Fig 2. Alanine scanning mutagenesis reveals E2 residues important for mAb binding.

(A) Amino acid sequence of E2 from the RRV T48 strain, indicating loss-of-binding residues determined through alanine scanning mutagenesis. Each amino acid residue is numbered according to its position within the E2 protein, with the A, B, and C domains along with the arch regions [22,26] color coded (grey, arch; dark blue, domain A; green, domain B; magenta, domain C). A circle above the sequence indicates the position of residues for which alanine substitution disrupts mAb binding, with each circle color corresponding to a different mAb. (B) Summary table with residues disrupted by alanine scanning mutagenesis, including the E2 domain in which they are found and the competition group to which the mAb belongs (see Fig 3) Two independent experiments were performed and values were averaged for loss-of-binding determination. A cutoff value of 10% was used, with the requirement that two other mAbs have binding of 50% or greater. (C) Loss-of-binding residues mapped onto the crystal structure of the CHIKV E1/E2 heterodimer (PDB 3N42), with three heterodimers subunits combined to represent the viral spike trimer. Top and side views of the trimer are shown, with residues important for mAb binding color coded as in (A) and shown as space-filling forms. The E2 protein is shown in green and the E1 protein in light brown, and each of the domains is labeled as in (A). A side view of a single heterodimer subunit is also shown (bottom).