The P-113 peptide: new experimental evidences on its biological activity and conformational insights from Molecular Dynamics simulations Alessia Di Giampaoloa, Carla Luzib, Bruno Casciaroc, Argante Bozzib, Maria Luisa Mangonic, Massimiliano Aschia * a
Department of Physical and Chemical Sciences, University of L'Aquila. Via Vetoio snc 67100 l'Aquila - Italy b
Department of Biotechnological and Clinical Sciences, University of L'Aquila. Via Vetoio snc 67100 l'Aquila - Italy
c
Department of Biochemical Sciences, Sapienza University of Rome. P.le A. Moro 00185 Rome Italy
Running Title: P-113: New Biological Evidences and Structural Features. Key Words: cationic peptide; yeasts; MIC; Molecular Dynamics
Abstract In this paper we report novel and additional results, both experimental and computational, obtained in our laboratories on the peptide P-113. In particular our experimental results indicate that this peptide is endowed with a high target-cell selectivity towards yeast species, suggesting its potential development as a new drug against oral microbial infections. To provide additional structural insights we performed several Molecular Dynamics simulations in different conditions. Results suggest that P-113 is a rather compact species presumably because of its highly charged state as emerged from the dramatic increase of internal fluctuation occurring upon point-mutation. The peptide turns out to adopt, in water, a beta-hairpin-like This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/bip.22452 © 2013 Wiley Periodicals, Inc.
conformation and, in a more hydrophobic environment, is found to be in a (probably slow) equilibrium between α-helix and hairpin conformations. Complexation with Zn2+ induces a drastic mechanical stabilization which prevents any conformational organization of the peptide into a biologically active state.
Introduction. P-113 is a fragment of the parental peptide histatin-5, containing 12 of the 24 amino acid residues, which retains full antibacterial activity and exhibits a wide spectrum of activity in vitro against both bacteria and yeasts [1,2]. The aminoacidic sequence of P-113 AKRHHGYKRKFH results in a calculated net charge +6 at neutral pH. In addition, three histidines and the N-terminal group are responsible for the divalent cations binding. In particular, Cu2+ [3] and Zn2+ [4] have been shown to be strongly complexed. Concerning the antimicrobial activity, Zn2+-P-113 and Cu2+-P-113 have been shown to have a quite similar behaviour. Previous studies [1,4] have demonstrated that P-113 is especially active against the yeast C. albicans, one of the major infectious agents in humans. However, binding of Zn2+ or of Cu2+ causes higher minimal inhibitory concentration (MIC) values compared with those observed with metal-free P-113. The lower activity exhibited by the metal-bound P-113 is possibly ascribed to drastic conformational transitions induced by the metal and, hence by the lack of an α-helix conformation which has been proven to be essential for antimicrobial activity of either P-113 or histatin [1,5,6]. The activity of P-113 against several strains of Candida makes this peptide a potential drug for the treatment of the more common oral candidiasis [1]. Moreover, it has also been shown that the peptide displays in vivo activity in the prevention of gingivitis and, interestingly, its use does not result in causing side effects of other dental components [7,8]. In this paper we report novel and additional results obtained in our laboratories 2
on P-113. In particular we will show: (i) new data concerning antimicrobial activity against some Gram-positive/negative bacterial strains and yeast species not tested sofar and (ii) long time-scale Molecular Dynamics (MD) simulations designed to better understand the intrinsic structural features of aqueous P-113 and the structural role of the complexed metal (Zn2+). Additional MD simulations were also carried out on the aqueous R9I mutant in order to shed some light on the importance of the charged residues in the architecture and also in trifluoroethanol (TFE) solution mimicking the hydrophobic membrane environment in which the peptide is supposed to play a key role for its biological action.
Materials and Methods Microorganisms The strains used for the antimicrobial assays were the following: the Gram-negative bacteria Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Yersinia pseudotuberculosis YPIII and the clinical isolate P. aeruginosa 1; the Grampositive bacteria Bacillus megaterium Bm11, Bacillus thuringiensis B15, Enterococcus faecalis ATCC 29212, the clinical isolate Staphylococcus capitis 1; and the yeasts Candida guiller mondii, Saccharomyces cerevisiae and S. pombe.
Antimicrobial assay Susceptibility testing was performed by adapting the microbroth dilution method outlined by the Clinical and Laboratory Standards Institute, using sterile 96-well plates (Falcon NJ, USA). The growth of the bacterial and yeast cells was aseptically measured by absorbance at 590 nm with a spectrophotometer (UV-1700 Pharma Spec Shimadzu, Tokyo, Japan). Afterwards, aliquots (50 μl) of microorganisms in mid-log 3
phase of growth at a concentration of 2x105 colony-forming units (CFU)/mL in culture medium (Mueller-Hinton, MH) were added to 50 μl of water containing the peptide in serial 2-fold dilutions ranging from 0.125 to 64 μM. The same procedure was followed with yeasts in Winge medium [9]. Inhibition of microbial growth was determined after an incubation of 18 h at 37 °C (30 °C for yeasts), by measuring the absorbance at 590 nm with a microplate reader (Infinite M200; Tecan, Salzburg, Austria). Antimicrobial activities were expressed as the minimal inhibitory concentration (MIC), the concentration of peptide at which 100% inhibition of microbial growth is observed after 18 h of incubation.
Molecular Dynamics simulations. The Molecular Dynamics (MD) simulations were performed utilizing the Gromacs package [10]. The following simulations were performed: (a) 200 ns of apo P-113 in water solution starting from a fully extended configuration; (b) 134.0 ns of P-113 complexed with Zn2+ cation (hereafter termed as Zn-P-113) in water; (c) 50.0 ns of Zn-P-113 in TFE solution (d) 224.0 ns of R9I mutant in water solution starting from a fully extended configuration; (e) 300.0 ns of apo P-113 in TFE solution starting with the fully extended configuration; (f) 100.0 ns of apo P-113 in TFE starting with α-helix configuration; (g) 20.0 ns of apo P-113 in water starting with α-helix configuration. 4
The initial coordinates of Zn-P-113 were constructed on the basis of previous studies [3-4]. Each peptide was put in a dodecahedral box whose dimension prevents selfinteraction. For the simulations in water 2512 molecules of water, at the typical density of water at 298K and 1.0 atm, utilizing the single point charge (SPC) model. [11] For TFE simulations using the all-atom fore field contained in the Gromacs package with the charges calculated by Van Buuren and Berendsen [12] All the simulations were performed adopting the protocol as described below. After an energy minimization, the whole system was slowly heated up to 300 K using short (100.0 ps) MD runs. (ii) The isothermal/isochoric ensemble was adopted in all the simulations making use of the the Berendsen thermostat [13]. On the basis of the backbone Root Mean Square Deviation (RMSD) we disregarded the first 100 ns for P-113, the first 20.0 ns for Zn-P-113, the first 20.0 ns for the simulations of P-113 in TFE starting both with the fully extended and with the α-helix configuration. The simulated peptides were described using the OPLS force field [14], the LINCS algorithm was adopted to constrain all bond lengths [15], and the long range electrostatics were computed by Particle Mesh Ewald method [16] with 34 wave vectors in each dimension and a 4th order cubic interpolation. For Zn-P-113 the charged were readjusted on the basis of point charges recalculated by standard quantum-chemical fitting [17] carried out on a tetrahedral complex between Zn+2, three imidazole rings and one methyl-amino group. The calculations were performed with the Gamess package [18] using Density Functional Theory with the B3LYP functional [19,20] and 6-311G* basis set. Much of the collective analysis used in this work are based on Essential Dynamics (ED) [21]. On the purpose the covariance matrix of the atomic positional fluctuations (either all-atoms or backbone, see Results section) was built from the MD trajectory and then diagonalized producing an orthonormal set of eigenvectors defining a new set of generalized coordinates along which the peptide fluctuations occur. The trace of the covariance matrix provides us with a direct measure of the extent of peptide fluctuation. The M eigenvectors with 5
the largest eigenvalues, i.e. with the largest associated fluctuation (large amplitude motions) allow to define the essential M-dimensional subspace onto which the trajectory is projected. From the obtained projection it is possible to easily identify the conformations sampled by the peptides and, hence, it might be relatively straightforward to concisely compare the conformational space spanned by the three investigated systems.
Results
1) Experimental results: Antimicrobial activity To expand our knowledge on the antimicrobial activity of P-113, several bacterial and yeast strains, not studied so far, were analyzed by the microbroth dilution assay, to determine their susceptibility to this peptide. As indicated in Table 1, a MIC higher than 64 μM was found against all the tested Gram-positive and Gram-negative bacteria, with the exception of B. megaterium. This is in line with what was previously reported on this derivative of Histatin 5 [4]. However, when P-113 was assayed against S. cerevisiae, S. pombe and C. guiller mondii, a very strong antimicrobial activity was observed, as indicated by the corresponding MIC values, ranging from 0.25 to 4 μM. Since the poor antimicrobial activity of P-113 shown towards the majority of the Gram+/- bacterial strains, but the strong activity displayed against different yeasts tested in our laboratories (see Table 1), we decided to investigate the preferential structures adopted by this peptide in aqueous solution by means of Molecular Dynamics simulations to achieve more information on its structure/activity 6
correlation. This is explained in the next subsection
2) MD simulations.
Our computational analysis is specifically devoted to achieve information as to: - what is the preferred conformation of P-113 in water; - what is the role of the charged residues in the conformational properties of aqueous apo P-113; - what are the conformational effects produced by the incorporation of Zn2+ in P-113 both in aqueous and TFE solution; - how is the α-helix conformation accessible to apo P-113 in water and in TFE;
2a - Simulation in water from a fully-extended configuration. As already stated in the Methods section, apo P-113 in water is considered as converged to a relatively stable conformation only in the last 100.0 ns of simulation as emerged by the backbone RMSD below reported in the Figure 1. In the figure it is, in fact, clear that after an initial random search, P-113 collapses into a relatively stable folded structure with a RMSD fluctuation much lower than 0.1 nm. In order to more carefully inspecting the structural features of P-113 in this stable region we carried out ED analysis as explained in the Methods section. Diagonalization of the all-atom covariance matrix has produced an eigenvalue spectrum characterized by the first two eigenvectors accounting for more than 55% of the whole system fluctuation. Hence, the projection of the trajectory onto the plane 7
formed by the first two eigenvectors might provide a well exhaustive conformational landscape. The result is reported in the Figure 2. In the Figure using red boxes, we have delimited regions (hereafter termed as basins) of arbitrary dimension representative of the different conformational states of the peptide. The configurations representative of each conformational basin, extracted from the center of the relative basin, are represented in the same Figure. According to our findings it is clear that P-113 adopts a rather stable hairpin-like conformation. Interestingly, in all these conformational states, we have systematically found an electrostatic interaction involving the side-chain of Arginine-9 (R9 in Figure) and the side-chains of Histidine-5 and Phenylalanine-11. Hence, although other stabilizing factors are certainly present, such an interaction does definitely play a key role and, therefore we decided to apply a point mutation by replacing the charged R9 residue with an uncharged residue such as Isoleucine. The effect of this mutation as well as the effect of the incorporation of Zn2+ into the apo P-113have been analyzed and the results reported in the next subsection.
2b - Comparative structural and mechanical analysis between P-113, Zn-P-113 and R9I in water solution. The results of our MD simulations of the three systems, summarized from the previously reported Figure 2 and the following Figure 3, Figure 4 and Table 2 concisely indicate that: i- apo P-113 is a relatively stable and compact structure (relatively low RMSF in 8
Figure 3b and lowest inertia moments in Table 2) characterized by a bent structure showing two antiparallel beta-sheets (residues Lysine-10, Phenylalanine-11, Histidine-4, Arginine-3 as emerged from the time course of secondary structure in Figure 4a) ii – inclusion of zinc-ion produces a collapse into a very stable structure (lowest RMSF in Figure 3b and lowest trace of the covariance matrix in Table 2) characterized by a loss of intra-peptide hydrogen bonds and a consequent loss of the beta-sheet organization (Figure 4b). Moreover from the differences found in the RMSD time courses between apo-P113 and Zn-P-113 in Figure 3a, it is also worth to mention that the most stable Zn-P-113 conformation turns out to be rather different from apo-P-113 structure. Most importantly the presence of Zn2+ inhibits any P-113 fluctuation preventing the possibility of adopting other conformations including the ones supposed to be biologically active. At the same time, the presence of the Zn2+ induces a slight but significant increase of moments of inertia and solvent accessible surface (SAS) clearly indicating a loss of the structural compactness observed for the apo-P-113. It is interesting to observe the role of the change of the environment in the mechanical and structural properties of Zn-P-113. In Figure 5 we compare the RMSD per residue (5a) and RMSF per residue (5b) in water and in TFE. At the same time we also compare the related radius of gyration of Zn-P-113 in water (0.75+0.03 nm) and in TFE (0.72+0.01). The almost superimposable RMSD in Figure 5a and the similar values of average radii of gyration, indicate that when Zn-P-113 is transferred from water to TFE the system does not undergo any relevant modification of the average structure (see Figure 4b). On the other hand the drastic reduction of RMSF in TFE (Figure 5b) sharply demonstrates a strong mechanical stabilization induced by TFE. This finding allows us to hypothesize that Zn-P-113 is a very stable, although fully unstructured, 9
species in rather different environments. iii- the point mutation produces a drastic geometrical and mechanical variation. In fact R9I system is characterized by a very noisy RMSD and, consequently by the loss of secondary structure and a very high RMSF and trace of backbone covariance matrix (Figure 3, Figure 4 and Table 2). Moreover it is also interesting to note, in the same table, the very large trace of the all-atom trace of the covariance matrix indicating a basically complete freedom of the atoms belonging to the peptide side chains. All these observations clearly suggest that, differently from the apo-P-113 and Zn-P-113, the mutant species is not able to easily find a single stable conformation and that, consequently, and it is in very rapid equilibrium between a large number of conformational states. Moreover, the complete loss of secondary structure and intrapeptide H-bonds together with the significant increment of peptide-solvent H-bonds, the significant increase of the moments of inertia and SAS do indicate a scarcely compact and basically unfolded structure. In order to better quantify the conformational differences we finally performed an additional Essential Dynamics analysis. In this regard the three trajectories were first linked. On the concatenated trajectory we have built and diagonalized the backbone covariance matrix. The trace of the covariance matrix and the spectrum of the relative eigenvalues have revealed that the first two eigenvectors, accounting for more than 60% of the whole fluctuation could be safely utilized for detecting any overlaps between the conformational basins. The result, shown in Figure 6, does indicate the absolute absence of any overlap between the conformational basins, indicating a marked difference between the conformational repertoire of the three systems. In any case, in water solution, apparently no α-helix conformation is sampled by apo-P-113. For this reason we investigated the plausible occurrence of α-helix formation in TFE solution. 10
2c – MD simulations of apo P-113 in TFE. Stability of α-helix. The first simulation of the apo P-113, initiated from a fully extended configuration and propagated up to 300 ns, did not reveal any α-helix organization. In particular, comparison of the RMSD between water and TFE simulation reported in Figure 7, indicates that in TFE apo P-113 after a few ns reaches a conformation with a low fluctuation pattern and structurally different from the one previously observed in water. In fact, the time course of the secondary structure, reported in the same figure, witnesses the complete absence of the antiparallel β strend observed in water (Figure 4a). In order to check whether the absence of α-helix might be due to its actual instability or to an incomplete sampling of the trajectory we repeated the apo P-113 simulation in TFE starting from an α-helix configuration. The same simulation has been also repeated in water in order to reinforce the result. The emerged picture is reported in Figure 8 where we have linked the two trajectories and projected them onto the plane formed by the related first two eigenvectors of the backbone covariance atom. The result clearly indicates that in TFE apo-P113 permanently remains in a relatively small region of the configurational space characterized by an α-helix conformation as also confirmed by the secondary structure evolution below reported. On the other hand the same structure in water essentially loses its secondary structure after just 5.0 ns. This finding qualitatively suggests that apo P-113 in TFE can adopt at least two stable conformations: the α-helix conformation of Figure 8 and the less structured bent conformation pictorially reported in Figure 7. In Figure 9 we also report the potential energy differences calculated for apo-P-112 in TFE and water. It is important to remark that the average potential energy refer to the whole system and, hence, only the comparison within the systems with the same number of particles 11
The average total potential energies indicate that in water, within the error, we do not observe any significant difference between the two conformational states. On the other hand in TFE, the α-helix conformation appears as energetically more stable. Hence, on the basis of the above results and in the lack of a quantitative estimation of the relative free energies of the different conformational states preventing any evaluation of their actual relative stability, we can definitely although qualitatively asses that in TFE α-helix and an unstructured conformation (hairpin-like) are both accessible and probably separated by a relatively high barrier. On the other hand, in water, α-helix represents a high free-energy inaccessible state and the peptide permanently resides in a beta hairpin conformation.
Discussion The antimicrobial assays performed in our laboratories and here described showed that P-113 is endowed with a high target-cell selectivity towards yeast species, suggesting its potential development as a new drug against oral candidiasis. In particular, the MIC values found ranged from 0.25 μM (C. guiller-mondii) to 4 μM (S. cerevisiae). These results are in line with those previously reported [1,2]. In order to gain further information of this peptide at molecular level, which may help in understanding its possible mechanism of action at molecular level its biophysical processes on biological membranes, structural studies were performed by means of MD simulations in solution. The computational results can be summarized as follows: i- apo P-113 is, in principle, a rather rigid species which in water turns out to collapse into a very stable and compact structure characterized by a hairpin-like motif while in 12
TFE exists in two rigid structures characterized by α-helix and hairpin-like conformational motifs probably separated by a relatively high free-energy barrier. ii- the P-113 multiply-charged nature turns out to be essential for its stability and compactness, as a matter of fact one point mutation with reduction of the charge severely alters the structural-mechanical feature of the peptide dramatically enhancing its fluctuation pattern. iii- upon complexation of Zn2+, P-113 undergoes a further mechanical stabilization and turns out to be conformationally hindered preventing any possibility of finding alternative secondary structures even in the presence of more hydrophobic environments. Such a stabilization is greatly reinforced when the Zn-P-113 is in TFE solution. iv- both Zn2+complexation and point-mutation drive the system in conformational states not accessible by the apo P-113. Previous data from literature have shown that P-113, having in its sequence three His residues and the N-terminal amino acting as the binding donors for either Cu2+ or Zn2+ , is a very attractive ligand for both metal ions [3,4]. In addition, the His-His pair results to play a major role in stabilizing the peptide-ion complex. A comparison between the structures of the two metal complexes indicates that although Zn-P-113 and Cu-P-113 share the same metal donor domains, they adopt different conformations as a consequence of the dissimilar geometries of the coordination shell. In fact, Zn-P-113 adopts preferentially a tetrahedral structure while Cu-P-113 stays better in tetragonal conformation [4]. As far as the antimicrobial activity concerns, Zn-P-113 and Cu-P-113 have been described to behave rather similarly. Reports from different laboratories [4,22] confirm that P-113 exhibits the strongest activity against C. albicans, one of the major fungal pathogens in humans. However, the P-113 complex with Zn2+ or Cu2+ ions does not improve the fungicidal power of the peptide devoid of the metals. Since Cu2+ and Zn2+ complexes with P-113 show 13
different geometries, the low antimicrobial activity exhibited by the metal-bound P113 is better ascribed to the lack of an α-helix conformation rather than to specific effects of the metals [4]. In fact, metal binding to the N-terminal group, to the HisHis pair and to the last His makes the peptide less prone to adopt α-helix conformation domains, already shown to be crucial for antimicrobial activity of either P-113 and histatin [5,6,22]. In support of this hypothesis, the CD spectra of P-113 in TFE/H2O (9:1) show the drastic reduction in α-elix content upon metal ions binding [4].
Conclusions. In conclusion, it can be speculated that in water solution apo-P-113 adopts a very compact structure characterized by a hairpin-like motif that, upon reaching the hydrophobic space inside the lipid bilayer of the membrane, may undergo α-helical arrangement which might plausibly favor the pore formation or even the translocation of
P-113 across the membrane. When P-113 is complexed with Zn2+ the
conformational repertoire of the peptide, both in water and TFE solution, is drastically reduced and no a-helix is formed. This latter finding, in the light of the experimentally observed reduced biological activity of the complexed peptide, allows us to hypothesize the strict correlation between antimicrobial efficacy and α-helix conformation for a peptide.
ACKNOWLEDGMENTS: The Authors wish to thank Dr. R. Petruzzelli for critical reviewing the manuscript and helpful suggestions during the experimental part of the work.
14
FIGURES AND FIGURES CAPTIONS
16
17
Figure 1. Time course of all-atom RMSD for apo-P-113 in water solution. The cartoon-like picture of the initial structure and the structure at 100.0 ns are also reported.
18
19
Figure 2. Projection of the trajectory onto the plane formed by the first all-atom covariance matrix eigenvector for apo-P-113 in water solution. Argine 9 is highlighted with R9. Conformational basins are highlighted with red boxes. Representative structures of each basin are also schematically reported.
20
21
Figure 3. Panel (a) : Time course of the backbone RMSD evaluated for the three systems in water solution: P-113 (red), Zn-P-113 (blue) and R9I (black). Note that all the reported RMSD were evaluated taking as reference structure the initial P-113 conformation. Panel (b) : backbone RMSF (same color of panel a) evaluated on the equilibrated portions of the trajectories (from 100.0 ns for P-113, from 20.0 ns for both Zn-P-113 and R9I).
22
23
Figure 4. Time course of secondary structure for apo P-113 (a), Zn-P-113 (b) and R9I. Configurations representative of the last conformations are also schematically reported.
24
Figure 5. Root Mean Square Deviation per residue (panel a) and Root Mean Square Fluctuation per residue (panel b) of Zn-P-113 in TFE (black lines) and in water (red lines). Both the Root Mean Square Deviations were evaluated with respect to Zn-P-113 initial structure in the water simulation.
25
Figure 6. Projection of the three linked trajectories in water solution (P-113, black; Zn-P-113, blue; R9I red) onto the plane formed by the first two eigenvectors of the relative backbone covariance-matrix.
27
28
Figure 7. Comparison of the RMSD time course of the simulations of apo P-113 in water solution starting from a fully extended configuration in water (black) and in TFE (red). The representative configurations of the final conformations are reported.
29
Figure 8 Projection of the concatenated trajectories of apo P-113 in TFE (red) and in water (black) starting from the same α-helix initial conformation. In blue we have also marked a qualitative pathway followed by the two trajectories. The secondary structure evolution is also reported in the lower side of the Figure .
30
32
Figure 9. Average total potential energies for the simulations in water and in TFE as described in the text. The values (from left to right in the Figure) have been evaluated as: averages in the last 100 ns of the trajectory of apo P-113 in water, in the first 6.0 ns of the trajectory starting from α-helix in water, in the last 200 ns of the trajectory of apo P113 in TFE and in the 100 ns of the trajectory of apo P-113 starting from α-helix in TFE. Error bars are standard errors evaluated along the same trajectories.
33
REFERENCES 1. Rothstein, D. M.; Spacciapoli, P. L.; Tran, T.; Xu, T.; Roberts, F. D.; Dalla Serra, M.; Buxton, D. K.; Oppenheim, F. G.; Friden, P. Antimicrob Agents Chemother 2001, 45, 1367-1373. 2. Sajjan, U. S.; Tran, L. T.; Sole, N.; Rovaldi, C.; AkiYama, A.; Friden, P. M.; Forstner, J. F.; Rothstein, D. M. Antimicrob Agents Chemother 2001, 45, 3437-3444. 3. Kulon, K.; Valensin, D.; Kamysz, W.; Valensin, G.; Nadolski, P.; Porciatti, E.; Gaggelli, E.; Kozlowski, H. J Inorg Biochem 2008, 102, 960-972. 4. Porciatti, E.; Milenkovic, M.; Gaggelli, E.; Valensin, G.; Kozlowski, H.; Kamysz, W.; Valensin, D. Inorg Chem 2010, 49, 8690-8698. 5. Raj, P. A.; Soni, S. D.; Levine, M. J. J Biol Chem 1994, 269, 9610-9619. 6. Ramalingam, K. T.; Gururaja, L.; Ramasubbu, N.; Levine, M. J. Biochem Biophys Res Commun 1996, 225, 47-53. 7. Mickels, N.; McManus, C.; Massaro, J.; Friden, P. M.; Braman, V.; D'Agostino, R.; Oppenheim, F. G.; Warbington, M.; Dibart, S.; Van Dyke, T. J Clin Periodontol 2001, 28, 404-410. 8. Paquette, D. W.; Waters, G. S.; Stefanidou, V. L.; Lawrence, H. P.; Friden, P. M.; O'Connor, S. M.; Sperati, J. D.; Oppenheim, F. G.; Hutchens, L. H.; Williams, R. C. J Clin Periodontol 1997, 24, 216-222. 9. Valenti P.; Visca P.; Antonini G.; Orsi N. A Mycopathologia 1985, 89, 169-175 10. Van Der Spoel D.; Lindahl E.; Hess B.; Groenhof G.; Mark A.E.; Berendsen H.J.C. J Comput Chem 2005, 26, 1701. 11. Berendsen, H.J.C. ; Postma, J.P.M.; van Gunsteren, W.F.; Hermans J. in Intermolecular Forces Pullmann B Editor. Reider Publishing Company: Dordrecht, 1981, pp. 331-342 12 Van Buuren, A.R.; Berendsen, H.J.C. Biopolymers 1993, 33, 1159-1166. 13. Berendsen, H.J.C.; Postma, J.P.M.; van Gunsteren, W.F.; Di Nola, A. J. Chem. Phys 1984, 81 3684. 14. Jorgensen, W.L.; Maxwell, D.S.; Tirado-Rives, J. J. Am. Chem. Soc. 1996, 118, 11225. 15. Hess, B.; Bekker, H.; Berendsen, H.J.C.; Frajie, J.C.E.M. J. Comput. Chem 1997, 18, 1463. 16. Darden, T.A.; York, D.M.; Pedersen, L-G. J. Chem. Phys. 1993, 98, 10089. 34
17. Breneman, C. M.; Wiberg, K. B. J. Comput. Chem 1990, 11, 361. 18. Schmidt, M.W.; Baldridge, K.K.; Boatz, J.A.; Elbert, S-T.; Gordon, M.S.; Jensen, J.H.; Koseki,
S.; Matsunaga, N.; Nguyen, K.A.; Su, S.; Windus, T.L.; Dupuis, M.; Montgomery, J.A. General Atomic and Molecular Electronic Structure System J. Comput. Chem., 1993, 14, 1347-1363. 19. Becke A.D. J. Chem. Phys. 1993, 98, 1372-1377. 20. Lee, C.; Yang, W.; Parr, R.G. Phys. Rev. B, 1998, 37, 785-789. 21. A. Amadei, A.B.M. Linssen, H.J.C. Proteins: Struct. Funct. Genet. 1993, 17, 412. 22. Ahmad, M.; Piludu, M.; Oppenheim, F.G.; Helmerhorst, E.J.; Hand, A.R. J. Histochem. Cytochem. 2004, 52, 361-370.
35
Table 1. Antimicrobial activity of P113 against different bacterial and yeast strains MIC (μM) Gram-negatives
Peptide
Gram-positives
E. coli ATCC 25922
P. aeruginosa
Y. pseudotuberculosis YPIII
E.coli ATCC 25922
B. megaterrium Bm11
B. thuringiensis B15
E. faecalis ATCC 29212
S. capiti 1
>64
>64
>64
>64
8
>64
>64
>64
P113
Each MIC value is the average of at least three independent experiments .
Table 2. Relevant structural and mechanical properties. When present, error bars are standard errors evaluated along the equilibrated portion of the trajectory: 100 ns for P-113, 20.0 ns for Zn-P113 and 20.0 ns for R9I. Property
P-113
Zn-P113
R9I
ITOT (amu nm2) I1 (amu nm2) I2 (amu nm2) I3 (amu nm2) Peptide-solvent H-bonds Average number of Intra-peptide H-bonds Hydrophobic area (nm2) Hydrophylic area (nm2) Trace of backbone covariance matrix (nm2) Trace all-atom covariance matrix (nm2)
1029.8±0.2 246.3±0.1 682.2±0.2 730.4±0.1 42 5 7.08±0.01 7.97±0.01 0.42 14.65
1136.1±0.3 303.9±0.1 727.1±0.2 817.0±0.2 42 2 8.17±0.01 8.09±0.01 0.23 10.23
1586.8±0.9 299.6±0.2 1045.3±0.9 1148.4±0.8 47 1 9.38±0.01 8.60±0.01 3.73 65.60
Inertia moment:
36
Department of Physical and Chemical Sciences, University of L'Aquila. Via Vetoio snc 67100 l'Aquila - Italy b
Department of Biotechnological and Clinical Sciences, University of L'Aquila. Via Vetoio snc 67100 l'Aquila - Italy
c
Department of Biochemical Sciences, Sapienza University of Rome. P.le A. Moro 00185 Rome Italy
Running Title: P-113: New Biological Evidences and Structural Features. Key Words: cationic peptide; yeasts; MIC; Molecular Dynamics
Abstract In this paper we report novel and additional results, both experimental and computational, obtained in our laboratories on the peptide P-113. In particular our experimental results indicate that this peptide is endowed with a high target-cell selectivity towards yeast species, suggesting its potential development as a new drug against oral microbial infections. To provide additional structural insights we performed several Molecular Dynamics simulations in different conditions. Results suggest that P-113 is a rather compact species presumably because of its highly charged state as emerged from the dramatic increase of internal fluctuation occurring upon point-mutation. The peptide turns out to adopt, in water, a beta-hairpin-like This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/bip.22452 © 2013 Wiley Periodicals, Inc.
conformation and, in a more hydrophobic environment, is found to be in a (probably slow) equilibrium between α-helix and hairpin conformations. Complexation with Zn2+ induces a drastic mechanical stabilization which prevents any conformational organization of the peptide into a biologically active state.
Introduction. P-113 is a fragment of the parental peptide histatin-5, containing 12 of the 24 amino acid residues, which retains full antibacterial activity and exhibits a wide spectrum of activity in vitro against both bacteria and yeasts [1,2]. The aminoacidic sequence of P-113 AKRHHGYKRKFH results in a calculated net charge +6 at neutral pH. In addition, three histidines and the N-terminal group are responsible for the divalent cations binding. In particular, Cu2+ [3] and Zn2+ [4] have been shown to be strongly complexed. Concerning the antimicrobial activity, Zn2+-P-113 and Cu2+-P-113 have been shown to have a quite similar behaviour. Previous studies [1,4] have demonstrated that P-113 is especially active against the yeast C. albicans, one of the major infectious agents in humans. However, binding of Zn2+ or of Cu2+ causes higher minimal inhibitory concentration (MIC) values compared with those observed with metal-free P-113. The lower activity exhibited by the metal-bound P-113 is possibly ascribed to drastic conformational transitions induced by the metal and, hence by the lack of an α-helix conformation which has been proven to be essential for antimicrobial activity of either P-113 or histatin [1,5,6]. The activity of P-113 against several strains of Candida makes this peptide a potential drug for the treatment of the more common oral candidiasis [1]. Moreover, it has also been shown that the peptide displays in vivo activity in the prevention of gingivitis and, interestingly, its use does not result in causing side effects of other dental components [7,8]. In this paper we report novel and additional results obtained in our laboratories 2
on P-113. In particular we will show: (i) new data concerning antimicrobial activity against some Gram-positive/negative bacterial strains and yeast species not tested sofar and (ii) long time-scale Molecular Dynamics (MD) simulations designed to better understand the intrinsic structural features of aqueous P-113 and the structural role of the complexed metal (Zn2+). Additional MD simulations were also carried out on the aqueous R9I mutant in order to shed some light on the importance of the charged residues in the architecture and also in trifluoroethanol (TFE) solution mimicking the hydrophobic membrane environment in which the peptide is supposed to play a key role for its biological action.
Materials and Methods Microorganisms The strains used for the antimicrobial assays were the following: the Gram-negative bacteria Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Yersinia pseudotuberculosis YPIII and the clinical isolate P. aeruginosa 1; the Grampositive bacteria Bacillus megaterium Bm11, Bacillus thuringiensis B15, Enterococcus faecalis ATCC 29212, the clinical isolate Staphylococcus capitis 1; and the yeasts Candida guiller mondii, Saccharomyces cerevisiae and S. pombe.
Antimicrobial assay Susceptibility testing was performed by adapting the microbroth dilution method outlined by the Clinical and Laboratory Standards Institute, using sterile 96-well plates (Falcon NJ, USA). The growth of the bacterial and yeast cells was aseptically measured by absorbance at 590 nm with a spectrophotometer (UV-1700 Pharma Spec Shimadzu, Tokyo, Japan). Afterwards, aliquots (50 μl) of microorganisms in mid-log 3
phase of growth at a concentration of 2x105 colony-forming units (CFU)/mL in culture medium (Mueller-Hinton, MH) were added to 50 μl of water containing the peptide in serial 2-fold dilutions ranging from 0.125 to 64 μM. The same procedure was followed with yeasts in Winge medium [9]. Inhibition of microbial growth was determined after an incubation of 18 h at 37 °C (30 °C for yeasts), by measuring the absorbance at 590 nm with a microplate reader (Infinite M200; Tecan, Salzburg, Austria). Antimicrobial activities were expressed as the minimal inhibitory concentration (MIC), the concentration of peptide at which 100% inhibition of microbial growth is observed after 18 h of incubation.
Molecular Dynamics simulations. The Molecular Dynamics (MD) simulations were performed utilizing the Gromacs package [10]. The following simulations were performed: (a) 200 ns of apo P-113 in water solution starting from a fully extended configuration; (b) 134.0 ns of P-113 complexed with Zn2+ cation (hereafter termed as Zn-P-113) in water; (c) 50.0 ns of Zn-P-113 in TFE solution (d) 224.0 ns of R9I mutant in water solution starting from a fully extended configuration; (e) 300.0 ns of apo P-113 in TFE solution starting with the fully extended configuration; (f) 100.0 ns of apo P-113 in TFE starting with α-helix configuration; (g) 20.0 ns of apo P-113 in water starting with α-helix configuration. 4
The initial coordinates of Zn-P-113 were constructed on the basis of previous studies [3-4]. Each peptide was put in a dodecahedral box whose dimension prevents selfinteraction. For the simulations in water 2512 molecules of water, at the typical density of water at 298K and 1.0 atm, utilizing the single point charge (SPC) model. [11] For TFE simulations using the all-atom fore field contained in the Gromacs package with the charges calculated by Van Buuren and Berendsen [12] All the simulations were performed adopting the protocol as described below. After an energy minimization, the whole system was slowly heated up to 300 K using short (100.0 ps) MD runs. (ii) The isothermal/isochoric ensemble was adopted in all the simulations making use of the the Berendsen thermostat [13]. On the basis of the backbone Root Mean Square Deviation (RMSD) we disregarded the first 100 ns for P-113, the first 20.0 ns for Zn-P-113, the first 20.0 ns for the simulations of P-113 in TFE starting both with the fully extended and with the α-helix configuration. The simulated peptides were described using the OPLS force field [14], the LINCS algorithm was adopted to constrain all bond lengths [15], and the long range electrostatics were computed by Particle Mesh Ewald method [16] with 34 wave vectors in each dimension and a 4th order cubic interpolation. For Zn-P-113 the charged were readjusted on the basis of point charges recalculated by standard quantum-chemical fitting [17] carried out on a tetrahedral complex between Zn+2, three imidazole rings and one methyl-amino group. The calculations were performed with the Gamess package [18] using Density Functional Theory with the B3LYP functional [19,20] and 6-311G* basis set. Much of the collective analysis used in this work are based on Essential Dynamics (ED) [21]. On the purpose the covariance matrix of the atomic positional fluctuations (either all-atoms or backbone, see Results section) was built from the MD trajectory and then diagonalized producing an orthonormal set of eigenvectors defining a new set of generalized coordinates along which the peptide fluctuations occur. The trace of the covariance matrix provides us with a direct measure of the extent of peptide fluctuation. The M eigenvectors with 5
the largest eigenvalues, i.e. with the largest associated fluctuation (large amplitude motions) allow to define the essential M-dimensional subspace onto which the trajectory is projected. From the obtained projection it is possible to easily identify the conformations sampled by the peptides and, hence, it might be relatively straightforward to concisely compare the conformational space spanned by the three investigated systems.
Results
1) Experimental results: Antimicrobial activity To expand our knowledge on the antimicrobial activity of P-113, several bacterial and yeast strains, not studied so far, were analyzed by the microbroth dilution assay, to determine their susceptibility to this peptide. As indicated in Table 1, a MIC higher than 64 μM was found against all the tested Gram-positive and Gram-negative bacteria, with the exception of B. megaterium. This is in line with what was previously reported on this derivative of Histatin 5 [4]. However, when P-113 was assayed against S. cerevisiae, S. pombe and C. guiller mondii, a very strong antimicrobial activity was observed, as indicated by the corresponding MIC values, ranging from 0.25 to 4 μM. Since the poor antimicrobial activity of P-113 shown towards the majority of the Gram+/- bacterial strains, but the strong activity displayed against different yeasts tested in our laboratories (see Table 1), we decided to investigate the preferential structures adopted by this peptide in aqueous solution by means of Molecular Dynamics simulations to achieve more information on its structure/activity 6
correlation. This is explained in the next subsection
2) MD simulations.
Our computational analysis is specifically devoted to achieve information as to: - what is the preferred conformation of P-113 in water; - what is the role of the charged residues in the conformational properties of aqueous apo P-113; - what are the conformational effects produced by the incorporation of Zn2+ in P-113 both in aqueous and TFE solution; - how is the α-helix conformation accessible to apo P-113 in water and in TFE;
2a - Simulation in water from a fully-extended configuration. As already stated in the Methods section, apo P-113 in water is considered as converged to a relatively stable conformation only in the last 100.0 ns of simulation as emerged by the backbone RMSD below reported in the Figure 1. In the figure it is, in fact, clear that after an initial random search, P-113 collapses into a relatively stable folded structure with a RMSD fluctuation much lower than 0.1 nm. In order to more carefully inspecting the structural features of P-113 in this stable region we carried out ED analysis as explained in the Methods section. Diagonalization of the all-atom covariance matrix has produced an eigenvalue spectrum characterized by the first two eigenvectors accounting for more than 55% of the whole system fluctuation. Hence, the projection of the trajectory onto the plane 7
formed by the first two eigenvectors might provide a well exhaustive conformational landscape. The result is reported in the Figure 2. In the Figure using red boxes, we have delimited regions (hereafter termed as basins) of arbitrary dimension representative of the different conformational states of the peptide. The configurations representative of each conformational basin, extracted from the center of the relative basin, are represented in the same Figure. According to our findings it is clear that P-113 adopts a rather stable hairpin-like conformation. Interestingly, in all these conformational states, we have systematically found an electrostatic interaction involving the side-chain of Arginine-9 (R9 in Figure) and the side-chains of Histidine-5 and Phenylalanine-11. Hence, although other stabilizing factors are certainly present, such an interaction does definitely play a key role and, therefore we decided to apply a point mutation by replacing the charged R9 residue with an uncharged residue such as Isoleucine. The effect of this mutation as well as the effect of the incorporation of Zn2+ into the apo P-113have been analyzed and the results reported in the next subsection.
2b - Comparative structural and mechanical analysis between P-113, Zn-P-113 and R9I in water solution. The results of our MD simulations of the three systems, summarized from the previously reported Figure 2 and the following Figure 3, Figure 4 and Table 2 concisely indicate that: i- apo P-113 is a relatively stable and compact structure (relatively low RMSF in 8
Figure 3b and lowest inertia moments in Table 2) characterized by a bent structure showing two antiparallel beta-sheets (residues Lysine-10, Phenylalanine-11, Histidine-4, Arginine-3 as emerged from the time course of secondary structure in Figure 4a) ii – inclusion of zinc-ion produces a collapse into a very stable structure (lowest RMSF in Figure 3b and lowest trace of the covariance matrix in Table 2) characterized by a loss of intra-peptide hydrogen bonds and a consequent loss of the beta-sheet organization (Figure 4b). Moreover from the differences found in the RMSD time courses between apo-P113 and Zn-P-113 in Figure 3a, it is also worth to mention that the most stable Zn-P-113 conformation turns out to be rather different from apo-P-113 structure. Most importantly the presence of Zn2+ inhibits any P-113 fluctuation preventing the possibility of adopting other conformations including the ones supposed to be biologically active. At the same time, the presence of the Zn2+ induces a slight but significant increase of moments of inertia and solvent accessible surface (SAS) clearly indicating a loss of the structural compactness observed for the apo-P-113. It is interesting to observe the role of the change of the environment in the mechanical and structural properties of Zn-P-113. In Figure 5 we compare the RMSD per residue (5a) and RMSF per residue (5b) in water and in TFE. At the same time we also compare the related radius of gyration of Zn-P-113 in water (0.75+0.03 nm) and in TFE (0.72+0.01). The almost superimposable RMSD in Figure 5a and the similar values of average radii of gyration, indicate that when Zn-P-113 is transferred from water to TFE the system does not undergo any relevant modification of the average structure (see Figure 4b). On the other hand the drastic reduction of RMSF in TFE (Figure 5b) sharply demonstrates a strong mechanical stabilization induced by TFE. This finding allows us to hypothesize that Zn-P-113 is a very stable, although fully unstructured, 9
species in rather different environments. iii- the point mutation produces a drastic geometrical and mechanical variation. In fact R9I system is characterized by a very noisy RMSD and, consequently by the loss of secondary structure and a very high RMSF and trace of backbone covariance matrix (Figure 3, Figure 4 and Table 2). Moreover it is also interesting to note, in the same table, the very large trace of the all-atom trace of the covariance matrix indicating a basically complete freedom of the atoms belonging to the peptide side chains. All these observations clearly suggest that, differently from the apo-P-113 and Zn-P-113, the mutant species is not able to easily find a single stable conformation and that, consequently, and it is in very rapid equilibrium between a large number of conformational states. Moreover, the complete loss of secondary structure and intrapeptide H-bonds together with the significant increment of peptide-solvent H-bonds, the significant increase of the moments of inertia and SAS do indicate a scarcely compact and basically unfolded structure. In order to better quantify the conformational differences we finally performed an additional Essential Dynamics analysis. In this regard the three trajectories were first linked. On the concatenated trajectory we have built and diagonalized the backbone covariance matrix. The trace of the covariance matrix and the spectrum of the relative eigenvalues have revealed that the first two eigenvectors, accounting for more than 60% of the whole fluctuation could be safely utilized for detecting any overlaps between the conformational basins. The result, shown in Figure 6, does indicate the absolute absence of any overlap between the conformational basins, indicating a marked difference between the conformational repertoire of the three systems. In any case, in water solution, apparently no α-helix conformation is sampled by apo-P-113. For this reason we investigated the plausible occurrence of α-helix formation in TFE solution. 10
2c – MD simulations of apo P-113 in TFE. Stability of α-helix. The first simulation of the apo P-113, initiated from a fully extended configuration and propagated up to 300 ns, did not reveal any α-helix organization. In particular, comparison of the RMSD between water and TFE simulation reported in Figure 7, indicates that in TFE apo P-113 after a few ns reaches a conformation with a low fluctuation pattern and structurally different from the one previously observed in water. In fact, the time course of the secondary structure, reported in the same figure, witnesses the complete absence of the antiparallel β strend observed in water (Figure 4a). In order to check whether the absence of α-helix might be due to its actual instability or to an incomplete sampling of the trajectory we repeated the apo P-113 simulation in TFE starting from an α-helix configuration. The same simulation has been also repeated in water in order to reinforce the result. The emerged picture is reported in Figure 8 where we have linked the two trajectories and projected them onto the plane formed by the related first two eigenvectors of the backbone covariance atom. The result clearly indicates that in TFE apo-P113 permanently remains in a relatively small region of the configurational space characterized by an α-helix conformation as also confirmed by the secondary structure evolution below reported. On the other hand the same structure in water essentially loses its secondary structure after just 5.0 ns. This finding qualitatively suggests that apo P-113 in TFE can adopt at least two stable conformations: the α-helix conformation of Figure 8 and the less structured bent conformation pictorially reported in Figure 7. In Figure 9 we also report the potential energy differences calculated for apo-P-112 in TFE and water. It is important to remark that the average potential energy refer to the whole system and, hence, only the comparison within the systems with the same number of particles 11
The average total potential energies indicate that in water, within the error, we do not observe any significant difference between the two conformational states. On the other hand in TFE, the α-helix conformation appears as energetically more stable. Hence, on the basis of the above results and in the lack of a quantitative estimation of the relative free energies of the different conformational states preventing any evaluation of their actual relative stability, we can definitely although qualitatively asses that in TFE α-helix and an unstructured conformation (hairpin-like) are both accessible and probably separated by a relatively high barrier. On the other hand, in water, α-helix represents a high free-energy inaccessible state and the peptide permanently resides in a beta hairpin conformation.
Discussion The antimicrobial assays performed in our laboratories and here described showed that P-113 is endowed with a high target-cell selectivity towards yeast species, suggesting its potential development as a new drug against oral candidiasis. In particular, the MIC values found ranged from 0.25 μM (C. guiller-mondii) to 4 μM (S. cerevisiae). These results are in line with those previously reported [1,2]. In order to gain further information of this peptide at molecular level, which may help in understanding its possible mechanism of action at molecular level its biophysical processes on biological membranes, structural studies were performed by means of MD simulations in solution. The computational results can be summarized as follows: i- apo P-113 is, in principle, a rather rigid species which in water turns out to collapse into a very stable and compact structure characterized by a hairpin-like motif while in 12
TFE exists in two rigid structures characterized by α-helix and hairpin-like conformational motifs probably separated by a relatively high free-energy barrier. ii- the P-113 multiply-charged nature turns out to be essential for its stability and compactness, as a matter of fact one point mutation with reduction of the charge severely alters the structural-mechanical feature of the peptide dramatically enhancing its fluctuation pattern. iii- upon complexation of Zn2+, P-113 undergoes a further mechanical stabilization and turns out to be conformationally hindered preventing any possibility of finding alternative secondary structures even in the presence of more hydrophobic environments. Such a stabilization is greatly reinforced when the Zn-P-113 is in TFE solution. iv- both Zn2+complexation and point-mutation drive the system in conformational states not accessible by the apo P-113. Previous data from literature have shown that P-113, having in its sequence three His residues and the N-terminal amino acting as the binding donors for either Cu2+ or Zn2+ , is a very attractive ligand for both metal ions [3,4]. In addition, the His-His pair results to play a major role in stabilizing the peptide-ion complex. A comparison between the structures of the two metal complexes indicates that although Zn-P-113 and Cu-P-113 share the same metal donor domains, they adopt different conformations as a consequence of the dissimilar geometries of the coordination shell. In fact, Zn-P-113 adopts preferentially a tetrahedral structure while Cu-P-113 stays better in tetragonal conformation [4]. As far as the antimicrobial activity concerns, Zn-P-113 and Cu-P-113 have been described to behave rather similarly. Reports from different laboratories [4,22] confirm that P-113 exhibits the strongest activity against C. albicans, one of the major fungal pathogens in humans. However, the P-113 complex with Zn2+ or Cu2+ ions does not improve the fungicidal power of the peptide devoid of the metals. Since Cu2+ and Zn2+ complexes with P-113 show 13
different geometries, the low antimicrobial activity exhibited by the metal-bound P113 is better ascribed to the lack of an α-helix conformation rather than to specific effects of the metals [4]. In fact, metal binding to the N-terminal group, to the HisHis pair and to the last His makes the peptide less prone to adopt α-helix conformation domains, already shown to be crucial for antimicrobial activity of either P-113 and histatin [5,6,22]. In support of this hypothesis, the CD spectra of P-113 in TFE/H2O (9:1) show the drastic reduction in α-elix content upon metal ions binding [4].
Conclusions. In conclusion, it can be speculated that in water solution apo-P-113 adopts a very compact structure characterized by a hairpin-like motif that, upon reaching the hydrophobic space inside the lipid bilayer of the membrane, may undergo α-helical arrangement which might plausibly favor the pore formation or even the translocation of
P-113 across the membrane. When P-113 is complexed with Zn2+ the
conformational repertoire of the peptide, both in water and TFE solution, is drastically reduced and no a-helix is formed. This latter finding, in the light of the experimentally observed reduced biological activity of the complexed peptide, allows us to hypothesize the strict correlation between antimicrobial efficacy and α-helix conformation for a peptide.
ACKNOWLEDGMENTS: The Authors wish to thank Dr. R. Petruzzelli for critical reviewing the manuscript and helpful suggestions during the experimental part of the work.
14
FIGURES AND FIGURES CAPTIONS
16
17
Figure 1. Time course of all-atom RMSD for apo-P-113 in water solution. The cartoon-like picture of the initial structure and the structure at 100.0 ns are also reported.
18
19
Figure 2. Projection of the trajectory onto the plane formed by the first all-atom covariance matrix eigenvector for apo-P-113 in water solution. Argine 9 is highlighted with R9. Conformational basins are highlighted with red boxes. Representative structures of each basin are also schematically reported.
20
21
Figure 3. Panel (a) : Time course of the backbone RMSD evaluated for the three systems in water solution: P-113 (red), Zn-P-113 (blue) and R9I (black). Note that all the reported RMSD were evaluated taking as reference structure the initial P-113 conformation. Panel (b) : backbone RMSF (same color of panel a) evaluated on the equilibrated portions of the trajectories (from 100.0 ns for P-113, from 20.0 ns for both Zn-P-113 and R9I).
22
23
Figure 4. Time course of secondary structure for apo P-113 (a), Zn-P-113 (b) and R9I. Configurations representative of the last conformations are also schematically reported.
24
Figure 5. Root Mean Square Deviation per residue (panel a) and Root Mean Square Fluctuation per residue (panel b) of Zn-P-113 in TFE (black lines) and in water (red lines). Both the Root Mean Square Deviations were evaluated with respect to Zn-P-113 initial structure in the water simulation.
25
Figure 6. Projection of the three linked trajectories in water solution (P-113, black; Zn-P-113, blue; R9I red) onto the plane formed by the first two eigenvectors of the relative backbone covariance-matrix.
27
28
Figure 7. Comparison of the RMSD time course of the simulations of apo P-113 in water solution starting from a fully extended configuration in water (black) and in TFE (red). The representative configurations of the final conformations are reported.
29
Figure 8 Projection of the concatenated trajectories of apo P-113 in TFE (red) and in water (black) starting from the same α-helix initial conformation. In blue we have also marked a qualitative pathway followed by the two trajectories. The secondary structure evolution is also reported in the lower side of the Figure .
30
32
Figure 9. Average total potential energies for the simulations in water and in TFE as described in the text. The values (from left to right in the Figure) have been evaluated as: averages in the last 100 ns of the trajectory of apo P-113 in water, in the first 6.0 ns of the trajectory starting from α-helix in water, in the last 200 ns of the trajectory of apo P113 in TFE and in the 100 ns of the trajectory of apo P-113 starting from α-helix in TFE. Error bars are standard errors evaluated along the same trajectories.
33
REFERENCES 1. Rothstein, D. M.; Spacciapoli, P. L.; Tran, T.; Xu, T.; Roberts, F. D.; Dalla Serra, M.; Buxton, D. K.; Oppenheim, F. G.; Friden, P. Antimicrob Agents Chemother 2001, 45, 1367-1373. 2. Sajjan, U. S.; Tran, L. T.; Sole, N.; Rovaldi, C.; AkiYama, A.; Friden, P. M.; Forstner, J. F.; Rothstein, D. M. Antimicrob Agents Chemother 2001, 45, 3437-3444. 3. Kulon, K.; Valensin, D.; Kamysz, W.; Valensin, G.; Nadolski, P.; Porciatti, E.; Gaggelli, E.; Kozlowski, H. J Inorg Biochem 2008, 102, 960-972. 4. Porciatti, E.; Milenkovic, M.; Gaggelli, E.; Valensin, G.; Kozlowski, H.; Kamysz, W.; Valensin, D. Inorg Chem 2010, 49, 8690-8698. 5. Raj, P. A.; Soni, S. D.; Levine, M. J. J Biol Chem 1994, 269, 9610-9619. 6. Ramalingam, K. T.; Gururaja, L.; Ramasubbu, N.; Levine, M. J. Biochem Biophys Res Commun 1996, 225, 47-53. 7. Mickels, N.; McManus, C.; Massaro, J.; Friden, P. M.; Braman, V.; D'Agostino, R.; Oppenheim, F. G.; Warbington, M.; Dibart, S.; Van Dyke, T. J Clin Periodontol 2001, 28, 404-410. 8. Paquette, D. W.; Waters, G. S.; Stefanidou, V. L.; Lawrence, H. P.; Friden, P. M.; O'Connor, S. M.; Sperati, J. D.; Oppenheim, F. G.; Hutchens, L. H.; Williams, R. C. J Clin Periodontol 1997, 24, 216-222. 9. Valenti P.; Visca P.; Antonini G.; Orsi N. A Mycopathologia 1985, 89, 169-175 10. Van Der Spoel D.; Lindahl E.; Hess B.; Groenhof G.; Mark A.E.; Berendsen H.J.C. J Comput Chem 2005, 26, 1701. 11. Berendsen, H.J.C. ; Postma, J.P.M.; van Gunsteren, W.F.; Hermans J. in Intermolecular Forces Pullmann B Editor. Reider Publishing Company: Dordrecht, 1981, pp. 331-342 12 Van Buuren, A.R.; Berendsen, H.J.C. Biopolymers 1993, 33, 1159-1166. 13. Berendsen, H.J.C.; Postma, J.P.M.; van Gunsteren, W.F.; Di Nola, A. J. Chem. Phys 1984, 81 3684. 14. Jorgensen, W.L.; Maxwell, D.S.; Tirado-Rives, J. J. Am. Chem. Soc. 1996, 118, 11225. 15. Hess, B.; Bekker, H.; Berendsen, H.J.C.; Frajie, J.C.E.M. J. Comput. Chem 1997, 18, 1463. 16. Darden, T.A.; York, D.M.; Pedersen, L-G. J. Chem. Phys. 1993, 98, 10089. 34
17. Breneman, C. M.; Wiberg, K. B. J. Comput. Chem 1990, 11, 361. 18. Schmidt, M.W.; Baldridge, K.K.; Boatz, J.A.; Elbert, S-T.; Gordon, M.S.; Jensen, J.H.; Koseki,
S.; Matsunaga, N.; Nguyen, K.A.; Su, S.; Windus, T.L.; Dupuis, M.; Montgomery, J.A. General Atomic and Molecular Electronic Structure System J. Comput. Chem., 1993, 14, 1347-1363. 19. Becke A.D. J. Chem. Phys. 1993, 98, 1372-1377. 20. Lee, C.; Yang, W.; Parr, R.G. Phys. Rev. B, 1998, 37, 785-789. 21. A. Amadei, A.B.M. Linssen, H.J.C. Proteins: Struct. Funct. Genet. 1993, 17, 412. 22. Ahmad, M.; Piludu, M.; Oppenheim, F.G.; Helmerhorst, E.J.; Hand, A.R. J. Histochem. Cytochem. 2004, 52, 361-370.
35
Table 1. Antimicrobial activity of P113 against different bacterial and yeast strains MIC (μM) Gram-negatives
Peptide
Gram-positives
E. coli ATCC 25922
P. aeruginosa
Y. pseudotuberculosis YPIII
E.coli ATCC 25922
B. megaterrium Bm11
B. thuringiensis B15
E. faecalis ATCC 29212
S. capiti 1
>64
>64
>64
>64
8
>64
>64
>64
P113
Each MIC value is the average of at least three independent experiments .
Table 2. Relevant structural and mechanical properties. When present, error bars are standard errors evaluated along the equilibrated portion of the trajectory: 100 ns for P-113, 20.0 ns for Zn-P113 and 20.0 ns for R9I. Property
P-113
Zn-P113
R9I
ITOT (amu nm2) I1 (amu nm2) I2 (amu nm2) I3 (amu nm2) Peptide-solvent H-bonds Average number of Intra-peptide H-bonds Hydrophobic area (nm2) Hydrophylic area (nm2) Trace of backbone covariance matrix (nm2) Trace all-atom covariance matrix (nm2)
1029.8±0.2 246.3±0.1 682.2±0.2 730.4±0.1 42 5 7.08±0.01 7.97±0.01 0.42 14.65
1136.1±0.3 303.9±0.1 727.1±0.2 817.0±0.2 42 2 8.17±0.01 8.09±0.01 0.23 10.23
1586.8±0.9 299.6±0.2 1045.3±0.9 1148.4±0.8 47 1 9.38±0.01 8.60±0.01 3.73 65.60
Inertia moment:
36
