Rational Design of High Affinity Interaction Between CC Chemokine Binding Protein vCCI and CCL17/TARC.

Area of Science:

• Immunology
• Biochemistry
• Computational Biology

Background:

• The poxvirus protein vCCI inhibits pro-inflammatory CC chemokines with high affinity.
• CCL17 (TARC) exhibits unusually weak micromolar binding to vCCI, limiting its therapeutic potential.
• Understanding vCCI-CCL17 interactions is crucial for developing targeted anti-inflammatory strategies.

Purpose of the Study:

• To elucidate the molecular basis for the weak binding between vCCI and CCL17.
• To identify mutations in CCL17 that enhance binding affinity to vCCI.
• To provide a foundation for engineering chemokine inhibitors for anti-inflammatory therapies.

Main Methods:

• Sequence analysis of CCL17 and related chemokines.
• Molecular simulations to predict mutation sites impacting vCCI binding.
• Site-directed mutagenesis to create CCL17 variants.
• Biochemical assays to measure binding affinity (K_d) of vCCI to CCL17 mutants.

Main Results:

• Sequence and simulation analyses identified key residues in CCL17 for vCCI interaction.
• Single point mutants (V44K, Q45R) increased vCCI binding affinity 2-3 fold.
• A triple mutant (G17R/V44K/Q45R) demonstrated a 68-fold affinity improvement (K_d = 0.25 μM).
• A quadruple mutant showed high affinity, though slightly lower than the triple mutant.

Conclusions:

• Sequence comparisons and molecular simulations effectively predict mutations enhancing chemokine-inhibitor binding.
• Engineered CCL17 mutants exhibit significantly improved affinity for vCCI.
• This study represents a critical step towards developing novel anti-inflammatory therapeutics based on modified chemokines.