bi050099s_si_001.pdf (167.89 kB)
NMR Structure of the Amino-Terminal Domain from the Tfb1 Subunit of TFIIH and Characterization of Its Phosphoinositide and VP16 Binding Sites†,‡
journal contribution
posted on 2005-05-31, 00:00 authored by Paola Di Lello, Bao D. Nguyen, Tamara N. Jones, Krzysztof Potempa, Michael S. Kobor, Pascale Legault, James G. OmichinskiGeneral transcription factor IIH (TFIIH) is recruited to the preinitiation complex (PIC) through
direct interactions between its p62 (Tfb1) subunit and the carboxyl-terminal domain of TFIIEα. TFIIH
has also been shown to interact with a number of transcriptional activator proteins through interactions
with the same p62 (Tfb1) subunit. We have determined the NMR solution structure of the amino-terminal
domain from the Tfb1 subunit of yeast TFIIH (Tfb11-115). Like the corresponding domain from the human
p62 protein, Tfb11-115 contains a PH domain fold despite a low level of sequence identity between the
two functionally homologous proteins. In addition, we have performed in vitro binding studies that
demonstrate that the PH domains of Tfb1 and p62 specifically bind to monophosphorylated inositides
[PtdIns(5)P and PtdIns(3)P]. NMR chemical shift mapping demonstrated that the PtdIns(5)P binding site
on Tfb1 (p62) is located in the basic pocket formed by β-strands β5−β7 of the PH domain fold.
Interestingly, the structural composition of the PtdIns(5)P binding site is different from the composition
of the binding sites for phosphoinositides on prototypic PH domains. We have also determined that the
PH domains from Tfb1 and p62 are sufficient for binding to the activation domain of VP16. NMR chemical
shift mapping demonstrated that the VP16 binding site within the PH domain of Tfb1 (p62) overlaps with
the PtdIns(5)P binding site on Tfb1 (p62). These results provide new information about the recognition
of phosphoinositides by PH domains, and point to a potential role for phosphoinositides in VP16 regulation.