In-Depth Mutational Analysis of the Promyelocytic Leukemia Zinc Finger BTB/POZ Domain Reveals Motifs and Residues Required for Biological and Transcriptional Functions

Abstract

The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor disrupted in patients with t(11;17)(q23;q21)-associated acute promyelocytic leukemia. PLZF contains an N-terminal BTB/POZ domain which is required for dimerization, transcriptional repression, formation of high-molecular-weight DNAprotein complexes, nuclear sublocalization, and growth suppression. X-ray crystallographic data show that the PLZF BTB/POZ domain forms an obligate homodimer via an extensive interface. In addition, the dimer possesses several highly conserved features, including a charged pocket, a hydrophobic monomer core, an exposed hydrophobic surface on the floor of the dimer, and two negatively charged surface patches. To determine the role of these structures, mutational analysis of the BTB/POZ domain was performed. We found that point mutations in conserved residues that disrupt the dimer interface or the monomer core result in a misfolded nonfunctional protein. Mutation of key residues from the exposed hydrophobic surface suggests that these are also important for the stability of PLZF complexes. The integrity of the charged-pocket region was crucial for proper folding of the BTB/POZ domain. In addition, the pocket was critical for the ability of the BTB/POZ domain to repress transcription. Alteration of charged-pocket residue arginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimerize but activates rather than represses transcription. In the context of full-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions. However, PLZF with the single pocket mutation R49Q repressed transcription, while the double mutant D35N/R49Q could not, despite its ability to dimerize. These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression.

Genomic and transcriptomic analyses of the medicinal fungusAntrodia cinnamomeafor its metabolite biosynthesis and sexual development

“…The BTB/POZ domain has been reported in mediation of protein-protein dimerization, regulation of cytoskeleton dynamics, and transcriptional regulation (41)(42)(43). The mutations at the dimer interface were found to result in misfolded proteins (the single-pocket mutation R49Q repressed transcription, whereas the double-mutant D35N/R49Q could not) (44).…”

The SCAN Domain of ZNF174 Is a Dimer

“…Furthermore, the molecule used here was well behaved biophysically as evidenced by reversible thermal denaturation. The high degree of stability of the SCAN dimer, as indicated by the T m of over 70°C, is ideal for a protein that may serve as a dimerization domain, as is the case with BTB/POZ (21) and with the leucine zippers of GCN4, FOS, and JUN (22,23).…”

Differentiation induction as a treatment for hematologic malignancies

“…PLZF and Bcl-6 both transcription repressors contain within their protein a POZ/BTB structure domain (Dhordain et al, 2000) that is required for the recruitment of N-CoR (Deltour et al, 1999). PLZF uses the BTB domain to form homodimers, the crystal structure of which has been solved and the key sites of the BTB domain that are required for direct co-repressor binding has been identi®ed (Melnick et al, 2000a). This site represents an attractive therapeutic target to overcome transcriptional repression in some acute leukemias and di use large cell lymphoma.…”