A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIa
Abstract Proteases play a major role in many vital physiological processes. Trypsin-like serine proteases (TLPs), in particular, are paramount in proteolytic cascade systems such as blood coagulation and complement activation. The structural topology of TLPs is highly conserved, with the trypsin fol...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-03-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-07620-7 |
| _version_ | 1819236800084836352 |
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| author | Anders B. Sorensen Per Jr. Greisen Jesper J. Madsen Jacob Lund Gorm Andersen Pernille G. Wulff-Larsen Anette A. Pedersen Prafull S. Gandhi Michael T. Overgaard Henrik Østergaard Ole H. Olsen |
| author_facet | Anders B. Sorensen Per Jr. Greisen Jesper J. Madsen Jacob Lund Gorm Andersen Pernille G. Wulff-Larsen Anette A. Pedersen Prafull S. Gandhi Michael T. Overgaard Henrik Østergaard Ole H. Olsen |
| author_sort | Anders B. Sorensen |
| collection | DOAJ |
| description | Abstract Proteases play a major role in many vital physiological processes. Trypsin-like serine proteases (TLPs), in particular, are paramount in proteolytic cascade systems such as blood coagulation and complement activation. The structural topology of TLPs is highly conserved, with the trypsin fold comprising two β-barrels connected by a number of variable surface-exposed loops that provide a surprising capacity for functional diversity and substrate specificity. To expand our understanding of the roles these loops play in substrate and co-factor interactions, we employ a systematic methodology akin to the natural truncations and insertions observed through evolution of TLPs. The approach explores a larger deletion space than classical random or directed mutagenesis. Using FVIIa as a model system, deletions of 1–7 amino acids through the surface exposed 170 loop, a vital allosteric regulator, was introduced. All variants were extensively evaluated by established functional assays and computational loop modelling with Rosetta. The approach revealed detailed structural and functional insights recapitulation and expanding on the main findings in relation to 170 loop functions elucidated over several decades using more cumbersome crystallization and single deletion/mutation methodologies. The larger deletion space was key in capturing the most active variant, which unexpectedly had a six-amino acid truncation. This variant would have remained undiscovered if only 2–3 deletions were considered, supporting the usefulness of the methodology in general protease engineering approaches. Our findings shed further light on the complex role that surface-exposed loops play in TLP function and supports the important role of loop length in the regulation and fine-tunning of enzymatic function throughout evolution. |
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| format | Article |
| id | doaj.art-cd179ec03b8a4207afd8a746fa3e6331 |
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| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-23T13:10:11Z |
| publishDate | 2022-03-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-cd179ec03b8a4207afd8a746fa3e63312022-12-21T17:45:46ZengNature PortfolioScientific Reports2045-23222022-03-0112111210.1038/s41598-022-07620-7A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIaAnders B. Sorensen0Per Jr. Greisen1Jesper J. Madsen2Jacob Lund3Gorm Andersen4Pernille G. Wulff-Larsen5Anette A. Pedersen6Prafull S. Gandhi7Michael T. Overgaard8Henrik Østergaard9Ole H. Olsen10Global Research, Novo Nordisk A/SGlobal Research, Novo Nordisk A/SGlobal and Planetary Health, College of Public Health, University of South FloridaGlobal Research, Novo Nordisk A/SGlobal Research, Novo Nordisk A/SGlobal Research, Novo Nordisk A/SGlobal Research, Novo Nordisk A/SGlobal Research, Novo Nordisk A/SDepartment of Chemistry and Bioscience, Aalborg UniversityGlobal Research, Novo Nordisk A/SGlobal Research, Novo Nordisk A/SAbstract Proteases play a major role in many vital physiological processes. Trypsin-like serine proteases (TLPs), in particular, are paramount in proteolytic cascade systems such as blood coagulation and complement activation. The structural topology of TLPs is highly conserved, with the trypsin fold comprising two β-barrels connected by a number of variable surface-exposed loops that provide a surprising capacity for functional diversity and substrate specificity. To expand our understanding of the roles these loops play in substrate and co-factor interactions, we employ a systematic methodology akin to the natural truncations and insertions observed through evolution of TLPs. The approach explores a larger deletion space than classical random or directed mutagenesis. Using FVIIa as a model system, deletions of 1–7 amino acids through the surface exposed 170 loop, a vital allosteric regulator, was introduced. All variants were extensively evaluated by established functional assays and computational loop modelling with Rosetta. The approach revealed detailed structural and functional insights recapitulation and expanding on the main findings in relation to 170 loop functions elucidated over several decades using more cumbersome crystallization and single deletion/mutation methodologies. The larger deletion space was key in capturing the most active variant, which unexpectedly had a six-amino acid truncation. This variant would have remained undiscovered if only 2–3 deletions were considered, supporting the usefulness of the methodology in general protease engineering approaches. Our findings shed further light on the complex role that surface-exposed loops play in TLP function and supports the important role of loop length in the regulation and fine-tunning of enzymatic function throughout evolution.https://doi.org/10.1038/s41598-022-07620-7 |
| spellingShingle | Anders B. Sorensen Per Jr. Greisen Jesper J. Madsen Jacob Lund Gorm Andersen Pernille G. Wulff-Larsen Anette A. Pedersen Prafull S. Gandhi Michael T. Overgaard Henrik Østergaard Ole H. Olsen A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIa Scientific Reports |
| title | A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIa |
| title_full | A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIa |
| title_fullStr | A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIa |
| title_full_unstemmed | A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIa |
| title_short | A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIa |
| title_sort | systematic approach for evaluating the role of surface exposed loops in trypsin like serine proteases applied to the 170 loop in coagulation factor viia |
| url | https://doi.org/10.1038/s41598-022-07620-7 |
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