Solution structure of the HMG protein NHP6A and its interaction with DNA reveals the structural determinants for non-sequence-specific binding

Abstract

NHP6A is a chromatin-associated protein fromSaccharomyces cerevisiae belonging to the HMG1/2 family of non-specific DNA binding proteins. NHP6A has only one HMG DNA binding domain and forms relatively stable complexes with DNA. We have determined the solution structure of NHP6A and constructed an NMR-based model structure of the DNA complex. The free NHP6A folds into an L-shaped three α-helix structure, and contains an unstructured 17 amino acid basic tail N-terminal to the HMG box. Intermolecular NOEs assigned between NHP6A and a 15 bp 13 C, 15 Nlabeled DNA duplex containing the SRY recognition sequence have positioned the NHP6A HMG domain onto the minor groove of the DNA at a site that is shifted by 1 bp and in reverse orientation from that found in the SRY-DNA complex. In the model structure of the NHP6A-DNA complex, the N-terminal basic tail is wrapped around the major groove in a manner mimicking the C-terminal tail of LEF1. The DNA in the complex is severely distorted and contains two adjacent kinks where side chains of methionine and phenylalanine that are important for bending are inserted. The NHP6A-DNA model structure provides insight into how this class of architectural DNA binding proteins may select preferential binding sites.

Sequence-specific HMG box factors recognize 10-12 base pair minor groove motifs

“…Upon binding to DNA however, the N-terminus adopts structure, wrapping around the DNA (17). It has been shown that this N-terminal tail is indispensable for the stable interaction with DNA and for proper bending (17). A similar phenomenon has been demonstrated for the B-domain of HMG-1 (42).…”

“…When not bound to DNA, the acidic N-terminal tail is relatively unstructured. Upon binding to DNA however, the N-terminus adopts structure, wrapping around the DNA (17). It has been shown that this N-terminal tail is indispensable for the stable interaction with DNA and for proper bending (17).…”

“…Moreover it has been shown for NHP6A and HMG-1D that these non-sequence specific HMG domain proteins occupy over 11 base pairs (17,43).…”

“…The non-sequence specific HMG boxes of HMG-1 (15,16), S. cerevisiae NHP6A (17) and Drosophila-HMG (18) were found to adopt highly similar structures consisting of three α-helices, arranged in an unusual L-shape or arrow head. Based on the presence of very similar secondary structural elements, an analogous structure was suggested for the sequence-specific HMG box of Sox-5 (12).…”

Multiple Nhp6 Molecules Are Required to Recruit Spt16-Pob3 to Form yFACT Complexes and to Reorganize Nucleosomes

“…This suggests that Nhp6 converts nucleosomes to an altered form in which the DNA is locally more accessible to DNase I or is a more suitable substrate for this nuclease and that unlike free nucleosomes this form is competent to bind Spt16-Pob3. DNA is dramatically bent by Nhp6 binding (27), and this could alter nucleosomes by disrupting local histone-DNA contacts. Nhp6 might bind to linker DNA and disturb contacts at the entry-exit points (linkers are defined here as the DNA extending beyond the 146-bp sea urchin 5 S rDNA nucleosome positioning sequence, Ref.…”