BBP5 E347 side chain tends to make van der Waals contacts together with the
BBP5 E347 side chain makes van der Waals contacts with the backbone of Ash2L residues forming the b1 two loop, though the R348 side chain is solvent-exposed. In stark contrast, the E349 side chain binds inside a deep pocket formed by the side chains of Tyr313 and Arg367 (Fig. 1A, C). The primary chain carbonyl of E349 tends to make a hydrogen bond with the Ash2L Tyr313 hydroxyl group, although its carboxylate group engages in quite a few hydrogen bonds using the guanidium group of Arg367. Positioned within the bulge with the S-shaped conformation, the F352 phenyl side chain makes hydrophobic contacts with Tyr313, Pro356, and Tyr359 side chains. Equivalent to E349, the D353 carboxylate group tends to make two hydrogen bonds together with the Arg343 guanidium group, suggesting that the Ash2LSPRY positively charged cleft is important for HDAC11 medchemexpress binding this area predominantly occupied by CXCR6 Storage & Stability glutamic acid and aspartic acid residues (subsequently referred to as the DE box) of RbBP5 (Fig. 1B,C). Disruption of Ash2LRbBP5 interaction impairs MLL1 enzymatic stimulation and delays erythroid cell terminal differentiation Following structural evaluation of the Ash2LRbBP5 complex, we first sought to determine Ash2L residues which might be key for binding to RbBP5. Utilizing isothermal titration calorimetry (ITC) (Fig. 2A; Supplemental Fig. S3A), we found that replacement of Tyr313 and Arg343–twoGENES DEVELOPMENTFigure 1. The ASH2L SPRY domain binds a DE box on RbBP5. (A) Cartoon representation in the Ash2L SPRY domain (green) in complicated with RbBP5 (yellow) and also a zoomed view on the interactions in between the ASH2L SPRY domain and RbBP5. Ash2L and RbBP5 carbon atoms are highlighted in light green and yellow, respectively. Essential hydrogen bonds are rendered as red dashed lines. For clarity, only a subset of interactions is shown. (B) Electrostatic potentials are contoured from 0 kbTe (red) to 10 kbTe (blue). (e) Charge of an electron; (kb) Bolzmann’s continuous; (T) temperature in Kelvin. Zoomed view is around the positively charged cleft of Ash2L. (C) Schematic representation in the interactions stabilizing RbBP5 into the Ash2L SPRY peptide-binding pocket. Yellow spheres represent RbBP5 residues. Ash2L residues generating hydrogen bonds (filled boxes), hydrophobic contacts, or van der Waals contacts (empty boxes) with RbBP5 are rendered in blue. Hydrogen bonds are highlighted as orange dashed lines. For clarity, some interactions have been omitted in the figure.residues lining the base of the Ash2LSPRY DE-binding pocket and interacting with RbBP5 E347 and D353, respectively–with alanine severely impaired binding of RbBP5. Accordingly, enzymatic assays performed with all the similar mutants resulted in an about fivefold reduction of MLL1 methyltransferase activity compared with wild-type Ash2L (Fig. 2B; Supplemental Fig. S3B). Mutation of Pro356 and Arg367, residues interacting using the hydrophobic bulge and E349 with the RbBP5 DE box, resulted in sixfold and 13-fold reduction in binding, respectively. Accordingly, reconstitution with the complex together with the Ash2L Pro356Ala and Arg367Ala mutants failed to stimulate MLL1 methyltransferase activity for the similar extent as wild-type Ash2L, demonstrating that an Ash2L positively charged pocket lined by hydrophobic residues is essential for WRAD assembly and MLL1 methyltransferase activity (Fig. 2A,B).RbBP5 phosphorylation regulates H3K4 methylationof Flag-ASH2LTyr359Val, a mutant that exhibited activity related to Ash2LWT, restored H3K4me3 and b-globin gene expression levels comparable to Ash2LWT. Togethe.