| 1. |
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| 2. |
- Plan, Manuel Rey R, et al.
(författare)
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The cyclotide fingerprint in Oldenlandia affinis : elucidation of chemically modified, linear and novel macrocyclic peptides
- 2007
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 8:9, s. 1001-1011
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Tidskriftsartikel (refereegranskat)abstract
- The complete suite of cyclotides present in Oldenlandia affinis (Rubiaceae), the plant that was originally found to contain this unique family of circular proteins, has been characterised. This study expands the number of known cyclotides in this plant to 17, of which nine new sequences (kalata B9-B17) were characterised in this work. In addition, five derivatives that contain oxidation products of the conserved tryptophan were identified, and it was shown that the formation of these derivatives is catalysed by exposure to sunlight. Furthermore, we describe two linear cyclotide analogues. These acyclic peptides have three intact disulfide bonds, and their N and C termini coincide with the hypothesised cleavage sites from the precursor protein. This work increases our knowledge about the sequence variation that is accommodated by the cyclic cystine knot scaffold, confirms its applicability as a template for drug design, and also shows the first natural degradation pathways for cyclotides. These pathways have important implications for the persistence and environmental fate of the cyclotides if used as crop-protection agents.
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| 3. |
- Andersson, Håkan S., et al.
(författare)
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The alpha-defensin salt-bridge induces backbone stability to facilitate folding and confer proteolytic resistance
- 2012
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Ingår i: Amino Acids. - : Springer Science and Business Media LLC. - 0939-4451 .- 1438-2199. ; 43:4, s. 1471-1483
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Tidskriftsartikel (refereegranskat)abstract
- Salt-bridge interactions between acidic and basic amino acids contribute to the structural stability of proteins and to protein-protein interactions. A conserved salt-bridge is a canonical feature of the alpha-defensin antimicrobial peptide family, but the role of this common structural element has not been fully elucidated. We have investigated mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) and peptide variants with mutations at Arg(7) or Glu(15) residue positions to disrupt the salt-bridge and assess the consequences on Crp4 structure, function, and stability. NMR analyses showed that both (R7G)-Crp4 and (E15G)-Crp4 adopt native-like structures, evidence of fold plasticity that allows peptides to reshuffle side chains and stabilize the structure in the absence of the salt-bridge. In contrast, introduction of a large hydrophobic side chain at position 15, as in (E15L)-Crp4 cannot be accommodated in the context of the Crp4 primary structure. Regardless of which side of the salt-bridge was mutated, salt-bridge variants retained bactericidal peptide activity with differential microbicidal effects against certain bacterial cell targets, confirming that the salt-bridge does not determine bactericidal activity per se. The increased structural flexibility induced by salt-bridge disruption enhanced peptide sensitivity to proteolysis. Although sensitivity to proteolysis by MMP7 was unaffected by most Arg(7) and Glu(15) substitutions, every salt-bridge variant was degraded extensively by trypsin. Moreover, the salt-bridge facilitates adoption of the characteristic alpha-defensin fold as shown by the impaired in vitro refolding of (E15D)-proCrp4, the most conservative salt-bridge disrupting replacement. In Crp4, therefore, the canonical alpha-defensin salt-bridge facilitates adoption of the characteristic alpha-defensin fold, which decreases structural flexibility and confers resistance to degradation by proteinases.
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| 4. |
- Haugaard-Kedström, Linda M., et al.
(författare)
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Solution Structure, Aggregation Behavior, and Flexibility of Human Relaxin-2.
- 2015
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Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 10:3, s. 891-900
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Tidskriftsartikel (refereegranskat)abstract
- Relaxin is a member of the relaxin/insulin peptide hormone superfamily and is characterized by a two-chain structure constrained by three disulfide bonds. Relaxin is a pleiotropic hormone and involved in a number of physiological and pathogenic processes, including collagen and cardiovascular regulation and tissue remodelling during pregnancy and cancer. Crystallographic and ultracentrifugation experiments have revealed that the human form of relaxin, H2 relaxin, self-associates into dimers, but the significance of this is poorly understood. Here, we present the NMR structure of a monomeric, amidated form of H2 relaxin and compare its features and behavior in solution to those of native H2 relaxin. The overall structure of H2 relaxin is retained in the monomeric form. H2 relaxin amide is fully active at the relaxin receptor RXFP1 and thus dimerization is not required for biological activity. Analysis of NMR chemical shifts and relaxation parameters identified internal motion in H2 relaxin at the pico-nanosecond and milli-microsecond time scales, which is commonly seen in other relaxin and insulin peptides and might be related to function.
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| 5. |
- Haugaard-Kedström (published under the name Haugaard-Jönsson), Linda M., et al.
(författare)
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Structural Properties of Relaxin Chimeras: NMR Characterization of the R3/I5 Relaxin Peptide
- 2009
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Ingår i: Annals of the New York Academy of Sciences. - : Wiley. - 0077-8923 .- 1749-6632. ; 1160, s. 27-30
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Tidskriftsartikel (refereegranskat)abstract
- Relaxin-3 interacts with high potency with three relaxin family peptide receptors (RXFP1, RXFP3, and RXFP4). Therefore, the development of selective agonist and antagonist analogs is important for in vivo studies characterizing the biological significance of the different receptor-ligand systems and for future pharmaceutical applications. Recent reports demonstrated that a peptide selective for RXFP3 and RXFP4 over RXFP1 can be generated by the combination of the relaxin-3 B chain with the A chain from insulin-like peptide 5 (INSL5), creating an R3/I5 chimera. We have used NMR spectroscopy to determine the three-dimensional structure of this peptide to gain structural insights into the consequences of combining chains from two different relaxins. The R3/I5 structure reveals a similar backbone conformation for the relaxin-3 B chain compared to native relaxin-3, and the INSL5 A chain displays a relaxin/insulin-like fold with two parallel helices. The findings indicate that binding and activation of RXFP3 and RXFP4 mainly require the B chain and that the A chain functions as structural support. RXFP1, however, demonstrates a more complex binding mechanism, involving both the A chain and the B chain. The creation of chimeras is a promising strategy for generating new structure-activity data on relaxins.
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| 6. |
- Haugaard-Kedström (published under the name Haugaard-Jönsson), Linda M., et al.
(författare)
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Structure of the human insulin-like peptide 5 and characterization of conserved hydrogen bonds and electrostatic interactions within the relaxin framework
- 2009
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 419, s. 619-627
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Tidskriftsartikel (refereegranskat)abstract
- INSL5 (insulin-like peptide 5) is a two-chain peptide hormone related to insulin and relaxin. It was recently discovered through searches of expressed sequence tag databases and, although the fulfil biological significance of INSL5 is still being elucidated, high expression in peripheral tissues such as the colon, as well as in the brain and hypothalamus, suggests roles in gut contractility and neuroendocrine signalling. INSL5 activates the relaxin family peptide receptor 4 with high potency and appears to be the endogenous ligand for this receptor, on the basis of overlapping expression profiles and their apparent co-evolution. In the present Study, we have used solution-state NMR to characterize the three-dimensional structure of synthetic human INSL5. The structure reveals an insulin/relaxin-like fold with three helical segments that are braced by three disulfide bonds and enclose a hydrophobic core. Furthermore, we characterized in detail the hydrogen-bond network and electrostatic interactions between charged groups in INSL5 by NMR-monitored temperature and pH titrations and Undertook a comprehensive structural comparison with other members of the relaxin family, thus identifying the conserved structural features of the relaxin fold. The B-chain helix, which is the primary receptor-binding site of the relaxins, is longer in INSL5 than in its close relative relaxin-3. As this feature results in a different positioning of the receptor-activation domain Arg(B23) and Trp(B24), it may be an important contributor to the difference in biological activity observed for these two peptides. Overall, the structural Studies provide mechanistic insights into the receptor selectivity of this important family of hormones.
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| 7. |
- Haugaard-Kedström (published under the name Haugaard-Jönsson), Linda M., et al.
(författare)
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Structure of the R3/I5 chimeric relaxin peptide, a selective GPCR135 and GPCR142 agonist
- 2008
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 283:35, s. 23811-23818
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Tidskriftsartikel (refereegranskat)abstract
- The human relaxin family comprises seven peptide hormones with various biological functions mediated through interactions with G-protein-coupled receptors. Interestingly, among the hitherto characterized receptors there is no absolute selectivity toward their primary ligand. The most striking example of this is the relaxin family ancestor, relaxin-3, which is an agonist for three of the four currently known relaxin receptors: GPCR135, GPCR142, and LGR7. Relaxin-3 and its endogenous receptor GPCR135 are both expressed predominantly in the brain and have been linked to regulation of stress and feeding. However, to fully understand the role of relaxin-3 in neurological signaling, the development of selective GPCR135 agonists and antagonists for in vivo studies is crucial. Recent reports have demonstrated that such selective ligands can be achieved by making chimeric peptides comprising the relaxin-3 B-chain combined with the INSL5 A-chain. To obtain structural insights into the consequences of combining A-and B-chains from different relaxins we have determined the NMR solution structure of a human relaxin-3/INSL5 chimeric peptide. The structure reveals that the INSL5 A-chain adopts a conformation similar to the relaxin-3 A-chain, and thus has the ability to structurally support a native-like conformation of the relaxin-3 B-chain. These findings suggest that the decrease in activity at the LGR7 receptor seen for this peptide is a result of the removal of a secondary LGR7 binding site present in the relaxin-3 A-chain, rather than conformational changes in the primary B-chain receptor binding site.
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| 8. |
- Hossain, M. Akhter, et al.
(författare)
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The A-chain of the human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors
- 2008
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 283:25, s. 17287-17297
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Tidskriftsartikel (refereegranskat)abstract
- The relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the relaxin family peptide (RXFP) receptors 1-4, respectively. Although key binding residues have been identified in the B-chain of the relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human relaxin-2 and -3 (H2 and H3) relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 relaxin. In contrast, A-chain-truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. Our results provide new insights into the action of relaxins and demonstrate that the role of the A-chain for relaxin activity is both peptide- and receptor-dependent.
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| 9. |
- Koehbach, Johannes, et al.
(författare)
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Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design
- 2013
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:52, s. 21183-21188
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Tidskriftsartikel (refereegranskat)abstract
- Cyclotides are plant peptides comprising a circular backbone and three conserved disulfide bonds that confer them with exceptional stability. They were originally discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Recently, cyclotides have been identified in numerous plant species of the coffee, violet, cucurbit, pea, potato, and grass families. Their unique structural topology, high stability, and tolerance to sequence variation make them promising templates for the development of peptide-based pharmaceuticals. However, the mechanisms underlying their biological activities remain largely unknown; specifically, a receptor for a native cyclotide has not been reported hitherto. Using bioactivity-guided fractionation of an herbal peptide extract known to indigenous healers as "kalata-kalata," the cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V-1a receptors, members of the G protein-coupled receptor family, as molecular targets for this cyclotide. Furthermore, we show that cyclotides can serve as templates for the design of selective G protein-coupled receptor ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. These observations provide a proof of concept for the development of cyclotide-based peptide ligands.
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| 10. |
- Nilsson, K Peter R, et al.
(författare)
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Solution structure of chi-conopeptide MrIA, a modulator of the human norepinephrine transporter.
- 2005
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Ingår i: Biopolymers. - : Wiley. - 0006-3525 .- 1097-0282. ; 80:6, s. 815-823
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Tidskriftsartikel (refereegranskat)abstract
- The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a beta-hairpin with the two strands connected by an inverse gamma-turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I. A.; Palant, E.; Schroder, C. I.; Kaye, D. M.; Adams, D. J.; Alewood, P. F.; Lewis, R. J. J Biol Chem 2003, 278, 40317-40323), which appears to be more flexible than the beta-strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N-terminal truncations indicate that the N-terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, noncovalent interactions between Val3 and Hyp12 are likely to be involved in maintaining a stable conformation. The N-terminus also affects activity, as a single N-terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced chi-conopeptide potency.
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| 11. |
- Plan, Manuel R, et al.
(författare)
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Structural and biochemical characteristics of the cyclotide kalata B5 from Oldenlandia affinis.
- 2010
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Ingår i: Biopolymers. - : Wiley. - 0006-3525 .- 1097-0282. ; 94:5, Sp. Iss. SI, s. 647-658
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Tidskriftsartikel (refereegranskat)abstract
- Cyclotides are a large family of plant-derived proteins typified by their head-to-tail cyclic backbone and knotted arrangement of three disulfide bonds. Although they display a diverse range of biological activities, their native function is thought to be plant defense. Here we characterized the expression, three-dimensional structure, and hemolytic activity of the cyclotide kalata B5 from the African plant Oldenlandia affinis. Kalata B5 shows an interesting seasonal variation in its expression and can only be isolated during certain times of the year, when the plant is flowering. It displays a typical tightly folded cyclic cystine knot structure. A range of pH and temperature titrations reveal that a conserved glutamic acid in loop 1 of the structure forms a key hydrogen bond network, similar to that reported previously for other cyclotides. However, specific line broadening in the NMR spectra of kalata B5 suggests that the hydrogen bonding network in this peptide is less rigid than in other cyclotides. Notably, the pK(a) of Glu6 of 4.5 is higher than the values for other cyclotides studied so far, which range from 3.0 to 4.0, providing a further indication of a weaker hydrogen bond network. Kalata B5 has only moderate hemolytic activity compared with other highly expressed cyclotides, and this reduced activity probably reflects its more flexible structure. As is the case with other cyclotides, kalata B5 has an exposed hydrophobic region on its surface, supporting suggestions that this hydrophobic patch is a key feature for membrane binding and biological activity of cyclotides. (c) 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2010.
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| 12. |
- Rosengren, K. Johan, et al.
(författare)
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Structural insights into the function of relaxins
- 2009
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Ingår i: Annals of the New York Academy of Sciences. - : Wiley. - 0077-8923 .- 1749-6632. ; 1160, s. 20-26
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Tidskriftsartikel (refereegranskat)abstract
- The relaxin peptide hormones are members of the insulin superfamily and share a structural fold that is characterized by two peptide chains which are cross-braced by three disulfide bonds. On this framework, various amino acid side chains are presented, allowing specific interactions with different receptors. The relaxin receptors belong to two unrelated classes of G-protein-coupled receptors, but interestingly they are not selective for a single relaxin peptide. Relaxin-3, which is considered to be an extreme example of the relaxin family, can activate receptors from both classes and in fact interacts to some degree with all four receptors identified to date. To deduce how changes in the primary sequence can fine-tune the overall structure and thus the ability to interact with the various receptors, we have studied a range of relaxin-like peptides using solution nuclear magnetic resonance analysis. Three-dimensional structures of relaxin-3, insulin-like peptide 3 (INSL3), and INSL5 were determined and revealed a number of interesting features. All peptides showed a significant amount of line-broadening in certain regions, in particular around the intra-A-chain disulfide bond, suggesting that despite the disulfide bonds the fold is rather dynamic. Although the peptides share a common structural core there are significant differences, particularly around the termini. The structural data in combination with mutational studies provide valuable insights into the structure-activity relationships of relaxins.
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| 13. |
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