Crystal packing interaction that blocks crystallization of a site‐specific DNA binding protein–DNA complex

Abstract

We present here three high-resolution crystal structures of complexes between the DNA-binding domain of the heat-shock transcription factor (HSF) and DNA oligomers. Although the DNA oligomers contain HSF's specific binding sequence, called a heat-shock element, the crystal structures do not contain the specific protein-DNA complex. In one crystal structure, the 10 base pair DNA oligomer is statically disordered. In the other two related structures, the 12 base pair DNA oligomers are in unique positions, but the protein-DNA contacts in these two crystals are not sequence specific. In all three structures, the DNA appears to act as a rigid, polyanion scaffold to support columns of proteins in a crystalline lattice. A robust crystal packing interface between protein monomers obscures the true DNA-binding surface, known from previous genetic and biochemical studies. By redesigning the protein to interfere with the crystal lattice contacts, we were able to obtain physiologically relevant crystals in a specific protein-DNA complex. Thus, a crystal-packing interface was able to prevent the weak, but physiological relevant interactions between a protein and DNA.

Crystal Structure of the Heterodimeric Complex of the Adaptor, ClpS, with the N-domain of the AAA+ Chaperone, ClpA

“…Alternatively, the surfaces of ClpS or N-domain involved in these interfaces might have other binding functions in solution. It is not uncommon for substrate-binding sites and protein-protein interfaces to provide sites for crystal lattice contacts (43). In fact, in our structure of the full-length ClpA (5), the N-D1 interface employs the same region of the N-domain that makes interface B in the trigonal crystal form of ClpS-N. Also, the limited but rather precise fit producing contact C in the ClpS-N crystal may mimic another substrate or adaptor binding site that specifically recognizes an exposed Asp-Tyr sequence motif.…”

De Novo Appearance and “Strain” Formation of Yeast Prion [PSI+] Are Regulated by the Heat-Shock Transcription Factor

“…HSFs are structurally and functionally conserved from yeast to humans, containing a winged helix-turn-helix DNA-binding domain, a hydrophobic stretch necessary for homotrimerization, followed by a transcription activation domain that is also conserved (Wu 1995;Littlefield and Nelson 2001). HSF binds to cis-acting DNA promoter elements known as heat-shock elements (HSEs), which are highly conserved as well (Amin et al 1988).…”

Study of Cytochrome c-DNA Interaction – Evaluation of Binding Sites on the Redox Protein

“…with enzymes 2,3 and transcription factors. 4,5 This kind of interaction can be accompanied by intercalation of residues between the base pairs (after unwinding of the DNA) or protein binding to DNA secondary structures such as loops or G-quadruplexes. [6][7][8] Nonspecific binding, which often precedes specific binding, arises from charge-charge interaction between cationic residues and the anionic phosphate backbone.…”