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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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Analysis of Human Chemokines Binding to A41 and Modelling of the A41:CCL26 ComplexThe structure of CCL4 (A) coloured green, and the N-terminal loop, 20s loop, and 40s loop are coloured yellow, blue and magenta, respectively. (B) The NMR complex of CCL4 with the RPXV: vCCI is shown with CCL4 depicted as in (A) and RPXV vCCI is shown with its electrostatic surface charge. The position of the 2–4 loop is labelled and highlighted by a grey oval. The views are orientated facing the vCCI β sheet II (180° to the view in Figure 3A). (C) The structure of CCL26 coloured as (A). The positions of insertions present in the 20s and 40′s loops of the CC chemokines that bind to A41 are shown with spheres coloured blue and magenta, respectively. (D) A model of the interaction between A41 and CCL26 generated using SHP [28], in an equivalent representation and view to (B). (E) Sequence alignment of CC chemokines that bind to A41 and to vCCIs, created using CLUSTALW [67]. CC chemokines that bind to A41 are ordered in descending order of affinity to A41E. coli. Those CC chemokines that do not bind to A41, but bind to vCCIs are shaded grey. The secondary structure of CCL26 is shown above the alignment. Sequence numbering is according to the CCL26 sequence.
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ppat-0040005-g005: Analysis of Human Chemokines Binding to A41 and Modelling of the A41:CCL26 ComplexThe structure of CCL4 (A) coloured green, and the N-terminal loop, 20s loop, and 40s loop are coloured yellow, blue and magenta, respectively. (B) The NMR complex of CCL4 with the RPXV: vCCI is shown with CCL4 depicted as in (A) and RPXV vCCI is shown with its electrostatic surface charge. The position of the 2–4 loop is labelled and highlighted by a grey oval. The views are orientated facing the vCCI β sheet II (180° to the view in Figure 3A). (C) The structure of CCL26 coloured as (A). The positions of insertions present in the 20s and 40′s loops of the CC chemokines that bind to A41 are shown with spheres coloured blue and magenta, respectively. (D) A model of the interaction between A41 and CCL26 generated using SHP [28], in an equivalent representation and view to (B). (E) Sequence alignment of CC chemokines that bind to A41 and to vCCIs, created using CLUSTALW [67]. CC chemokines that bind to A41 are ordered in descending order of affinity to A41E. coli. Those CC chemokines that do not bind to A41, but bind to vCCIs are shaded grey. The secondary structure of CCL26 is shown above the alignment. Sequence numbering is according to the CCL26 sequence.

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)

Analysis of Human Chemokines Binding to A41 and Modelling of the A41:CCL26 ComplexThe structure of CCL4 (A) coloured green, and the N-terminal loop, 20s loop, and 40s loop are coloured yellow, blue and magenta, respectively. (B) The NMR complex of CCL4 with the RPXV: vCCI is shown with CCL4 depicted as in (A) and RPXV vCCI is shown with its electrostatic surface charge. The position of the 2–4 loop is labelled and highlighted by a grey oval. The views are orientated facing the vCCI β sheet II (180° to the view in Figure 3A). (C) The structure of CCL26 coloured as (A). The positions of insertions present in the 20s and 40′s loops of the CC chemokines that bind to A41 are shown with spheres coloured blue and magenta, respectively. (D) A model of the interaction between A41 and CCL26 generated using SHP [28], in an equivalent representation and view to (B). (E) Sequence alignment of CC chemokines that bind to A41 and to vCCIs, created using CLUSTALW [67]. CC chemokines that bind to A41 are ordered in descending order of affinity to A41E. coli. Those CC chemokines that do not bind to A41, but bind to vCCIs are shaded grey. The secondary structure of CCL26 is shown above the alignment. Sequence numbering is according to the CCL26 sequence.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-0040005-g005: Analysis of Human Chemokines Binding to A41 and Modelling of the A41:CCL26 ComplexThe structure of CCL4 (A) coloured green, and the N-terminal loop, 20s loop, and 40s loop are coloured yellow, blue and magenta, respectively. (B) The NMR complex of CCL4 with the RPXV: vCCI is shown with CCL4 depicted as in (A) and RPXV vCCI is shown with its electrostatic surface charge. The position of the 2–4 loop is labelled and highlighted by a grey oval. The views are orientated facing the vCCI β sheet II (180° to the view in Figure 3A). (C) The structure of CCL26 coloured as (A). The positions of insertions present in the 20s and 40′s loops of the CC chemokines that bind to A41 are shown with spheres coloured blue and magenta, respectively. (D) A model of the interaction between A41 and CCL26 generated using SHP [28], in an equivalent representation and view to (B). (E) Sequence alignment of CC chemokines that bind to A41 and to vCCIs, created using CLUSTALW [67]. CC chemokines that bind to A41 are ordered in descending order of affinity to A41E. coli. Those CC chemokines that do not bind to A41, but bind to vCCIs are shaded grey. The secondary structure of CCL26 is shown above the alignment. Sequence numbering is according to the CCL26 sequence.
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