- Short Report
- Open Access
SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family
© Antonets et al; licensee BioMed Central Ltd. 2010
- Received: 17 June 2010
- Accepted: 27 October 2010
- Published: 27 October 2010
Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein.
De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins.
Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.
- Root Mean Square Deviation
- Myxoma Virus
- Variola Virus
- Cowpox Virus
- Similar Tertiary Structure
Chemokines form a large family of chemoattractant cytokines with low molecular weight (~7-14 kDa) and their sequence similarity range from 20 to 90%. More than 50 distinct chemokines and over 19 different chemokine receptors were discovered to date. All known cellular chemokine receptors are type III transmembrane proteins associated with G-proteins. According to the arrangement of conservative N-terminal cysteine residues chemokines are divided into CC-, CXC-, C- and CX3C-chemokines . Chemokines play important roles in regulation of innate as well as acquired immunity orchestrating leukocyte migration. Through binding to cell surface or intercellular matrix glycosaminoglycans (GAGs) chemokines form chemotactic gradient attracting leukocytes to the sites of injury and inflammation. Chemokines are also involved in embryonic development, organogenesis and other processes [1, 2]. Despite low sequence identity between different chemokines all of them share remarkably similar tertiary structure with elongated N-terminal loop followed by three β-chains folded in a "greek key" moiety .
During long-lasting coevolution with their hosts poxviruses have developed efficient mechanisms to evade host immune reactions initiated in response to infection. In particular, one of the most important viral immunomodulatory strategies are viroceptors - virally encoded proteins secreted from infected cells - which bind and inhibit biological activity of tumour necrosis factor (TNF), different kinds of interferons, interleukine 18, chemokines and other mediators of host immune system [4–6]. Such viroceptors may be non-homologous to cellular proteins with correspondent biological activity, as it was shown for poxviral chemokine-binding proteins . Some poxviral immunomodulatory proteins possess pleiotropic activity, as it was shown for M-T7 protein of myxoma virus, which is able to bind γIFN as well as chemokines . Recently additional biological activity was discovered for orthopoxviral CrmB and CrmD proteins. Previously known only as TNF-binding proteins, they were shown to bind with high affinity and inhibit CCL25, CCL28, CCL25, CXCL12β, CXCL13 and CXCL14 chemokines. Chemokine-binding activity of CrmB and CrmD proteins was shown to be mediated by their unique C-terminal domain lacking substantial homology to any other known proteins. This domain was named SECRET - Smallpox virus-Encoded Chemokine Receptor .
Since C-terminal domain of VARV-CrmB lacks substantial sequence homology with other proteins (excepting orthologous proteins of other orthopoxviruses), and since its spatial structure is not known, we decided to predict it to find related proteins sharing similar structural features with VARV-CrmB SECRET domain. Predicted spatial structure of VARV CrmB SECRET domain was found to be closely related to cowpox virus vCCI protein, belonging to the family of poxviral type II chemokine-binding proteins.
Although spatial structures of vCkBPII proteins are remarkably similar, different members of the family have notable characteristic structural differences. Thus VACV A41 lacks long extended loop which connects the 2nd and the 3d β strands of vCCI (Figure 2A). This loop is conserved in vCCI and its orthologs and was shown to make these proteins able to bind wide spectrum of CC-chemokines with high affinity [3, 13, 17]. Comparison of known tertiary structure of VACV A41 with predicted spatial structure of the SECRET domain of VARV CrmB revealed that SECRET probably also lacks this loop (Figure 2). This finding gives a bit of support to hypothesis of Ruiz-Arguello and his colleagues  that high selectivity and similar specificity towards chemokines, observed for VACV A41 and ectromelia virus (ECTV) E163 protein (orthologous to A41) and SECRET-domain containing proteins, should be underlaid by common structural traits of these vCkBPs. VACV A41 and ECTV E163 bind with high affinity CCL21, 25, 26, 28, CXCL12α, CXCL12β, CXCL13 and CXCL14 chemokines [3, 14]; and SECRET domains of VARV CrmB, ECTV CrmD and some other SECRET-containing proteins were shown to bind with high affinity CCL25, 28, CXCL12β, CXCL13 and CXCL14 chemokines .
Our prediction that VARV CrmB SECRET domain belongs to the family of poxviral type II chemokine-binding proteins is supported by the following: 1. SECRET was shown to bind several chemokines with high affinity ; 2. its ligand-binding surface was predicted to have prominent electronegative charge required for binding to positively charged conservative amino-acid residues of chemokines, as it was shown for other members of vCkBPsII [3, 7, 13, 17]; 3. its selectivity towards chemokines is likely to be associated with lacking long extended negatively charged loop (present in vCCI and its orthologs [3, 7, 13, 17]) as it was shown for A41 protein . Thus the predicted structural similarity of SECRET with other vCkBPsII indicates that despite low similarity of their sequences they were most likely derived from the common ancestor. It's curiously that in genomes of Leporipoxvirus and Orthopoxvirus genera members genes coding for CrmB orthologs are situated in immediate proximity to the genes coding for vCCI orthologs, and we think, it would be of interest to examine phylogenetic relations between the SECRET domain of CrmB and vCCI orthologs.
Prediction of secondary structure of VARV-CrmB SECRET domain was done using PSIPRED server . Spatial structure of SECRET domain was modeled using I-TASSER web-server . The server selected the structure of cowpox virus (CPXV) vCCI (viral CC-chemokine inhibitor) protein [PDB:1CQ3] as the best template for modelling SECRET domain. Electrostatic potential of molecular surfaces of all compared proteins was calculated using DelPhi software . Superpositions of the structures were made using CEalign . All molecular graphic images were produced using PyMOL . VARV-CrmB amino acid sequence used in this work was taken from [Swiss-Prot:P34015]. Amino acid sequences of CPXV vCCI [PDB:1CQ3], vaccinia virus (VACV) A41 chemokine-binding protein [PDB:2VGA] and VARV-CrmB SECRET domain were structurally aligned using TM-align server . Alignment was manually edited taking in mind the secondary structures (shown above corresponding sequences) of the proteins using Aline software .
This work was supported by Russian Foundation for Basic Research (grant #09-04-00055a).
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