DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels.

The Cox protein from bacteriophage P2 forms oligomeric filaments and it has been proposed that DNA can be wound up around these filaments, similar to how histones condense DNA. We here use fluorescence microscopy to study single DNA-Cox complexes in nanofluidic channels and compare how the Cox homologs from phages P2 and WΦ affect DNA. By measuring the extension of nanoconfined DNA in absence and presence of Cox we show that the protein compacts DNA and that the binding is highly cooperative, in agreement with the model of a Cox filament around which DNA is wrapped. Furthermore, comparing microscopy images for the wild-type P2 Cox protein and two mutants allows us to discriminate between compaction due to filament formation and compaction by monomeric Cox. P2 and WΦ Cox have similar effects on the physical properties of DNA and the subtle, but significant, differences in DNA binding are due to differences in binding affinity rather than binding mode. The presented work highlights the use of single DNA molecule studies to confirm structural predictions from X-ray crystallography. It also shows how a small protein by oligomerization can have great impact on the organization of DNA and thereby fulfill multiple regulatory functions.

Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2.

The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 Å resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.

Crystal structures of OrfX2 and P47 from a Botulinum neurotoxin OrfX-type gene cluster.

Botulinum neurotoxins are highly toxic substances and are all encoded together with one of two alternative gene clusters, the HA or the OrfX gene cluster. Very little is known about the function and structure of the proteins encoded in the OrfX gene cluster, which in addition to the toxin contains five proteins (OrfX1, OrfX2, OrfX3, P47, and NTNH). We here present the structures of OrfX2 and P47, solved to 2.1 and 1.8 Å, respectively. We show that they belong to the TULIP protein superfamily, which are often involved in lipid binding. OrfX1 and OrfX2 were both found to bind phosphatidylinositol lipids.

Crystal structure of the bacteriophage P2 integrase catalytic domain.

Bacteriophage P2 is a temperate phage capable of integrating its DNA into the host genome by site-specific recombination upon lysogenization. Integration and excision of the phage genome requires P2 integrase, which performs recognition, cleavage and joining of DNA during these processes. This work presents the high-resolution crystal structure of the catalytic domain of P2 integrase, and analysis of the structure-function relationship of several previously identified non-functional P2 integrase mutants. The DNA binding area is characterized by a large positively charged patch, harboring key residues. The structure reveals potential for large dimer flexibility, likely essential for rearrangement of DNA strands upon integration and excision of the phage DNA.

An in vitro selection strategy for conferring protease resistance to ligand binding peptides.

One drawback to the use of peptides as therapeutics has been their susceptibility to proteolysis. Here, we have used an in vitro display technology, CIS display, to enhance the proteolytic resistance of ligand-binding peptides by selection of protecting motifs from a large peptide library. The premise to this selection was that certain linear peptides within a library could form structures capable of preventing the access of proteases to defined cleavage sites without affecting ligand binding. A diverse 12-mer peptide library was inserted between a FLAG epitope motif and a thrombin cleavage site and this construct was fused to the bacterial initiator protein RepA for CIS display selection. After five rounds of selection, protection motifs were isolated that were capable of preventing proteolytic cleavage of the adjacent thrombin site. Some of the selected peptides were also resistant to more promiscuous proteases, such as chymotrypsin and trypsin, which were not used in the selection. The observed resistance to thrombin, trypsin and chymotrypsin translated into increased resistance to plasma proteases in vitro and to an increase in circulating half-lives in rats. This method can be applied to enhancing the in vivo stability of therapeutic peptides.

Identification of a gene encoding a functional reverse transcriptase within a highly variable locus in the P2-like coliphages.

The P2-like coliphages are highly similar; the structural genes show at least 96% identity. However, at two loci they have genes believed to be horizontally transferred. We show that the genetic content at the second loci, the TO region, contains six completely different sequences with high AT contents and with different open reading frames. The product of one of them exhibits reverse transcriptase activity and blocks infection of phage T5.

CIS display: In vitro selection of peptides from libraries of protein-DNA complexes.

We describe the development of an in vitro library selection system (CIS display) that exploits the ability of a DNA replication initiator protein (RepA) to bind exclusively to the template DNA from which it has been expressed, a property called cis-activity. A diverse peptide library is created by ligation of DNA fragments of random sequence to a DNA fragment that encodes RepA. After in vitro transcription and translation, a pool of protein-DNA complexes is formed where each protein is stably associated with the DNA that encodes it. These complexes are amenable to the affinity selection of ligands to targets of interest. Here we show that RepA is a highly faithful cis-acting DNA-binding protein and demonstrate that libraries encoding >10(12) random 18-mer peptides can be constructed and used to isolate peptides that bind specifically to disparate targets. The use of DNA to encode the displayed peptides offers advantages over in vitro peptide display systems that use mRNA.