An iron fist in a velvet glove: The cooperation of a novel pyoverdine from Pseudomonas donghuensis P482 with 7-hydroxytropolone is pivotal for its antibacterial activity.

Pseudomonas donghuensis P482 exhibits broad antimicrobial activity against phytopathogens, including the soft rot bacteria of the Dickeya genus. Here, we report that under limited nutrient availability, the antibacterial activity of P. donghuensis P482 against Dickeya solani requires the reciprocal action of two iron scavengers: 7-hydroxytropolone (7-HT) and a newly characterized pyoverdine (PVDP482 ) and is quenched in the iron-augmented environment. Further, we show that the biosynthesis of pyoverdine and 7-HT is metabolically coordinated, and the functional BV82_4709 gene involved in 7-HT synthesis is pivotal for expressing the BV82_3755 gene, essential for pyoverdine biosynthesis and vice versa. The synthesis of both scavengers is under the control of Gac/Rsm, but only PVD is controlled by Fur. The isoelectric focusing profile of the P482 siderophore differs from that of the other Pseudomonas spp. tested. This finding led to the unveiling of the chemical structure of the new pyoverdine PVDP482 . To summarize, the antibacterial activity of P. donghuensis P482 is attributed to 7-HT and PVDP482 varies depending on the nutrient and iron availability, highlighting the importance of these factors in the competition between P482 and D. solani.

A Novel Cryptic Clostridial Peptide That Kills Bacteria by a Cell Membrane Permeabilization Mechanism.

This work reports detailed characteristics of the antimicrobial peptide Intestinalin (P30), which is derived from the LysC enzyme of Clostridium intestinale strain URNW. The peptide shows a broader antibacterial spectrum than the parental enzyme, showing potent antimicrobial activity against clinical strains of Gram-positive staphylococci and Gram-negative pathogens and causing between 3.04 ± 0.12 log kill for Pseudomonas aeruginosa PAO1 and 7.10 ± 0.05 log kill for multidrug-resistant Acinetobacter baumannii KPD 581 at a 5 µM concentration. Moreover, Intestinalin (P30) prevents biofilm formation and destroys 24-h and 72-h biofilms formed by Acinetobacter baumannii CRAB KPD 205 (reduction levels of 4.28 and 2.62 log CFU/mL, respectively). The activity of Intestinalin is combined with both no cytotoxicity and little hemolytic effect against mammalian cells. The nuclear magnetic resonance and molecular dynamics (MD) data show a high tendency of Intestinalin to interact with the bacterial phospholipid cell membrane. Although positively charged, Intestinalin resides in the membrane and aggregates into small oligomers. Negatively charged phospholipids stabilize peptide oligomers to form water- and ion-permeable pores, disrupting the integrity of bacterial cell membranes. Experimental data showed that Intestinalin interacts with negatively charged lipoteichoic acid (logK based on isothermal titration calorimetry, 7.45 ± 0.44), causes membrane depolarization, and affects membrane integrity by forming large pores, all of which result in loss of bacterial viability. IMPORTANCE Antibiotic resistance is rising rapidly among pathogenic bacteria, becoming a global public health problem that threatens the effectiveness of therapies for many infectious diseases. In this respect, antimicrobial peptides appear to be an interesting alternative to combat bacterial pathogens. Here, we report the characteristics of an antimicrobial peptide (of 30 amino acids) derived from the clostridial LysC enzyme. The peptide showed killing activity against clinical strains of Gram-positive and Gram-negative pathogens. Experimental data and computational modeling showed that this peptide forms transmembrane pores, directly engaging the negatively charged phospholipids of the bacterial cell membrane. Consequently, dissipation of the electrochemical gradient across cell membranes affects many vital processes, such as ATP synthesis, motility, and transport of nutrients. This kind of dysfunction leads to the loss of bacterial viability. Our firm conviction is that the presented study will be a helpful resource in searching for novel antimicrobial peptides that could have the potential to replace conventional antibiotics.

A general method for the derivation of the functional forms of the effective energy terms in coarse-grained energy functions of polymers. III. Determination of scale-consistent backbone-local and correlation potentials in the UNRES force field and force-field calibration and validation.

The general theory of the construction of scale-consistent energy terms in the coarse-grained force fields presented in Paper I of this series has been applied to the revision of the UNRES force field for physics-based simulations of proteins. The potentials of mean force corresponding to backbone-local and backbone-correlation energy terms were calculated from the ab initio energy surfaces of terminally blocked glycine, alanine, and proline, and the respective analytical expressions, derived by using the scale-consistent formalism, were fitted to them. The parameters of all these potentials depend on single-residue types, thus reducing their number and preventing over-fitting. The UNRES force field with the revised backbone-local and backbone-correlation terms was calibrated with a set of four small proteins with basic folds: tryptophan cage variant (TRP1; α), Full Sequence Design (FSD; α + ß), villin headpiece (villin; α), and a truncated FBP-28 WW-domain variant (2MWD; ß) (the NEWCT-4P force field) and, subsequently, with an enhanced set of 9 proteins composed of TRP1, FSD, villin, 1BDC (α), 2I18 (α), 1QHK (α + ß), 2N9L (α + ß), 1E0L (ß), and 2LX7 (ß) (the NEWCT-9P force field). The NEWCT-9P force field performed better than NEWCT-4P in a blind-prediction-like test with a set of 26 proteins not used in calibration and outperformed, in a test with 76 proteins, the most advanced OPT-WTFSA-2 version of UNRES with former backbone-local and backbone-correlation terms that contained more energy terms and more optimizable parameters. The NEWCT-9P force field reproduced the bimodal distribution of backbone-virtual-bond angles in the simulated structures, as observed in experimental protein structures.

Maximum Likelihood Calibration of the UNRES Force Field for Simulation of Protein Structure and Dynamics.

By using the maximum likelihood method for force-field calibration recently developed in our laboratory, which is aimed at achieving the agreement between the simulated conformational ensembles of selected training proteins and the corresponding ensembles determined experimentally at various temperatures, the physics-based coarse-grained UNRES force field for simulations of protein structure and dynamics was optimized with seven small training proteins exhibiting a variety of secondary and tertiary structures. Four runs of optimization, in which the number of optimized force-field parameters was gradually increased, were carried out, and the resulting force fields were subsequently tested with a set of 22 α-, 12 ß-, and 12 α + ß-proteins not used in optimization. The variant in which energy-term weights, local, and correlation potentials, side-chain radii, and anisotropies were optimized turned out to be the most transferable and outperformed all previous versions of UNRES on the test set.

Characteristics of C-terminal, ß-amyloid peptide binding fragment of neuroprotective protease inhibitor, cystatin C.

Cystatin C originally identified as a cysteine proteases inhibitor has a broad spectrum of biological roles ranging from inhibition of extracellular cysteine protease activities, bone resorption, and modulation of inflammatory responses to stimulation of fibroblasts proliferation. There is an increasing number of evidence to suggest that human cystatin C (hCC) might play a protective role in the pathophysiology of sporadic Alzheimer's disease. In vivo and in vitro results well documented the association of hCC with Aß and the hCC-induced inhibition of Aß fibril formation. In our earlier work, using a combination of selective proteolytic methods and MS spectroscopy, C-terminal fragment hCC(101-117) was identified as the Aß-binding region. The fragment of Aß peptide responsible for the complex formation with hCC was found in the middle, highly hydrophobic part, Aß(17-24). Structures and affinities of both Aß and hCC binding sites were characterized by the enzyme-linked immunosorbent assay-like assay, by surface plasmon resonance, and by nano-ESI-FTICR MS of the hCC-Aß-binding peptide complexes. In the in vitro inhibition studies, the binding cystatin sequence, hCC(101-117), revealed the highest relative inhibitory effect toward Aß-fibril formation. Herein, we present further studies on molecular details of the hCC-Aß complex. With Ala substitution, affinity experiments, and enzyme-linked immunosorbent assay-like assays for the Aß-binding fragment, hCC(101-117), and its variants, the importance of individual amino acid residues for the protein interaction was evaluated. The results were analyzed using hCC(101-117) nuclear magnetic resonance structural data with molecular dynamics calculations and molecular modeling of the complexes. The results point to conformational requirements and special importance of some amino acid residues for the protein interaction. The obtained results might be helpful for the design of low molecular compounds modulating the biological role of both proteins. Copyright © 2016 John Wiley & Sons, Ltd.

Probing the binding of Cu(2+) ions to a fragment of the Aß(1-42) polypeptide using fluorescence spectroscopy, isothermal titration calorimetry and molecular dynamics simulations.

Steady-state and time-resolved fluorescence quenching measurements supported by isothermal titration calorimetry (ITC) and molecular dynamics simulations (MD), with the NMR-derived restraints, were used to investigate the interactions of Cu(2+) ions with a fragment of the Aß(1-42) polypeptide, Aß(5-16) with the following sequence: Ac-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-NH2, denoted as HZ1. The studies presented in this paper, when compared with our previous results (Makowska et al., Spectrochim. Acta A 153: 451-456), show that the affinity of the peptide to metal ions is conformation-dependent. All the measurements were carried out in 20mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, pH6.0. The Stern-Volmer equations, along with spectroscopic observations, were used to determine the quenching and binding parameters. The obtained results unequivocally suggest that Cu(2+) ions quench the fluorescence of HZ1 only through a static quenching mechanism, in contrast to the fragment from the N-terminal part of the FPB28 protein, with sequence Ac-Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr- NH2 (D9) and its derivative with a single point mutation: Ac-Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr- NH2 (D9_M), where dynamic quenching occurred. The thermodynamic parameters (ΔITCH, ΔITCS) for the interactions between Cu(2+) ions and the HZ1 peptide were determined from the calorimetric data. The conditional thermodynamic parameters suggest that, under the experimental conditions, the formation of the Cu(2+)-HZ1 complex is both an enthalpy and entropy driven process.

A Maximum-Likelihood Approach to Force-Field Calibration.

A new approach to the calibration of the force fields is proposed, in which the force-field parameters are obtained by maximum-likelihood fitting of the calculated conformational ensembles to the experimental ensembles of training system(s). The maximum-likelihood function is composed of logarithms of the Boltzmann probabilities of the experimental conformations, calculated with the current energy function. Because the theoretical distribution is given in the form of the simulated conformations only, the contributions from all of the simulated conformations, with Gaussian weights in the distances from a given experimental conformation, are added to give the contribution to the target function from this conformation. In contrast to earlier methods for force-field calibration, the approach does not suffer from the arbitrariness of dividing the decoy set into native-like and non-native structures; however, if such a division is made instead of using Gaussian weights, application of the maximum-likelihood method results in the well-known energy-gap maximization. The computational procedure consists of cycles of decoy generation and maximum-likelihood-function optimization, which are iterated until convergence is reached. The method was tested with Gaussian distributions and then applied to the physics-based coarse-grained UNRES force field for proteins. The NMR structures of the tryptophan cage, a small α-helical protein, determined at three temperatures (T = 280, 305, and 313 K) by Halabis et al. ( J. Phys. Chem. B 2012 , 116 , 6898 - 6907 ), were used. Multiplexed replica-exchange molecular dynamics was used to generate the decoys. The iterative procedure exhibited steady convergence. Three variants of optimization were tried: optimization of the energy-term weights alone and use of the experimental ensemble of the folded protein only at T = 280 K (run 1); optimization of the energy-term weights and use of experimental ensembles at all three temperatures (run 2); and optimization of the energy-term weights and the coefficients of the torsional and multibody energy terms and use of experimental ensembles at all three temperatures (run 3). The force fields were subsequently tested with a set of 14 α-helical and two α + ß proteins. Optimization run 1 resulted in better agreement with the experimental ensemble at T = 280 K compared with optimization run 2 and in comparable performance on the test set but poorer agreement of the calculated folding temperature with the experimental folding temperature. Optimization run 3 resulted in the best fit of the calculated ensembles to the experimental ones for the tryptophan cage but in much poorer performance on the training set, suggesting that use of a small α-helical protein for extensive force-field calibration resulted in overfitting of the data for this protein at the expense of transferability. The optimized force field resulting from run 2 was found to fold 13 of the 14 tested α-helical proteins and one small α + ß protein with the correct topologies; the average structures of 10 of them were predicted with accuracies of about 5 Å C(α) root-mean-square deviation or better. Test simulations with an additional set of 12 α-helical proteins demonstrated that this force field performed better on α-helical proteins than the previous parametrizations of UNRES. The proposed approach is applicable to any problem of maximum-likelihood parameter estimation when the contributions to the maximum-likelihood function cannot be evaluated at the experimental points and the dimension of the configurational space is too high to construct histograms of the experimental distributions.

A study of the influence of charged residues on ß-hairpin formation by nuclear magnetic resonance and molecular dynamics.

Chain reversals are often nucleation sites in protein folding. The ß-hairpins of FBP28 WW domain and IgG are stable and have been proved to initiate the folding and are, therefore, suitable for studying the influence of charged residues on ß-hairpin conformation. In this paper, we carried out NMR examination of the conformations in solution of two fragments from the FPB28 protein (PDB code: 1E0L) (N-terminal part) namely KTADGKT-NH2 (1E0L 12-18, D7) and YKTADGKTY-NH2 (1E0L 11-19, D9), one from the B3 domain of the protein G (PDB code: 1IGD), namely DDATKT-NH2 (1IGD 51-56) (Dag1), and three variants of Dag1 peptide: DVATKT-NH2 (Dag2), OVATKT-NH2 (Dag3) and KVATKT-NH2 (Dag4), respectively, in which the original charged residue were replaced with non-polar residues or modified charged residues. It was found that both the D7 and D9 peptides form a large fraction bent conformations. However, no hydrophobic contacts between the terminal Tyr residues of D9 occur, which suggests that the presence of a pair of like-charged residues stabilizes chain reversal. Conversely, only the Dag1 and Dag2 peptides exhibit some chain reversal; replacing the second aspartic-acid residue with a valine and the first one with a basic residue results in a nearly extended conformation. These results suggest that basic residues farther away in sequence can result in stabilization of chain reversal owing to screening of the non-polar core. Conversely, smaller distance in sequence prohibits this screening, while the presence oppositely-charged residues can stabilize a turn because of salt-bridge formation.

Conformational dynamics of the trp-cage miniprotein at its folding temperature.

The folding temperature of the trp-cage mini-protein was determined to be in the range 311-317 K depending on the method used. Our study is focused on determining the structure and dynamics of the polypeptide chain close to its unfolding or melting temperature. At T = 305 K, Trp6-Arg16 and Trp6-Pro12 long-range interactions are observed, and at T = 313 K, only the Trp6-Arg16 interactions remain, while all of mentioned interactions are observed in the native state of the protein. Partial (at T = 305 K) and complete (at T = 313 K) melting of the N-terminal α-helix is observed, manifested by the appearance of minor sets of signals in NMR spectra. Our key findings are: (i) conformational phase transition (melting point) could be described as a cooperative breaking of the Trp6-Pro12 long-range hydrophobic interaction and the melting of the N-terminal α-helix; (ii) many ROE signals corresponding to local or short-range interactions vanish rapidly with temperature increase; however, long-range interaction such as Trp6-Arg16 remains until 313 K. The presence of the native long-range interaction at 313 K makes that conformational ensemble resemble a very diffuse native state structure, but it is not a simple mixture of the folded and unfolded states, as could be expected on the basis of the common two-state folding mechanism.

Like-charged residues at the ends of oligoalanine sequences might induce a chain reversal.

We have examined the effect of like-charged residues on the conformation of an oligoalanine sequence. This was facilitated by circular dichroism (CD) and NMR spectroscopic and differential scanning calorimetric (DSC) measurements, and molecular dynamics calculations of the following three alanine-based peptides: Ac-K-(A)(5) -K-NH(2) (KAK5), Ac-K-(A)(4) -K-NH(2) (KAK4), Ac-K-(A)(3) -K-NH(2) (KAK3), where A and K denote alanine and lysine residues, respectively. Our earlier studies suggested that the presence of like-charged residues at the end of a short polypeptide chain composed of nonpolar residues can induce a chain reversal. For all three peptides, canonical molecular dynamics simulations with NMR-derived restraints demonstrate the presence of ensembles of structures with a tendency to form a chain reversal. The KAK3 peptide exhibits a bent shape with its ends close to each other, while KAK4 and KAK5 are more extended. In the KAK5 peptide, the lysine residues do not have any influence on each other and are very mobile. Nevertheless, the tendency to form a more or less pronounced chain reversal is observed and it seems to be stable in all three peptides. This chain reversal seems to be caused by screening of the nonpolar core from the solvent by the hydrated charged residues.

Mechanism of formation of the C-terminal beta-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. II. Interplay of local backbone conformational dynamics and long-range hydrophobic interactions in hairpin formation.

Two peptides, corresponding to the turn region of the C-terminal beta-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus, consisting of residues 51-56 [IG(51-56)] and 50-57 [IG(50-57)], respectively, were studied by circular dichroism and NMR spectroscopy at various temperatures and by differential scanning calorimetry. Our results show that the part of the sequence corresponding to the beta-turn in the native structure (DDATKT) of the B3 domain forms bent conformations similar to those observed in the native protein. The formation of a turn is observed for both peptides in a broad range of temperatures (T = 283-323 K), which confirms the conclusion drawn from our previous studies of longer sequences from the C-terminal beta-hairpin of the B3 domain of the immunoglobulin binding protein G (16, 14, and 12 residues), that the DDATKT sequence forms a nucleation site for formation of the beta-hairpin structure of peptides corresponding to the C-terminal part of all the B domains of the immunoglobulin binding protein G. We also show and discuss the role of long-range hydrophobic interactions as well as local conformational properties of polypeptide chains in the mechanism of formation of the beta-hairpin structure.