The study is focusing on modulation of murine gammaherpesvirus 68 M3 protein via site-direct mutagenesis. Study encompasses the site-direct mutagenesis of M3 protein, chemokine binding modulation, biophysical studies and homology modelling of the mature M3 protein and its E70A and K282A mutants.. Immunomodulatory M3 Protein of Murine Herpesvirus 68: Chemokine Binding Affinity, Molecular Recognition and Stability

The M3 protein encoded by murine gammaherpesvirus 68 is a unique viral immunomodulatory protein capable of binding a wide spectrum of chemokines with distinct affinity. The ability of M3 protein to modulate inflammatory diseases and cancers arises from host chemokine network dysregulation. Here, we present a pilot study encompassing chemokine binding modulation, biophysical studies and homology modelling of the mature M3 protein and its E70A and K282A mutants prepared in a Baculovirus expression system. We examined inhibition of CCL2, CCL5, CCL11 and CXCL8 chemokines by all three M3 proteins and characterized their thermal stability. The affinity of wild-type M3 for chemokines decreased in the order CCL5 > CCL11 > CXCL8 > CCL2. The highest chemokine binding modulation by E70A and K282A mutations was observed towards CXCL8. Both mutations stimulated binding to CXCL8 by 2–4 fold however weakened the affinity for CCL2. Only modest modulation was observed towards CCL5 and CCL11. Circular dichroism and nanoscale differential scanning fluorimetry confirmed that all three proteins adopted a native-like fold in pH 7.4 and have very good thermal stability with Tm ≥ 64°C in mild-basic pH (7.4−8.0). Homology modeling of the M3–CCL11 complex confirmed the fundamental role of charge interactions for M3–chemokine complex recognition. An analysis of all crystal and in silico constructed M3–chemokine complexes strongly indicated a crucial role of E105 in M3−chemokine promiscuity.