Figure 8. Target binding of parent and mutationally stabilized nanobodies.

(A) Representative images of nanobody-TagBFP expression in HeLa cells in the presence and absence of nuclearly localized target. Wild-type (WT) nanobodies, partial consensus (PC) mutants, and full-consensus (FC) mutants are depicted. Red nuclear signal is from co-transfected CAG-H2B-mCherry plasmid. Transfected DNA and nuclear protein amounts were standardized by addition of off-target nuclear localization sequence (NLS) plasmid to transfection mix for the ‘no target’ condition. Scale bar is 25 µm. (B) Crystal structures of two nanobodies that lose target binding when mutated to adhere to a full-consensus framework are shown. Non-consensus framework residues directly contributing to target interface are depicted. (C) Target-interfacing surface area values in square angstroms (rounded to whole numbers) across distinct regions for nanobodies tested for target binding are shown. Values are taken from buried surface area interface values made available through PDBE-PISA.

Figure 8—figure supplement 1. Positional interactions and intensity measurements.

(A) Total number of target-interfacing positions across 75 nanobodies. Positions of interaction were determined based on buried surface area for crystal structure interfaces made available through PDBE-PISA. Black bars labeled ‘A, B, and C’ denote positions across CDRs 1, 2, and 3, respectively. Colored bars describe sequence-level conservation across target-interfacing framework positions. (B) Nanobody-TagBFP signal intensities normalized to co-transfected H2B-mCherry signal intensities from captured fluorescence images (n = 3 replicate experiments for each condition, error bars denote standard deviation). (C) Nanobody-TagBFP signal fraction colocalized with H2B-mCherry signal in co-transfected cells from captured fluorescence images (n = 3 replicate experiments for each condition, error bars denote standard deviation). Red-dashed line represents average nuclear colocalization between nanobody-TagBFP signals and H2B-mCherry signals in the absence of nuclear target. Cells without both nanobody-TagBFP and H2B-mCherry signal were excluded from analysis.