Figure 5. Crystal Structures Reveal the Molecular Basis for Rare Y33P Mutation Requirement.

(A) Development of somatic hypermutations along the maturation pathway of VRC34.01. SHMs of the VRC34 intermediates are mapped on the structure of VRC34.01 shown in ribbon representation. The location of each SHM is indicated with a sphere and colored according to the paired intermediates. (B) Effect of single Ala or Gly mutation on the binding affinity of mutant peptides to VRC34 UCA and intermediates was measured by BLI. Fitted binding curves of the Ala/Gly mutants and parent peptide to VRC34-UCA, VRC34-pI1, VRC34-pI2 demonstrating absence of binding are shown on the left; binding response of individual mutant peptides (alanine (grey bars) and glycine (white bars)) normalized to parent FP for VRC34-pI3, VRC34-pI4 and VRC34.01 are shown on the right. (C) Binding interaction of FP residues with intermediates VRC34-pI2 (left panel), VRC34-pI3 (middle), and VRC34-pI4 (right). VRC34-pI2 was a homology model based on VRC34-pI3. FP is colored red, pI2 mutations are colored blue, pI3 mutations are colored orange, and pI4 mutations are colored purple. (D) Binding kinetics by BLI of chimeric antibodies, VRC34 pI2_HC/pI3_LC and VRC34 pI3_HC/pI2_LC, and of single point mutants, pI2 H33P, pI3 P33H and pI3 P33A, with His-tagged FP8_v1 (upper row) and diverse FP-KLH conjugates (lower row). We note that in the lower row, interactions between bivalent antibodies and the multivalent FP-KLH conjugates reduce off rate, leading to very high apparent affinities, which in these BLI measurements “max out” at ~K_D=10⁻¹².

See also Figure S6 and Table S5.