Structures of replication initiation proteins from staphylococcal antibiotic resistance plasmids reveal protein asymmetry and flexibility are necessary for replication - PubMed (original) (raw)
Structures of replication initiation proteins from staphylococcal antibiotic resistance plasmids reveal protein asymmetry and flexibility are necessary for replication
Stephen B Carr et al. Nucleic Acids Res. 2016.
Abstract
Antibiotic resistance in pathogenic bacteria is a continual threat to human health, often residing in extrachromosomal plasmid DNA. Plasmids of the pT181 family are widespread and confer various antibiotic resistances to Staphylococcus aureus. They replicate via a rolling circle mechanism that requires a multi-functional, plasmid-encoded replication protein to initiate replication, recruit a helicase to the site of initiation and terminate replication after DNA synthesis is complete. We present the first atomic resolution structures of three such replication proteins that reveal distinct, functionally relevant conformations. The proteins possess a unique active site and have been shown to contain a catalytically essential metal ion that is bound in a manner distinct from that of any other rolling circle replication proteins. These structures are the first examples of the Rep_trans Pfam family providing insights into the replication of numerous antibiotic resistance plasmids from Gram-positive bacteria, Gram-negative phage and the mobilisation of DNA by conjugative transposons.
© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
Figures
Figure 1.
Overview of Rep mediated rolling circle replication. (A)(1) Rep protein binds to a hairpin loop at the origin of replication and nicks the (+) strand (red), forming a covalent adduct with the DNA. (2) The Rep protein recruits PcrA helicase to the nick site and following strand separation DNA synthesis commences from the 3′-end of the nick. (3) Synthesis of a new (+) strand (green) displaces the original (+) strand (blue) and on completion the Rep protein nicks the newly synthesized origin and religates the ends of the displaced strand, producing a single stranded product. A new (−) strand is synthesized by the host cell replication machinery. (4) Replication continues 9–11 bases (magenta) beyond the nick site to regenerate the hairpin loop which the Rep protein nicks via one active site while religating the ends of the newly synthesized (+) strand with the other completing synthesis. The 11 base-pair oligonucleotide remains covalently attached to the Rep protein to produce a catalytically inactive Rep* molecule. (B) The DNA sequence at the origin of replication of S. aureus plasmid pC221. The two inverted repeats are ICRII, which forms a conserved stem–loop structure presenting the nick site at the tip in the (+) strand, and ICRIII, a plasmid-specific repeat which spans the Rep protein binding region and permits discrimination of cognate plasmids by their Rep proteins in vivo (highlighted green).
Figure 2.
X-ray crystal structure of RepSTK1 from Geobacillus stearothermophilus. (A) Cartoon representation of RepSTK1 with the catalytic tyrosine residues displayed as green sticks. (B) A monomer of RepSTK1 colored blue at the N-terminus through to red at the C-terminus with the location of each strand and helix identified. The structure is also shown schematically with the two 5-strand modules found in the major β-sheet coloured magenta and cyan to highlight the pseudo-symmetry within this part of the structure.
Figure 3.
X-ray crystal structure of RepDE from Staphylococcus aureus. (A) Cartoon representation of RepDE with the location of the catalytic tyrosine residues shown as green sticks. The 2-fold, non-crystallographic symmetry axes of both the catalytic and DNA binding domains (DBD) are indicated, highlighting the tilted conformation of the DBD with respect the catalytic domain. The 15° rotation of the catalytic domain relative to its position in RepSTK1 is also shown. RepDN is not shown, but is structurally very similar to RepDE. (B) Monomer of RepDE with rainbow colouring from blue at the N-terminus to red at the C-terminus with each strand and helix identified.
Figure 4.
RepDE and RepSTK1 share a common active site architecture. (A) The active site of RepDE. The structure reveals that the conserved residues of the Rep_trans family cluster around the catalytic tyrosine residue to form the active site. (B) The catalytic centre of RepSTK1 shows a remarkable conservation of structure between the two proteins with all of the side chains adopting identical conformations except R189 (R177 in RepSTK1). The electron density shown is anomalous difference density calculated from diffraction data collected at the Mn _K_-edge from a RepSTK1 crystal soaked in 10 mM MnCl2, revealing the position of the metal ion in the active site. The structure shown was crystallized from a metal free solution where a water molecule (red sphere) occupies the metal binding site.
Figure 5.
The location of DNA and PcrA binding interfaces. (A) The structure of RepDE with the DNA binding interface shown as red sticks, and the location of pcrA3 suppressor mutations shown as blue sticks. The catalytic tyrosine is also indicated. (B) Electrostatic surface representation of RepDE with the location of the protease K sensitive loop indicated by an arrow. (C) Electrostatic surface representation of RepSTK1, with the two basic loops which take the place of the DBD indicated with arrows.
Figure 6.
Recognition of the Origin of replication by RepDE. (A) Electrostatic surface of RepDE with a B-DNA duplex docked onto the DNA binding region, the white arrow shows the possible path the DNA would need to take in order to access the active site. (B) The origin of replication modelled as a Holliday junction, where the coaxial arrangement of DNA duplexes allows the DNA binding domain of the protein to interact with the target sequence while positioning the nick site close to the catalytic tyrosine (green spheres). This orientation of the Rep protein also presents the PcrA interaction interface (blue spheres) immediately upstream of the nick site presumably aiding recruitment of the helicase (green) to the DNA. The schematic of the origin of replication inset shows the conformation the DNA strands in the Holliday junction.
Figure 7.
RepDE binds PcrA helicase to form a processive complex. (A) Model of DNA encircled by the RepDE catalytic domains (yellow and purple) after DNA nicking based on the binding of DNA by TATA-binding protein (green). The inner face of the catalytic domain is basic in character and could interact non-specifically with the DNA backbone. With the DNA bound in this manner the interaction of PcrA with the supposed contact residues (blue spheres) would ‘lock’ the helicase onto the substrate. (B) Positioning the duplex DNA present in the PcrA-DNA complex (pdb 3pjr) suggests how the Rep protein might interact with PcrA (gray). Two wide grooves, between domains 2A and 1/2B on the helicase, could represent the Rep binding interface. This interaction creates a ternary complex in which the two proteins completely encircle the DNA, with the Rep protein stabilising the complex between the helicase and its substrate.
References
- Novick R.P. Staphylococcal plasmids and their replication. Annu. Rev. Microbiol. 1989;43:537–565. - PubMed
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