Abstract
The M3 protein (M3) encoded by murine gammaherpesvirus 68 (MHV-68) is a unique viral immunomodulator with a high-affinity for a broad spectrum of chemokines, key mediators responsible for the migration of immune cells to sites of inflammation. M3 is currently being studied as a very attractive and desirable tool for blocking the chemokine signaling involved in some inflammatory diseases and cancers. In this study, we elucidated the role of M3 residues E70 and T272 in binding to chemokines by examining the effects of the E70A and T272G mutations on the ability of recombinant M3, prepared in Escherichia coli cells, to bind the human chemokines CCL5 and CXCL8. We found that the E70A mutation enhanced binding of M3 to CCL5 two-fold but had little effect on its binding to CXCL8. In contrast, the T272G mutation was found to be important for the thermal stability of M3 and significantly decreased M3's binding to both CCL5 (by about 4×) and CXCL8 (by about 5×). We also constructed in silico models of the wild-type M3–CCL5 and M3–CCL8 complexes and found substantial differences in their physical and chemical properties. M3 models with single mutation E70A and T272G suggested the role of E70 and T272 in binding M3 protein to chemokines. In sum, we have confirmed that site-directed mutagenesis could be an effective tool for modulating the blockade of particular chemokines by M3, as desired in therapeutic treatments for severe inflammatory illnesses arising from chemokine network dysregulation.
Highlights
Chemokines are low molecular weight, chemoattractant cytokines that modulate the migration of immune cells from blood vessels to sites of infection and inflammation, an important phenomenon in host defense (Zlotnik and Yoshie, 2000)
E70 is in the M3 protein (M3) N-terminal domain (NTD) (1–234), and T272 is in the C-terminal domain (CTD) (235–406) (Figures 1A,B); both are close to sequences known to be involved in binding chemokine CCL2 (E104–I110, A261, and Y290–A299) (Diaz et al, 2009)
Testing the binding of recombinant M3’s with concentration ranges on the order of 10−10 to 10−7 mol/l to both chemokines we found that wild-type M3 protein (wtM3) and M3-E70A, but not M3-T272G, were able to inhibit more than 50% of CCL5 (Figure 3A)
Summary
Chemokines are low molecular weight, chemoattractant cytokines (classified into four subfamilies—CC, CXC, CX3C, and C according to the position of their N-terminal cysteine residues) that modulate the migration of immune cells from blood vessels to sites of infection and inflammation, an important phenomenon in host defense (Zlotnik and Yoshie, 2000). Viruses have acquired and optimized molecules that interact with the host chemokine network They express a repertoire of proteins which interfere with the host immune response in order to avoid being eliminated from the organism. Virus-encoded immunomodulatory proteins target or bind to chemokines and their receptors and modulate chemokine gradient formation and ligand-receptor recognition; they even have the potential to completely block chemokine-mediated responses to viral infection. They are used to promote cell entry, facilitate dissemination of infected cells, and interfere with extracellular chemokines (Alcami, 2003)
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