02460nas a2200241   4500008004100000022001400041245014600055210006900201260000900270300001100279490000800290520157900298653001901877653002901896653002301925653003201948653001401980653002701994100002402021700002202045700002002067856013102087       2024                        eng d  a0006-291X00aEngineering inhibitory repeat domains of Pin-II type proteinase inhibitors indicate their high structural-functional tolerance to mutagenesis0 aEngineering inhibitory repeat domains of PinII type proteinase i  cNOV   a1508080 v7353 a<p>
	Plant proteinase inhibitors (PIs) are critical in defending against biotic stress. Most PIs contain an inhibitory repeat domain (IRD), which serves as the functional component, displaying a high degree of sequence and structural conservation. In this study, we examined the structural and functional resilience of IRDs using a combination of computational modeling and experimental validation. We have taken an evolution-based approach to enhance the PIs effectiveness of two previously identified Capsicum annuum IRDs, IRD4 and IRD10. Through in silico site-saturation mutagenesis of IRD4 and IRD10, we identified key sites associated with enhanced PI activity for targeted mutagenesis. Binding energy predictions for a mutant IRD library, tested against target proteases, suggested that positions R11 and N32 in IRD4 and N32 and H33 in IRD10 were promising candidates for further modification to improve inhibitory potential. Subsequent experimental validation revealed that the mutant proteins IRD4_R11K and IRD4_N32S exhibited stronger chymotrypsin inhibition than the wild-type (WT) IRD4. Similarly, the mutants IRD10_N32S and IRD10_H33 N demonstrated improved trypsin inhibition relative to the WT IRD10. These findings indicate that engineered IRD variants can tolerate structural changes while maintaining or enhancing their inhibitory activity against target proteases. Overall, this study demonstrates the potential of engineering PIs to increase their structural and functional resilience, offering new opportunities for biotechnological applications.</p>
10aCapsicum annum10aInhibitory repeat domain10amolecular modeling10aplant proteinase inhibitors10aProteases10aSaturation mutagenesis1 aBarbole, Ranjit, S.1 aJoshi, Rakesh, S.1 aGiri, Ashok, P.  uhttps://library.ncl.res.in/content/engineering-inhibitory-repeat-domains-pin-ii-type-proteinase-inhibitors-indicate-their-high