Humanized HDAC10 Crystallization Protocol

A “humanized” version of HDAC10 was designed by making the A24E and D94A substitutions in Danio rerio (zebrafish) HDAC10 so as to more closely resemble the active site of human HDAC10. The preparation of this HDAC10 construct using standard PCR mutagenesis techniques will be described separately; purification was achieved as described for the wild-type enzyme.^{[32 (link)–33 (link)]} For crystallization, the protein solution [10 mg/mL HDAC10, 50 mM HEPES pH 7.5, 300 mM KCl, 5% glycerol (v/v), and 1 mM tris-(2-carboxyethyl)phosphine (TCEP) was augmented with 2 mM 3a and incubated for 1 h on ice. Trypsin was added (1:1000 trypsin:HDAC10) and the mixture was allowed to digest at ambient temperature for 1 h and then filtered using a 0.22 μm centrifuge filter. Utilizing a Mosquito crystallization robot (TTP Labtech), a 100 nL drop of protein solution was added to a 100 nL drop of precipitant solution [0.168 M KH₂PO₄, 0.032 M K₂HPO₄, and 20% PEG 3350] and microseeded with crystals of the HDAC10-Tubastatin A complex. The 200 nL sitting drop was equilibrated against 80 μL of precipitant buffer in the well reservoir at 4 °C. Crystals appeared within one day.