Background: Sortase A (SrtA) is a transpeptidase capable of catalyzing the formation of amide bonds. Results: SrtA was used to backbone-cyclize disulfide-rich peptides, including kalata B1, ␣-conotoxin Vc1.1, and SFTI-1. Conclusion: SrtA-mediated cyclization is applicable to small disulfide-rich peptides. Significance: SrtA-mediated cyclization is an alternative to native chemical ligation for the cyclization of small peptides of therapeutic interest.

Scorpion α-toxins are invaluable pharmacological tools for studying voltage-gated sodium channels, but few structure-function studies have been undertaken due to their challenging synthesis. To address this deficiency, we report a chemical engineering strategy based upon native chemical ligation. The chemical synthesis of α-toxin OD1 was achieved by chemical ligation of three unprotected peptide segments. A high resolution X-ray structure (1.8 Å) of synthetic OD1 showed the typical βαββ α-toxin fold and revealed important conformational differences in the pharmacophore region when compared with other α-toxin structures. Pharmacological analysis of synthetic OD1 revealed potent α-toxin activity (inhibition of fast inactivation) at Nav1.7, as well as Nav1.4 and Nav1.6. In addition, OD1 also produced potent β-toxin activity at Nav1.4 and Nav1.6 (shift of channel activation in the hyperpolarizing direction), indicating that OD1 might interact at more than one site with Nav1.4 and Nav1.6. Investigation of nine OD1 mutants revealed that three residues in the reverse turn contributed significantly to selectivity, with the triple OD1 mutant (D9K, D10P, K11H) being 40-fold more selective for Nav1.7 over Nav1.6, while OD1 K11V was 5-fold more selective for Nav1.6 than Nav1.7. This switch in selectivity highlights the importance of the reverse turn for engineering α-toxins with altered selectivity at Nav subtypes.

Background: Mechanistic insight into allosteric modulation of GPCRs can facilitate antagonist design. Results: Extracellular surface residues (ECS) of the ␣ 1B -adrenoceptor at the base of extracellular loop 3 interact with the allosteric antagonist TIA. Conclusion:The identified ECS pharmacophore provides the first structural constraints for allosteric antagonist design at ␣ 1 -adrenoceptors. Significance: Binding to the ECS of a GPCR can allosterically inhibit agonist signaling.

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We report the total chemical synthesis of human C3a by onepot native chemical ligation of three unprotected peptide segments followed by efficient in-vitro folding, which yielded the target molecule in high yield and excellent purity. The 10 synthetic material was fully active and facilitated determination of the C3a crystal structure at 2.1Å resolution.The anaphylatoxins C3a and C5a are key mediators of the complement system, which represents the first line of immunological defense for the recognition and elimination of 15 microbes and pathogens. 1 They selectively bind to their respective G protein-coupled receptors (C3aR and C5aR) triggering a variety of pro-inflammatory processes and have recently been linked to a number of infectious, inflammatory, neurodegenerative and autoimmune diseases. 2 Currently, C3a is 20 commercially produced in relatively low yields by biological means (recombinant expression or purification from plasma) 3, 4 resulting in a market value of approximately US$ 5000 per mg of protein. In addition, these approaches often require protein purification tags and additional steps for their removal and suffer 25 from the inherent lability of C3a in biological fluids. C3a is rapidly inactivated within seconds by carboxypeptidase-mediated cleavage of a single C-terminal arginine residue (Arg 77 ). 5,6 The resulting protein, C3a-desArg (also termed acylation-stimulating protein, ASP), does not bind C3aR, lacks any pro-inflammatory 30 activity but instead has been shown to stimulate triglyceride synthesis and glucose uptake in adipose tissue. [7][8][9] To circumvent the problems associated with the isolation of C3a from biological sources we sought a total chemical synthesis approach enabling preparation of homogenous full length C3a. 35Human C3a is a 77 residue protein containing three intramolecular disulfide bonds between C22-C49, C23-C56, and C36-C57. 10, 11 Because polypeptides of this size are difficult to obtain in high purity by standard stepwise solid phase peptide synthesis (SPPS), 12 we envisioned a fragment ligation approach 40 by employing Kent's native chemical ligation (NCL). 13 The 77 amino acid polypeptide chain is retro-synthetically split into three peptide segments of about similar length and the three polypeptides are then joined consecutively in the C-to Nterminal direction by NCL as shown in Scheme complete after 6h after which methoxyamine HCl was added and the pH adjusted to 4.0. After completion of the deprotection reaction, the mixture was readjusted to pH 7.1 and the second ligation initiated by adding C3a[1-22]-α-thioester (complete after 6 h). This one-pot approach afforded fully reduced C3a 60 without purification of intermediate products in short time (~24 h), good yield (41%) and high purity (SI Figure 1). The final folding of a cysteine-rich polypeptide chain into its 3D structure can often be problematic because they tend to yield multiple disulfide isomers that are difficult to separate and result 65 in lower overall yield. This often necessitates optimizati...