FIGURE 5.

Mutational and RDC analysis to evaluate the MyT1-DNA interaction. A, weighted chemical shift changes for backbone H and N from Tyr-817/Tyr-861 in the absence and presence of 1 molar eq of RARE DNA. The number on top of the bars (average from n = 3–4 ± S.E.) indicates binding association constants from SPR experiments carried out with immobilized biotinylated double-stranded RARE DNA and mutant F4F5 constructs in solution. Green bars indicate equal or higher affinity constants as compared with wild-type F4F5. Red bars indicate mutations that result in significantly lower association constants and are therefore important for the interaction. B, graphic (left) and space-filling representation (middle and right) of F4 with mutations that result in equal/stronger and weaker DNA binding colored in green and red (as in panel A), respectively. The second ZF domain (F5, not shown) shows an identical pattern (with corresponding mutations). C, space-filling representation of F4 from run I and run II in the same orientation as in the middle of panel B with residues that make specific contacts to the DNA colored in dark gray. Note that the DNA-binding residues in run I match red residues (important for binding) in B (in contrast to run II), in agreement with our structural model. D, portion of the ¹⁵N HSQC of ¹⁵N-labeled double ZF mutant Y817F/Y861F (top) and S824D/S868D (bottom) in the absence (gray) and presence (black) of 1 molar eq of RARE DNA (as in Fig. 2A). Note the significant difference between the two mutants. E, representative SPR binding curves for the interaction of the Y817F/Y861F double mutant (0.2–6 μm) in solution with immobilized biotinylated double-stranded RARE DNA. F, plot of calculated (from refined structures) versus measured one-bond HN-N RDCs (average from the 10 lowest SANI energy HADDOCK structures).