On the importance of oxidative folding in the evolution of conotoxins: cysteine codon preservation through gene duplication and adaptation.

Conotoxin genes are among the most rapidly evolving genes currently known; however, despite the well-established hypervariability of the intercysteine loops, the cysteines demonstrate significant conservation, with a site-specific codon bias for each cysteine in a family of conotoxins. Herein we present a novel rationale behind the codon-level conservation of the cysteines that comprise the disulfide scaffold. We analyze cysteine codon conservation using an internal reference and phylogenetic tools; our results suggest that the established codon conservation can be explained as the result of selective pressures linked to the production efficiency and folding of conotoxins, driving the conservation of cysteine at the amino-acid level. The preservation of cysteine has resulted in maintenance of the ancestral codon in most of the daughter lineages, despite the hypervariability of adjacent residues. We propose that the selective pressures acting on the venom components of cone snails involve an interplay of biosynthetic efficiency, activity at the target receptor and the importance of that activity to effective prey immobilization. Functional redundancy in the venom can thus serve as a buffer for the energy expenditure of venom production.

Modulation of conotoxin structure and function is achieved through a multienzyme complex in the venom glands of cone snails.

The oxidative folding of large polypeptides has been investigated in detail; however, comparatively little is known about the enzyme-assisted folding of small, disulfide-containing peptide substrates. To investigate the concerted effect of multiple enzymes on the folding of small disulfide-rich peptides, we sequenced and expressed protein-disulfide isomerase (PDI), peptidyl-prolyl cis-trans isomerase, and immunoglobulin-binding protein (BiP) from Conus venom glands. Conus PDI was shown to catalyze the oxidation and reduction of disulfide bonds in two conotoxins, α-GI and α-ImI. Oxidative folding rates were further increased in the presence of Conus PPI with the maximum effect observed in the presence of both enzymes. In contrast, Conus BiP was only observed to assist folding in the presence of microsomes, suggesting that additional co-factors were involved. The identification of a complex between BiP, PDI, and nascent conotoxins further suggests that the folding and assembly of conotoxins is a highly regulated multienzyme-assisted process. Unexpectedly, all three enzymes contributed to the folding of the ribbon isomer of α-ImI. Here, we identify this alternative disulfide-linked species in the venom of Conus imperialis, providing the first evidence for the existence of a "non-native" peptide isomer in the venom of cone snails. Thus, ER-resident enzymes act in concert to accelerate the oxidative folding of conotoxins and modulate their conformation and function by reconfiguring disulfide connectivities. This study has evaluated the role of a number of ER-resident enzymes in the folding of conotoxins, providing novel insights into the enzyme-guided assembly of these small, disulfide-rich peptides.

Dissecting a role of evolutionary-conserved but noncritical disulfide bridges in cysteine-rich peptides using ω-conotoxin GVIA and its selenocysteine analogs.

Conotoxins comprise a large group of peptidic neurotoxins that use diverse disulfide-rich scaffolds. Each scaffold is determined by an evolutionarily conserved pattern of cysteine residues. Although many structure-activity relationship studies confirm the functional and structural importance of disulfide crosslinks, there is growing evidence that not all disulfide bridges are critical in maintaining activities of conotoxins. To answer the fundamental biological question of what the role of noncritical disulfide bridges is, we investigated function and folding of disulfide-depleted analogs of ω-conotoxin GVIA (GVIA) that belongs to an inhibitory cystine knot motif family and blocks N-type calcium channels. Removal of a noncritical Cys1-Cys16 disulfide bridge in GVIA or its selenopeptide analog had, as predicted, rather minimal effects on the inhibitory activity on calcium channels, as well as on in vivo activity following intracranial administration. However, the disulfide-depleted GVIA exhibited significantly lower folding yields for forming the remaining two native disulfide bridges. The disulfide-depleted selenoconotoxin GVIA analog also folded with significantly lower yields, suggesting that the functionally noncritical disulfide pair plays an important cooperative role in forming the native disulfide scaffold. Taken together, our results suggest that distinct disulfide bridges may be evolutionarily preserved by the oxidative folding or/and stabilization of the bioactive conformation of a disulfide-rich scaffold.

Selective purification of recombinant neuroactive peptides using the flagellar type III secretion system.

UNLABELLED: The structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233-2243, 2006). The T3SS is reported to secrete proteins at rates of up to 10,000 amino acid residues per second. In this work, we showed that the flagellar T3SS of Salmonella enterica serovar Typhimurium could be manipulated to export recombinant nonflagellar proteins through the flagellum and into the surrounding medium. We translationally fused various neuroactive peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria to the flagellar secretion substrate FlgM. We found that all tested peptides of various sizes were secreted via the bacterial flagellar T3SS. We subsequently purified the recombinant µ-conotoxin SIIIA (rSIIIA) from Conus striatus by affinity chromatography and confirmed that T3SS-derived rSIIIA inhibited mammalian voltage-gated sodium channel Na(V)1.2 comparably to chemically synthesized SIIIA. IMPORTANCE: Manipulation of the flagellar secretion system bypasses the problems of inclusion body formation and cellular degradation that occur during conventional recombinant protein expression. This work serves as a proof of principle for the use of engineered bacterial cells for rapid purification of recombinant neuroactive peptides and proteins by exploiting secretion via the well-characterized flagellator type III secretion system.

Optimization of oxidative folding methods for cysteine-rich peptides: a study of conotoxins containing three disulfide bridges.

The oxidative folding of small, cysteine-rich peptides to selectively achieve the native disulfide bond connectivities is critical for discovery and structure-function studies of many bioactive peptides. As the propensity to acquire the native conformation greatly depends on the peptide sequence, numerous empirical oxidation methods are employed. The context-dependent optimization of these methods has thus far precluded a generalized oxidative folding protocol, in particular for peptides containing more than two disulfides. Herein, we compare the efficacy of optimized solution-phase and polymer-supported oxidation methods using three disulfide-bridged conotoxins, namely µ-SIIIA, µ-KIIIA and ω-GVIA. The use of diselenide bridges as proxies for disulfide bridges is also evaluated. We propose the ClearOx-assisted oxidation of selenopeptides as a fairly generalized oxidative folding protocol.

Sin resolvase catalytic activity and oligomerization state are tightly coupled.

Serine recombinases promote specific DNA rearrangements by a cut-and-paste mechanism that involves cleavage of all four DNA strands at two sites recognized by the enzyme. Dissecting the order and timing of these cleavage events and the steps leading up to them is difficult because the cleavage reaction is readily reversible. Here, we describe assays using activated Sin mutants and a DNA substrate with a 3'-bridging phosphorothiolate modification that renders Sin-mediated DNA cleavage irreversible. We find that activating Sin mutations promote DNA cleavage rather than simply stabilize the cleavage product. Cleavage events at the scissile phosphates on complementary strands of the duplex are tightly coupled, and the overall DNA cleavage rate is strongly dependent on Sin concentration. When combined with analytical ultracentrifugation data, these results suggest that Sin catalytic activity and oligomerization state are tightly linked, and that activating mutations promote formation of a cleavage-competent oligomeric state that is normally formed only transiently within the full synaptic complex.

Addition-hydroamination reactions of propargyl cyanamides: rapid access to highly substituted 2-aminoimidazoles.

A valuable pharmacophore, the 2-aminoimidazole moiety, can be accessed with a variety of substitution patterns through an addition-hydroamination-isomerization sequence (see scheme; R(1),R(4),R(5)=alkyl; R(3)=alkyl, aryl; R(2)=H, alkyl, aryl). The synthesis of the propargyl cyanamide precursors through a three-component coupling enables the preparation of this important heterocyclic core structure in just three steps.

Interactions of the Mcm1 MADS box protein with cofactors that regulate mating in yeast.

The yeast Mcm1 protein is a member of the MADS box family of transcriptional regulatory factors, a class of DNA-binding proteins that control numerous cellular and developmental processes in yeast, Drosophila melanogaster, plants, and mammals. Although these proteins bind DNA on their own, they often combine with different cofactors to bind with increased affinity and specificity to their target sites. To understand how this class of proteins functions, we have made a series of alanine substitutions in the MADS box domain of Mcm1 and examined the effects of these mutations in combination with its cofactors that regulate mating in yeast. Our results indicate which residues of Mcm1 are essential for viability and transcriptional regulation with its cofactors in vivo. Most of the mutations in Mcm1 that are lethal affect DNA-binding affinity. Interestingly, the lethality of many of these mutations can be suppressed if the MCM1 gene is expressed from a high-copy-number plasmid. Although many of the alanine substitutions affect the ability of Mcm1 to activate transcription alone or in combination with the alpha 1 and Ste12 cofactors, most mutations have little or no effect on Mcm1-mediated repression in combination with the alpha 2 cofactor. Even nonconservative amino acid substitutions of residues in Mcm1 that directly contact alpha 2 do not significantly affect repression. These results suggest that within the same region of the Mcm1 MADS box domain, there are different requirements for interaction with alpha 2 than for interaction with either alpha1 or Ste12. Our results suggest how a small domain, the MADS box, interacts with multiple cofactors to achieve specificity in transcriptional regulation and how subtle differences in the sequences of different MADS box proteins can influence the interactions with specific cofactors while not affecting the interactions with common cofactors.