Structural Analysis of the SANT/Myb Domain of FLASH and YARP Proteins and Their Complex with the C-Terminal Fragment of NPAT by NMR Spectroscopy and Computer Simulations

Abstract

FLICE-associated huge protein (FLASH), Yin Yang 1-Associated Protein-Related Protein(YARP) and Nuclear Protein, Ataxia-Telangiectasia Locus (NPAT) localize to discrete nuclearstructures called histone locus bodies (HLBs) where they control various steps in histone geneexpression.Near the C-terminus, FLASH and YARP contain a highly homologous domainthat interacts with the C-terminal region of NPAT. Structural aspects of the FLASH–NPAT andYARP–NPAT complexes and their role in histone gene expression remain largely unknown. In thisstudy, we used multidimensional NMR spectroscopy andin silicomodeling to analyze the C-terminaldomain in FLASH and YARP in an unbound form and in a complex with the last 31 amino acidsof NPAT. Our results demonstrate that FLASH and YARP domains share the same fold of a tripleα-helical bundle that resembles the DNA binding domain of Myb transcriptional factors and theSANT domain found in chromatin-modifying and remodeling complexes. The NPAT peptide containsa singleα-helix that makes multiple contacts withα-helices I and III of the FLASH and YARP domains.Surprisingly, in spite of sharing a significant amino acid similarity, each domain likely binds NPATusing a unique network of interactions, yielding two distinct complexes.In silicomodeling suggeststhat both complexes are structurally compatible with DNA binding, raising the possibility that theymay function in identifying specific sequences within histone gene clusters, hence initiating theassembly of HLBs and regulating histone gene expression during cell cycle progression.