Structure of microcin J25, a peptide inhibitor of bacterial RNA polymerase, is a lassoed tail

Microcin J25 (MccJ25) is a 21-amino acid peptide inhibitor active against the DNA-dependent RNA polymerase of Gram negative bacteria. Previously, the structure of MccJ25 was reported to be a head-to-tail circle, cyclo(-G(1)GAGHVPEYF(10)VGIGTPISFY(20)G-). On the basis of biochemical studies, mass spectrometry, and NMR, we show that this structure is incorrect, and that the peptide has an extraordinary structural fold. MccJ25 contains an internal lactam linkage between the alpha-amino group of Gly1 and the gamma-carboxyl of Glu8. The tail (Tyr9-Gly21) passes through the ring (Gly1-Glu8), with Phe19 and Tyr20 straddling each side of the ring, sterically trapping the tail in a noncovalent interaction we call a lassoed tail.     

Single Peptide Backbone Surrogate Mutations to Regulate Angiotensin GPCR Subtype Selectivity

Mutating the side-chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half-life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y]⁶-Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4-disubstituted 1,2,3-triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT₂R/AT₁R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross-peaks. The most potent analogue, compound 2 , has been explored regarding its neurotrophic potential and resulted in an enhanced neurite growth with respect to the established agent C21.     

Peptide AS-48: prototype of a new class of cyclic bacteriocins

After the discovery of bacteriocin AS-48, a 70-residue cyclic peptide produced by Enterococcus faecalis subsp. liquefaciens, some naturally-occurring cyclic proteins from bacteria have been reported. AS-48 is encoded by the 68-kb pheromone-responsive plasmid pMB2, and the gene cluster involved in production and immunity has been identified and sequenced. This peptide exerts a bactericidal action on sensitive cells (most of the Gram-positive and some Gram-negative bacteria). Its target is the cytoplasmic membrane, in which it opens pores, leading to the dissipation of the proton motive force and cell death, a mechanism similar to that proposed for the action of defensins or, most generally, cationic antibacterial peptides. This fact, together with its remarkable stability and solubility over a wide pH range, suggest that this bacteriocin could be a good candidate as a natural food preservative. The amino acid composition of purified AS-48 shows the absence of modified or dehydrated residues, making it clearly different from lantibiotics. Bacteriocin AS-48 also differs from defensins in that it does not contain cysteines and consequently no disulfide bridges, which makes is high stability even more remarkable. Composition analysis of AS-48 shows a high proportion of basic to acidic amino acids, conferring to this peptide a strong basic character, with an isoelectric point close to 10.5. Determination of the AS-48 structural gene DNA sequence, together with the sequences of AS-48 protease digestion fragments and mass spectrometry determinations, allowed us to determine unambiguously the cyclic structure of the molecule, being the first example of a posttranslational modification in which a cyclic structure arises from a "head-to-tail" linkage. We have solved the three-dimensional structure of AS-48 in solution, and it consists of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. Interestingly, the head-to-tail peptide link between Trp-70 and Met-1 lies in the middle of alpha-helix 5, which is shown to have a pronounced effect on the stability of the three-dimensional structure. Analysis of structure-function relationship allowed us to propose models to understand the aspects of the molecular function of AS-48. The purpose of this work is to review recent developments in our understanding about the biochemical and biological characteristics and structure of this unusual type of bacteriocin.     

Structure of gramicidin a in a lipid bilayer environment determined using molecular dynamics simulations and solid-state NMR data

Two different high-resolution structures recently have been proposed for the membrane-spanning gramicidin A channel: one based on solid-state NMR experiments in oriented phospholipid bilayers (Ketchem, R. R.; Roux, B.; Cross, T. A. Structure 1997, 5, 1655-1669; Protein Data Bank, PDB:1MAG); and one based on two-dimensional NMR in detergent micelles (Townsley, L. E.; Tucker, W. A.; Sham, S.; Hinton, J. F. Biochemistry 2001, 40, 11676-11686; PDB:1JNO). Despite overall agreement, the two structures differ in peptide backbone pitch and the orientation of several side chains; in particular that of the Trp at position 9. Given the importance of the peptide backbone and Trp side chains for ion permeation, we undertook an investigation of the two structures using molecular dynamics simulation with an explicit lipid bilayer membrane, similar to the system used for the solid-state NMR experiments. Based on 0.1 micros of simulation, both backbone structures converge to a structure with 6.25 residues per turn, in agreement with X-ray scattering, and broad agreement with SS backbone NMR observables. The side chain of Trp 9 is mobile, more so than Trp 11, 13, and 15, and undergoes spontaneous transitions between the orientations in 1JNO and 1MAG. Based on empirical fitting to the NMR results, and umbrella sampling calculations, we conclude that Trp 9 spends 80% of the time in the 1JNO orientation and 20% in the 1MAG orientation. These results underscore the utility of molecular dynamics simulations in the analysis and interpretation of structural information from solid-state NMR.     

Transporter Protein-Guided Genome Mining for Head-to-Tail Cyclized Bacteriocins

Head-to-tail cyclized bacteriocins are ribosomally synthesized antimicrobial peptides that are defined by peptide backbone cyclization involving the N- and C- terminal amino acids. Their cyclic nature and overall three-dimensional fold confer superior stability against extreme pH and temperature conditions, and protease degradation. Most of the characterized head-to-tail cyclized bacteriocins were discovered through a traditional approach that involved the screening of bacterial isolates for antimicrobial activity and subsequent isolation and characterization of the active molecule. In this study, we performed genome mining using transporter protein sequences associated with experimentally validated head-to-tail cyclized bacteriocins as driver sequences to search for novel bacteriocins. Biosynthetic gene cluster analysis was then performed to select the high probability functional gene clusters. A total of 387 producer strains that encode putative head-to-tail cyclized bacteriocins were identified. Sequence and phylogenetic analyses revealed that this class of bacteriocins is more diverse than previously thought. Furthermore, our genome mining strategy captured hits that were not identified in precursor-based bioprospecting, showcasing the utility of this approach to expanding the repertoire of head-to-tail cyclized bacteriocins. This work sets the stage for future isolation of novel head-to-tail cyclized bacteriocins to serve as possible alternatives to traditional antibiotics and potentially help address the increasing threat posed by resistant pathogens.     

Alamethicin channels in a membrane: molecular dynamics simulations

Alamethicin (Alm) is a 20 residue peptide which forms a kinked alpha-helix in membrane and membrane-mimetic environments. Ion channels formed by intramembraneous aggregates of Alm are thought to be formed by bundles of approximately parallel Alm helices surrounding a central bilayer pore. Different channel conductance levels correspond to different numbers of helices per bundle, ranging from N = 5 to N > 8. Calculation of the predicted pKA values of the ring of Glu18 sidechains at the C-terminal mouth of the pore suggests that at neutral pH most or all of these sidechains will remain protonated. Nanosecond molecular dynamics (MD) simulations of N = 5, 6, 7 and 8 bundles of Alm helices in a POPC bilayer have been run, corresponding to a total simulation time of 4 ns. These simulations explore the stability and conformational dynamics of these helix bundle channels when embedded in a full phospholipid bilayer in an aqueous environment. The structural and dynamic properties of water in these model channels are examined. As in earlier in vacuo simulations (J. Breed, R. Sankararamakrishnan, I. D. Kerr and M. S. P. Sansom, Biophys. J., 1996, 70, 1643) the dipole moments of water molecules within the pores are aligned antiparallel to the helix dipoles. This helps to contribute to the stability of the helix bundles.     

Chemical synthesis of a cyclotide via intramolecular cyclization of peptide O-esters

Cyclotides constitute a fascinating family of circular proteins containing ca.30 amino acid residues.They have a unique cyclic cysteine knot topology and exhibit remarkable thermal,chemical and enzymatic stabilities.These characteristics enable them to have a range of biological activities and promising pharmaceutical and agricultural applications.Here,we present a practical strategy for the chemical synthesis of cyclotides through the intramolecular ligation of fully unprotected peptide O-esters.This strategy involves the mild Fmoc solid-phase peptide synthesis of the peptide O-ester backbone,the head-to-tail cyclization of the cyclotide backbone by native chemical ligation,and the oxidative refolding to yield the natural knot protein.The simplicity and high efficiency of the strategy can be employed in the synthesis of artificial cyclotides for pharmaceutical applications.     

Relative strength of cation-pi vs salt-bridge interactions: the Gtalpha(340-350) peptide/rhodopsin system

Interactions between cationic and aromatic side chains of amino acid residues, the so-called cation-pi interaction, are thought to contribute to the overall stability of the folded structure of peptides and proteins. The transferred NOE NMR structure of the G(t)alpha(340-350) peptide bound to photoactivated rhodopsin (R*) geometrically suggests a cation-pi interaction stabilizing the structure between the epsilon-amine of Lys341 and the aromatic ring of the C-terminal residue, Phe350. This interaction has been explored by varying substituents on the phenyl ring to alter the electron density of the aromatic ring of Phe350 and observing the impact on binding of the peptide to R*. The results suggest that while a cation-pi interaction geometrically exists in the G(t)alpha(340-350) peptide when bound to R*, its energetic contribution to the stability of the receptor-bound structure is relatively insignificant, as it was not observed experimentally. The presence of an adjacent and competing salt-bridge interaction between the epsilon-amine of Lys341 and the C-terminal carboxylate of Phe350 effectively shields the charge of the ammonium group. Experimental data supporting a significant cation-pi interaction can be regained through a series of Phe350 analogues where the C-terminal carboxyl has been converted to the neutral carboxamide, thus eliminating the shielding salt-bridge. TrNOE NMR experiments confirmed the existence of the cation-pi interaction in the carboxamide analogues. Various literature estimates of the strength of cation-pi interactions, including some that estimate strengths in excess of salt-bridges, are compromised by omission of the relevant anion in the calculations.     

Recent progress in computational approaches to studying the M2 proton channel and its implication to drug design against influenza viruses

For quite a long period of time in history, many intense efforts have been made to determine the 3D (three-dimensional) structure of the M2 proton channel. The reason why the M2 proton channel has attracted so many attentions is because (1) it is the key for really understanding the life cycle of influenza viruses, and (2) it is indispensable for conducting rational drug design against the flu viruses. Recently, the long-sough 3D structures of the M2 proton channels for both influenza A and B viruses were consecutively successfully determined by the high-resolution NMR spectroscopy (Schnell J.R. and Chou, J.J., Nature, 2008, 451: 591-595; Wang, J., Pielak, R.M., McClintock, M.A., and Chou, J.J., Nature Structural & Molecular Biology, 2009,16: 1267-1271). Such a milestone work has provided a solid structural basis for in-depth understanding the action mechanism of the M2 channel and rationally designing effective drugs against influenza viruses. This review is devoted to, with the focus on the M2 proton channel of influenza A, addressing a series of relevant problems, such as how to correctly understand the novel allosteric inhibition mechanism inferred from the NMR structure that is completely different from the traditional view, what the possible impacts are to the previous functional studies in this area, and what kind of new strategy can be stimulated for drug development against influenza.     

Structure and chemistry of apicidins, a class of novel cyclic tetrapeptides without a terminal alpha-keto epoxide as inhibitors of histone deacetylase with potent antiprotozoal activities

Apicidins are a class of cyclic tetrapeptides that do not contain the classical electrophilic alpha-keto epoxide yet are potent (nM) inhibitors of histone deacetylase and antiprotozoal agents. These compounds showed broad-spectrum activities against the apicomplexan family of protozoa including Plasmodium sp (malarial parasite), Toxoplasma gondii, Cryptosporidium sp., and Eimeria sp. These cyclic peptides contain a beta-turn amino acid (R)-Pip or (R)-Pro, (S)-N-methoxy Trp, (S)-Ile, or (S)-Val, and either (S)-2-amino-8-oxodecanoic acid or a modified (S)-2-amino-8-oxodecanoic acid. The isolation and structure elucidation of new apicidins from two Fusarium species, temperature-dependent NMR studies of apicidin, NMR and molecular modeling based conformation of the 12-membered macrocyclic ring, and selected chemical modifications of apicidin have been detailed in this paper. The cyclic nature of the peptide, the C-8 keto group, and the tryptophan are all critical for the biological activity.     

A reverse turn structure induced by a D,L-alpha-aminoxy acid dimer

Our previous work revealed that two adjacent D-alpha-aminoxy acids could form two homochiral N-O turns, with the backbone folding into an extended helical structure (1.8(8)-helix). Here, we report the conformational studies of linear peptides 3-6, which contain a D,L-alpha-aminoxy acid dimer segment. The NMR and X-ray analysis of 3 showed that it folded into a loop conformation with two heterochiral N-O turns. This loop segment can be used to constrain tetrapeptides 4 and 6 to form a reverse turn structure. (1)H NMR dilution studies, DMSO-d6 addition studies, and 2D-NOESY data indicated that tetrapeptides 4 and 6 folded into reverse turn conformations featured by a head-to-tail 16-membered-ring intramolecular hydrogen bond. In contrast, tetrapeptide 5 with L-Ala instead of Gly or D-Ala as the N-terminal amino acid could not form the desired reverse turn structure for steric reasons. Quantum mechanics calculations showed that model pentamide 7, with the same substitution pattern of 4, adopted a novel reverse turn conformation featuring two heterochiral N-O turns (each of an 8-membered ring hydrogen bond), a cross-strand 16-membered ring hydrogen bond, and a 7-membered ring gamma-turn.     

Side-chain conformation of the M2 transmembrane peptide proton channel of influenza a virus from 19F solid-state NMR

The M2 transmembrane peptide (M2TMP) of the influenza A virus forms a tetrameric helical bundle that acts as a proton-selective channel important in the viral life cycle. The side-chain conformation of the peptide is largely unknown and is important for elucidating the proton-conducting mechanism and the channel stability. Using a 19F spin diffusion NMR technique called CODEX, we have measured the oligomeric states and interhelical side chain-side chain 19F-19F distances at several residues using singly fluorinated M2TMP bound to DMPC bilayers. 19F CODEX data at a key residue of the proton channel, Trp41, confirm the tetrameric state of the peptide and yield a nearest-neighbor interhelical distance of approximately 11 A under both neutral and acidic pH. Since the helix orientation is precisely known from previous 15N NMR experiments and the backbone channel diameter has a narrow allowed range, this 19F distance constrains the Trp41 side-chain conformation to t90 (chi1 approximately 180 degrees , chi2 approximately 90 degrees ). This Trp41 rotamer, combined with a previously measured 15N-13C distance between His37 and Trp411, suggests that the His37 rotamer is t-160. The implication of the proposed (His37, Trp41) rotamers to the gating mechanism of the M2 proton channel is discussed. Binding of the antiviral drug amantadine to the peptide does not affect the F-F distance at Trp41. Interhelical 19F-19F distances are also measured at residues 27 and 38, each mutated to 4-19F-Phe. For V27F-M2TMP, the 19F-19F distances suggest a mixture of dimers and tetramers, whereas the L38F-M2TMP data indicate two tetramers of different sizes, suggesting side chain conformational heterogeneity at this lipid-facing residue. This work shows that 19F spin diffusion NMR is a valuable tool for determining long-range intermolecular distances that shed light on the mechanism of action and conformational heterogeneity of membrane protein oligomers.     

Structure of a cyclic peptide with a catalytic triad, determined by computer simulation and NMR spectroscopy

Summary We report the design of a cyclic, eight-residue peptide that possesses the catalytic triad residues of the serine proteases. A manually built model has been relaxed by 0.3 ns of molecular dynamics simulation at room temperature, during which no major changes occurred in the peptide. The molecule has been synthesised and purified. Two-dimensional NMR spectroscopy provided 35 distance and 7 torsion angle constraints, which were used to determine the three-dimensional structure. The experimental conformation agrees with the predicted one at the -turn, but deviates in the arrangement of the disulphide bridge that closes the backbone to a ring. A 1.2 ns simulation at 600 K provided extended sampling of conformation space. The disulphide bridge reoriented into the experimental arrangement, producing a minimum backbone rmsd from the experimental conformation of 0.8 . At a later stage in the simulation, a transition at Ser³ produced more pronounced high-temperature behaviour. The peptide hydrolyses p-nitrophenyl acetate about nine times faster than free histidine.     

Dinuclear azolato-bridged complexes of the nickel group metals with haloaryl ligands: a reinvestigation of their behavior in solution

A reinvestigation of the NMR spectra of the complexes (NBu4)2[M2(mu-LL)2R4] (M = Pd, Ni, Pt, LL = pyrazolate (pz), 3,5-dimethylpyrazolate (dmpz), 3-methylpyrazolate (mpz), indazolate (indz), R = C6F5; M = Pd, LL = pz, dmpz, mpz, indz, R = 2,4,6-C6F3H2) shows that the boat-shaped dimeric structures of their anions are quite stable in solution, and the previously proposed fast equilibria or dissociations to give species such as [R2M(N-N)(acetone)]-, [R2M(acetone)2] + 2dmpz-, or [R2M(N1-N2)(acetone)]- + [R2M(N2-N1)(acetone)]- in no case occur. A mixture of the two diastereoisomers (head-to-head, HH, and head-to-tail, HT) is present for the asymmetrically substituted azolates (mpz and indz), in a ratio ranging from 1:7 to 1:30 for the different complexes. Strong through-space coupling between the endo ortho fluorine nuclei of different MR2 fragments is observed in the 19F NMR spectra of these diastereoisomers whose boatlike structures place these atoms at short distances.     

Patterns in Protein Flexibility: A Comparison of NMR “Ensembles”, MD Trajectories,and Crystallographic B-Factors

Proteins are molecular machines requiring flexibility to function. Crystallographic B-factors and Molecular Dynamics (MD) simulations both provide insights into protein flexibility on an atomic scale. Nuclear Magnetic Resonance (NMR) lacks a universally accepted analog of the B-factor. However, a lack of convergence in atomic coordinates in an NMR-based structure calculation also suggests atomic mobility. This paper describes a pattern in the coordinate uncertainties of backbone heavy atoms in NMR-derived structural “ensembles” first noted in the development of FindCore2 (previously called Expanded FindCore: DA Snyder, J Grullon, YJ Huang, R Tejero, GT Montelione, Proteins: Structure, Function, and Bioinformatics 82 (S2), 219–230) and demonstrates that this pattern exists in coordinate variances across MD trajectories but not in crystallographic B-factors. This either suggests that MD trajectories and NMR “ensembles” capture motional behavior of peptide bond units not captured by B-factors or indicates a deficiency common to force fields used in both NMR and MD calculations.     

Dead-end street of protein folding: thermodynamic rationale of amyloid fibril formation

An increasing number of diseases, including Alzheimer's, have been found to be a result of the formation of amyloid aggregates that are practically independent of the original primary sequence of the protein(s). (Eakin, C. M.; Berman, A. J.; Miranker, A. D. Nat. Struct. Mol. Biol. 2006, 13, 202-208.) Consequently, the driving force of the transformation from original to disordered amyloid fold is expected to lie in the protein backbone, which is common to all proteins. (Nelson, R.; Sawaya, M. R.; Balbirnie, M.; Madsen, A. O.; Riekel, C.; Grothe, R.; Eisenberg, D. Nature 2005, 435, 773-778. Wright, C. F.; Teichmann, S. A.; Clarke, J.; Dobson, C. M. Nature 2005, 438, 878-881.) However, the exact explanation for the existence of such a "dead-end" structure is still unknown. Using systematic first principle calculations on carefully selected but large enough systems modeling the protein backbone we show that the beta-pleated sheet structure, the building block of amyloid fibers, is the thermodynamically most stable supramolecular arrangement of all the possible peptide dimers and oligomers both in vacuum and in aqueous environments. Even in a crystalline state (periodical, tight peptide attechment), the beta-pleated sheet assembly remains the most stable superstructure. The present theoretical study provides a quantum-level explanation for why proteins can take the amyloid state when local structural preferences jeopardize the functional native global fold and why it is a beta-pleated sheetlike structure they prefer.     

Structure, assembly, and topology of the G185R mutant of the fourth transmembrane domain of divalent metal transporter

The mammalian iron transporter, divalent metal transporter (DMT1), is a 12-transmembrane domain integral protein, responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues. Two disease-causing mutants in animals have been found and attributed to the same missense mutation (G185R), which occurs within the putative transmembrane domain 4 (TM4) of DMT1. We have characterized a synthetic 24-mer peptide, corresponding to the sequence of the TM4 of DMT1 with G185R mutation using circular dichroism (CD) and NMR spectroscopy and show that the G185R peptide assumes mainly alpha-helical conformations in various membrane-mimetic environments. Solution structures derived from NMR and molecular dynamics/simulated annealing calculations demonstrate that the peptide exhibits a highly defined alpha-helix in its middle portion, flanked by a highly flexible N-terminus and a relatively ordered C-terminus. Both the folding and location of the C-terminus in SDS micelles are regulated by pH values. Paramagnetic broadening on peptide NMR signals by spin-labeled 5- and 16-doxylstearic acids and Mn(2+) ion suggests that both the N-terminus and the helical region of the peptide are embedded in SDS micelles. Surprisingly, self-association of the peptides for both the wild type and the G185R mutant studied by CD, electrospray ionization mass spectrometry, and NMR diffusion-ordered spectroscopy demonstrated that mutation of the Gly185 to a bulky and positively charged arginine causes a different self-assembly of the peptide, e.g., from a trimer to a hexamer, which implies that the quaternary structure of integral DMT1 may be crucial for its function in vivo.     

Design principles for Brownian molecular machines: how to swim in molasses and walk in a hurricane

Protein molecular motors-perfected over the course of millions of years of evolution-play an essential role in moving and assembling biological structures. Recently chemists have been able to synthesize molecules that emulate in part the remarkable capabilities of these biomolecular motors (for extensive reviews see the recent papers: E. R. Kay, D. A. Leigh and F. Zerbetto, Angew. Chem., Int. Ed., 2006, 46, 72-191; W. R. Browne and B. L. Feringa, Nat. Nanotechnol., 2006, 1, 25-35; M. N. Chatterjee, E. R. Kay and D. A. Leigh, J. Am. Chem. Soc., 2006, 128, 4058-4073; G. S. Kottas, L. I. Clarke, D. Horinek and J. Michl, Chem. Rev., 2005, 105, 1281-1376; M. A. Garcia-Garibay, Proc. Natl. Acad. Sci., U. S. A., 2005, 102, 10771-10776)). Like their biological counterparts, many of these synthetic machines function in an environment where viscous forces dominate inertia-to move they must "swim in molasses". Further, the thermal noise power exchanged reversibly between the motor and its environment is many orders of magnitude greater than the power provided by the chemical fuel to drive directed motion. One might think that moving in a specific direction would be as difficult as walking in a hurricane. Yet biomolecular motors (and increasingly, synthetic motors) move and accomplish their function with almost deterministic precision. In this Perspective we will investigate the physical principles that govern nanoscale systems at the single molecule level and how these principles can be useful in designing synthetic molecular machines.     

Tetrazole peptides as copper(II) ion chelators

The 1,5-disubstituted tetrazole ring a mimetic of the cis-amide bond is an unique element modifying the ability of peptides to chelate copper(II) ions. The position of the tetrazole ring system in the peptide backbone plays a critical role in the stabilization of the metallopeptide molecule. The insertion of a tetrazole between amide groups leads to enhancing the stability of the complex and to obtaining a very effective peptide chelating agent. These findings can provide important information for modeling biologically relevant peptide–metal binding sites. Some aspects of biological activity of tetrazole modified exogenous opioid peptides in the presence of copper(II) ions are also presented in this review.