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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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The Structure of VACV Protein A41(A) Cartoon representation of A41 in stereo view, coloured from blue at the N terminus to red at the C terminus, with the four disulphide bonds shown as yellow sticks. (B) View at 90° to (A), showing the 9–11 loop in yellow. (C) Superposition of A41 and CPXV vCCI in stereo view, with A41 coloured blue and CPXV vCCI coloured green. (D) CPXV vCCI in the same orientation as (B), with the 2–4 loop coloured green and the 9–11 loop coloured yellow. (E) Structure-based sequence alignment between VACV A41 and the vCCIs of CPXV, RPXV and ECTV. Structurally equivalent residues defined by SHP [28] were used to generate a clustalw alignment using shp2clustalw (unpublished program). Identical residues are boxed in blue, and conserved residues are boxed in white. The secondary structure of A41 is shown above the alignment. The 2–4 loop insertion in the vCCIs, which is absent in A41, is highlighted in green, and the 9–11 loop in yellow. With the exception of these loops, residues for the vCCIs that are not matched by A41 are omitted and the position and number of residues removed is indicated under the alignment. This figure was generated with ESPript [64]. PDB IDs 1CQ3, 2FFK and 2GRK were used for the CPXV vCCI, RPXV vCCI, and ECTV EVM1 proteins, respectively. All molecular representations were generated in PyMOL [65].
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ppat-0040005-g003: The Structure of VACV Protein A41(A) Cartoon representation of A41 in stereo view, coloured from blue at the N terminus to red at the C terminus, with the four disulphide bonds shown as yellow sticks. (B) View at 90° to (A), showing the 9–11 loop in yellow. (C) Superposition of A41 and CPXV vCCI in stereo view, with A41 coloured blue and CPXV vCCI coloured green. (D) CPXV vCCI in the same orientation as (B), with the 2–4 loop coloured green and the 9–11 loop coloured yellow. (E) Structure-based sequence alignment between VACV A41 and the vCCIs of CPXV, RPXV and ECTV. Structurally equivalent residues defined by SHP [28] were used to generate a clustalw alignment using shp2clustalw (unpublished program). Identical residues are boxed in blue, and conserved residues are boxed in white. The secondary structure of A41 is shown above the alignment. The 2–4 loop insertion in the vCCIs, which is absent in A41, is highlighted in green, and the 9–11 loop in yellow. With the exception of these loops, residues for the vCCIs that are not matched by A41 are omitted and the position and number of residues removed is indicated under the alignment. This figure was generated with ESPript [64]. PDB IDs 1CQ3, 2FFK and 2GRK were used for the CPXV vCCI, RPXV vCCI, and ECTV EVM1 proteins, respectively. All molecular representations were generated in PyMOL [65].

Mentions: To understand the structural basis of the action of A41, its crystal structure was determined using protein expressed in E. coli and refolded from inclusion bodies (Materials and Methods). Phase determination was accomplished by MAD analysis of Seleno-methione (SeMet)-labelled crystals. Electron density was observed for residues 26–219 (numbering for mature protein begins at one) and the structure was refined to 1.9 Å (final R = 20.4 and Rfree = 25.2, Table 3). A41 is a single domain protein with the distinctive β sandwich fold seen in the vCCI class of poxvirus chemokine binding proteins. The two β sheets that form the β sandwich, lie parallel to each other (Figure 3A), and are linked by an array of large loops. Five anti-parallel β strands (6, 7, 1, 12 and 13) form β sheet I and define the core of the structure (the naming of secondary structure is as defined in Carfi et al, 1999). The second β sheet (sheet II) is also composed of 5 β strands; 2, 4, 5 and 9 are anti-parallel whilst 11 is adjacent to and parallel with 9. Sheet I is largely buried from solvent by two long enveloping loops on one side and sheet II on the other (the other face of sheet II is exposed to solvent). One of these loops (the 9–11 loop, residues 113–144) wraps around the molecule and connects β strands 9 and 11 (Figure 3B), whilst the second comprises the C terminus and packs tightly against the face of sheet I, and bears strands 14 and 15. Two short β strands (10 and 14) from these loops clip together in front of sheet I. There are also two short helical segments, the first of which (α1) packs against the back of sheet II and is preceded by strand 7. The second, α2, helix comprises a single turn prior to strand 10. Eight cysteine residues in A41 form four disulphide bridges (C6-C166, C33-C199, C58-C104, and C112-C152).


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)

The Structure of VACV Protein A41(A) Cartoon representation of A41 in stereo view, coloured from blue at the N terminus to red at the C terminus, with the four disulphide bonds shown as yellow sticks. (B) View at 90° to (A), showing the 9–11 loop in yellow. (C) Superposition of A41 and CPXV vCCI in stereo view, with A41 coloured blue and CPXV vCCI coloured green. (D) CPXV vCCI in the same orientation as (B), with the 2–4 loop coloured green and the 9–11 loop coloured yellow. (E) Structure-based sequence alignment between VACV A41 and the vCCIs of CPXV, RPXV and ECTV. Structurally equivalent residues defined by SHP [28] were used to generate a clustalw alignment using shp2clustalw (unpublished program). Identical residues are boxed in blue, and conserved residues are boxed in white. The secondary structure of A41 is shown above the alignment. The 2–4 loop insertion in the vCCIs, which is absent in A41, is highlighted in green, and the 9–11 loop in yellow. With the exception of these loops, residues for the vCCIs that are not matched by A41 are omitted and the position and number of residues removed is indicated under the alignment. This figure was generated with ESPript [64]. PDB IDs 1CQ3, 2FFK and 2GRK were used for the CPXV vCCI, RPXV vCCI, and ECTV EVM1 proteins, respectively. All molecular representations were generated in PyMOL [65].
© Copyright Policy
Related In: Results  -  Collection

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

ppat-0040005-g003: The Structure of VACV Protein A41(A) Cartoon representation of A41 in stereo view, coloured from blue at the N terminus to red at the C terminus, with the four disulphide bonds shown as yellow sticks. (B) View at 90° to (A), showing the 9–11 loop in yellow. (C) Superposition of A41 and CPXV vCCI in stereo view, with A41 coloured blue and CPXV vCCI coloured green. (D) CPXV vCCI in the same orientation as (B), with the 2–4 loop coloured green and the 9–11 loop coloured yellow. (E) Structure-based sequence alignment between VACV A41 and the vCCIs of CPXV, RPXV and ECTV. Structurally equivalent residues defined by SHP [28] were used to generate a clustalw alignment using shp2clustalw (unpublished program). Identical residues are boxed in blue, and conserved residues are boxed in white. The secondary structure of A41 is shown above the alignment. The 2–4 loop insertion in the vCCIs, which is absent in A41, is highlighted in green, and the 9–11 loop in yellow. With the exception of these loops, residues for the vCCIs that are not matched by A41 are omitted and the position and number of residues removed is indicated under the alignment. This figure was generated with ESPript [64]. PDB IDs 1CQ3, 2FFK and 2GRK were used for the CPXV vCCI, RPXV vCCI, and ECTV EVM1 proteins, respectively. All molecular representations were generated in PyMOL [65].
Mentions: To understand the structural basis of the action of A41, its crystal structure was determined using protein expressed in E. coli and refolded from inclusion bodies (Materials and Methods). Phase determination was accomplished by MAD analysis of Seleno-methione (SeMet)-labelled crystals. Electron density was observed for residues 26–219 (numbering for mature protein begins at one) and the structure was refined to 1.9 Å (final R = 20.4 and Rfree = 25.2, Table 3). A41 is a single domain protein with the distinctive β sandwich fold seen in the vCCI class of poxvirus chemokine binding proteins. The two β sheets that form the β sandwich, lie parallel to each other (Figure 3A), and are linked by an array of large loops. Five anti-parallel β strands (6, 7, 1, 12 and 13) form β sheet I and define the core of the structure (the naming of secondary structure is as defined in Carfi et al, 1999). The second β sheet (sheet II) is also composed of 5 β strands; 2, 4, 5 and 9 are anti-parallel whilst 11 is adjacent to and parallel with 9. Sheet I is largely buried from solvent by two long enveloping loops on one side and sheet II on the other (the other face of sheet II is exposed to solvent). One of these loops (the 9–11 loop, residues 113–144) wraps around the molecule and connects β strands 9 and 11 (Figure 3B), whilst the second comprises the C terminus and packs tightly against the face of sheet I, and bears strands 14 and 15. Two short β strands (10 and 14) from these loops clip together in front of sheet I. There are also two short helical segments, the first of which (α1) packs against the back of sheet II and is preceded by strand 7. The second, α2, helix comprises a single turn prior to strand 10. Eight cysteine residues in A41 form four disulphide bridges (C6-C166, C33-C199, C58-C104, and C112-C152).

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