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• W3126435539 abstract "•Simulations of polymyxin B1 in the crowded environment of the E. coli periplasm•LolA may be able to carry PMB1 through the periplasm The periplasm of Gram-negative bacteria is a complex, highly crowded molecular environment. Little is known about how antibiotics move across the periplasm and the interactions they experience. Here, atomistic molecular dynamics simulations are used to study the antibiotic polymyxin B1 within models of the periplasm, which are crowded to different extents. We show that PMB1 is likely to be able to “hitchhike” within the periplasm by binding to lipoprotein carriers—a previously unreported passive transport route. The simulations reveal that PMB1 forms both transient and long-lived interactions with proteins, osmolytes, lipids of the outer membrane, and the cell wall, and is rarely uncomplexed when in the periplasm. Furthermore, it can interfere in the conformational dynamics of native proteins. These are important considerations for interpreting its mechanism of action and are likely to also hold for other antibiotics that rely on diffusion to cross the periplasm. The periplasm of Gram-negative bacteria is a complex, highly crowded molecular environment. Little is known about how antibiotics move across the periplasm and the interactions they experience. Here, atomistic molecular dynamics simulations are used to study the antibiotic polymyxin B1 within models of the periplasm, which are crowded to different extents. We show that PMB1 is likely to be able to “hitchhike” within the periplasm by binding to lipoprotein carriers—a previously unreported passive transport route. The simulations reveal that PMB1 forms both transient and long-lived interactions with proteins, osmolytes, lipids of the outer membrane, and the cell wall, and is rarely uncomplexed when in the periplasm. Furthermore, it can interfere in the conformational dynamics of native proteins. These are important considerations for interpreting its mechanism of action and are likely to also hold for other antibiotics that rely on diffusion to cross the periplasm. The periplasm of Gram-negative bacteria is a crowded aqueous compartment bounded by the inner membrane (IM) and outer membrane (OM). The cell wall is contained within the periplasm, as well as hundreds of proteins including chaperones, transporters, proteases, and nucleases (Weiner and Li, 2008Weiner J.H. Li L. Proteome of the Escherichia coli envelope and technological challenges in membrane proteome analysis.Biochim. Biophys. Acta. 2008; 1778: 1698-1713Crossref PubMed Scopus (63) Google Scholar; Goemans et al., 2014Goemans C. Denoncin K. Collet J.-F.F. Folding mechanisms of periplasmic proteins.Biochim. Biophys. Acta. 2014; 1843: 1517-1528Crossref PubMed Scopus (78) Google Scholar). The periplasm also contains a range of osmolytes, including urea, sugars, spermidine, and putrescine. Thus, it is a complex and crowded environment. Very little is known about the spatial arrangement of the myriad molecules within the periplasm. It is still not known whether the proteins and osmolytes are evenly distributed or whether there is some degree of organization. This makes it very difficult to predict the interactions experienced by molecules within the periplasm. This extends to molecules that are not native to the bacteria, such as antibacterial agents. Thus, we have little information regarding which moieties of antibiotics are available to carry out the desired functions, and which are unavailable as they are involved in interactions with native proteins/osmolytes/cell wall. To this end, we have conducted a study of polymyxin B1 (PMB1) within models of the Escherichia coli periplasm. PMB1 is a lipopeptide antibiotic used as a “last-resort” drug for the treatment of infections caused by Gram-negative bacteria (Vaara, 2019Vaara M. Polymyxins and their potential next generation as therapeutic antibiotics.Front. Microbiol. 2019; 10: 1689Crossref PubMed Scopus (33) Google Scholar). It is composed of a cyclic, cationic polypeptide ring connected to a branched fatty acid tail. The cationic ring contains five residues of the irregular amino acid α,γ-diaminobutyric acid (DAB), each of which contributes a charge of +1 e giving PMB1 an overall charge of +5 e. The cationic ring enables solubility in aqueous solvents, whereas the lipid tail facilitates insertion into bacterial membranes (Morrison and Jacobs, 1976Morrison D.C. Jacobs D.M. Binding of polymyxin B to the lipid A portion of bacterial lipopolysaccharides.Immunochemistry. 1976; 13: 813-818Crossref PubMed Scopus (640) Google Scholar; Bader and Teuber, 1973Bader J. Teuber M. Action of polymyxin B on bacterial membranes, I. Binding to the O-antigenic lipopolysaccharide of Salmonella typhimurium.Z. Naturforsch. C. 1973; 28: 422-430Crossref PubMed Scopus (5) Google Scholar; Evans, 1999Evans M. Polymyxin B sulfate and colistin: old antibiotics for emerging multiresistant Gram-negative bacteria.Ann. Pharmacother. 1999; 33: 960-967Crossref PubMed Scopus (431) Google Scholar; Trimble et al., 2016Trimble M.J. Mlynarcik P. Kolar M. Hancock R.E.W. Polymyxin: alternative mechanisms of action and resistance.Cold Spring Harb. Perspect. Med. 2016; 6: a025288Crossref PubMed Scopus (156) Google Scholar). While PMB1 along with colistin (polymyxin E) were for many years last-resort antibiotics, in recent years bacterial strains that are resistant to both antibiotics have emerged in a number of countries (Gales et al., 2011Gales A.C. Jones R.N. Sader H.S. Contemporary activity of colistin and polymyxin B against a worldwide collection of Gram-negative pathogens: results from the SENTRY Antimicrobial Surveillance Program (2006-09).J. Antimicrob. Chemother. 2011; 66: 2070-2074Crossref PubMed Scopus (238) Google Scholar; Li et al., 2019Li Z. Cao Y. Yi L. Liu J.H. Yang Q. Emergent polymyxin resistance: end of an era?.Open Forum Infect. Dis. 2019; 6: ofz368Crossref PubMed Scopus (39) Google Scholar). Thus, to either modify these drugs or develop completely novel antibiotics, it is timely to establish a molecular-level understanding of each stage of the process via which they bring about bacterial cell death. To date, mechanistic studies of PMB1 have focused almost entirely on the two membranes of Gram-negative bacteria (Trimble et al., 2016Trimble M.J. Mlynarcik P. Kolar M. Hancock R.E.W. Polymyxin: alternative mechanisms of action and resistance.Cold Spring Harb. Perspect. Med. 2016; 6: a025288Crossref PubMed Scopus (156) Google Scholar; Li and Velkov, 2019Li Z. Velkov T. Polymyxins: mode of action.Adv. Exp. Med. Biol. 2019; 1145: 37-54Crossref PubMed Scopus (13) Google Scholar). From a simulation perspective, the slowly diffusing nature of the lipopolysaccharide (LPS) molecules in the outer leaflet of the OM renders it difficult to achieve the timescales required to observe penetration into, let alone crossing of, the OM at atomistic or even coarse-grained resolution (Berglund et al., 2015Berglund N.A. Piggot T.J. Jefferies D. Sessions R.B. Bond P.J. Khalid S. Interaction of the antimicrobial peptide polymyxin B1 with both membranes of E. coli: a molecular dynamics study.PLoS Comput. Biol. 2015; 11: e1004180Crossref PubMed Scopus (101) Google Scholar; Jefferies et al., 2017Jefferies D. Hsu P.S. Khalid S. Through the lipopolysaccharide glass: a potent antimicrobial peptide induces phase changes in membranes.Biochemistry. 2017; 56: 1672-1679Crossref PubMed Scopus (27) Google Scholar). The studies of the action of PMB1 on the membranes leave unaddressed the question of how PMB1 crosses the periplasm to get from the OM to the IM. Here, a series of atomistic molecular dynamics simulations (Table 1) were performed of models of portions of the E. coli cell envelope. The simulation systems contain an asymmetric model of the OM composed of LPS and phospholipids, a single-layered cell wall, various proteins/lipoproteins, osmolytes, and PMB1, with system sizes ranging from 200,000 to 760,000 atoms. The proteins are a combination of Braun's lipoprotein (BLP), LolA, LolB, OmpA, and Pal (Figure 1). BLP is the most abundant protein in E. coli (there are an estimated 105 copies of BLP in each E. coli) (Vollmer and Holtje, 2004Vollmer W. Holtje J.V. The architecture of the murein (peptidoglycan) in gram-negative bacteria: vertical scaffold or horizontal layer(s)?.J. Bacteriol. 2004; 186: 5978-5987Crossref PubMed Scopus (174) Google Scholar). It exists as a coiled-coil trimer that is essential for compartment stability (Hirota et al., 1977Hirota Y. Suzuki H. Nishimura Y. Yasuda S. On the process of cellular division in Escherichia coli: a mutant of E. coli lacking a murein-lipoprotein.Proc. Natl. Acad. Sci. U S A. 1977; 74: 1417-1420Crossref PubMed Scopus (169) Google Scholar). It is anchored in the OM via a lipidated moiety at its N terminus, whereas it is covalently bound to peptidoglycan via its C terminus. LolA and LolB are small soluble proteins that carry lipoproteins (Matsuyama et al., 1995Matsuyama S. Tajima T. Tokuda H. A novel periplasmic carrier protein involved in the sorting and transport of Escherichia coli lipoproteins destined for the outer membrane.EMBO J. 1995; 14: 3365-3372Crossref PubMed Scopus (161) Google Scholar, Matsuyama et al., 1997Matsuyama S.I. Yokota N. Tokuda H. A novel outer membrane lipoprotein, LolB (HemM), involved in the LolA (p20)-dependent localization of lipoproteins to the outer membrane of Escherichia coli.EMBO J. 1997; 16: 6947-6955Crossref PubMed Scopus (164) Google Scholar). They are largely similar in structure, although LolB is anchored to the OM via a lipidated moiety whereas LolA is free to diffuse across the cell envelope. OmpA is composed of an eight-stranded barrel located in the OM, and is connected via a linker to the soluble domain that can bind peptidoglycan in the periplasm (Smith et al., 2007Smith S.G. Mahon V. Lambert M.A. Fagan R.P. A molecular Swiss army knife: OmpA structure, function and expression.FEMS Microbiol. Lett. 2007; 273: 1-11Crossref PubMed Scopus (278) Google Scholar; Marcoux et al., 2014Marcoux J. Politis A. Rinehart D. Marshall D.P. Wallace M.I. Tamm L.K. Robinson C.V. Mass spectrometry defines the C-terminal dimerization domain and enables modeling of the structure of full-length OmpA.Structure. 2014; 22: 781-790Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar; Boags et al., 2019bBoags A. Samsudin F. Khalid S. Binding from both sides: TolR and OmpA bind and maintain the local structure of the E. coli cell wall.Structure. 2019; 27: 713-724Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Pal also has a lipidated anchor in the OM like LolB, while its C-terminal domain resembles the OmpA soluble domain. Like OmpA, Pal has a linker that can extend into the periplasm enabling the protein to bind non-covalently to peptidoglycan, thereby assisting with maintaining compartment integrity (Mizuno, 1979Mizuno T. A novel peptidoglycan-associated lipoprotein found in the cell envelope of Pseudomonas aeruginosa and Escherichia coli.J. Biochem. 1979; 86: 991-1000Crossref PubMed Scopus (83) Google Scholar; Lazzaroni and Portalier, 1992Lazzaroni J.C. Portalier R. The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein (PAL).Mol. Microbiol. 1992; 6: 735-742Crossref PubMed Scopus (92) Google Scholar). The most compositionally complex system studied here also contained a range of osmolytes in order to better represent the crowded environment that these molecules encounter in the periplasm.Table 1Summary of all simulated systemsSystemaAll systems were simulated using GROMOS 54a7 force field other than PMBcharmm, which was simulated with CHARMM36.,bOuter membrane composition is Ra-LPS in the outer leaflet and PE (90%), PG (5%), and cardiolipin (5%) in the inner leaflet (see STAR methods for details of cell wall); 1,328 Mg2+ ions are also included.ProteinsNo. of atomsPMB1 moleculesOsmolytes and ionscMolar concentration: Na+, Cl− (200 mM), glycerol (35 mM), urea (30 mM), trehalose (10 mM), spermidine (0.2 mM), putrescine (30 mM), osmoregulated periplasmic glucans (OPG, 20 mM).LengthPMBonlynone237,11930Na+, Cl−2 μsPMBcharmmBLP (×1)225,6508K+, Cl−250 nsPMBdilBLP (×4)759,09530Na+, Cl−2 × 500 nsPMBprotBLP (×4), LolA, LolB, Pal, OmpA655,55230Na+, Cl−2 × 500 nsPMBcrowdBLP (×4), LolA, LolB, Pal, OmpA632,39330Na+, Cl−, glycerol, urea, trehalose, spermidine, putrescine, OPG2 × 500 nsa All systems were simulated using GROMOS 54a7 force field other than PMBcharmm, which was simulated with CHARMM36.b Outer membrane composition is Ra-LPS in the outer leaflet and PE (90%), PG (5%), and cardiolipin (5%) in the inner leaflet (see STAR methods for details of cell wall); 1,328 Mg2+ ions are also included.c Molar concentration: Na+, Cl− (200 mM), glycerol (35 mM), urea (30 mM), trehalose (10 mM), spermidine (0.2 mM), putrescine (30 mM), osmoregulated periplasmic glucans (OPG, 20 mM). Open table in a new tab The osmolytes were selected on the basis of a combination of their abundance and chemical diversity. Importantly, all of these osmolytes have their concentrations in the periplasm either documented or estimated in the literature (Cayley et al., 2000Cayley D.S. Guttman H.J. Record Jr., M.T. Biophysical characterization of changes in amounts and activity of Escherichia coli cell and compartment water and turgor pressure in response to osmotic stress.Biophys. J. 2000; 78: 1748-1764Abstract Full Text Full Text PDF PubMed Google Scholar; Boos et al., 1987Boos W. Ehmann U. Bremer E. Middendorf A. Postma P. Trehalase of Escherichia coli. Mapping and cloning of its structural gene and identification of the enzyme as a periplasmic protein induced under high osmolarity growth conditions.J. Biol. Chem. 1987; 262: 13212-13218Abstract Full Text PDF PubMed Google Scholar; Cohen, 1997Cohen S.S. A Guide to the Polyamines. Oxford University Press, 1997Google Scholar; Shah and Swiatlo, 2008Shah P. Swiatlo E. A multifaceted role for polyamines in bacterial pathogens.Mol. Microbiol. 2008; 68: 4-16Crossref PubMed Scopus (239) Google Scholar; Wang et al., 2019Wang D. Weng J. Wang W. Glycerol transport through the aquaglyceroporin GlpF: bridging dynamics and kinetics with atomic simulation.Chem. Sci. 2019; 10: 6957-6965Crossref PubMed Google Scholar; Krishnamurthy et al., 1998Krishnamurthy P. Parlow M. Zitzer J.B. Vakil N.B. Mobley H.L.T. Levy M. Phadnis S.H. Dunn B.E. Helicobacter pylori containing only cytoplasmic Urease is susceptible to acid.Infect. Immun. 1998; 66: 5060-5066Crossref PubMed Google Scholar), and these concentrations are reproduced here: osmoregulated periplasmic glucans (OPG) (20 mM), trehalose (10 mM), putrescine (30 mM), spermidine (3 mM), glycerol (36 mM), and urea (20 mM). Both OPG and trehalose are widely distributed in bacteria, with OPG having a prominent role on regulating osmotic pressure and virulence (Bontemps-Gallo and Lacroix, 2015Bontemps-Gallo S. Lacroix J.M. New insights into the biological role of the osmoregulated periplasmic glucans in pathogenic and symbiotic bacteria.Environ. Microbiol. Rep. 2015; 7: 690-697Crossref PubMed Scopus (23) Google Scholar), whereas trehalose is mainly involved in response to stress conditions (Ruhal et al., 2013Ruhal R. Kataria R. Choudhury B. Trends in bacterial trehalose metabolism and significant nodes of metabolic pathway in the direction of trehalose accumulation.Microb. Biotechnol. 2013; 6: 493-502Crossref PubMed Scopus (76) Google Scholar). The polyamines, putrescine and spermidine, are the two most common in all bacteria, with functions that include supporting bacterial growth, incorporation into the cell wall, and biosynthesis of siderophores (Wortham et al., 2007Wortham B.W. Oliveira M.A. Patel C.N. Polyamines in bacteria: pleiotropic effects yet specific mechanisms.Adv. Exp. Med. Biol. 2007; 603: 106-115Crossref PubMed Scopus (110) Google Scholar). Glycerol is metabolized in E. coli cells for different applications, both aerobically and anaerobically (Murarka et al., 2008Murarka A. Dharmadi Y. Yazdani S.S. Gonzalez R. Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals.Appl. Environ. Microbiol. 2008; 74: 1124-1135Crossref PubMed Scopus (264) Google Scholar; Martínez-Gómez et al., 2012Martínez-Gómez K. Flores N. Castañeda H.M. Martínez-Batallar G. Hernández-Chávez G. Ramírez O.T. Gosset G. Encarnación S. Bolivar F. New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol.Microb. Cell Fact. 2012; 11: 46Crossref PubMed Scopus (106) Google Scholar). Urea is a source of nitrogen after its breakdown (Beckers et al., 2004Beckers G. Bendt A.K. Krämer R. Burkovski A. Molecular identification of the urea uptake system and transcriptional analysis of urea transporter- and urease-encoding genes in Corynebacterium glutamicum.J. Bacteriol. 2004; 186: 7645-7652Crossref PubMed Scopus (74) Google Scholar). Simulations were initiated by placing PMB1 molecules randomly in the aqueous region between the OM and the cell wall. The osmolyte concentrations are derived from literature values and the number of proteins is selected to reproduce crowding volume fraction of ϕ ∼ 0.21 as estimated from experimental studies (Cayley et al., 2000Cayley D.S. Guttman H.J. Record Jr., M.T. Biophysical characterization of changes in amounts and activity of Escherichia coli cell and compartment water and turgor pressure in response to osmotic stress.Biophys. J. 2000; 78: 1748-1764Abstract Full Text Full Text PDF PubMed Google Scholar). One simulation of PMB1 in just water and ions was also performed for comparison. Table 1 provides a summary of the simulations performed in this study. Initial observations focused on general mobility and aggregation of PMB1 followed by in-depth analyses probing the causes of these observations. The crowded nature of the systems had a clear impact upon the solvent-accessible surface area (SASA) of PMB1 (Figure 1). The SASA is lower when PMB1 molecules are just in water and counter ions (PMBonly) compared with when in the protein-containing systems (PMBdil, PMBprot, and PMBcrowd). Tracking the PMB1 motion within the xy plane (Figure S1) of the protein-containing systems shows the movement of polymyxins in the crowded systems (PMBcrowd and PMBprot) is more confined compared with PMBdil, in which BLP is the only protein. Additionally, in the latter system more PMB1 molecules moved toward the OM and the cell wall rather than remaining in the solution area between these two large structures, compared with PMBcrowd and PMBprot. Another effect observed with increasing system complexity is the slower diffusion of PMB1 (Figures 2A and S2), by calculation of the translational diffusion coefficients (Dt) from two different time regimes. For the PMBonly system, the Dt from the longer time regime was estimated to be 4.2 ± 0.4 × 10−6 cm2/s, while for PMBdil, PMBprot, and PMBcrowd systems, the values were 4.1 ± 0.4 × 10−8 cm2/s, 3.5 ± 0.4 × 10−8 cm2/s, and 2.8 ± 0.3 × 10−8 cm2/s, respectively, demonstrating a major reduction when compared with PMBonly (100-fold). The slowest diffusion is recorded for the most crowded system. The dynamics of water was also affected by crowding (Figure 2B), with a Dt rate of 4.8 ± 0.3 × 10−5 cm2/s in PMBonly compared with 2.4 ± 0.1 × 10−5 cm2/s in PMBcrowd for the 10- to 50-ns time period. The values for systems in the presence of the OM are similar to those of a previous report (Lima et al., 2019Lima M.P.M. Nader M. Santos D.E.S. Soares T.A. Compatibility of GROMOS-derived atomic parameters for lipopolysaccharide membranes with the SPC/E water model and alternative long-range electrostatic treatments using single nonbonded cutoff and atom-based charge schemes.J. Braz. Chem. Soc. 2019; 30: 2219-2230Google Scholar) of simulations of the OM in water using the simple point charge (SPC) water model (Berendsen et al., 1981Berendsen H.J. Postma J.P.M. van Gunsteren W.F. Hermans J. Interaction models for water in relation to protein hydration.in: Pullman A. Intermolecular Forces. Reidel Publishing, 1981: 331-342Crossref Google Scholar), and with crowded simulations (Harada et al., 2012Harada R. Sugita Y. Feig M. Protein crowding affects hydration structure and dynamics.J. Am. Chem. Soc. 2012; 134: 4842-4849Crossref PubMed Scopus (138) Google Scholar) using a different water model. Protein diffusion rates were also calculated for the PMBdil, PMBprot, and PMBcrowd systems, showing Dt values that also decrease with increasing crowding volume fraction ϕ. Although LolA is neither bound to the cell wall nor anchored/embedded in the membrane, its calculated Dt falls in the same range as the other proteins, indicating that overall protein motion is quite restricted in the crowded systems for all proteins. While the environment simulated here is more complex due to the presence of membrane and cell wall, the diffusion rates for proteins calculated here are comparable with those of previous reports involving simulations of crowded environments (von Bülow et al., 2019von Bülow S. Siggel M. Linke M. Hummer G. Dynamic cluster formation determines viscosity and diffusion in dense protein solutions.Proc. Natl. Acad. Sci. U S A. 2019; 116: 9843-9852Crossref PubMed Scopus (59) Google Scholar) and cytoplasm models (Yu et al., 2016Yu I. Mori T. Ando T. Harada R. Jung J. Sugita Y. Feig M. Biomolecular interactions modulate macromolecular structure and dynamics in atomistic model of a bacterial cytoplasm.Elife. 2016; 5: e19274Crossref PubMed Scopus (160) Google Scholar; Bortot et al., 2020Bortot L.O. Bashardanesh Z. van der Spoel D. Making soup: preparing and validating models of the bacterial cytoplasm for molecular simulation.J. Chem. Inf. Model. 2020; 60: 322-331Crossref PubMed Scopus (6) Google Scholar), as well as with experimental data from GFP proteins at the periplasm and cytoplasm (Mullineaux et al., 2006Mullineaux C.W. Nenninger A. Ray N. Robinson C. Diffusion of green fluorescent protein in three cell environments in Escherichia coli.J. Bacteriol. 2006; 188: 3442-3448Crossref PubMed Scopus (155) Google Scholar). To check for any impact of the force field on the calculated diffusion rates, we performed an additional simulation (PMBcharmm). The crowding volume fraction φ for that simulation was 0.12 and the diffusion rates for PMB1 and water were 2.3 ± 0.5 × 10−7 cm2/s and 3.25 ± 0.2 × 10−5 cm2/s, respectively, fitting very well with the trends from the GROMOS simulations (Figures 2A and 2B). Here we characterize the molecular interactions that underpin the aforementioned SASA, lateral motion, and translational diffusion profiles. The complexity of the system composition is such that a vast amount of data regarding molecular interactions is generated from these simulations. To facilitate interpretation of the observations, we have presented the results from the perspective of PMB1 interactions, namely PMB1 interactions with itself, osmolytes, proteins, and the cell wall. In PMBonly (PMB1 in solution), differently sized aggregates (dimers to pentamers) formed during the simulations. The lifetimes of interactions between PMB1 molecules ranged from short periods (a few nanoseconds) to longer-term interactions (200–400 ns) leading to formation of aggregates, as shown in the example in Figures 3A and 3B . A range of configurations were observed during the simulations. The majority of interactions occurred via the hydrophobic portions of PMB1, namely Lip1, DPhe7, and Leu8, while the charged sites remained largely exposed to water and ions (Figures 3A and 3B). In the example of a tetrameric association as shown in Figure 3A, four of the PMB1 molecules had Lip1 tails buried in the middle of the aggregate along with two DPhe7 and three Leu8 moieties, thus forming a structure that resembled a micelle. Due to exposure to the aqueous environment of the positive charges and polar residues in this tetramer, the surface of the micelle-like structure was decorated by Cl− ions, which interacted mostly with the NH3+ groups from Dab residues. The center of the micelle was largely protected from exposure to water (Figure 3D). This self-assembly behavior has previously been reported for other similar amphiphilic antibiotics, such as colistin and colistin methanesulfonate, but shown not to occur for the non-amphiphilic polymyxin B nonapeptide, an analog that lacks the hydrophobic tail (Wallace et al., 2011Wallace S.J. Li J. Nation R.L. Prankerd R.J. Velkov T. Boyd B.J. Self-assembly behaviour of colistin and its prodrug colistin methanesulfonate: implications for solution stability and solubilization.J. Phys. Chem. B. 2011; 114: 4836-4840Crossref Scopus (52) Google Scholar). In the cases previously reported, aggregate diameters were calculated to have a Z-average of around 2 ± 0.3 nm, which correlates well with the tetrameric aggregate observed in our simulations (2.2 ± 0.5 nm). Thus, as predicted for colistin and its analog (Wallace et al., 2011Wallace S.J. Li J. Nation R.L. Prankerd R.J. Velkov T. Boyd B.J. Self-assembly behaviour of colistin and its prodrug colistin methanesulfonate: implications for solution stability and solubilization.J. Phys. Chem. B. 2011; 114: 4836-4840Crossref Scopus (52) Google Scholar), PMB1 micelle formation followed a “closed association” model, in which the number of monomers per micelle, in general, does not exceed five in our simulations. In the PMBdil, PMBprot, and PMBcrowd systems, interactions between PMB1s resulted in smaller aggregates, generally involving dimerization (but with the additional participation of other molecular species, as discussed in the next section). Extension of the simulation to 2 μs resulted in the formation of a hexamer, which was stable from 1.2 μs to the end of the simulation. The interaction of PMB1 with osmolytes and ions was firstly characterized by measuring the proximity of each osmolyte type to PMB1. The radial distribution function (RDF) of each osmolyte with PMB1 molecules as a reference (Figure 4) showed a clear preference for glycerol and OPG. This is reasonable considering the number of polar groups on both osmolytes and the negative charge (−1 e) on the phosphate group of OPGs. Putrescine, spermidine, and Na+ ions were found furthest from PMB1, which correlates with both being positively charged (putrescine = +2 e, spermidine = +3 e). It has been discussed previously (von Bülow et al., 2019von Bülow S. Siggel M. Linke M. Hummer G. Dynamic cluster formation determines viscosity and diffusion in dense protein solutions.Proc. Natl. Acad. Sci. U S A. 2019; 116: 9843-9852Crossref PubMed Scopus (59) Google Scholar; Ando and Skolnick, 2010Ando T. Skolnick J. Crowding and hydrodynamic interactions likely dominate in vivo macromolecular motion.Proc. Natl. Acad. Sci. U S A. 2010; 107: 18457-18462Crossref PubMed Scopus (296) Google Scholar; Minton, 1980Minton A.P. 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J. 2019; 116: 1075-1084Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) that in crowded environments non-specific binding occurs constantly, generating transient clusters that affect the structure and dynamics of the molecules in this environment. In our simulations, we observed formation of small osmolyte-PMB1 clusters which had an average size ∼2.5–3.0 nm (slightly larger than PMB1 micelles described in the previous section). These clusters generally contained PMB1 monomers interacting directly with OPG (via -OH groups and cyclohexane rings) and glycerol (via -OH groups), although participation of other osmolytes such as putrescine and urea was also observed, but usually without directly interacting with PMB1. The association between PMB1 molecules and OPG was particularly prevalent (see RDF plot in Figure 4). For example, in one case, four OPG molecules bound around the surface of a PMB1 dimer (Figure 4A), while a fifth OPG molecule mediated the interaction between the PMB1 dimer and a third PMB1 molecule. Four additional molecules of glycerol, one putrescine molecule, and two urea molecules also participated in this cluster, effectively bridging the PMB1 dimer to the third PMB1 (Figure 4D), stabilizing the complex. This cluster took ∼100 ns to stabilize in terms of number of components, apart from one urea molecule that only joined the cluster after 400 ns (Figures 4D and 4E). The largest cluster in all simulations was ∼4.2 nm (diameter) and composed of four PMB1 molecules and ∼20 osmolytes (one trehalose, five putrescine, seven glycerol, and seven OPG). In this cluster, only two of the PMB1 molecules are directly associated with each other, interacting via their DPhe7 residues. The formation of the cluster was initiated by many of the molecules binding to the cell wall (within 30 ns of the start of the simulation). The full cluster had formed after ∼100 ns and lasted for ∼240 ns. Despite showing a higher preference for cluster formation in the cell wall area, a few aggregates were also observed on th" @default.
• W3126435539 created "2021-02-15" @default.
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• W3126435539 creator A5079670006 @default.
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• W3126435539 date "2021-05-01" @default.
• W3126435539 modified "2023-09-26" @default.
• W3126435539 title "The hitchhiker's guide to the periplasm: Unexpected molecular interactions of polymyxin B1 in E. coli" @default.
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