The structure of a DNA unwinding protein and its complexes with oligodeoxynucleotides by x-ray diffraction

Abstract

The structure of the gene 5 DNA unwinding protein from bacteriophage fd has been solved to 2.3 A resolution by x-ray diffraction techniques. The molecule contains an extensive cleft region that we have identified as the DNA binding site on the basis of the residues that comprise its surface. The interior of the groove has a rather large number of basic amino acid residues that serve to draw the polynucleotide backbone into the cleft. Arrayed along the external edges of the groove are a number of aromatic amino acid side groups that are in position to stack upon the bases of the DNA and fix it in place. The cleft then acts as an elongated pair of jaws that draws the DNA between them by charge interactions involving the phosphates with the interior lysines and arginines. The jaws then close on the DNA strand through small conformation changes and the rotation of aromatic side-chains into position to stack upon the purines and pyrimidines. Complexes of the gene 5 protein with a variety of oligodeoxynucleotides have been formed and crystallized for x-ray diffraction analysis. The crystallographic parameters of four different unit cells indicate that the fundamental unit of the complex is composed of six gene 5 protein dimers. We believe this aggregate has 622 point group symmetry and is a ring formed by end to end closure of a linear array of six dimers. From our results we have proposed a double helical model for the gene 5 protein-DNA complex in which the protein forms a spindle or core around which the DNA is spooled. 5.0-A x-ray diffraction data from one of the crystalline complexes is currently being analyzed by molecular replacement techniques to obtain what we believe will be the first direct visualization of a protein-deoxyribonucleic acid complex approaching atomic resolution.

500 MHz 1H NMR study of the role of lysines and arginines in the binding of gene‐5 protein to oligoadenylic acids

“…From X-ray diffraction data [20] it was concluded that 2 lysyl residues (24 and 26) and 3 arginyl residues (21,80 and 82) may be situated at the inner surface of the DNA binding groove. In [21 ] DNA binding was inhibited upon acetylation of the lysyl residues.…”

Cooperative Interactions of the Gene 5 Protein

“…These aggregates were shown to be not present in the absence of nucleic acid. It has also been found that when gene 5 protein is combined with oligomers of four to eight in length, essentially irrespective of sequence, and subsequently crystallized, that these crystals invariably contain an aggregate of twelve gene 5 monomers as their asymmetric unit (11). These are in addition to the obvious and direct observation that in the presence of linear single stranded DNA contiguous aggregates appear and in its absence they do not.…”

“…The structural mechanism for these attractive interactions must be assumed absent or inoperative when no DNA is at hand, for the protein does not spontaneously aggregate in solution even at high concentrations but exists only as a discrete dimer specie. Thus linear strands of DNA must play a direct role in the cooperative phenomena, and indeed it appears that even short fragments of DNA, such as oligonucleotides, may also promote aggregate formation ( 10,11 ).…”

Primary Structure of Escherichia coli Ribosomal Protein S1 and Features of Its Functional Domains

“…It is nevertheless interesting to note the similarity between the predicted secondary structure of the central, RNA-binding domain of SI and the secondary structure of phage fd gene-5 protein (a small DNA-binding and unwinding protein whole structure has been determined at 0.23 nm resolution [64], composed entirely of antiparallel p-structure). Gene-5 protein contains two identical DNA-binding sites (of opposite orientations) in the diineric native state [64]. By analogy, it is tempting to speculate that the two homologous stretches in the central region of SI are involved in the RNA-binding and helix-destabilizing functions of this protein.…”