Design of novel peptide inhibitors against the conserved bacterial transcription terminator, Rho
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100653
ISSN1083-351X
AutoresGairika Ghosh, Pankaj Sharma, Amit Kumar, Sriyans Jain, Ranjan Sen,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe transcription terminator Rho regulates many physiological processes in bacteria, such as antibiotic sensitivity, DNA repair, RNA remodeling, and so forth, and hence, is a potential antimicrobial target, which is unexplored. The bacteriophage P4 capsid protein, Psu, moonlights as a natural Rho antagonist. Here, we report the design of novel peptides based on the C-terminal region of Psu using phenotypic screening methods. The resultant 38-mer peptides, in addition to containing mutagenized Psu sequences, also contained plasmid sequences, fused to their C termini. Expression of these peptides inhibited the growth of Escherichia coli and specifically inhibited Rho-dependent termination in vivo. Peptides 16 and 33 exhibited the best Rho-inhibitory properties in vivo. Direct high-affinity binding of these two peptides to Rho also inhibited the latter's RNA-dependent ATPase and transcription termination functions in vitro. These two peptides remained functional even if eight to ten amino acids were deleted from their C termini. In silico modeling and genetic and biochemical evidence revealed that these two peptides bind to the primary RNA-binding site of the Rho hexamer near its subunit interfaces. In addition, the gene expression profiles of these peptides and Psu overlapped significantly. These peptides also inhibited the growth of Mycobacteria and inhibited the activities of Rho proteins from Mycobacterium tuberculosis, Xanthomonas, Vibrio cholerae, and Salmonella enterica. Our results showed that these novel anti-Rho peptides mimic the Rho-inhibition function of the ∼42-kDa dimeric bacteriophage P4 capsid protein, Psu. We conclude that these peptides and their C-terminal deletion derivatives could provide a basis on which to design novel antimicrobial peptides. The transcription terminator Rho regulates many physiological processes in bacteria, such as antibiotic sensitivity, DNA repair, RNA remodeling, and so forth, and hence, is a potential antimicrobial target, which is unexplored. The bacteriophage P4 capsid protein, Psu, moonlights as a natural Rho antagonist. Here, we report the design of novel peptides based on the C-terminal region of Psu using phenotypic screening methods. The resultant 38-mer peptides, in addition to containing mutagenized Psu sequences, also contained plasmid sequences, fused to their C termini. Expression of these peptides inhibited the growth of Escherichia coli and specifically inhibited Rho-dependent termination in vivo. Peptides 16 and 33 exhibited the best Rho-inhibitory properties in vivo. Direct high-affinity binding of these two peptides to Rho also inhibited the latter's RNA-dependent ATPase and transcription termination functions in vitro. These two peptides remained functional even if eight to ten amino acids were deleted from their C termini. In silico modeling and genetic and biochemical evidence revealed that these two peptides bind to the primary RNA-binding site of the Rho hexamer near its subunit interfaces. In addition, the gene expression profiles of these peptides and Psu overlapped significantly. These peptides also inhibited the growth of Mycobacteria and inhibited the activities of Rho proteins from Mycobacterium tuberculosis, Xanthomonas, Vibrio cholerae, and Salmonella enterica. Our results showed that these novel anti-Rho peptides mimic the Rho-inhibition function of the ∼42-kDa dimeric bacteriophage P4 capsid protein, Psu. We conclude that these peptides and their C-terminal deletion derivatives could provide a basis on which to design novel antimicrobial peptides. The Rho-dependent transcription termination plays a major role in the regulation of gene expression in bacteria. The transcription termination of about half of the operons in Escherichia coli is controlled by this termination process. The Rho protein, a homohexamer with a protomer of 46.8 kDa, is a highly conserved protein found in most bacteria. It is an RNA/DNA helicase or translocase that dissociates RNA polymerase from the DNA template using its RNA-dependent ATPase activity to bring about the transcription termination (1Richardson J.P. Rho-dependent termination and ATPases in transcript termination.Biochim. Biophys. Acta. 2002; 1577: 251-260Crossref PubMed Scopus (142) Google Scholar, 2Richardson J.P. Loading Rho to terminate transcription.Cell. 2003; 114: 157-159Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 3Banerjee S. Chalissery J. Bandey I. Sen R. Rho-dependent transcription termination: More questions than answers.J. Microbiol. 2006; 44: 11-22PubMed Google Scholar, 4Mitra P. Ghosh G. Hafeezunnisa M. Sen R. Rho protein: Roles and mechanisms.Annu. Rev. Microbiol. 2017; 71: 687-709Crossref PubMed Scopus (55) Google Scholar). It binds to the rut site (Rho utilization; a C-rich unstructured region) of the exiting nascent RNA, and this interaction is a prerequisite for its termination function (5Kalyani B.S. Muteeb G. Qayyum M.Z. Sen R. Interaction with the nascent RNA Is a prerequisite for the recruitment of Rho to the transcription elongation complex in vitro.J. Mol. Biol. 2011; 413: 548-560Crossref PubMed Scopus (24) Google Scholar). This termination function regulates many physiological processes (4Mitra P. Ghosh G. Hafeezunnisa M. Sen R. Rho protein: Roles and mechanisms.Annu. Rev. Microbiol. 2017; 71: 687-709Crossref PubMed Scopus (55) Google Scholar, 6Jain S. Gupta R. Sen R. Rho-dependent transcription termination in bacteria recycles RNA polymerases stalled at DNA lesions.Nat. Commun. 2019; 10: 1207Crossref PubMed Scopus (5) Google Scholar, 7Hafeezunnisa M. Sen R. The Rho-dependent transcription termination is involved in broad-spectrum antibiotic susceptibility in Escherichia coli.Front. Microbiol. 2020; 11: 605305Crossref PubMed Scopus (2) Google Scholar), and the conserved nature of the Rho protein in a wide range of bacteria makes it an ideal target for bactericidal agents. Psu (polarity suppression) is an unconventional capsid protein of the E. coli bacteriophage P4 that moonlights as a specific and efficient inhibitor of Rho (8Linderoth N.A. Calendar R.L. The Psu protein of bacteriophage P4 is an antitermination factor for Rho-dependent transcription termination.J. Bacteriol. 1991; 173: 6722-6731Crossref PubMed Google Scholar, 9Pani B. Banerjee S. Chalissery J. Abhishek M. Ramya M.L. Suganthan R. Sen R. Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu.J. Biol. Chem. 2006; 281: 26491-26500Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). It binds and antagonizes Rho in trans by creating a mechanical hindrance to the Rho translocation process (10Pani B. Ranjan A. Sen R. Interaction surface of bacteriophage P4 protein Psu required for Complex formation with the transcription terminator rho.J. Mol. Biol. 2009; 389: 647-660Crossref PubMed Scopus (18) Google Scholar, 11Ranjan A. Sharma S. Banerjee R. Sen U. Sen R. Structural and mechanistic basis of anti-termination of Rho-dependent transcription termination by bacteriophage P4 capsid protein Psu.Nucleic Acids Res. 2013; 41: 6839-6856Crossref PubMed Scopus (13) Google Scholar) upon the formation of a V-shaped cap-like knotted homodimer structure at the RNA exit point of the central channel of Rho (11Ranjan A. Sharma S. Banerjee R. Sen U. Sen R. Structural and mechanistic basis of anti-termination of Rho-dependent transcription termination by bacteriophage P4 capsid protein Psu.Nucleic Acids Res. 2013; 41: 6839-6856Crossref PubMed Scopus (13) Google Scholar, 12Banerjee R. Nath S. Ranjan A. Khamrui K. Pani B. Sen R. Sen U. The first structure of polarity suppression protein, Psu from enterobacteria phage P4, reveals a novel fold and a knotted dimer.J. Biol. Chem. 2012; 287: 44667-44675Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Its solvent-exposed flexible C-terminal domain (CTD) (helices 6 and 7) (12Banerjee R. Nath S. Ranjan A. Khamrui K. Pani B. Sen R. Sen U. The first structure of polarity suppression protein, Psu from enterobacteria phage P4, reveals a novel fold and a knotted dimer.J. Biol. Chem. 2012; 287: 44667-44675Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) interacts directly with Rho, and its N-terminal domain (NTD) imparts stability to the protein (9Pani B. Banerjee S. Chalissery J. Abhishek M. Ramya M.L. Suganthan R. Sen R. Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu.J. Biol. Chem. 2006; 281: 26491-26500Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 10Pani B. Ranjan A. Sen R. Interaction surface of bacteriophage P4 protein Psu required for Complex formation with the transcription terminator rho.J. Mol. Biol. 2009; 389: 647-660Crossref PubMed Scopus (18) Google Scholar, 12Banerjee R. Nath S. Ranjan A. Khamrui K. Pani B. Sen R. Sen U. The first structure of polarity suppression protein, Psu from enterobacteria phage P4, reveals a novel fold and a knotted dimer.J. Biol. Chem. 2012; 287: 44667-44675Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Psu is also capable of antagonizing the Rho proteins from different bacterial pathogens (13Ghosh G. Reddy J. Sambhare S. Sen R. A bacteriophage capsid protein is an inhibitor of a conserved transcription terminator of various bacterial pathogens.J. Bacteriol. 2018; 200e00380-17Crossref PubMed Scopus (10) Google Scholar). We hypothesize that the Rho-interacting C-terminal region or its derivatives in isolation might show Rho-inhibitory activities, which could be further developed into antimicrobials targeting the Rho protein. Alternative strategies to design new-generation antimicrobials, such as antimicrobial peptides (AMPs), are essential in the wake of the emergence of many multidrug-resistant and extensively drug-resistant pathogenic strains. Efforts to design AMPs from different phage proteins such as endolysins, LysAB2 (14Peng S.Y. You R.I. Lai M.J. Lin N.T. Chen L.K. Chang K.C. Highly potent antimicrobial modified peptides derived from the Acinetobacter baumannii phage endolysin LysAB2.Sci. Rep. 2017; 7: 11477Crossref PubMed Scopus (40) Google Scholar), and PflyF307 (15Thandar M. Lood R. Winer B.Y. Deutsch D.R. Euler C.W. Fischetti V.A. Novel engineered peptides of a phage lysin as effective antimicrobials against multidrug-resistant Acinetobacter baumannii.Antimicrob. Agents Chemother. 2016; 60: 2671-2679Crossref PubMed Scopus (60) Google Scholar) have been reported earlier. Here, we report the design of peptides from the mutagenized CTD (helix 7) of Psu, using a phenotypic screening method. We screened peptides based on their ability to induce growth defects and inhibiting Rho-dependent termination in vivo. These peptides not only had the mutagenized sequence from Psu helix 7 but also contained an extra region from the adjacent nucleotide sequence of the plasmid that got appended to their C-terminal region because of frame-shift mutations. In vitro, the peptides inhibited the RNA release and ATPase activities of the E. coli Rho via a direct interaction. The molecular docking and genetic and biochemical evidence revealed that they bind near the primary RNA-binding region of Rho at the interface of its two subunits. Both the peptides and Psu exerted similar genome-wide upregulation upon in vivo expressions. The expressions of these peptides caused lethality in Mycobacteria and inhibited the in vitro functions of the Rho proteins from various other pathogens. The bacteriophage P4 capsid protein, Psu, has been shown to act as an inhibitor of the transcription terminator Rho of E. coli and various bacterial pathogens (9Pani B. Banerjee S. Chalissery J. Abhishek M. Ramya M.L. Suganthan R. Sen R. Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu.J. Biol. Chem. 2006; 281: 26491-26500Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 13Ghosh G. Reddy J. Sambhare S. Sen R. A bacteriophage capsid protein is an inhibitor of a conserved transcription terminator of various bacterial pathogens.J. Bacteriol. 2018; 200e00380-17Crossref PubMed Scopus (10) Google Scholar). The C-terminal helix 7 of the Psu protein (Fig. 1A) interacts directly with the Rho (9Pani B. Banerjee S. Chalissery J. Abhishek M. Ramya M.L. Suganthan R. Sen R. Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu.J. Biol. Chem. 2006; 281: 26491-26500Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 11Ranjan A. Sharma S. Banerjee R. Sen U. Sen R. Structural and mechanistic basis of anti-termination of Rho-dependent transcription termination by bacteriophage P4 capsid protein Psu.Nucleic Acids Res. 2013; 41: 6839-6856Crossref PubMed Scopus (13) Google Scholar). Hence, we hypothesized that this helix could be used for developing anti-Rho peptides. The overexpression of the isolated 21-mer Psu helix 7 did not induce any toxic effect in E. coli, unlike the full-length Psu (Fig. 2A). Therefore, to obtain gain-of-function peptide(s) from this WT helix 7, we used a phenotypic screening strategy by randomly mutagenizing specific amino acids of this 21-mer peptide. We used different synthetic DNA oligonucleotides containing one or more degenerated codons at the positions corresponding to each of the targeted residues. The residues that were already shown (11Ranjan A. Sharma S. Banerjee R. Sen U. Sen R. Structural and mechanistic basis of anti-termination of Rho-dependent transcription termination by bacteriophage P4 capsid protein Psu.Nucleic Acids Res. 2013; 41: 6839-6856Crossref PubMed Scopus (13) Google Scholar) to be important for the interaction with Rho were selected. The amino acid positions of Psu, N174, F177, S181, and L184 were targeted, and the oligonucleotides were designed to mutagenize the first, the second, or both the positions of each of the codon of the targeted residues (Fig. 1B). The library of mutant peptides was cloned under the control of an IPTG-inducible Ptac promoter in the pNL150 vector and was expressed in the RS734 strain having a lacZ-reporter cassette fused downstream of a Rho-dependent terminator, tR1 (Plac-nutR/tR1-lacZYA). In this setup, gain-of-function peptide clones would yield blue colonies on the LB X-gal plates. We screened ∼80,000 colonies. The colonies appearing deep blue were screened and were further checked for their growth-inhibition properties (Fig. 1C). The mutant clones obtained above were expressed in a WT MG1655 strain in the presence of 100 μM of the inducer, IPTG, and the growth assays were followed (Fig. 2, A and B). We observed significant growth defects upon expressions of several peptide clones. Among the nine peptide clones, peptides 16 and 33 exhibited severe growth defects and strong in vivo antitermination of Rho-dependent termination (Fig. 3). The in vivo Rho inhibitory properties of these two peptides were comparable with that of Psu. Upon nucleotide sequencing of these peptide clones, we observed that all the peptides, in addition to the desired point mutations, acquired novel sequences, which evolved because of a frameshift mutation in the helix-7 sequence (Figs. 2C and S1). The addition of new sequences was from the adjacent vector sequences at their C-terminal region, which converted these gain-of-function peptides into 38-mer peptides from the original 21-mer helix 7 of Psu. In silico modeling of these two peptides, using the threading method in the Iterative Threading ASSEmbly Refinement (I-TASSER ) server, revealed that the C-terminal new adjacent sequence was predicted to fold into a helical structure, whereas a part of the N-terminal region formed weak antiparallel β-sheets that were stabilized as random coils after a 1-μs molecular dynamics (MD) simulation (Fig. 2, D and E, and Fig. S2). This adjacent sequence might be responsible for structurally stabilizing the peptides in vivo that were absent in the 21-mer WT helix 7 rendering the latter unstable and nonfunctional. The quality of the predicted structures was high with a confidential score for peptides 16 and 33 of −1.79 and −1.72, respectively. The structure validation by the Ramachandran plot showed that both the peptides have no residues in the disallowed regions (Fig. 2, D and E). To understand the structural basis of the variations in their functional efficiencies, we modeled and performed dynamic simulations of all the other peptides and compared their RMSD structural deviations from peptide 33 (Fig. S1B). We observed that the changes in the amino acid sequences in the helix-7 regions induced structural variations ranging from ∼6 Å to ∼11 Å as compared with peptide 33. Unlike peptides 16 and 33, in most of the other peptides, the N-terminal β-sheets remained intact after the MD simulation. Also, the spatial orientations of the C-terminal helixes of these other peptides were significantly different compared with peptide 33. We reasoned that these structural changes could have affected the functions of these peptides. CD spectra of peptides 16 and 33 also revealed that they are predominantly α-helical (Fig. S3). We tested the in vivo anti-Rho function potentials of peptides 16 and 33. For this purpose, we first performed in vivo Rho-dependent termination assays using an MC4100 strain having a single-copy galEP3 reporter in the chromosome. This reporter cassette consists of a series of Rho-dependent terminators present in the IS2 element inserted at the beginning of the galactose operon. If the expression of Psu or the peptides (cloned in pNL150, under an IPTG-inducible promoter, Ptac) inhibits the Rho function, antitermination will occur through these terminators, leading to expression of the galactose operon that would be manifested as red or pink colonies on MacConkey–galactose indicator plates. We observed the appearance of red colonies upon overexpression of the WT Psu and the peptides indicating the inhibition of Rho-dependent termination in vivo (Fig. S4A). Next, we monitored the effects of the in vivo expressions of Psu and the peptides on Rho-dependent termination at two well-known terminators, trpt′ and trac, using the lacZ reporter system. They are Plac-trpt′-lacZAY (RS2045) and Plac-H19B nutR/tR1-lacZAY (RS2047), where the terminators are fused upstream of the lacZ genes and are inserted in the chromosome of E. coli MG1655 Δrac Δlac. The colonies would appear blue on the LB–X-gal indicator plates if the Rho-dependent termination is inhibited. We observed that upon expressions of Psu and the peptides (induction by 50 μM IPTG), deep blue colonies appeared on the indicator plates. Colonies were white or pale blue in the presence of an empty pNL150 vector (Fig. 3A). We repeated the in vivo termination assays using quantitative reverse transcription PCR (qRT-PCR) to get more quantitative data by measuring the level of lacZ expression using the same terminator-lacZAY fusion templates described above (Fig. 3B). The strains described in Figure. 3A having these two lacZ fusions in the chromosome and harboring the pNL150 vector alone or expressing Psu or the peptides were used. Upon induction by IPTG, the expression levels of the Plac-trac-lacZ fusion were observed to be enhanced by 28-fold when Psu was expressed, whereas in the presence of the peptides, this level was increased by 6- to 12-folds. In the case of the trpt′-lacZ fusion, these enhanced levels of expression were less but still were significant. These results further confirmed that like Psu, peptides are capable of inhibiting Rho-dependent termination in vivo efficiently. Earlier, we have shown that in vitro, the binding of Psu to Rho reduced the rate of RNA-dependent ATP hydrolysis of the latter (9Pani B. Banerjee S. Chalissery J. Abhishek M. Ramya M.L. Suganthan R. Sen R. Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu.J. Biol. Chem. 2006; 281: 26491-26500Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 13Ghosh G. Reddy J. Sambhare S. Sen R. A bacteriophage capsid protein is an inhibitor of a conserved transcription terminator of various bacterial pathogens.J. Bacteriol. 2018; 200e00380-17Crossref PubMed Scopus (10) Google Scholar). We used an in vitro–synthesized RNA having the λtR1 terminator sequence to induce the in vitro ATPase function of the E. coli Rho. We observed that like Psu, both the peptides inhibited the ATPase activity of the E. coli Rho (Fig. 3C). Owing to higher affinity, peptide 33 was more efficient than peptide 16 in inhibiting this Rho function in vitro. However, about 10 times more molar amount of peptides than Psu was required to observe this inhibitory activity. The requirement of a higher concentration of peptides to exert their effect is consistent with the requirement of a higher level of IPTG induction in the in vivo experiments. It should be noted that the peptides used in the in vitro studies were chemically synthesized. Psu is capable of inhibiting E. coli Rho-dependent termination in an in vitro–purified system (9Pani B. Banerjee S. Chalissery J. Abhishek M. Ramya M.L. Suganthan R. Sen R. Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu.J. Biol. Chem. 2006; 281: 26491-26500Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Next, we assayed the peptide-mediated inhibition of the in vitro transcription termination functions of the Rho. We used a linear DNA template where a trpt′ terminator is fused downstream of a strong T7A1 promoter. On this template, efficient transcription termination over a terminator zone is usually observed in the presence of Rho (Fig. 3D; (16Chalissery J. Banerjee S. Bandey I. Sen R. Transcription termination defective mutants of rho: Role of different functions of rho in releasing RNA from the elongation complex.J. Mol. Biol. 2007; 371: 855-872Crossref PubMed Scopus (46) Google Scholar)). The amount of run-off transcripts at the end of this template gives the measure of Rho inhibition by the peptides. We observed that the amount of run-off transcripts increased to ∼28% at higher concentrations of peptide 33 from ∼6% in its absence. A similar level of in vitro Rho inhibition was earlier observed in the presence of Psu (9Pani B. Banerjee S. Chalissery J. Abhishek M. Ramya M.L. Suganthan R. Sen R. Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu.J. Biol. Chem. 2006; 281: 26491-26500Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Peptide 33 did not have any effect on transcription when Rho was absent (lane 2 from left). The effect of peptide 16 in this type of transcription termination assays was observed to be much less, which could be due to its weaker affinity for Rho (data not shown). Next, we measured the inhibition of Rho-mediated RNA release from a stalled elongation complex (EC) by the peptides. We designed a setup where the EC is stalled on a template bound to magnetic beads at a particular position inside the trpt′ terminator region using the lac repressor as a roadblock (RB) (Fig. 3E, top panel). On this template also, the transcription is initiated from the T7A1 promoter. In this setup, the RNA released because of the Rho-dependent transcription termination would be visible in the supernatant (S). We observed that in the presence of either Psu (Fig. S4B) or the peptides (Fig. 3E for peptide 33 and Fig. S4B for peptide 16), the Rho-induced RNA release from the stalled EC was significantly inhibited. Peptide 16 could efficiently inhibit the RNA release of Rho only when stalled EC was used (Fig. S4B). Similar to the ATPase assays, higher concentrations of peptides than Psu were required to bring about this inhibition function. Peptide 33 exerted its action at a lower concentration than peptide 16, indicating a tighter binding to Rho by the former (Fig. S4B). It should be noted that the peptides do not have any adverse effect on the in vitro transcription process in the absence of Rho (Fig. 3E, no Rho lane). In general, at ≥50 μM, peptide 33 exerted its maximal effects. However, as these peptides were synthesized and commercially purchased, the concentration requirements to exert the maximum effect varied between different lots. These observations strongly indicate that the peptides are capable of inhibiting the in vitro functions of Rho protein in a manner comparable with that observed for the WT Psu. Earlier, we have demonstrated the formation of a specific Psu–E. coli Rho complex in vivo by overexpressing both the proteins together in the same strain (9Pani B. Banerjee S. Chalissery J. Abhishek M. Ramya M.L. Suganthan R. Sen R. Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu.J. Biol. Chem. 2006; 281: 26491-26500Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). However, we failed to obtain a stable in vitro Rho–Psu complex with the WT Rho. We observed that an in vitro stable complex between Psu and Rho could form if a mutant Rho, P167L, was used (11Ranjan A. Sharma S. Banerjee R. Sen U. Sen R. Structural and mechanistic basis of anti-termination of Rho-dependent transcription termination by bacteriophage P4 capsid protein Psu.Nucleic Acids Res. 2013; 41: 6839-6856Crossref PubMed Scopus (13) Google Scholar). This mutant Rho was obtained as a suppressor of a Psu mutant that was defective in binding to WT Rho (11Ranjan A. Sharma S. Banerjee R. Sen U. Sen R. Structural and mechanistic basis of anti-termination of Rho-dependent transcription termination by bacteriophage P4 capsid protein Psu.Nucleic Acids Res. 2013; 41: 6839-6856Crossref PubMed Scopus (13) Google Scholar). This Rho mutant appeared to form a stable hexamer at a lower concentration unlike the WT Rho, which could have improved its affinity for Psu. We assumed that this Rho mutant would also have a higher affinity for the peptides. Hence, to demonstrate peptide–Rho interactions in vitro, we have used P167L Rho. We performed the pull-down assays by using His-tagged versions of Psu (Fig. S4C) and peptides 16 and 33 (Fig. 4A) and the non–His-tagged P167L Rho, where peptides and Psu were bound to nickel nitrilotriacetic acid (Ni-NTA) agarose beads. In this assay, unbound Rho would be visible in the flow-through and the wash fractions, and the bound Rho would be in the eluted fraction. In all these cases, ∼ 40 to 70% of Rho proteins were found to be associated with the peptides (Fig. 4A), a level comparable with what we observed for the Psu–Rho complex (Fig. S4C). The complexes were observed to be formed at the ratios of Rho hexamer:peptide 16::1:6, Rho hexamer:peptide 33::1:4, and Rho hexamer:Psu:: 1:3. In all these cases, ratios were calculated from the amounts of Rho/Psu/peptides expressed in micrograms (Fig. 4A). These results indicated a direct interaction of the peptides with Rho, which induced the anti-Rho functions. To understand the mode of interactions of the peptides with P167L Rho, we performed the isothermal titration calorimetry (ITC) analysis of peptides 16– and 33–Rho interactions. The binding and thermodynamic parameters are listed in Figure. 4B. We observed the following. (1) Peptide 33 binds to P167L Rho with a fairly high affinity (Kd = 17 nM) with a Rho hexamer:peptide 33::1:4 binding stoichiometry. Peptide 16 binds with much weaker affinity and with significantly reduced stoichiometry. This weaker binding of peptide 16 is consistent with its requirement of higher concentrations in the functional assays (Fig. 3). (2) The thermodynamic parameters indicated that the binding of both the peptides is highly enthalpy driven, most likely because of the formation of specific H-bonds between the amino acids of the peptides and Rho (17London N. Movshovitz-Attias D. Schueler-Furman O. The structural basis of peptide-protein binding strategies.Structure. 2010; 18: 188-199Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), which is common for smaller peptides fitting into specific pockets of larger macromolecules via an "induced-fit" mechanism. The aforementioned results showed that the functional peptides contain an N-terminal His-tag sequence and an additional sequence from the vector appended to their C-terminal region in addition to that derived from Psu helix 7. To understand the importance of additional N- and C-terminal regions, we made a series of deletions in these regions and performed growth assays in similar ways as described in Figure. 2A. We observed that the N-terminal 6XHis-tagged WT Psu helix 7 sequence either in the presence or absence of the C-terminal adjacent vector sequence did not cause a growth defect in the presence of IPTG (Fig. 5A; see the plots, helix 7 peptide, and helix 7 + adjacent sequence). The presence of C-terminal vector sequences (shown in green) in peptides 16 and 33 was found to be essential for their function in vivo (Fig. 5A). This suggests that in addition to the C-terminal vector sequence, the mutations present in the helix-7 region (shown in red) of peptides 16 and 33 are also important. The vector sequence might be providing structural stability to the mutated helix-7 region of the peptides. The length of these two peptides is too long to be functional as AMPs as cells would not allow their entry when added exogenously. So, we explored the minimal length of these peptides required to remain functional. We constructed a series of truncated versions of the peptides with the deletions of amino acids from their C-terminal region and expressed them in vivo in the presence of IPTG and monitored their abilities to induce growth defects in a similar way as described in Figure. 2 (Fig. 5B). We observed that up to eight amino acids could be deleted from the C-terminal region of peptide 33, without incurring significant functional defects
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