FIGURE 4.

Binding site mapping. (A) Fifty-six single or double alanine variants of hsPrp38^NTD are clustered according to the degree of similarity in their effects on the interactions with the 12 identified hsPrp38^NTD binding partners (clusters I–VII on the right). As a measure of relative binding strength, the growth signal of Y2H hits was quantified, normalized to the growth signal of the hsPrp38^NTD–hsMFAP1 interaction and presented in a heat map by a white-to-red color gradient. White boxes—no growth; red boxes—strong growth. The wt binding profile is boxed in cluster I. (B) Growth of representative Y2H colonies of the 12 hsPrp38^NTD binding partners with respective hsPrp38^NTD variants of all seven clusters on selective agar (I–VII). Red or green boxes indicate reduction or increase of the growth signal relative to wt hsPrp38^NTD, respectively. V↑—subgroup of cluster V with increased interaction to hsSnu66; V↓—subgroup of cluster V with reduced interaction to hsSnu66. (C) Cartoon representation of hsPrp38^NTD with clusters IV–VII mapped to the protein surface. Residues mutated in clusters IV–VII that negatively affect binding of an interaction partner are colored in red; the residue that upon mutation positively affects binding of hsSnu66 is colored in green.