The SH3 domains of the protein kinases ITK and LCK compete for adjacent sites on T cell-specific adapter protein.

T-cell activation requires stimulation of specific intracellular signaling pathways in which protein-tyrosine kinases, phosphatases, and adapter proteins interact to transmit signals from the T-cell receptor to the nucleus. Interactions of LCK proto-oncogene, SRC family tyrosine kinase (LCK), and the IL-2-inducible T cell kinase (ITK) with the T cell-specific adapter protein (TSAD) promotes LCK-mediated phosphorylation and thereby ITK activation. Both ITK and LCK interact with TSAD's proline-rich region (PRR) through their Src homology 3 (SH3) domains. Whereas LCK may also interact with TSAD through its SH2 domain, ITK interacts with TSAD only through its SH3 domain. To begin to understand on a molecular level how the LCK SH3 and ITK SH3 domains interact with TSAD in human HEK293T cells, here we combined biochemical analyses with NMR spectroscopy. We found that the ITK and LCK SH3 domains potentially have adjacent and overlapping binding sites within the TSAD PRR amino acids (aa) 239-274. Pulldown experiments and NMR spectroscopy revealed that both domains may bind to TSAD aa 239-256 and aa 257-274. Co-immunoprecipitation experiments further revealed that both domains may also bind simultaneously to TSAD aa 242-268. Accordingly, NMR spectroscopy indicated that the SH3 domains may compete for these two adjacent binding sites. We propose that once the associations of ITK and LCK with TSAD promote the ITK and LCK interaction, the interactions among TSAD, ITK, and LCK are dynamically altered by ITK phosphorylation status.

Interactions of a class IIb bacteriocin with a model lipid bilayer, investigated through molecular dynamics simulations.

The emergence of antibiotic resistant microorganisms poses an alarming threat to global health. Antimicrobial peptides (AMPs) are considered a possible effective alternative to conventional antibiotic therapies. An understanding of the mechanism of action of AMPs is needed in order to better control and optimize their bactericidal activity. Plantaricin EF is a heterodimeric AMP, consisting of two peptides Plantaricin E (PlnE) and Plantaricin F (PlnF). We studied the behavior of these peptides on the surface of a model lipid bilayer. We identified the residues that facilitate peptide-peptide interactions. We also identified residues that mediate interactions of the dimer with the membrane. PlnE interacts with the membrane through amino acids at both its termini, while only the N terminus of PlnF approaches the membrane. By comparing the activity of single-site mutants of the two-peptide bacteriocin and the simulations of the bacteriocin on the surface of a model lipid bilayer, structure activity relationships are proposed. These studies allow us to generate hypotheses that relate biophysical interactions observed in simulations with the experimentally measured activity. We find that single-site amino acid substitutions result in markedly stronger antimicrobial activity when they strengthen the interactions between the two peptides, while, concomitantly, they weaken peptide-membrane association. This effect is more pronounced in the case of the PlnE mutant (G20A), which interacts the strongest with PlnF and the weakest with the membrane while displaying the highest activity.

Selective membrane disruption by the cyclotide kalata B7: complex ions and essential functional groups in the phosphatidylethanolamine binding pocket.

The cyclic cystine knot plant peptides called cyclotides are active against a wide variety of organisms. This is primarily achieved through membrane binding and disruption, in part deriving from a high affinity for phosphatidylethanolamine (PE) lipids. Some cyclotides, such as kalata B7 (kB7), form complexes with divalent cations in a pocket associated with the tyrosine residue at position 15 (Tyr15). In the current work we explore the effect of cations on membrane leakage caused by cyclotides kB1, kB2 and kB7, and we identify a functional group that is essential for PE selectivity. The presence of PE-lipids in liposomes increased the membrane permeabilizing potency of the cyclotides, with the potency of kB7 increasing by as much as 740-fold. The divalent cations Mn(2+), Mg(2+) and Ca(2+) had no apparent effect on PE selectivity. However, amino acid substitutions in kB7 proved that Tyr15 is crucial for PE-selective membrane permeabilization on various liposome systems. Although the tertiary structure of kB7 was maintained, as reflected by the NMR solution structure, mutating Tyr into Ser at position 15 resulted in substantially reduced PE selectivity. Ala substitution at the same position produced a similar reduction in PE selectivity, while substitution with Phe maintained high selectivity. We conclude that the phenyl ring in Tyr15 is critical for the high PE selectivity of kB7. Our results suggest that PE-binding and divalent cation coordination occur in the same pocket without adverse effects of competitive binding for the phospholipid.

Structure-Function Analysis of the Two-Peptide Bacteriocin Plantaricin EF.

Plantaricin EF is a two-peptide bacteriocin that depends on the complementary action of two different peptides (PlnE and PlnF) to function. The structures of the individual peptides have previously been analyzed by nuclear magnetic resonance spectroscopy ( Fimland, N. et al. ( 2008 ) , Biochim. Biophys. Acta 1784 , 1711 - 1719 ), but the bacteriocin structure and how the two peptides interact have not been determined. All two-peptide bacteriocins identified so far contain GxxxG motifs. These motifs, together with GxxxG-like motifs, are known to mediate helix-helix interactions in membrane proteins. We have mutated all GxxxG and GxxxG-like motifs in PlnE and PlnF in order to determine if any of these motifs are important for antimicrobial activity and thus possibly for interactions between PlnE and PlnF. Moreover, the aromatic amino acids Tyr and Trp in PlnE and PlnF were substituted, and four fusion polypeptides were constructed in order to investigate the relative orientation of PlnE and PlnF in target cell membranes. The results obtained with the fusion polypeptides indicate that PlnE and PlnF interact in an antiparallel manner and that the C-terminus of PlnE and N-terminus of PlnF are on the outer part of target cell membranes and the N-terminus of PlnE and C-terminus of PlnF are on the inner part. The preference for an aromatic residue at position 6 in PlnE suggests a positioning of this residue in or near the membrane interface on the cells inside. Mutations in the GxxxG motifs indicate that the G5xxxG9 motif in PlnE and the S26xxxG30 motif in PlnF are involved in helix-helix interactions. Atomistic molecular dynamics simulation of a structural model consistent with the results confirmed the stability of the structure and its orientation in membranes. The simulation approved the anticipated interactions and revealed additional interactions that further increase the stability of the proposed structure.

Nuclear Magnetic Resonance Structure and Mutational Analysis of the Lactococcin A Immunity Protein.

The class IId bacteriocin lactococcin A and the pediocin-like bacteriocins induce membrane leakage and cell death by specifically binding the mannose phophotransferase system (man-PTS) on their target cells. The bacteriocins' cognate immunity proteins that protect the producer cell from its own bacteriocin recognize and bind to the bacteriocin-man-PTS complex and thereby block membrane leakage. In this study, we have determined the three-dimensional structure of the lactococcin A immunity protein (LciA) by the use of nuclear magnetic resonance spectroscopy. LciA forms a four-helix bundle structure with a flexible C-terminal tail. Despite the low degree of sequence similarity between LciA and the pediocin-like immunity proteins, they share the same fold. However, there are certain differences between the structures. The C-terminal helix in LciA is considerably shorter than that observed in the pediocin-like immunity proteins, and the surface potentials of the immunity proteins differ. Truncated variants of LciA in which 6 or 10 of the C-terminal residues were removed yielded a reduced degree of protection, indicating that the unstructured C-terminal tail is important for the functionality of the immunity proteins.

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.

Solubility of recombinant Src homology 2 domains expressed in E. coli can be predicted by TANGO.

BACKGROUND: Signalling proteins often contain several well defined and conserved protein domains. Structural analyses of such domains by nuclear magnetic spectroscopy or X-ray crystallography may greatly inform the function of proteins. A limiting step is often the production of sufficient amounts of the recombinant protein. However, there is no particular way to predict whether a protein will be soluble when expressed in E.coli. Here we report our experience with expression of a Src homology 2 (SH2) domain. RESULTS: The SH2 domain of the SH2D2A protein (or T cell specific adapter protein, TSAd) forms insoluble aggregates when expressed as various GST-fusion proteins in Escherichia coli (E. coli). Alteration of the flanking sequences, or growth temperature influenced expression and solubility of TSAd-SH2, however overall yield of soluble protein remained low. The algorithm TANGO, which predicts amyloid fibril formation in eukaryotic cells, identified a hydrophobic sequence within the TSAd-SH2 domain with high propensity for beta-aggregation. Mutation to the corresponding amino acids of the related HSH2- (or ALX) SH2 domain increased the yield of soluble TSAd-SH2 domains. High beta-aggregation values predicted by TANGO correlated with low solubility of recombinant SH2 domains as reported in the literature. CONCLUSIONS: Solubility of recombinant proteins expressed in E.coli can be predicted by TANGO, an algorithm developed to determine the aggregation propensity of peptides. Targeted mutations representing corresponding amino acids in similar protein domains may increase solubility of recombinant proteins.

A poly-proline II helix in YadA from Yersinia enterocolitica serotype O:9 facilitates heparin binding through electrostatic interactions.

Poly-proline II helices are secondary structure motifs frequently found in ligand-binding sites. They exhibit increased flexibility and solvent exposure compared to the strongly hydrogen-bonded α-helices or ß-strands and can therefore easily be misinterpreted as completely unstructured regions with an extremely high rotational freedom. Here, we show that the adhesin YadA of Yersinia enterocolitica serotype O:9 contains a poly-proline II helix interaction motif in the N-terminal region. The motif is involved in the interaction of YadAO:9 with heparin, a host glycosaminoglycan. We show that the basic residues within the N-terminal motif of YadA are required for electrostatic interactions with the sulfate groups of heparin. Biophysical methods including CD spectroscopy, solution-state NMR and SAXS all independently support the presence of a poly-proline helix allowing YadAO:9 binding to the rigid heparin. Lastly, we show that host cells deficient in sulfation of heparin and heparan sulfate are not targeted by YadAO:9 -mediated adhesion. We speculate that the YadAO:9 -heparin interaction plays an important and highly strain-specific role in the pathogenicity of Yersinia enterocolitica serotype O:9.

Structure analysis of the two-peptide bacteriocin lactococcin G by introducing D-amino acid residues.

The importance of 3D structuring in the N- and C-terminal ends of the two peptides (39-mer LcnG-alpha and 35-mer LcnG-beta) that constitute the two-peptide bacteriocin lactococcin G was analysed by replacing residues in the end regions with the corresponding D-isomeric residues. When assayed for antibacterial activity in combination with the complementary wild-type peptide, LcnG-alpha with four D-residues in its C-terminal region and LcnG-beta with four d-residues in either its N- or its C-terminal region were relatively active (two- to 20-fold reduction in activity). 3D structuring of the C-terminal region in LcnG-alpha and the C- and N-terminal regions in LcnG-beta is thus not particularly critical for retaining antibacterial activity, indicating that the 3D structure of these regions is not vital for interpeptide interactions or for interactions between the peptides and cellular components. The 3D structure of the N-terminal region in LcnG-alpha may be more important, as LcnG-alpha with four N-terminal D-residues was the least active of these four peptides (10- to 100-fold reduction in activity). The results are consistent with a proposed structural model of lactococcin G in which LcnG-alpha and -beta form a transmembrane parallel helix-helix structure involving approximately 20 residues in each peptide, starting near the N terminus of LcnG-alpha and at about residue 13 in LcnG-beta. Upon expressing the lactococcin G immunity protein, sensitive target cells became resistant to all of these D-residue-containing peptides. The end regions of the two lactococcin G peptides are consequently not involved in essential structure-dependent interactions with the immunity protein. The relatively high activity of most of the D-residue-containing peptides suggests that bacteriocins with increased resistance to exopeptidases may be generated by replacing their N- and C-terminal residues with d-residues.

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.

Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin plantaricin EF.

The three-dimensional structures of the two peptides plantaricin E (plnE; 33 residues) and plantaricin F (plnF; 34 residues) constituting the two-peptide bacteriocin plantaricin EF (plnEF) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles. PlnE has an N-terminal alpha-helix (residues 10-21), and a C-terminal alpha-helix-like structure (residues 25-31). PlnF has a long central alpha-helix (residues 7-32) with a kink of 38+/-7 degrees at Pro20. There is some flexibility in the helix in the kink region. Both helices in plnE are amphiphilic, while the helix in plnF is polar in its N-terminal half and amphiphilic in its C-terminal half. The alpha-helical content obtained by NMR spectroscopy is in agreement with CD studies. PlnE has two GxxxG motifs which are putative helix-helix interaction motifs, one at residues 5 to 9 and one at residues 20 to 24, while plnF has one such motif at residues 30 to 34. The peptides are flexible in these GxxxG regions. It is suggested that the two peptides lie parallel in a staggered fashion relative to each other and interact through helix-helix interactions involving the GxxxG motifs.

NMR structures and mutational analysis of the two peptides constituting the bacteriocin plantaricin S.

The structure of the individual peptides of the two-peptide bacteriocin plantaricin S, an antimicrobial peptide produced by a Lactobacillus plantarum strain, has been determined in DPC micelles. The two peptides of plantaricin S, Pls-α and Pls-ß, form an α-helix from and including residue 8 to 24 with a less structured region around residue 16-19 and an amphiphilic α-helix from and including residue 7 to 23, respectively. Activity assays on single amino acid-substituted GxxxG and GxxxG-like motifs show that substituting the Ser and Gly residues in the G9xxxG13 motif in Pls-α and the S17xxxG21 motif in Pls-ß reduced or drastically reduced the antimicrobial activity. The two-peptide bacteriocin muricidin contains GxxxG-like motifs at similar positions and displays 40-50% amino acid identity with plantaricin S. Activity assays of combinations of the peptides that constitute the bacteriocins plantaricin S and muricidin show that some combinations are highly active. Furthermore, sequence alignments show that the motifs important for plantaricin S activity align with identical motifs in muricidin. Based on sequence comparison and activity assays, a membrane-inserted model of plantaricin S in which the two peptides are oriented antiparallel relative to each other and where the GxxxG and GxxxG-like motifs important for activity come close in space, is proposed.

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.

Mutational analysis of the class IIa bacteriocin curvacin A and its orientation in target cell membranes.

To analyze the orientation in target cell membranes of the pediocin-like bacteriocin (antimicrobial peptide) curvacin A, 55 variants were generated by site-directed mutagenesis and their potencies against four different target cells determined. The result suggest that the somewhat hydrophilic short central helix (residues 19 to 24), along with the N-terminal beta-sheet-like structure (residues 1 to 16), inserts in the interface region of the target cell membrane, with Ala22 close to the hydrophobic core of the membrane. The following hinge region, with Gly28 as an important residue, may then form a turn wherein Gly28 becomes positioned near the border between the interface and the hydrophobic regions, thus permitting the longer and more-hydrophobic C-terminal helix (residues 29 to 41) to insert into the hydrophobic core of the membrane. This helix contains three glycine residues (G33, G37, and G40) that form a putative helix-helix-interacting GxxxGxxG motif. The replacement of any of these glycines with a larger residue was very detrimental, suggesting their possible involvement in helix-helix interactions with a membrane-embedded receptor protein.

Mutational analysis and membrane-interactions of the beta-sheet-like N-terminal domain of the pediocin-like antimicrobial peptide sakacin P.

To gain insight into how the N-terminal three-stranded beta-sheet-like domain in pediocin-like antimicrobial peptides positions itself on membranes, residues in the well-conserved (Y)YGNGV-motif in the domain were substituted and the effect of the substitutions on antimicrobial activity and binding of peptides to liposomes was determined. Peptide-liposome interactions were detected by measuring tryptophan-fluorescence upon exposing liposomes to peptides in which a tryptophan residue had been introduced in the N-terminal domain. The results revealed that the N-terminal domain associates readily with anionic liposomes, but not with neutral liposomes. The electrostatic interactions between peptides and liposomes facilitated the penetration of some of the peptide residues into the liposomes. Measuring the antimicrobial activity of the mutated peptides revealed that the Tyr2Leu and Tyr3Leu mutations resulted in about a 10-fold reduction in activity, whereas the Tyr2Trp, Tyr2Phe, Tyr3Trp and Tyr3Phe mutations were tolerated fairly well, especially the mutations in position 3. The Val7Ile mutation did not have a marked detrimental effect on the activity. The Gly6Ala mutation was highly detrimental, consistent with Gly6 being in one of the turns in the beta-sheet-like N-terminal domain, whereas the Gly4Ala mutation was tolerated fairly well. All mutations involving Asn5, including the conservative mutations Asn5Gln and Asn5Asp, were very deleterious. Thus, both the polar amide group on the side chain of Asn5 and its exact position in space were crucial for the peptides to be fully active. Taken together, the results are consistent with Val7 positioning itself in the hydrophobic core of target membranes, thus forcing most of the other residues in the N-terminal domain into the membrane interface region: Tyr3 and Asn5 in the lower half with their side chains pointing downward and approaching the hydrophobic core, Tyr2, Gly4 and His8 and 12 in the upper half, Lys1 near the middle of the interface region, and the side chain of Lys11 pointing out toward the membrane surface.

The Leaderless Bacteriocin Enterocin K1 Is Highly Potent against Enterococcus faecium: A Study on Structure, Target Spectrum and Receptor.

Enterocin K1 (EntK1), enterocin EJ97 (EntEJ97), and LsbB are three sequence related leaderless bacteriocins. Yet LsbB kills only lactococci while EntK1 and EntEJ97 target wider spectra with EntK1 being particularly active against Enterococcus faecium, including nosocomial multidrug resistant isolates. NMR study of EntK1 showed that it had a structure very similar to LsbB - both having an amphiphilic N-terminal α-helix and an unstructured C-terminus. The α-helix in EntK1 is, however, about 3-4 residues longer than that of LsbB. Enterococcal mutants highly resistant to EntEJ97 and EntK1 were found to have mutations within rseP, a gene encoding a stress response membrane-bound Zn-dependent protease. Heterologous expression of the enterococcal rseP rendered resistant cells of Streptococcus pneumoniae sensitive to EntK1 and EntEJ97, suggesting that RseP likely serves as the receptor for EntK1 and EntEJ97. It was also shown that the conserved proteolytic active site in E. faecalis RseP is partly required for EntK1 and EntEJ97 activity, since alanine substitutions of its conserved residues (HExxH) reduced the sensitivity of the clones to the bacteriocins. RseP is known to be involved in bacterial stress response. As expected, the growth of resistant mutants with mutations within rseP was severely affected when they were exposed to higher (stressing) growth temperatures, e.g., at 45°C, at which wild type cells still grew well. These findings allow us to design a hurdle strategy with a combination of the bacteriocin(s) and higher temperature that effectively kills bacteriocin sensitive bacteria and prevents the development of resistant cells.

Double-quantum dipolar recoupling at high magic-angle spinning rates.

A full investigation of the possible homonuclear double-quantum recoupling sequences, based on the RN family of sequences with N < or = 20, is given. Several new RN sequences, R16(6)(5), R18(8)(5), and R18(10)(5), were applied at high magic-angle spinning rates and compared with theory. The R18(10)(5) technique can be used to recouple dipolar couplings at spinning rates up to 39 kHz, and the application of the sequence in an INADEQUATE experiment is shown for a spinning rate of 30 kHz.

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.

Three-dimensional structure of the two-peptide bacteriocin plantaricin JK.

The three-dimensional structures of the two peptides, PlnJ and PlnK, that constitutes the two-peptide bacteriocin plantaricin JK have been solved in water/TFE and water/DPC-micellar solutions using nuclear magnetic resonance (NMR) spectroscopy. PlnJ, a 25 residue peptide, has an N-terminal amphiphilic alpha-helix between Trp-3 and Tyr-15. The 32 residues long PlnK forms a central amphiphilic alpha-helix between Gly-9 and Leu-24. Measurements of the effect on anti-microbial activity of single glycine replacements in PlnJ and PlnK show that Gly-13 and Gly-17 in both peptides are very sensitive, giving more than a 100-fold reduction in activity when large residues replace glycine. In variants where other glycine residues, Gly-20 in PlnJ and Gly-7, Gly-9, Gly-24 and Gly-25 in PlnK, were replaced, the activity was reduced less than 10-fold. It is proposed that the detrimental effect on activity when exchanging Gly-13 and Gly-17 in PlnJ and PlnK is a result of reduced ability of the two peptides to interact through the GxxxG-motifs constituting Gly-13 and Gly-17.

Identification of a high affinity FcgammaRIIA-binding peptide that distinguishes FcgammaRIIA from FcgammaRIIB and exploits FcgammaRIIA-mediated phagocytosis and degradation.

FcgammaRIIA is a key activating receptor linking immune complex formation with cellular effector functions. FcgammaRIIA has 93% identity with an inhibitory receptor, FcgammaRIIB, which negatively regulates FcgammaRIIA. FcgammaRIIA is important in the therapeutic action of several monoclonal antibodies. Binding molecules that discriminate FcgammaRIIA from FcgammaRIIB may optimize receptor activity and serve as a lead for development of therapeutics with FcgammaRIIA as a key target. Here we report the use of phage display libraries to select short peptides with distinct FcgammaRIIA binding properties. An 11-mer peptide (WAWVWLTETAV) was characterized that bound FcgammaRIIA with a K(d) of 500 nm. It mediated cell internalization and degradation of a model antigen. The peptide-binding site on FcgammaRIIA was shown to involve Phe(163) and the IgG binding amino acids Trp(90) and Trp(113). It is thus overlapping but not identical to that of IgG. Neither activating receptors FcgammaRI and FcgammaRIII, nor FcgammaRIIB, all of which lack Phe(163), bound the peptide.