Protocol for Biochemical Analysis and Structure Determination of the ZZ Domain of the E3 Ubiquitin Ligase HERC2

Abstract

Summary Since its discovery, several ligands of the ZZ domain have been identified; however, molecular and structural information underlying binding of these ligands remains limited. Here, we describe a protocol for biochemical and structural analysis of the ZZ domain of human E3 ubiquitin ligase HERC2 (HERC2 ZZ ) and its interaction with its ligands: the N-terminal tails of histone H3 and SUMO1. This methodology could be applied for characterization of binding activities of other histone readers. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020) .

The ZZ domain of HERC2 is a receptor of arginylated substrates

“…Our data demonstrate that the ZZ domain of HERC2 plays critical roles in function of both nuclear and cytoplasmic pools of HERC2. In the nucleus, HERC2 ZZ binds to the amino-terminal sequences of histone H3 and SUMO1 23 , 24 , which is essential in mediating conformational changes, DNA binding activity, chromatin localization and catalytic function of HERC2. In the cytoplasm, HERC2 ZZ recognizes the Nt-R degradation signal, and this interaction suggests that cytosolic HERC2 could act as a selective cargo degradation or recycling receptor.…”

“…Although HERC2 was identified in 1998 22 , progress in defining biological functions of the HERC2 domains remains slow, likely, due to its gigantic size. Our recent work shows that the ZZ domain of nuclear HERC2 binds to the amino-terminal sequences of histone H3 and SUMO1 23 , 24 . Here, we demonstrate that the ZZ domain of HERC2 (HERC2 ZZ ) recognizes the Nt-R degradation signal, which suggests a role of cytosolic HERC2 in the selective cargo degradation pathways.…”

Structural and biophysical characterization of the nucleosome-binding PZP domain

“… In the tube containing the beads, bring the volume up to 15–20 mL with PZP Purification Buffer B. To cleave the GST-tag, add TEV enzyme and follow protocol from the manufacture or use home purified enzyme (14 mg/mL, 30 μL) ( Liu et al., 2020a ) and gently rock on a platform shaker set to level 2-3, (Microplate Titer Plate Shaker, LAB-LINE INSTRUMENTS, Inc.) for 15–20 h at 4°C. Note: Fast or rough agitation at this stage in the purification may not be good for the protein stability.…”

Protein purification, crystallization, and structure determination of human DEAD-box RNA helicase DDX21 in different unwinding states

“… Alternatives: Sonication could be used to lyse the E.coli cells. For details also refer to Liu et al. (2020) .…”