Per Eugen Kristiansen

The Two-Peptide Class II Bacteriocins: Structure, Production, and Mode of Action

The two-peptide class II bacteriocins consist of two different unmodified peptides, both of which must be present in about equal amounts in order for these bacteriocins to exert optimal antimicrobial activity. These bacteriocins render the membrane of target cells permeable to various small molecules. The genes encoding the two peptides of two-peptide bacteriocins are adjacent to each other in the same operon and they are near the genes encoding (i) the immunity protein that protects the bacteriocin-producing bacteria from being killed by their own bacteriocin, (ii) a dedicated ABC transporter that transports the bacteriocin out of the bacteriocin-producing bacteria, and (iii) an accessory protein whose specific role is not known, but which also appears to be required for secretion of the bacteriocin. The production of some two-peptide bacteriocins is transcriptionally regulated through a three-component regulatory system that consists of a membrane-interacting peptide pheromone, a membrane-associated histidine protein kinase, and response regulators. Structure analysis of three two-peptide bacteriocins (plantaricin E/F, plantaricin J/K, and lactococcin G) by CD (and in part by NMR) spectroscopy reveal that these bacteriocins contain long amphiphilic α-helical stretches and that the two complementary peptides interact and structure each other when exposed to membrane-like entities. Lactococcin G shares about 55% sequence identity with enterocin 1071, but these two bacteriocins nevertheless kill different types of bacteria. The target-cell specificity of lactococcin G-enterocin 1071 hybrid bacteriocins that have been constructed by site-directed mutagenesis suggests that the β-peptide is important for determining the target-cell specificity.

Structure and Mode-of-Action of the Two-Peptide (Class-IIb) Bacteriocins

This review focuses on the structure and mode-of-action of the two-peptide (class-IIb) bacteriocins that consist of two different peptides whose genes are next to each other in the same operon. Optimal antibacterial activity requires the presence of both peptides in about equal amounts. The two peptides are synthesized as preforms that contain a 15–30 residue double-glycine-type N-terminal leader sequence that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter that concomitantly transfers the bacteriocin peptides across cell membranes. Two-peptide bacteriocins render the membrane of sensitive bacteria permeable to a selected group of ions, indicating that the bacteriocins form or induce the formation of pores that display specificity with respect to the transport of molecules. Based on structure–function studies, it has been proposed that the two peptides of two-peptide bacteriocins form a membrane-penetrating helix–helix structure involving helix–helix-interacting GxxxG-motifs that are present in all characterized two-peptide bacteriocins. It has also been suggested that the membrane-penetrating helix–helix structure interacts with an integrated membrane protein, thereby triggering a conformational alteration in the protein, which in turn causes membrane-leakage. This proposed mode-of-action is similar to the mode-of-action of the pediocin-like (class-IIa) bacteriocins and lactococcin A (a class-IId bacteriocin), which bind to a membrane-embedded part of the mannose phosphotransferase permease in a manner that causes membrane-leakage and cell death.

The CW domain, a new histone recognition module in chromatin proteins

Post‐translational modifications of the N‐terminal histone tails, including lysine methylation, have key roles in regulation of chromatin and gene expression. A number of protein modules have been identified that recognize differentially modified histone tails and provide their proteins with the capacity to sense such modifications. Here, we identify the CW domain of plant and animal chromatin‐related proteins as a novel module that recognizes different methylated states of lysine 4 on histone H3 (H3K4me). The solution structure of the CW domain of the Arabidopsis ASH1 HOMOLOG2 (ASHH2) histone methyltransferase provides insight into how different CW domains can distinguish different methylated histone tails. We provide evidence that ASHH2 is acting on H3K4me‐marked genes, allowing for ASHH2‐dependent H3K36 tri‐methylation, which contributes to sustained expression of tissue‐specific and developmentally regulated genes. This suggests that ASHH2 is a combined ‘reader’ and ‘writer’ of the histone code. We propose that different CW domains, dependent on their specificity for different H3K4 methylations, are important for epigenetic memory or participate in switching between permissive and repressive chromatin states.

Many‐body calculations of hyperfine constants in diatomic molecules. II. First‐row hydrides

Magnetic hyperfine parameters (Frosch and Foley parameters) have been computed for first‐row diatomic hydrides by use of many‐body perturbation theory. The computations are complete to third order in the many‐body expansion, which means that core polarization corrections are included through second and thrid order, and correlation effects by their leading third‐order corrections. Computed results are presented for the ground states, and in addition for the three excited states A 2Δ, A Π, and A 2Σ in CH, NH, and OH, respectively. The vibrational dependencies of the hyperfine parameters were also predicted, and even a hyperfine centrifugal distortion constant observed for CH was computed, in good agreement with experiment. Good agreements between computed and experimental parameters were generally obtained, in particular for the ground states, where the errors in the computed values are at most a few percent.

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin lactococcin G

The three-dimensional structures of the two peptides, lactococcin G-alpha (LcnG-alpha; contains 39 residues) and lactococcin G-beta (LcnG-beta, contains 35 residues), that constitute the two-peptide bacteriocin lactococcin G (LcnG) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles and TFE. In DPC, LcnG-alpha has an N-terminal alpha-helix (residues 3-21) that contains a GxxxG helix-helix interaction motif (residues 7-11) and a less well defined C-terminal alpha-helix (residues 24-34), and in between (residues 18-22) there is a second somewhat flexible GxxxG-motif. Its structure in TFE was similar. In DPC, LcnG-beta has an N-terminal alpha-helix (residues 6-19). The region from residues 20 to 35, which also contains a flexible GxxxG-motif (residues 18-22), appeared to be fairly unstructured in DPC. In the presence of TFE, however, the region between and including residues 23 and 32 formed a well defined alpha-helix. The N-terminal helix between and including residues 6 and 19 seen in the presence of DPC, was broken at residues 8 and 9 in the presence of TFE. The N-terminal helices, both in LcnG-alpha and -beta, are amphiphilic. We postulate that LcnG-alpha and -beta have a parallel orientation and interact through helix-helix interactions involving the first GxxxG (residues 7-11) motif in LcnG-alpha and the one (residues 18-22) in LcnG-beta, and that they thus lie in a staggered fashion relative to each other.

Structure and Mode of Action of the Membrane-permeabilizing Antimicrobial Peptide Pheromone Plantaricin A

The three-dimensional structure in dodecyl phosphocholine micelles of the 26-mer membrane-permeabilizing bacteriocin-like pheromone plantaricin A (PlnA) has been determined by use of nuclear magnetic resonance spectroscopy. The peptide was unstructured in water but became partly structured upon exposure to micelles. An amphiphilic α-helix stretching from residue 12 to 21 (possibly also including residues 22 and 23) was then formed in the C-terminal part of the peptide, whereas the N-terminal part remained largely unstructured. PlnA exerted its membrane-permeabilizing antimicrobial activity through a nonchiral interaction with the target cell membrane because the d-enantiomeric form had the same activity as the natural l-form. This nonchiral interaction involved the amphiphilic α-helical region in the C-terminal half of PlnA because a 17-mer fragment that contains the amphiphilic α-helical part of the peptide had antimicrobial potency that was similar to that of the l- and d-enantiomeric forms of PlnA. Also the pheromone activity of PlnA depended on this nonchiral interaction because both the l- and d-enantiomeric forms of the 17-mer fragment inhibited the pheromone activity. The pheromone activity also involved, however, a chiral interaction between the N-terminal part of PlnA and its receptor because high concentrations of the l-form (but not the d-form) of a 5-mer fragment derived from the N-terminal part of PlnA had pheromone activity. The results thus reveal a novel mechanism whereby peptide pheromones such as PlnA may function. An initial nonchiral interaction with membrane lipids induces α-helical structuring in a segment of the peptide pheromone. The peptide becomes thereby sufficiently structured and properly positioned in the membrane interface, thus enabling it to engage in a chiral interaction with its receptor in or near the membrane water interface. This membrane-interacting mode of action explains why some peptide pheromones/hormones such as PlnA sometimes display antimicrobial activity in addition to their pheromone activity.

Theory and applications of supercycled symmetry-based recoupling sequences in solid-state nuclear magnetic resonance

We present the theoretical principles of supercycled symmetry-based recoupling sequences in solid-state magic-angle-spinning NMR. We discuss the construction procedure of the SR26 pulse sequence, which is a particularly robust sequence for double-quantum homonuclear dipole-dipole recoupling. The supercycle removes destructive higher-order average Hamiltonian terms and renders the sequence robust over long time intervals. We demonstrate applications of the SR26 sequence to double-quantum spectroscopy, homonuclear spin counting, and determination of the relative orientations of chemical shift anisotropy tensors.

A Hydrophobic Patch in the Competence-Stimulating Peptide, a Pneumococcal Competence Pheromone, Is Essential for Specificity and Biological Activity

Induction of competence for natural genetic transformation in Streptococcus pneumoniae depends on pheromone-mediated cell-cell communication and a signaling pathway consisting of the competence-stimulating peptide (CSP), its membrane-embedded histidine kinase receptor ComD, and the cognate response regulator ComE. Extensive screening of pneumococcal isolates has revealed that two major CSP variants, CSP1 and CSP2, are found in members of this species. Even though the primary structures of CSP1 and CSP2 are about 50% identical, they are highly specific for their respective receptors, ComD1 and ComD2. In the present work, we have investigated the structural basis of this specificity by determining the three-dimensional structure of CSP1 from nuclear magnetic resonance data and comparing the agonist activity of a number of CSP1/CSP2 hybrid peptides toward the ComD1 and ComD2 receptors. Our results show that upon exposure to membrane-mimicking environments, the 17-amino-acid CSP1 pheromone adopts an amphiphilic alpha-helical configuration stretching from residue 6 to residue 12. Furthermore, the pattern of agonist activity displayed by the various hybrid peptides revealed that hydrophobic amino acids, some of which are situated on the nonpolar side of the alpha-helix, strongly contribute to CSP specificity. Together, these data indicate that the identified alpha-helix is an important structural feature of CSP1 which is essential for effective receptor recognition under natural conditions.

Many‐body calculations of hyperfine constants in diatomic molecules. I. The ground state of 16OH

Magnetic hyperfine parameters (Frosch and Foley parameters) have been computed for the 2Π ground state of 16OH by use of many‐body perturbation theory. The computations are complete to third order in the many‐body expansion, and they were repeated for a series of internuclear distances around re to reproduce the vibrational dependencies of the parameters. Parameters were computed for the three lowest vibrational levels, and were found to be within 1%–2% of the very accurate experimental values. The rather strong vibrational dependencies of the parameters were reproduced with accuracies of 80%–90%. Finally, centrifugal distortion corrections to the magnetic hyperfine parameters were also computed, and for the one parameter (dD) observed, the error was about 5%. The vibrational wave functions needed were obtained from a published accurate CI potential curve. Thus, no empirical results are incorporated in the present ab initio calculations.

Mutational analysis of putative helix-helix interacting GxxxG-motifs and tryptophan residues in the two-peptide bacteriocin lactococcin G.

The membrane-permeabilizing two-peptide bacteriocin lactococcin G consists of two different peptides, LcnG-alpha and LcnG-beta. The bacteriocin contains several tryptophan and tyrosine residues and three putative helix-helix interacting GxxxG-motifs, G 7xxxG 11 and G 18xxxG 22 in LcnG-alpha and G 18xxxG 22 in LcnG-beta. The tryptophan and tyrosine residues and residues in the GxxxG-motifs were altered by site-directed mutagenesis to analyze the structure and membrane-orientation of lactococcin G. Substituting the glycine residues at position 7 or 11 in the G 7xxxG 11-motif in LcnG-alpha with large hydrophobic or hydrophilic residues was highly detrimental, whereas small residues were tolerated. Qualitatively similar results were obtained for the G 18xxxG 22-motif in LcnG-beta. In contrast, replacement of the glycine residues in the middle of these two motifs with large hydrophilic residues was tolerated. All mutations in the G 18xxxG 22-motif in LcnG-alpha were relatively well-tolerated, indicating that this motif is not involved in helix-helix interactions. The four aromatic residues in the N-terminal part of LcnG-beta could individually be replaced by other aromatic residues, a hydrophilic positive residue, and a hydrophobic residue without a marked reduced activity, indicating that this region is structurally flexible and not embedded in a strictly hydrophobic or hydrophilic environment. The results are in accordance with a structural model where the G 7xxxG 11-motif in LcnG-alpha and the G 18xxxG 22-motif in LcnG-beta interact and allow the two peptides to form a parallel transmembrane helix-helix structure, with the tryptophan-rich N-terminal part of LcnG-beta positioned in the outer membrane interface and the cationic C-terminal end of LcnG-alpha inside the cell.