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 nuclear structures called histone locus bodies (HLBs) where they control various steps in histone gene expression. Near the C-terminus, FLASH and YARP contain a highly homologous domain that interacts with the C-terminal region of NPAT. Structural aspects of the FLASH–NPAT and YARP–NPAT complexes and their role in histone gene expression remain largely unknown. In this study, we used multidimensional NMR spectroscopy and in silico modeling to analyze the C-terminal domain in FLASH and YARP in an unbound form and in a complex with the last 31 amino acids of NPAT. Our results demonstrate that FLASH and YARP domains share the same fold of a triple α-helical bundle that resembles the DNA binding domain of Myb transcriptional factors and the SANT domain found in chromatin-modifying and remodeling complexes. The NPAT peptide contains a single α-helix that makes multiple contacts with α-helices I and III of the FLASH and YARP domains. Surprisingly, in spite of sharing a significant amino acid similarity, each domain likely binds NPAT using a unique network of interactions, yielding two distinct complexes. In silico modeling suggests that both complexes are structurally compatible with DNA binding, raising the possibility that they may function in identifying specific sequences within histone gene clusters, hence initiating the assembly of HLBs and regulating histone gene expression during cell cycle progression.

Histone locus bodies: a paradigm for how nuclear biomolecular condensates control cell cycle regulated gene expression

“…Interestingly, GON4L/YARP binds to the same 31 amino acids at the very C terminus of NPAT that FLASH does. GON4L/YARP and FLASH use similarly structured SANT/Myb domains containing a bundle of three alpha helices that each binds to NPAT in a slightly different orientation [ 40 ]. NMR structural studies indicate that these binding events are mutually exclusive, setting up an interesting possible scenario in vivo in which multiple different NPAT complexes with different functional roles reside with the HLB.…”

The Physical Chemistry and Chemical Physics (PCCP) Section of the International Journal of Molecular Sciences in Its Publications: The First 300 Thematic Articles in the First 3 Years

“…This area is the largest in terms of the number of related publications and includes 91 articles, namely [ 1 , 2 , 3 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 ,…”

Mxc, a Drosophila homolog of mental retardation-associated gene NPAT, maintains neural stem cell fate

“…Studies have shown that NPAT/Mxc acts as the scaffold protein of the HLB required for HLB assembly and transcription of canonical histone genes [ 5 ]. The N-terminal of NPAT/Mxc is crucial for self-interaction, while the C-terminal is required for recruiting other members of the HLB [ 9 , 10 ]. NPAT/Mxc is phosphorylated by cyclin E/Cdk2, such phosphorylation being a key factor in the formation of phase separation allowing the formation of HLB to be precisely and dynamically regulated [ 11 , 12 ].…”