Structure-Based Functional Analysis of the Replication Protein of Plasmid R6K: Key Amino Acids at the π/DNA Interface

Abstract

In previous work, we characterized the bases in an iteron of plasmid R6K that are important for the binding of protein monomers and dimers. Here we investigate the following six amino acids of , encoded by pir, hypothesized to be important for DNA contact: Ser71, Try74, Gly131, Gly211, Arg225, and Arg254.Rep proteins activate replication origins (ori's) by binding to tandem repeats in the DNA called iterons (3,8,13). When the sequences of iterons and rep genes are compared across families, striking similarities are observed (2,10,(16)(17)(18). A breakthrough in Rep/iteron studies came when Komori et al. published the crystal structure of a RepE monomer (from the F plasmid) in complex with its iteron DNA (10). The structure revealed that the Rep monomer contains two DNA-binding domains, an N-terminal WH1 (winged helix 1) and a C-terminal WH2 domain, with both domains used for iteron binding. A sequence alignment of several Reps presented by our group (10, 13) is a useful reference for identifying possible conserved amino acids in replication initiators, including RepE and the protein (described below). Based on the homologies observed in this class of proteins, Sharma et al. subsequently reported theoretical three-dimensional models for several Reps (18). The accuracy of these models remained to be tested.In a minimal R6K replicon, an ori called ␥ is activated by the Rep protein, , which is encoded by the pir gene ( Fig. 1A) (4,6,7,9,15). exists in both monomeric and dimeric forms, with monomers being activators of replication and dimers functioning both as replication inhibitors and as autorepressors of pir. The latter function is mediated by dimers binding to inverted repeats in the promoter that share sequence similarity with the iterons. monomers show little to no affinity for the inverted repeats but bind to an iteron better than dimers do (20). Insights into how achieves these similar but functionally distinct interactions were sought from analyses of Rep structure and the contacts that occur at the protein/DNA interface.In previous work from this lab, we determined the importance of individual bases in the iteron for the binding of monomers and dimers (14). Beyond this, we knew of no other experimental data addressing the contact residues of protein and DNA; a solved /iteron cocrystal structure was not available at that time. In the present study, we investigated the protein/DNA interface again, this time with an interest in identifying amino acids of that contact DNA. By analyzing the theoretical structural model of the monomer/iteron complex, we identified approximately 2 dozen amino acids as candidates for DNA contact and narrowed the list down to six for further study (Fig. 1B). Four (Ser71, Gly131, Arg225, and Arg254) appeared to be positionally conserved within the RepE/iteron cocrystal structure (Ser75, Gly125, Arg206, and Arg233, respectively). Tyr74 was strongly suspected to contact a thymine residue in the half of the iteron bound only by monomers (14). Lastly, Gly211 was of interest because it f...

Plasmid R6K replication control

“…Overall, the Rep proteins of pOLA54 and R6K display roughly 40% identity and >70% similarity. The data and conclusions presented by Kunnimalaiyaan et al (2004; 2007) and Swan et al (2006) are in good agreement but with some variation. For example, research by the former group did not investigate all of the amino acids implicated in DNA contact by the crystal structure, two of which (Arg75 and Asp226) received additional support in later mutation analyses (Saxena et al, 2010b).…”

“…Six amino acids (Tyr74, Ser71, Gly131, Gly211, Arg225 and Arg254) were deduced from the history of available biochemical data as being vital for π monomer–iteron contact (Kunnimalaiyaan et al, 2007). Four of these amino acids (Tyr74, Gly131, Gly211 and Arg254) are conserved and two are similar (Ser71 replaced by Thr68; Arg225 replaced by Lys 221) in the π protein encoded by plasmid pOLA54, mentioned earlier (Norman et al, 2008).…”

“…In the homology model by Kunnimalaiyaan et al, (2007), Arg254 adopts a compact form that accommodates binding with the DNA, however, its homolog in the crystallized RepE54 adopts a more extended conformation. Given the terminal location of the basic Arg254 on an unstructured loop, it is intriguing to hypothesize that the amino acid might interact with the acid-rich amino acids at the opposite end of the π monomer (e.g., Asp129 and Glu130) during cooperative binding of the protein to consecutive iterons.…”

Investigations of π Initiator Protein-mediated Interaction between Replication Origins α and γ of the Plasmid R6K

“…18). Chemical and enzymatic footprinting data are consistent with the structure (19,20). In contrast, the dimeric form of the protein contacts the bp located only in the 5Ј-half of the iteron probably through the C-terminal recognition helix; no contacts are seen with those located in the 3Ј-half (19).…”

Cooperative Binding Mode of the Inhibitors of R6K Replication, π Dimers

“…9,15-17 A π monomer contacts both the 5' half and the 3' half of the iteron through the C-terminal winged-helix (WH2) and the N-terminal winged helix (WH1), respectively, but a π dimer only contacts the 5' half of each iteron, including a highly conserved TGAGnG motif, with the WH2 of one of its subunits. [24][25][26][27] This potentially allows the WH2 motif of the second subunit of the dimer to contact another iteron-bearing sequence, 9,26 for instance, either of the other two functional oris of R6K, the α iteron or the β half-iteron, which are the active oris in vivo. 28,29 Such 'looping' is believed to transmit the replication signal from the internal iteron cluster at γ ori to the distant oris.…”