Structural insights into serine protease inhibition by a marine invertebrate BPTI Kunitz-type inhibitor

García-Fernández, Rossana; Pons, Tirso; Perbandt, Markus; Valiente, Pedro A.; Talavera, Ariel; González-González, Yamile; Roggenbuck, Dirk; Chávez, María Á.; Betzel, Christian; Redecke, Lars

doi:10.1016/j.jsb.2012.08.009

Cited by 19 publications

(38 citation statements)

References 45 publications

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“…The protein concentration was determined by absorbance at 280 nm using a theoretical extinction coefficient (E 280 nm 1% ) of 1.80 calculated for the complex with ProtParam (49) based on both protein sequences. Sample monodispersity and the hydrodynamic radius of the protein complex were evaluated by dynamic light scattering techniques as described previously (35,38).…”

Section: Methodsmentioning

confidence: 99%

“…Compared with the interface of trypsin-bound rShPI-1A (PDB code 3M7Q) (35), residues Gly 10 (P4), Tyr 15 (P2Ј), and Pro 16 (P3Ј) provide an increased contribution to the elastase interaction, whereas the impact of Arg 11 (P3) and Pro 17 (P4Ј) is slightly reduced. The rShPI-1/K13L⅐PPE interface is extended by one residue at both sites of the scissile bond compared with known complexes of BPTI and rShPI-1A with trypsin (32)(33)(34)(35). The accessible surface area of the outer residues, Lys 8 (P6) and Arg 18 (P5Ј), is buried by more than 30% after complex formation with PPE (Fig.…”

Section: Rshpi-1/k13l⅐ppe Interface and Comparison With The Rshpi1a⅐tmentioning

confidence: 97%

“…The accessible surface area of the outer residues, Lys 8 (P6) and Arg 18 (P5Ј), is buried by more than 30% after complex formation with PPE (Fig. 1c), whereas these res- idues do not show any contribution to the rShPI-1A⅐trypsin complex (35). An overall number of 156 direct enzyme-inhibitor contacts is shorter than 4 Å, including 144 hydrophobic contacts and 10 hydrogen bonds; these are involved in the stabilization of the rShPI-1/K13L⅐PPE interface (Table 3).…”

Section: Rshpi-1/k13l⅐ppe Interface and Comparison With The Rshpi1a⅐tmentioning

confidence: 99%

“…Although the structural details of trypsin and chymotrypsin inhibition by BPTI have been extensively investigated (32)(33)(34)(35), the structural basis of the elastase specificity has not been elucidated for this type of inhibitors. Together with the large number of available mutagenesis studies (25)(26)(27)(28), structural insights into the elastase interaction could provide important information for the design of novel potent elastase inhibitors exploiting the Kunitz-type scaffold.…”

mentioning

confidence: 99%

“…This molecule inhibits not only serine proteases but also cysteine and aspartic proteases such as papain and pepsin with K i values in the nanomolar range (21), qualifying ShPI-1 for biotechnological use (37). We recently presented the three-dimensional structure of free and trypsin-bound recombinant ShPI-1 (rShPI-1A) (35,38), revealing a high degree of conservation for the intermolecular interactions around the basic P1 residue (Lys 13 ) compared with homologous complexes of mammalian inhibitors. However, a prominent stabilizing role of arginine at the P3 position was identified as the most significant deviation, thus far unique within this inhibitor family.…”

mentioning

confidence: 99%

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Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme

García-Fernández

Perbandt

Roggenbuck³

et al. 2015

Journal of Biological Chemistry

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Background: Kunitz-type inhibitors provide a suitable scaffold for novel elastase inhibitors. Results: The inhibitor ShPI-1 was modified for pancreatic elastase binding, and the crystal structure of the complex was elucidated and analyzed. Conclusion: The extended protease-inhibitor interactions provide a potential switch to direct inhibitor selectivity toward elastases. Significance: These results will help to design novel elastase inhibitors for the treatment of tissue destruction diseases.

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Section: Methodsmentioning

confidence: 99%

Section: Rshpi-1/k13l⅐ppe Interface and Comparison With The Rshpi1a⅐tmentioning

confidence: 97%

Section: Rshpi-1/k13l⅐ppe Interface and Comparison With The Rshpi1a⅐tmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme

García-Fernández

Perbandt

Roggenbuck³

et al. 2015

Journal of Biological Chemistry

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Engineering trypsin for inhibitor resistance

et al. 2015

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The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [k cat /K M 5 (1.2 6 0.3) 3 10 7 M 21 s 21 . Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 ms combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.

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Secretion and assembly of calicivirus-like particles in high-cell-density yeast fermentations: strategies based on a recombinant non-specific BPTI-Kunitz-type protease inhibitor

Fernández

Toledo

Mansur

et al. 2014

Appl Microbiol Biotechnol

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The yeast Pichia pastoris is one of the most robust cell factories in use for the large-scale production of biopharmaceuticals with applications in the fields of human and animal health. Recently, intracellular high-level expression of rabbit hemorrhagic disease virus (RHDV) capsid protein (VP1) as a self-assembled multipurpose antigen/carrier was established as a production process from P. pastoris. Since recovery of VP1 from the culture media implies technological and economic advantages, the secretion of VP1 variants was undertaken in this work. Conversely, extensive degradation of VP1 was detected. Variations to culture parameters and supplementation with different classes of additives were unable to diminish degradation. Strategies were then conducted during fermentations using a recombinant variant of a non-specific BPTI-Kunitz-type protease inhibitor (rShPI-1A) isolated from the sea anemone Stichodactyla helianthus. The presence of the inhibitor in the culture medium at the recombinant protein induction phase, as well as co-culture of the yeast strains expressing VP1 and rShPI-1A, led to VP1 protection from proteolysis and to production of ordered virus-like particles. A yeast strain was also engineered to co-express the rShPI-1A inhibitor and intact VP1. Expression levels up to 116 mg L(-1) of VP1 were reached under these approaches. The antigen was characterized and purified in a single chromatography step, its immunogenic capacity was evaluated, and a detection test for specific antibodies was developed. This work provides feasible strategies for improvements in P. pastoris heterologous protein secretion and is the first report on co-expression of the ShPI-1A with a recombinant product otherwise subjected to proteolytic degradation.

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Structural insights into serine protease inhibition by a marine invertebrate BPTI Kunitz-type inhibitor

Cited by 19 publications

References 45 publications

Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme

Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme

Engineering trypsin for inhibitor resistance

Secretion and assembly of calicivirus-like particles in high-cell-density yeast fermentations: strategies based on a recombinant non-specific BPTI-Kunitz-type protease inhibitor

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