Efficient enzymatic cyclization of an inhibitory cystine knot-containing peptide.
Disulfide-rich peptides isolated from cone snails are of great interest as drug leads due to their high specificity and potency toward therapeutically relevant ion channels and receptors. They commonly contain the inhibitor cystine knot (ICK) motif comprising three disulfide bonds forming a knotted core. Here we report the successful enzymatic backbone cyclization of an ICK-containing peptide κ-PVIIA, a 27-amino acid conopeptide from Conus purpurascens, using a mutated version of the bacterial transpeptidase, sortase A. Although a slight loss of activity was observed compared to native κ-PVIIA, cyclic κ-PVIIA is a functional peptide that inhibits the Shaker voltage-gated potassium (Kv) channel. Molecular modeling suggests that the decrease in potency may be related to the loss of crucial, but previously unidentified electrostatic interactions between the N-terminus of the peptide and the Shaker channel. This hypothesis was confirmed by testing an N-terminally acetylated κ-PVIIA, which shows a similar decrease in activity. We also investigated the conformational dynamics and hydrogen bond network of cyc-PVIIA, both of which are important factors to be considered for successful cyclization of peptides. We found that cyc-PVIIA has the same conformational dynamics, but different hydrogen bond network compared to those of κ-PVIIA. The ability to efficiently cyclize ICK peptides using sortase A will enable future protein engineering for this class of peptides and may help in the development of novel therapeutic molecules. Biotechnol. Bioeng. 2016;113: 2202-2212. © 2016 Wiley Periodicals, Inc.
The University of Queensland, Institute for Molecular Bioscience, Brisbane, Qld, 4072, Australia.
Soohyun
S
Kwon
Department of Physiology and Solomon H Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Frank
F
Bosmans
The University of Queensland, Institute for Molecular Bioscience, Brisbane, Qld, 4072, Australia.
Quentin
Q
Kaas
The University of Queensland, Institute for Molecular Bioscience, Brisbane, Qld, 4072, Australia.
Olivier
O
Cheneval
The University of Queensland, Institute for Molecular Bioscience, Brisbane, Qld, 4072, Australia.
Anne C
AC
Conibear
The University of Queensland, School of Biomedical Sciences, Brisbane, Qld, Australia.
K Johan
KJ
Rosengren
The University of Queensland, Institute for Molecular Bioscience, Brisbane, Qld, 4072, Australia.
Conan K
CK
Wang
The University of Queensland, Institute for Molecular Bioscience, Brisbane, Qld, 4072, Australia. c.schroeder@imb.uq.edu.au.
Christina I
CI
Schroeder
The University of Queensland, Institute for Molecular Bioscience, Brisbane, Qld, 4072, Australia. d.craik@imb.uq.edu.au.
David J
DJ
Craik
D019881:Aminoacyltransferases / Q000737:chemistry / Q000648:ultrastructure*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000648:ultrastructure*; D001665:Binding Sites; D020916:Conotoxins / Q000737:chemistry*; D052078:Conus Snail / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000648:ultrastructure*; D003553:Cystine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004789:Enzyme Activation; D008956:Models, Chemical; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D015221:Potassium Channels / Q000737:chemistry / Q000648:ultrastructure*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding
D001426:Bacterial Proteins; D020916:Conotoxins; D004220:Disulfides; D010455:Peptides; D015221:Potassium Channels; D003553:Cystine; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
cyclization; inhibitory cystine knot; sortase A; κ-PVIIA