| 1. |
- de Veer, Simon J., et al.
(författare)
-
Engineered protease inhibitors based on sunflower trypsin inhibitor-1 (SFTI-1) provide insights into the role of sequence and conformation in Laskowski mechanism inhibition
- 2015
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 469:2, s. 243-253
-
Tidskriftsartikel (refereegranskat)abstract
- Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesizing a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor's resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor's potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor's sequence and structure relate to its binding kinetics and function. Using enzyme assays, MD simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show that the geometry of the binding loop mainly influences the inhibitor's potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor's sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and the dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin and two kallikrein-related peptidases (KLK5 and KLK14) that are implicated in various cancers and skin diseases.
|
|
| 2. |
- Sörensson, Carolin, et al.
(författare)
-
Determination of primary sequence specificity of Arabidopsis MAPKs MPK3 and MPK6 leads to identification of new substrates
- 2012
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 446, s. 271-278
-
Tidskriftsartikel (refereegranskat)abstract
- MAPKs (mitogen-activated protein kinases) are signalling components highly conserved among eukaryotes. Their diverse biological functions include cellular differentiation and responses to different extracellular stress stimuli. Although some substrates of MAPKs have been identified in plants, no information is available about whether amino acids in the primary sequence other than proline-directed phosphorylation (pS-P) contribute to kinase specificity towards substrates. in the present study, we used a random positional peptide library to search for consensus phosphorylation sequences for Arabidopsis MAPKs MPK3 and MPK6. These experiments indicated a preference towards the sequence L/P-P/X-S-P-R/K for both kinases. After bioinformatic processing, a number of novel candidate MAPK substrates were predicted and subsequently confirmed by in vitro kinase assays using bacterially expressed native Arabidopsis proteins as substrates. MPK3 and MPK6 phosphorylated all proteins tested more efficiently than did another MAPK, MPK4. These results indicate that the amino acid residues in the primary sequence surrounding the phosphorylation site of Arabidopsis MAPK substrates can contribute to MAPK specificity. Further characterization of one of these new substrates confirmed that AtIg80180.1 was phosphorylated in planta in a MAPK-dependent manner. Phenotypic analyses of Arabidopsis expressing phosphorylation site mutant forms of AtIg80180.1 showed clustered stomata and higher stomatal index in cotyledons expressing the phosphomimetic form of AtIg80180.1, providing a link between this new MAPK substrate and the defined role for MPK3 and MPK6 in stomatal patterning.
|
|
| 3. |
- Zavialov, Anton
(författare)
-
Caf1A usher possesses a Caf1 subunit-like domain that is crucial for Caf1 fibre secretion
- 2009
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 418, s. 541-551
-
Tidskriftsartikel (refereegranskat)abstract
- The chaperone/usher pathway controls assembly of fibres of adhesive organelles of Gram-negative bacteria. The final steps of fibre assembly and fibre translocation to the cell surface are co-ordinated by the outer membrane proteins, ushers. Ushers consist of several soluble periplasmic domains and a single transmembrane beta-barrel. Here we report isolation and structural/functional characterization of a novel middle domain of the Caf1A usher from Yersinia pestis. The isolated UMD (usher middle domain) is a highly soluble monomeric protein capable of autonomous folding. A 2.8 angstrom (1 angstrom = 0.1 nm) resolution crystal structure of UMD revealed that this domain has an immunoglobulin-like fold similar to that of donor-strand-complemented Caf1 fibre subunit. Moreover, these proteins displayed significant structural similarity. Although UMD is in the middle of the predicted amphipathic beta-barrel of Caf1A, the usher still assembled in the membrane in the absence of this domain. UMD did not bind Caf1M-Caf1 complexes, but its presence was shown to be essential for Caf1 fibre secretion. The study suggests that UMD may play the role of a subunit-substituting protein (dummy subunit), plugging or priming secretion through the channel in the Caf1A usher. Comparison of isolated UMD with the recent strcture of the corresponding domain of PapC usher revealed high similarity of the core structures, suggesting a universal structural adaptation of FGL (F(1)G(1) long) and FGS (F(1)G(1) short) chaperone/usher pathways for the secretion of different types of fibres. The functional role of two topologically different states of this plug domain suggested by structural and biochemical results is discussed.
|
|
