residues that bind to the ORFV CKBP chemokine binding site. ORFV CKBP residues forming the chemokine binding site are colored based on chemokine binding region. CKBP residues contributing to regions I and II of the chemokine binding site are colored blue and yellow, respectively, while residues contributing to region III are colored green. All noninteracting regions of ORFV CKBP and CCL2 are shown in white. Key ORFV CKBP residues are labeled and those forming the hydrophobic pocket are shown in dark green, while those from the negatively charged groove interacting with CCL2 residues R18 and R24 are colored orange and dark blue, respectively. CCL2 residues are colored according to the regions of the ORFV CKBP chemokine binding site to which they bind. CCL2 residues that interact with ORFV CKBP are shown as colored spheres where key residues (C12, Y13, I42, and C52) interacting with the ORFV CKBP hydrophobic pocket (region III) are shown in dark green, and key residues (R18 and R24) interacting with regions I and II of the ORFV CKBP negatively charged groove are shown in dark blue and orange, respectively. All other CCL2 residues interacting with ORFV CKBP are highlighted in yellow, light green, or light blue according to the regions of the ORFV CKBP chemokine binding site to which they bind.
(B) Electrostatic potential surface representations of the ORFV CKBP monomer (left) and bound CCL2 (right). The view is the same as that shown in (A). The chemokine binding site regions I, II, and III are emphasized with dashed lines. Glycosylation at the ORFV CKBP N80 glycosylation site is shown with a purple ball-and-stick representation. Solvent-accessible surfaces are colored by potential (kT/e) as indicated by the level bar shown at the bottom.
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7. Figure 5
Both Hydrophobic Interactions and Antiparallel β-Strand Hydrogen Bonding Contribute to CKBP-Chemokine Binding
(A) From the structure of the complex, CCL2 is shown in green with CKBP depicted in an electrostatic surface representation. The diagram shows Y13 within the hydrophobic recess formed by CKBP β strands 8–10 (yellow). Two key arginines (R18 and R24) from CCL2 are also shown.
(B) Extensive hydrogen bonding between CKBP β8 (yellow) and chemokine N loop (green). Electrostatic surfaces are colored by potential (kT/e) as indicated.
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8. Figure 6
Structural Comparison of the ORFV CKBP and Rabbitpox Virus vCCI Chemokine Binding Modes
(A) Electrostatic potential surface representation of the ORFV CKBP monomer and cartoon representations for bound CCL2 (green) and superposed CCL4 (yellow) resulting from superposition of the ORFV CKBP and rabbitpox virus (RPV) vCCI proteins. Chemokine residues R18 and R24 (CCL2 numbering, identified with an asterisk), and R18 and F24 (CCL4 numbering) are shown as ball-and-stick representations, and are labeled along with the N and C termini of the chemokines. Glycosylation at the ORFV CKBP N80 glycosylation site is shown with a purple ball-and-stick representation.
(B) Electrostatic surface potential representation of RPV vCCI and cartoon representation for bound CCL4 (yellow) (PDB: 2FFK). The extended RPV vCCI β2-β3 loop involved in chemokine binding is labeled. Chemokine residues R18 and F24 (CCL4 numbering) are shown as ball-and-stick representations, and the N and C termini of the chemokine are labeled. Electrostatic surfaces are colored by potential (kT/e) as indicated.
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9. Figure 7 Analysis of ORFV CKBP Binding to C- and CXC Class Chemokines
(A) Electrostatic surface potential representation of CCL2 from the ORFV CKBP-CCL2 crystal structure juxtaposed with a similar representation for chemokines (CXCL8, CXCL10, and CXCL12) that do not bind to ORFV CKBP. The view is similar as that shown in Figure 4B. Electrostatic surfaces are colored by potential (kT/e) as indicated. Key structural features of the chemokines that influence CCL2 binding to ORFV CKBP are labeled. Residues from CXCLs 8, 10, and 12 that may interfere with binding to CKBP are labeled.
(B) Structure-based sequence alignment of chemokines that interact with both vCCIs, and ORFV CKBP and those that interact with ORFV CKBP but not with vCCIs. Only residues in regions that interact with ORFV CKBP are shown in the alignment. Gaps in chemokine sequence are shown as dashed lines. Numbers shown are for CCL2. Chemokine binding regions on ORFV CKBP are indicated by blue (region I), yellow (region II), and green (region III). Red triangles indicate CCL2 residues shown to be important for ORFV CKBP binding.
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10. Figure 8 The ORFV CKBP Dimer Binds Two Chemokines
(A) MALLS-SEC analysis of CKBP (black) and CKBP-CCL2 complex (red). Measured masses (square boxes) are shown. The average mass is also indicated.
(B) SEC analysis of CKBP over a range of protein concentrations. A280 profiles (0.05, 0.5, 2.0 mg/ml CKBP).
(C) Native PAGE gel-shift assay for ORFV CKBP complexed with CCL2.
(D) The ORFV CKBP homodimer (β sheet II). (Left) Cartoon representation. β Strands and C termini are labeled. Each subunit of the ORFV CKBP dimer is colored in a gradient from the N terminus (blue) to the C terminus (red). (Right) Electrostatic potential surface representations. The molecular surface is shown with electrostatics depicted in color as indicated. The main-chain trace of two CCL2 molecules as bound in the CK-CKBP complex are shown in green. Glycosylation is shown in a purple ball-and-stick representation.
Structure  , DOI: ( /j.str )
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