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Structure and function of A41, a vaccinia virus chemokine binding protein.

Bahar MW, Kenyon JC, Putz MM, Abrescia NG, Pease JE, Wise EL, Stuart DI, Smith GL, Grimes JM - PLoS Pathog. (2008)

Bottom Line: Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors.Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding.The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (microM-nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine-chemokine receptor interactions.

View Article: PubMed Central - PubMed

Affiliation: The Division of Structural Biology and The Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

ABSTRACT
The vaccinia virus (VACV) A41L gene encodes a secreted 30 kDa glycoprotein that is nonessential for virus replication but affects the host response to infection. The A41 protein shares sequence similarity with another VACV protein that binds CC chemokines (called vCKBP, or viral CC chemokine inhibitor, vCCI), and strains of VACV lacking the A41L gene induced stronger CD8+ T-cell responses than control viruses expressing A41. Using surface plasmon resonance, we screened 39 human and murine chemokines and identified CCL21, CCL25, CCL26 and CCL28 as A41 ligands, with Kds of between 8 nM and 118 nM. Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors. However the interaction of A41 and chemokines was inhibited in a dose-dependent manner by heparin, suggesting that A41 and heparin bind to overlapping sites on these chemokines. To better understand the mechanism of action of A41 its crystal structure was solved to 1.9 A resolution. The protein has a globular beta sandwich structure similar to that of the poxvirus vCCI family of proteins, but there are notable structural differences, particularly in surface loops and electrostatic charge distribution. Structural modelling suggests that the binding paradigm as defined for the vCCI-chemokine interaction is likely to be conserved between A41 and its chemokine partners. Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding. The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (microM-nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine-chemokine receptor interactions.

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Model of the Interaction of A41 with CCL26, and Schematic Representation of the Biological Mechanism(A) The model of A41 interaction with CCL26 is shown orientated as in Figure 5D, with the structure based sequence conservation between A41 and vCCIs mapped onto the A41 surface (coloured as in Figure 3E, with dark blue patches representing strict sequence identity, the colour scheme for CCL26 is as for Figure 5A). (B) A close up of the interaction between the N-terminal loop of CCL26 and the Phe181 of A41. The A41 surface is shown semitransparent with a cartoon representation of A41 in pale green beneath the surface. (C) Model for mode of action of vCCI-like proteins and A41. The epitope for chemokine receptor on the chemokine is shown in yellow whilst the portion of the chemokine surface that interacts with GAG is coloured blue.
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ppat-0040005-g006: Model of the Interaction of A41 with CCL26, and Schematic Representation of the Biological Mechanism(A) The model of A41 interaction with CCL26 is shown orientated as in Figure 5D, with the structure based sequence conservation between A41 and vCCIs mapped onto the A41 surface (coloured as in Figure 3E, with dark blue patches representing strict sequence identity, the colour scheme for CCL26 is as for Figure 5A). (B) A close up of the interaction between the N-terminal loop of CCL26 and the Phe181 of A41. The A41 surface is shown semitransparent with a cartoon representation of A41 in pale green beneath the surface. (C) Model for mode of action of vCCI-like proteins and A41. The epitope for chemokine receptor on the chemokine is shown in yellow whilst the portion of the chemokine surface that interacts with GAG is coloured blue.

Mentions: The surface charge properties of A41 are broadly similar to those seen in other poxvirus vCCI proteins, although sheet I exhibits a large patch of positive charge (Figure 4A) whereas in CPXV vCCI sheet I is comparatively uncharged (Figure 4B). On the opposite face of A41 (sheet II) the dominant electrostatic feature is a negatively charged patch, and although this is not conserved in sequence between A41 and the vCCIs this region is negatively charged in the vCCIs and is conserved within that family (E46, D49, E125 and Y62 in particular, CPXV numbering). The complex of RPXV vCCI with chemokine CCL4 demonstrated that this negatively charged surface forms crucial electrostatic interactions with the positively charged 20s and 40s loop of the chemokine (Figure 5A, 5B). This charged surface includes the acidic 2–4 loop that harbours residues E46 and D49 and protrudes from sheet II to lock the chemokine in place. This loop differs in length in the vCCIs (it is 16 and 27 aa long in CPXV and RPXV respectively) and is absent in A41. Mapping structure based sequence alignment between A41 and the vCCIs onto the surface of A41 reveals that below this unconserved charged patch is a region of conservation (Figure 6A, 6B), central to which is a strictly conserved phenylalanine (F181 in A41) which forms a hydrophobic depression on the edge of the β sandwich in A41 and the vCCIs (Figure 5D).


Structure and function of A41, a vaccinia virus chemokine binding protein.

Bahar MW, Kenyon JC, Putz MM, Abrescia NG, Pease JE, Wise EL, Stuart DI, Smith GL, Grimes JM - PLoS Pathog. (2008)

Model of the Interaction of A41 with CCL26, and Schematic Representation of the Biological Mechanism(A) The model of A41 interaction with CCL26 is shown orientated as in Figure 5D, with the structure based sequence conservation between A41 and vCCIs mapped onto the A41 surface (coloured as in Figure 3E, with dark blue patches representing strict sequence identity, the colour scheme for CCL26 is as for Figure 5A). (B) A close up of the interaction between the N-terminal loop of CCL26 and the Phe181 of A41. The A41 surface is shown semitransparent with a cartoon representation of A41 in pale green beneath the surface. (C) Model for mode of action of vCCI-like proteins and A41. The epitope for chemokine receptor on the chemokine is shown in yellow whilst the portion of the chemokine surface that interacts with GAG is coloured blue.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211551&req=5

ppat-0040005-g006: Model of the Interaction of A41 with CCL26, and Schematic Representation of the Biological Mechanism(A) The model of A41 interaction with CCL26 is shown orientated as in Figure 5D, with the structure based sequence conservation between A41 and vCCIs mapped onto the A41 surface (coloured as in Figure 3E, with dark blue patches representing strict sequence identity, the colour scheme for CCL26 is as for Figure 5A). (B) A close up of the interaction between the N-terminal loop of CCL26 and the Phe181 of A41. The A41 surface is shown semitransparent with a cartoon representation of A41 in pale green beneath the surface. (C) Model for mode of action of vCCI-like proteins and A41. The epitope for chemokine receptor on the chemokine is shown in yellow whilst the portion of the chemokine surface that interacts with GAG is coloured blue.
Mentions: The surface charge properties of A41 are broadly similar to those seen in other poxvirus vCCI proteins, although sheet I exhibits a large patch of positive charge (Figure 4A) whereas in CPXV vCCI sheet I is comparatively uncharged (Figure 4B). On the opposite face of A41 (sheet II) the dominant electrostatic feature is a negatively charged patch, and although this is not conserved in sequence between A41 and the vCCIs this region is negatively charged in the vCCIs and is conserved within that family (E46, D49, E125 and Y62 in particular, CPXV numbering). The complex of RPXV vCCI with chemokine CCL4 demonstrated that this negatively charged surface forms crucial electrostatic interactions with the positively charged 20s and 40s loop of the chemokine (Figure 5A, 5B). This charged surface includes the acidic 2–4 loop that harbours residues E46 and D49 and protrudes from sheet II to lock the chemokine in place. This loop differs in length in the vCCIs (it is 16 and 27 aa long in CPXV and RPXV respectively) and is absent in A41. Mapping structure based sequence alignment between A41 and the vCCIs onto the surface of A41 reveals that below this unconserved charged patch is a region of conservation (Figure 6A, 6B), central to which is a strictly conserved phenylalanine (F181 in A41) which forms a hydrophobic depression on the edge of the β sandwich in A41 and the vCCIs (Figure 5D).

Bottom Line: Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors.Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding.The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (microM-nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine-chemokine receptor interactions.

View Article: PubMed Central - PubMed

Affiliation: The Division of Structural Biology and The Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

ABSTRACT
The vaccinia virus (VACV) A41L gene encodes a secreted 30 kDa glycoprotein that is nonessential for virus replication but affects the host response to infection. The A41 protein shares sequence similarity with another VACV protein that binds CC chemokines (called vCKBP, or viral CC chemokine inhibitor, vCCI), and strains of VACV lacking the A41L gene induced stronger CD8+ T-cell responses than control viruses expressing A41. Using surface plasmon resonance, we screened 39 human and murine chemokines and identified CCL21, CCL25, CCL26 and CCL28 as A41 ligands, with Kds of between 8 nM and 118 nM. Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors. However the interaction of A41 and chemokines was inhibited in a dose-dependent manner by heparin, suggesting that A41 and heparin bind to overlapping sites on these chemokines. To better understand the mechanism of action of A41 its crystal structure was solved to 1.9 A resolution. The protein has a globular beta sandwich structure similar to that of the poxvirus vCCI family of proteins, but there are notable structural differences, particularly in surface loops and electrostatic charge distribution. Structural modelling suggests that the binding paradigm as defined for the vCCI-chemokine interaction is likely to be conserved between A41 and its chemokine partners. Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding. The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (microM-nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine-chemokine receptor interactions.

Show MeSH
Related in: MedlinePlus