Structural and Mutational Analyses of the Interaction between the Barley α-Amylase/Subtilisin Inhibitor and the Subtilisin Savinase Reveal a Novel Mode of Inhibition

Micheelsen, Pernille O.; Vévodová, J.; Maria, Leonardo De; Østergaard, Per; Friis, Esben P.; Wilson, K.S.; Skjøt, Michael

doi:10.1016/j.jmb.2008.05.034

Cited by 48 publications

(41 citation statements)

References 44 publications

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“…From the analysis of the topology of the fold, a single molecule could potentially engage other enzymes units through 11 different loops. Indeed, the structures of PPI in complex with two trypsin units, BASI/WASI in complex with ␣-amylase and subtilisin (9,23), API-A in complex with trypsin (7), and clitocypin in complex with serine/cysteine proteases (10), show that a single inhibitor molecule is capable of interacting with two enzymes. Moreover, the position of the reactive loops varies within the families, with occurrences in loops ␤2-␤3, ␤4-␤5, ␤5-␤6, ␤6-␤7, and ␤10-␤11, and the relative specificities are also interchangeable with position ␤2-␤3 being used for the inhibition of serine and cysteine proteases.…”

Section: Discussionmentioning

confidence: 99%

“…It is commonly assumed that most members of this family have only a single reactive site loop, which for the archetypical soybean trypsin inhibitor (STI) is located between residues Ser-60 and Phe-66. However, several cases of inhibitors possessing two reactive sites, and thus binding two target molecules simultaneously, have been reported (7)(8)(9)(10). These have been dubbed "double-headed" or "Janus-type" inhibitors.…”

mentioning

confidence: 99%

“…However, despite the abundance of data obtained from x-ray crystallography for free inhibitors and complexes with serine proteases (7)(8)(9)(12)(13)(14), very little is known on the specifics of multiple target recognition by these Janus-type proteins with only one structure in the Protein Data Bank of a Kunitz-STI inhibitor bound to two protease molecules (7).…”

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confidence: 99%

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The Plasticity of the β-Trefoil Fold Constitutes an Evolutionary Platform for Protease Inhibition

Azarkan

Martínez‐Rodríguez

Buts

et al. 2011

Journal of Biological Chemistry

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Background:The Kunitz-STI family is a paradigm of protease-inhibitor interaction in particular and protein-protein recognition in general. Results: PPI is a versatile protease inhibitor that targets several subfamilies of serine proteases. Conclusion: The ␤-trefoil fold constitutes an evolutionary platform for protease inhibition and molecular recognition. Significance: Fold plasticity influences protein evolution toward multiple function and binding promiscuity.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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The Plasticity of the β-Trefoil Fold Constitutes an Evolutionary Platform for Protease Inhibition

Azarkan

Martínez‐Rodríguez

Buts

et al. 2011

Journal of Biological Chemistry

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“…Structural defects may be present in SAM-T08 models (Chavelas-Adame et al, 2011), and this model was refined using a slow-convergence molecular mechanics (Hyperchem 7.5, AMBER 99 forcefield) minimization scheme described elsewhere (Rosales-León et al, 2012). The resulting model was superimposed to the barley (Hordeum vulgare) subtilisin/a-amylase inhibitor (Micheelsen et al, 2008; Protein Data Bank entry 3BX1, chain C) using STAMP (Russell and Barton, 1992) as implemented in MULTISEC (Roberts et al, 2006). The structural alignment allowed us to predict two disulfide bonds (Cys-88 to ).…”

Section: Comparative Modeling Of the Nastep Three-dimensional Structurementioning

confidence: 99%

NaStEP: A Proteinase Inhibitor Essential to Self-Incompatibility and a Positive Regulator of HT-B Stability inNicotiana alataPollen Tubes

et al. 2012

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In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown.

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“…The BASI binds to the savinase in a substrate-like manner and forms a β -strand in the P5-P2 region alongside the active site (Figure 4 ). Numerous interactions between this region and the savinase active site are present, including seven hydrogen bonds between the main-chain atoms of the BASI and the savinase residues (Micheelsen et al , 2008 ). The P1 and P1 ′ residues do not interact with savinase because the loop is pulled out of the cleft by the disulfi de bond of the P1 residue (Cys90) formed with the Cys43 of the inhibitor.…”

Section: Inhibition Of the S8 Family Serine Proteasesmentioning

confidence: 99%

β-Trefoil inhibitors – from the work of Kunitz onward

Renko

Sabotič²,

Turk³

2012

Biological Chemistry

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Protein protease inhibitors are the tools of nature in controlling proteolytic enzymes. They come in different shapes and sizes. The β -trefoil protease inhibitors that come from plants, fi rst discovered by Kunitz, were later complemented with representatives from higher fungi. They inhibit serine (families S1 and S8) and cysteine proteases (families C1 and C13) as well as other hydrolases. Their versatility is the result of the plasticity of the loops coming out of the stable β -trefoil scaffold. For this reason, they display several different mechanisms of inhibition involving different positions of the loops and their combinations. Natural diversity, as well as the initial successes in de novo protein engineering, makes the β -trefoil proteins a promising starting point for the generation of strong, specifi c, multitarget inhibitors capable of inhibiting multiple types of hydrolytic enzymes and simultaneously interacting with different protein, carbohydrate, or DNA molecules. This pool of knowledge opens up new possibilities for the exploration of their naturally occurring as well as modifi ed properties for applications in many fi elds of medicine, biotechnology, and agriculture.

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Structural and Mutational Analyses of the Interaction between the Barley α-Amylase/Subtilisin Inhibitor and the Subtilisin Savinase Reveal a Novel Mode of Inhibition

Cited by 48 publications

References 44 publications

The Plasticity of the β-Trefoil Fold Constitutes an Evolutionary Platform for Protease Inhibition

The Plasticity of the β-Trefoil Fold Constitutes an Evolutionary Platform for Protease Inhibition

NaStEP: A Proteinase Inhibitor Essential to Self-Incompatibility and a Positive Regulator of HT-B Stability inNicotiana alataPollen Tubes

β-Trefoil inhibitors – from the work of Kunitz onward

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