Molecular Mechanisms of Interaction between Human Tumor Necrosis Factor and CrmB TNF-binding Proteins of Cowpox and Variola Viruses

Recombinant viral proteins inhibiting Tumor Necrosis Factor (TNF) activity could be used for development of new drugs for the inflammatory diseases treatment. Computer simulation of the complexes of interacting proteins and recognition of their interaction patterns allow to plan site directed mutagenesis studies and to predict modified forms of TNF-binding proteins of Orthopoxviruses with improved binding affinity and TNF neutralizing activity. Computer models based on the X-RAY structure of the mutant complex of human TNF (hTNF) with TNF receptor type II were used to analyze mechanisms of protein interactions and to identify key residues involved in the formation of complexes between hTNF and TNF-binding domains of cowpox virus (TNFBD-CPXV) and variola virus (TNFBD-VARV). Molecular dynamics simulation and MM-GBSA free energy calculations showed that hTNF binds with higher affinity to TNFBD-VARV in comparison with TNFBD-CPXV. The results of the calculations were confirmed by experimental binding affinity measurements using the SPR method. Amino acid substitution ASP63$\to$ASN63 was revealed to induce structural rearrangements favoring the interaction between TNFBD-VARV and hTNF. Additionally, free energy decomposition protocol of individual amino-acid residues allowed to identify key amino acids involved in the complex formation energy.