Molecular Basis for SUMOylation-dependent Regulation of DNA Binding Activity of Heat Shock Factor 2

Yukihiro Tateishi,Mariko Ariyoshi,Ryuji Igarashi,Hideyuki Hara,Kenji Mizuguchi,Azusa Seto,Akira Nakai,Tetsuro Kokubo,Hidehito Tochio,Masahiro Shirakawa

doi:10.1074/jbc.m806392200

Abstract

Heat shock factor 2 (HSF2) is a member of a vertebrate transcription factor family for genes of heat shock proteins and is involved in the regulation of development and cellular differentiation. The DNA binding property of HSF2 is modulated by the post-translational modification of a specific lysine residue in its DNA binding domain by small ubiquitin-like modifier (SUMO), but the consequences of SUMOylation and its underlying molecular mechanism remain unclear. Here we show the inhibitory effect of SUMOylation on the interaction between HSF2 and DNA based on biochemical analysis using isolated recombinant HSF2. NMR study of the SUMOylated DNA binding domain of HSF2 indicates that the SUMO moiety is flexible with respect to the DNA binding domain and has neither a noncovalent interface with nor a structural effect on the domain. Combined with data from double electron-electron resonance and paramagnetic NMR relaxation enhancement experiments, these results suggest that SUMO attachment negatively modulates the formation of the protein-DNA complex through a randomly distributed steric interference.

Highlights

The heat shock factor (HSF) members are characterized by a conserved domain structure that consists of a winged helix-turn-helix motif near the N terminus followed by an extended hydrophobic heptad repeat (HR-A/B)
The isolated DNA binding domain (DBD) of Kluyveromyces lactis have been shown to bind to heat shock elements (HSEs) in vitro [9]; it is suggested that isolated DBDs of HSFs retain a binding affinity for HSE repeats and that the trimer formation of HSF1 and Heat shock factor 2 (HSF2) has a regulative role in DNA binding
small ubiquitin-like modifier (SUMO)-1 Modification Abrogates the DNA Binding Activity of hHSF2 DBD—Combined with biochemical data, the crystal structure of the K. lactis HSF DBD in complex with DNA previously demonstrated a stable dimer formation of its DBD upon binding to two inverted HSEs arranged in the tail-to-tail orientation [9]

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The cDNA region encoding DBD of human HSF2␤ (hHSF2DBD) was amplified by PCR and cloned into the bacterial expression vector pGEX4T-3 (GE Healthcare) with an N-terminal GST tag. The reaction mixtures were incubated at room temperature for 4 h These SUMOylated proteins were purified in the same manner as described above. Each duplex oligonucleotide was mixed with the unmodified or SUMOylated protein in a binding reaction mixture containing 10 mM HEPES-KOH (pH 7.5), 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 5% glycerol. MTSL, dissolved in DMSO, was added to each SUMOylated hHSF2DBD mutant in a 4 M excess of cysteine residues, and the reaction mixture was incubated at room temperature for 12 h. A series of models of hHSF2DBD was generated with MODELLER [34] using each of the 28 NMR-derived models of Drosophila HSF DBD [35] deposited in the Protein Data Bank (PDB entry 1HKT) as a template. Lower limits were set based on the fact that difference in distances between two C-␣ atoms (R␣␣) of spin-labeled cysteines and between the two nitroxide moieties (RNONO) of a doubly MTSL-labeled protein has been shown to range from 0 to 10 Å

Response Units

NDa NDa

RESULTS

The absence of chemical shift differences for all other signals suggests

DISCUSSION