Screening, large-scale production and structure-based classification of cystine-dense peptides

Abstract

Peptides folded through interwoven disulfides display extreme biochemical properties and unique medicinal potential. Their exploitation was hampered by the limited amounts isolatable from natural sources and the expense of chemical synthesis. We developed reliable biological methods for high-throughput expression screening and large-scale production of these peptides. 46 were successfully produced in multimilligram quantities, and over 600 more were deemed expressible by stringent screening criteria. Many showed extreme resistance to temperature, proteolysis, and/or reduction, and all displayed inhibitory activity against at least one of 20 ion channels tested, confirming biological functionality. Crystal structures of 12 were determined, confirming proper cystine topology, and the utility of crystallography for studying these molecules, but highlighted the need for rational classification. Previous attempts at categorization have focused on limited subsets siloed around distinct motifs. Stepping back, we present a global definition, classification, and analysis of over 700 structures of cystine-dense peptides, unifying these molecules.

Dissecting Toxicity: The Venom Gland Transcriptome and the Venom Proteome of the Highly Venomous Scorpion Centruroides limpidus (Karsch, 1879)

“…CliKTxAlp15 codes for a peptide confirmed to be expressed in the venom of C. limpidus by the proteomic analysis here reported. It is similar to alpha-KTx4.5 from Tityus costatus (Q5G8B6), a toxin found to inhibit with low potency the Kv1.1, Kv1.2, Kv1.3, and Kv11.1 (ERG1) channels [42]. CliKTxAlp07 potentially codes for a peptide similar to Noxiustoxin-2 (Q9TXD1), having only one difference at the amino acid level.…”

Cytosolic expression, solution structures, and molecular dynamics simulation of genetically encodable disulfide‐rich de novo designed peptides

“…The temperature was reduced to 20 C and cultures were incubated with shaking at 225 rpm for an additional 12-16 h. Following expression, the fusion protein was purified from soluble cellular lysate by immobilized metal affinity chromatography. Purified fusion proteins were cleaved using the site-specific protease superTEV, 4 and mature peptides were purified to homogeneity by reverse-phase high-performance liquid chromatography on an Agilent 1260 HPLC equipped with a C-18 Zorbax SB-C18 4.6 × 150 mm column. Solvent A (water + 0.1%TFA) and solvent B (acetonitrile + 0.1%TFA) were run at a flow rate of 5 mL/min using the following gradient: 0%-5% solvent B (5 min), 5%-45% solvent B (40 min).…”

“…Constrained peptides are commonly employed by nature as signaling molecules or toxins, and the incorporation of multiple disulfide covalent crosslinks between pairs of cysteine residues is thought to be responsible for the stability and potent drug‐like properties of these molecules . Knottins and short‐chain scorpion toxins are two well‐characterized groups within this protein family, each characterized by conserved structural motifs and highly‐conserved disulfide bonds . However, the ability to generate stable structures adopting a wide range of specified sizes and shapes not observed in nature will be necessary to unlock the full pharmacological potential of peptide‐based drugs.…”

“…2 Knottins and short-chain scorpion toxins are two wellcharacterized groups within this protein family, each characterized by conserved structural motifs and highly-conserved disulfide bonds. 3,4 However, the ability to generate stable structures adopting a wide range of specified sizes and shapes not observed in nature will be necessary to unlock the full pharmacological potential of peptide-based drugs. Working toward this goal, we previously reported a computational method for the de novo design of genetically encodable cysteine-rich peptides of various topologies, which were validated by experiments.…”

Secreted Cysteine-Rich Repeat Proteins “SCREPs”: A Novel Multi-Domain Architecture

“…Hopefully this overview will provide sufficient details outlining both the features of this new source of biomolecules as well as a practical data mining approach necessary for further investigation. Interestingly, recent similar workflows aimed at the investigation of DRPs provide a very applicable demonstration, describing the implementation of large scale production and screening techniques of cysteine dense ion channel ligands ( Correnti et al, 2018 ). Furthermore, recent advances in both recombinant DRP production as well as techniques in obtaining correct disulfide bond connectivities ( Berkmen, 2012 ; Klint et al, 2013 ) provide more affordable methods that result in improved experimental accessibility toward the investigation of SCREPs.…”