BIOMAT Database Logo BIOMAT Database Logo

Selective removal of individual disulfide bonds within a potato type II serine proteinase inhibitor from Nicotiana alata reveals differential stabilization of the reactive-site loop. 2010

Horst Joachim Schirra, and Rosemary F Guarino, and Marilyn A Anderson, and David J Craik
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.

The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were individually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a K(i) of <5 nM, C4A/C41A-T1 has a weaker K(i) of approximately 350 nM, and the potency of the C8A/C37A variant is further decreased to a K(i) of approximately 1.8 microM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the amplitude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4-C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond-nanosecond) timescale. In contrast, no comparable interactions are present around the C8-C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8-C37 and C4-C41 disulfide bonds. The C8-C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4-C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond-nanosecond timescale.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010940 Plant Proteins Proteins found in plants (flowers, herbs, shrubs, trees, etc.). The concept does not include proteins found in vegetables for which PLANT PROTEINS, DIETARY is available. Plant Protein,Protein, Plant,Proteins, Plant
D011198 Solanum tuberosum A plant species of the genus SOLANUM, family SOLANACEAE. The starchy roots are used as food. SOLANINE is found in green parts. Potatoes,Potato,Solanum tuberosums,tuberosum, Solanum,tuberosums, Solanum
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D004220 Disulfides Chemical groups containing the covalent disulfide bonds -S-S-. The sulfur atoms can be bound to inorganic or organic moieties. Disulfide
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D001665 Binding Sites The parts of a macromolecule that directly participate in its specific combination with another molecule. Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining

Related Publications

Horst Joachim Schirra, and Rosemary F Guarino, and Marilyn A Anderson, and David J Craik
Characterization of potato proteinase inhibitor II reactive site mutants.
April 2000, European journal of biochemistry,
Horst Joachim Schirra, and Rosemary F Guarino, and Marilyn A Anderson, and David J Craik
Identification of the reactive site of potato proteinase inhibitor II-a.
May 1973, Journal of biochemistry,
Horst Joachim Schirra, and Rosemary F Guarino, and Marilyn A Anderson, and David J Craik
Selective loss of cysteine residues and disulphide bonds in a potato proteinase inhibitor II family.
April 2011, PloS one,
Horst Joachim Schirra, and Rosemary F Guarino, and Marilyn A Anderson, and David J Craik
Identification of the reactive site of potato proteinase inhibitor II-b for bovine chymotrypsin and a bacterial proteinase.
August 1973, Journal of biochemistry,
Horst Joachim Schirra, and Rosemary F Guarino, and Marilyn A Anderson, and David J Craik
Primary structure of potato kunitz-type serine proteinase inhibitor.
December 2000, Biological chemistry,
Horst Joachim Schirra, and Rosemary F Guarino, and Marilyn A Anderson, and David J Craik
The remarkable efficiency of a Pin-II proteinase inhibitor sans two conserved disulfide bonds is due to enhanced flexibility and hydrogen bond density in the reactive site loop.
January 2014, Journal of biomolecular structure & dynamics,
Horst Joachim Schirra, and Rosemary F Guarino, and Marilyn A Anderson, and David J Craik
Identification of a novel four-domain member of the proteinase inhibitor II family from the stigmas of Nicotiana alata.
January 2000, Plant molecular biology,
Horst Joachim Schirra, and Rosemary F Guarino, and Marilyn A Anderson, and David J Craik
Characterization of the protease processing sites in a multidomain proteinase inhibitor precursor from Nicotiana alata.
May 1995, European journal of biochemistry,
Horst Joachim Schirra, and Rosemary F Guarino, and Marilyn A Anderson, and David J Craik
Dual location of a family of proteinase inhibitors within the stigmas of Nicotiana alata.
April 2007, Planta,
Horst Joachim Schirra, and Rosemary F Guarino, and Marilyn A Anderson, and David J Craik
Identification of the reactive site of potato proteinase inhibitor I for various proteinases.
October 1973, Journal of biochemistry,
Copied contents to your clipboard!