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Mechanism of Staphylococcal Multiresistance Plasmid Replication Origin Assembly by the Repa Protein

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Mechanism of Staphylococcal Multiresistance Plasmid Replication Origin Assembly by the Repa Protein Mechanism of staphylococcal multiresistance plasmid replication origin assembly by the RepA protein Maria A. Schumachera,1, Nam K. Tonthata, Stephen M. Kwongb, Naga babu Chinnama, Michael A. Liub, Ronald A. Skurrayb, and Neville Firthb aDepartment of Biochemistry, Duke University Medical Center, Durham, NC 27710; and bSchool of Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia Edited by James M. Berger, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved May 15, 2014 (received for review April 1, 2014) The staphylococcal multiresistance plasmids are key contributors (16). In Gram-negative bacteria the primosome includes DnaG to the alarming rise in bacterial multidrug resistance. A conserved primase, the replicative helicase (DnaB), and DnaC (17). Rep- replication initiator, RepA, encoded on these plasmids is essential lication initiation in Gram-positive bacteria involves DnaG pri- for their propagation. RepA proteins consist of flexibly linked mase and helicase (DnaC) and the proteins DnaD, DnaI, and N-terminal (NTD) and C-terminal (CTD) domains. Despite their essen- DnaB (18–22). DnaD binds first to DnaA at the origin. This is tial role in replication, the molecular basis for RepA function is followed by binding of DnaI/DnaB and DnaG, which together unknown. Here we describe a complete structural and functional recruit the replicative helicase (23, 24). Instead of DnaA, plas- dissection of RepA proteins. Unexpectedly, both the RepA NTD and mids encode and use their own specific replication initiator CTD show similarity to the corresponding domains of the bacterial binding protein. Structures are only available for RepA proteins primosome protein, DnaD. Although the RepA and DnaD NTD both (F, R6K, and pPS10 Rep) harbored in Gram-negative bacteria. These proteins contain winged helix-turn-helix (winged HTH) contain winged helix-turn-helices, the DnaD NTD self-assembles domains and bind iteron DNA as monomers to, in some still into large scaffolds whereas the tetrameric RepA NTD binds DNA unclear manner, drive replicon assembly (25–27). iterons using a newly described DNA binding mode. Strikingly, Replication mechanisms used by plasmids harbored in Gram- structural and atomic force microscopy data reveal that the NTD positive bacteria are less well understood and are distinct from tetramer mediates DNA bridging, suggesting a molecular mecha- their Gram-negative counterparts. Indeed, most plasmid RepA BIOCHEMISTRY nism for origin handcuffing. Finally, data show that the RepA CTD proteins in Gram-negative and Gram-positive bacteria show interacts with the host DnaG primase, which binds the replicative no sequence homology and seem to be unrelated. The multi- helicase. Thus, these combined data reveal the molecular mechanism resistance RepA proteins are arguably among the most abundant by which RepA mediates the specific replicon assembly of staphy- of plasmid Rep proteins, yet how they function is not known. lococcal multiresistant plasmids. Data suggest that these proteins are composed of three main regions: an N-terminal domain (NTD) consisting of ∼120 aa, a long RepA_N | replication initiation | S. aureus | two hybrid and variable linker region (∼30–50 residues), and a C-terminal domain (CTD) of ∼120 residues (28–31). The NTD and CTD CHEMISTRY he emergence of multidrug-resistant bacteria is a mounting are both essential for replication. The NTD exhibits the highest global health crisis. In particular, multidrug-resistant Staph- level of sequence conservation, which has resulted in the desig- T nation of plasmids that encode these proteins as the RepA_N ylococcus aureus is a major cause of nosocomial and community- replicon family (10). Although not as well conserved as the NTD, acquired infections and is resistant to most antibiotics commonly RepA CTD regions show homology between plasmids found in used for patient treatment (1). Hospital intensive care units in many countries, including the United States, now report methi- Significance cillin-resistant S. aureus infection rates exceeding 50% (2, 3). Antibiotic resistance in contemporary infectious S. aureus strains, such as in hospitals, is often encoded by plasmids that can be The large staphylococcal multiresistance plasmids harbored in transmitted between strains via horizontal DNA transfer mech- Gram-positive pathogens contribute significantly to the spread anisms. These plasmids are typically classified as small (<5 kb) of multidrug-resistant bacteria and are typified by the presence multicopy plasmids, which usually encode only a single resistance of a highly conserved replication initiator protein, RepA, which gene; medium-sized (8–40 kb) multirestance plasmids that con- is required for plasmid retention. RepA proteins contain N-terminal (NTD) and C-terminal (CTD) domains, which are both fer resistance to multiple antibiotics, disinfectants, and/or heavy required for replication. We show that the RepA NTD and CTD metals; and large (>40 kb) conjugative multiresistance plasmids show striking homology to the host primosome protein DnaD that additionally encode a conjugative DNA transfer mechanism yet perform distinct functions; the NTD binds origin DNA in (4–6). Importantly, sequence analyses have shown that most a novel manner and the CTD recruits the replicative helicase. staphylococcal conjugative and nonconjugative multiresistance Moreover, NTD–DNA structures reveal the first mechanism of plasmids encode a highly conserved replication initiation protein, – origin handcuffing. Combined, the data unveil the minimal denoted RepA_N (5 15). RepA_N proteins are also encoded by mechanism by which multiresistance plasmids mediate origin plasmids from other Gram-positive bacteria as well as by some assembly via the highly conserved RepA protein. phage, underscoring their ubiquitous nature (10). These RepA proteins are essential for replication of multiresistance plasmids, Author contributions: M.A.S., S.M.K., R.A.S., and N.F. designed research; M.A.S., N.K.T., and hence plasmid carriage and dissemination, yet the mech- S.M.K., N.b.C., and M.A.L. performed research; M.A.S., N.K.T., and N.F. analyzed data; and anisms by which these proteins function in replication are cur- M.A.S. wrote the paper. rently unknown. The authors declare no conflict of interest. The DNA replication cycle can be divided into three stages: This article is a PNAS Direct Submission. initiation, elongation, and termination. Replication initiation Data deposition: Coordinates and structure factor amplitudes have been deposited in the proteins (RepA) mediate the crucial first step of initiation. Bac- Protein Data Bank (PDB), www.pdb.org (PDB ID codes 4PQK, 4PQL, 4PT7, and 4PTA). terial chromosome replication is initiated by the chromosomal 1To whom correspondence should be addressed. E-mail: [email protected]. replication initiator protein, DnaA, which binds the origin and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. recruits the replication components known as the primosome 1073/pnas.1406065111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1406065111 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 genus-specific clusters, suggesting that this domain may perform a host-specific role (28–32). Although the function of the RepA CTD remains enigmatic, recent studies have indicated that the NTD mediates DNA binding and interacts with iterons that re- side within the plasmid origin (30). The essential roles played by RepA proteins in multiresistance plasmid retention marks them as attractive targets for the development of specific chemo- therapeutics. However, the successful design of such com- pounds necessitates structural and mechanistic insight. Here, we describe a detailed dissection of the RepA proteins from the multiresistance plasmids pSK41 and pTZ2126. The combined data reveal the molecular underpinnings of a minimalist repli- cation assembly mediated by multiresistance RepA proteins. Results and Discussion Structure of the RepA NTD Contains a Winged HTH with a Unique Dimer-of-Dimers Arrangement. To gain insight into the function of the RepA NTD, which is conserved in more than 100 proteins, structures were determined of the pSK41 and pTZ2162 RepA NTDs. The pSK41 NTD structure was determined first to 2.60 Å (Table S1). The structure revealed that the RepA NTD has an overall fold composed of a central winged HTH (residues 33– 112) flanked by an N-terminal helix-strand-helix (residues 1–27) and C-terminal helix-loop motif (residues 116–132), with overall topology [α1(residues 8–14)-β1(16–20)-α2(21–27)-α3(33–53)- α4(67–76)-α5(79–94)-β2(94–100)-β3(106–112)-α6(116–129)] (Fig. 1A). The helix-strand-helix and C-terminal helix function as oligomerization elements to create an extensive dimer-of-dimers or tetramer (Fig. 1 B and C). To form dimers, the β1 strands from two NTDs interdigitate via antiparallel interactions. The dimer is further stabilized by multiple contacts between residues in helices α1andα6(Fig.1B). The resultant dimer interface is extensive and buries 2,005 Å2 of protein surface from solvent. The RepA tetramer is created by the orthogonal packing of two Fig. 1. Structure of the multiresistance RepA NTD. (A) Ribbon diagram of NTD dimers. Key to the formation of this compact tetramer is the pTZ2162 RepA NTD. The winged HTH is colored gray and secondary the deep insertion of the N-terminal tails of each subunit into structural elements and the N and C termini are labeled.
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