Solution Structure of the C-terminal Domain of TFIIH P44 Subunit Reveals a Novel Type of C4C4 Ring Domain Involved in Protein-Protein Interactions

Esther Kellenberger,Cyril Dominguez,Sébastien Fribourg,Emeric Wasielewski,Dino Moras,Arnaud Poterszman,Rolf Boelens,Bruno Kieffer

doi:10.1074/jbc.m412999200

Abstract

The human general transcription factor TFIIH is involved in both transcription and DNA nucleotide excision repair. Among the 10 subunits of the complex, p44 subunit plays a crucial role in both mechanisms. Its N-terminal domain interacts with the XPD helicase, whereas its C-terminal domain is involved specifically in the promoter escape activity. By mutating an exposed and non-conserved cysteine residue into a serine, we produced a soluble mutant of p44-(321-395) suitable for solution structure determination. The domain adopts a C4C4 RING domain structure with sequential organization of beta-strands that is related to canonical RING domains by a circular permutation of the beta-sheet elements. Analysis of the molecular surface and mutagenesis experiments suggests that the binding of p44-(321-395) to TFIIH p34 subunit is not mediated by electrostatic interactions and, thus, differs from previously reported interaction mechanisms involving RING domains.

Highlights

TFIIH is a multiprotein complex that is required both in DNA repair through the nucleotide excision repair pathway and transcription [1, 2]
The domain adopts a C4C4 RING domain structure with sequential organization of ␤-strands that is related to canonical RING domains by a circular permutation of the ␤-sheet elements
Analysis of the molecular surface and mutagenesis experiments suggests that the binding of p44-(321–395) to TFIIH p34 subunit is not mediated by electrostatic interactions and, differs from previously reported interaction mechanisms involving RING domains

Summary

MATERIALS AND METHODS

Sample Preparation—The C381S mutant of p44-(321–395) was cloned into a modified version of pGEX-4T2 (Amersham Biosciences) as previously described for other p44 mutants [14]. 0.5 mM unlabeled and 1 mM 15N-labeled proteins were dissolved in 20 mM deuterated Tris-HCl buffer, 20 mM NaCl, and 0.5 mM dithiothreitol at pH 7 (at 4 °C). Gin) and pACYC184 –11b (no tag, chloramphenicol resistance, and p15A origin), respectively Both vectors harbor compatible replication origins suitable for co-expression of the two subunits as described in Fribourg et al [13]. State of the imidazole rings of histidine residues was investigated using long range 1H,15N HSQC spectra recorded on the zinc- and cadmiumloaded proteins with a magnetization transfer delay (1/2J1H-15N) of 23 ms. The protonation state was deduced from the pattern of observed correlations as described [15]

Vicinal scalar

RESULTS AND DISCUSSION

Residues in generously allowed regions