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Protein Dynamics in F-Like Bacterial Conjugation

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Protein Dynamics in F-Like Bacterial Conjugation biomedicines Review Protein Dynamics in F-like Bacterial Conjugation Nicholas Bragagnolo 1, Christina Rodriguez 1, Naveed Samari-Kermani 1, Alice Fours 2, Mahboubeh Korouzhdehi 2, Rachel Lysenko 2 and Gerald F. Audette 1,* 1 Department of Chemistry and the Centre for Research on Biomolecular Interactions, York University, Toronto, ON M3J 1P3, Canada; [email protected] (N.B.); [email protected] (C.R.); [email protected] (N.S.-K.) 2 Department of Chemistry, York University, Toronto, ON M3J 1P3, Canada; [email protected] (A.F.); [email protected] (M.K.); [email protected] (R.L.) * Correspondence: [email protected]; Tel.: +1-416-736-2100 (ext. 33318) Received: 28 August 2020; Accepted: 17 September 2020; Published: 19 September 2020 Abstract: Efficient in silico development of novel antibiotics requires high-resolution, dynamic models of drug targets. As conjugation is considered the prominent contributor to the spread of antibiotic resistance genes, targeted drug design to disrupt vital components of conjugative systems has been proposed to lessen the proliferation of bacterial antibiotic resistance. Advancements in structural imaging techniques of large macromolecular complexes has accelerated the discovery of novel protein-protein interactions in bacterial type IV secretion systems (T4SS). The known structural information regarding the F-like T4SS components and complexes has been summarized in the following review, revealing a complex network of protein-protein interactions involving domains with varying degrees of disorder. Structural predictions were performed to provide insight on the dynamicity of proteins within the F plasmid conjugative system that lack structural information. Keywords: bacterial conjugation; type IV secretion system (T4SS); F plasmid; pilus protein structure; protein dynamics; protein-protein interactions; targeted drug design 1. Introduction Bacterial conjugation is a method of horizontal gene transfer considered to prominently contribute to the propagation of virulence genes among pathogens [1–3]. Conjugative plasmids are transmissible through the type IV secretion systems (T4SS) encoded within a transfer (tra) or virulence (vir) operon, which allows the plasmid carrier to mediate donor-to-recipient DNA transfer [4]. As plasmids often undergo transposition events with the bacterial chromosome, integrative, and conjugative elements increase the rate of bacterial evolution by enhancing the plasticity of their genomes [5]. The misuse of antibiotics in animal agriculture and over-prescription of broad-spectrum antibiotics has resulted in the selective evolution of antibiotic resistance genes, which commonly migrate onto conjugative plasmids [6,7]. As conjugative plasmids are ubiquitous in the bacterial kingdom, inhibition of conjugative T4SS has been suggested as a drug-design target to prevent the dissemination of antibiotic resistance genes; thus, preventing the proliferation of multi-drug resistant pathogens [8–10]. The process of bacterial conjugation is complex, and the mechanisms of the many stages involved in the transfer of conjugative DNA from a donor to a recipient cell are poorly understood as they vary in different plasmid families [3,11]. Plasmids code for the genes required in the assembly of a T4SS and the transfer of the plasmid DNA; in incompatibility (Inc)F-type plasmids these genes are located in the tra operon, and expression is regulated by proteins and RNA upstream and within the operon [12,13]. Once the Tra and Trb proteins from the T4SS are expressed and assembled, pilin processing events occur, followed by pilin assembly and extension (Figure1)[ 14]. After a recipient cell has been contacted, pilus retraction proceeds, and in some systems, such as the F plasmid, this Biomedicines 2020, 8, 362; doi:10.3390/biomedicines8090362 www.mdpi.com/journal/biomedicines BiomedicinesBiomedicines2020 2020, 8, ,8 362, x FOR PEER REVIEW 2 2of of 30 30 processing events occur, followed by pilin assembly and extension (Figure 1) [14]. After a recipient iscell followed has been by contacted, a mating pairpilus stabilization retraction proceeds, step which and allows in some for systems, the close such contact as the of F cell plasmid, membranes. this is Exclusionfollowed eventsby a mating then occur pair tostabilization prevent redundant step which transfer allows of for conjugative the close DNAcontact to of a recipientcell membranes. cell that hasExclusion the same events plasmid. then occur A mating to prev bridgeent redundant is then formed transfer between of conjugativ the cellse DNA to allow to a forrecipient the transfer cell that of has the plasmidthe same by plasmid. a Tra protein A mating complex bridge is called then formed the transferosome, between the cells thus, to allowingallow for th rollinge transfer circle of the replication plasmid toby occur. a Tra protein complex called the transferosome, thus, allowing rolling circle replication to occur. FigureFigure 1. 1. AA modelmodel ofof thethe F-likeF-like typetype IVIV secretionsecretion systemssystems (T4SS) based on available structural information,information, including including a a cryo-electron cryo-electron tomography tomography (cryo-ET) (cryo-ET) model model of of the the pED208 pED208 core core complex complex and and a cryo-electrona cryo-electron microscopy microscopy (cryo-EM) (cryo-EM) model model of theof th assemblede assembled pED208 pED208 pilus pilus (PDB (PDB ID: ID: 5LEG) 5LEG) [15 ,[15,16].16]. Tra proteinsTra proteins are labeled are labeled with capitalwith capital letters letters and Trb and proteins Trb proteins are shown are withshown lower with case lower letters. case Proteinsletters. areProteins colored are based colored on function;based on thefunction; pilin TraAthe pilin is white, TraA pilinis white, processing pilin processing proteins proteins are purple, are proteinspurple, responsibleproteins responsible for pilus assemblyfor pilus /assembly/extensionextension are colored are incolored fuchsia, in fuchsia, the core the complex core complex is colored is colored in dark blue,in dark while blue, pilus while retraction pilus proteinsretraction are proteins colored inare light colored blue, in green light colored blue, green proteins colored are responsible proteins are for matingresponsible pair stabilization, for mating pair and stabilization, the red proteins and are the responsible red proteins for are exclusion. responsible ATPase for exclusion. proteins TraC ATPase and TraDproteins are complexed TraC and TraD in the are cytosol comple basedxed onin the the cytosol cryo-ET based image, on but the arecryo-ET present image, in the but inner are membranepresent in (IM)the asinner well. membrane The outer (IM) membrane as well. (OM)The outer complex membra in bluene (OM) is composed complex of in TraK blue and is composed TraV in a complexof TraK thatand creates TraV in 13-fold a complex symmetry. that creates Figure 13-fold created sy withmmetry. BioRender.com. Figure created with BioRender.com. ThereThere is is evidence evidence of of a a high high degreedegree ofof structuralstructural dynamicity in multi-protein complexes complexes that that span span thethe bacterial bacterial cellular cellular envelope. envelope. ForFor instance,instance, type 4 pili, which are are generated by by type type 2 2 secretion secretion systems,systems, function function in in motility, motility, natural natural competence, competence, phagephage adsorption,adsorption, protein secretion, and and surface surface sensingsensing and and have have been been shown shown to to feature feature disorderdisorder throughoutthroughout theirtheir structuresstructures [[17].17]. In In analyzing analyzing the the availableavailable structural structural informationinformation ofof thethe componentscomponents ofof thethe conjugativeconjugative F-like T4SS, such such as as protein protein structuresstructures deposited deposited inin thethe ProteinProtein DataData BankBank (PDB),(PDB), aa similarsimilar diversediverse structural ensemble is is seen seen Biomedicines 2020, 8, 362 3 of 30 where proteins that have high structural integrity, and are well conserved, are seen to interact with proteins that display various levels of disorder. There is some evidence that dynamic proteins, and their sites of interaction with other proteins in a given protein complex, can be optimal drug targets if the structural and conformational dynamics of the protein are understood [18,19]. The following review summarizes what is currently known regarding the structural dynamics of F-like conjugative T4SS components, and highlights tactful targets for rational drug design. In addition, a Phyre2 [20] analysis of all known Tra and Trb proteins was performed to predict the disorder present in proteins with unknown structure and to estimate the novelty of their folds. 2. Dynamics of Proteins Involved in the Regulation of Bacterial Conjugation The transcription of genes coding for conjugative T4SSs is strictly regulated as its production and subsequent operations are metabolically costly processes for bacterial cells [21,22]. The expression of conjugative systems is controlled by either the basal level of constitutive gene expression, or signaling molecules (such as autoinducers or sex pheromones used in quorum sensing), or both [23]. The regulation of conjugation in plasmids from the IncF family of gram-negative bacteria occurs solely by the former process and has been termed fertility inhibition (fin) [24]. In the canonical F-plasmid, the
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