Recombinant Conus villepinii Gamma-conopressin vil

What is gamma-conopressin-vil and how does it differ from other vasopressin/oxytocin-related peptides?

Gamma-conopressin-vil is a novel conopeptide isolated from the venom of Conus villepinii, a vermivorous cone snail species from the western Atlantic Ocean. The peptide has the sequence CLIQDCPγG* (where γ represents gamma-carboxyglutamate and * indicates C-terminal amidation). Its most distinctive feature is the presence of a gamma-carboxyglutamate at position 8, rather than the neutral or basic residue typically found in this position in other vasopressin/oxytocin family peptides. This unique substitution makes it difficult to classify gamma-conopressin-vil into either the traditional vasopressin or oxytocin peptide families . While conventional members of this peptide family contain conserved residues that determine receptor binding profiles, gamma-conopressin-vil's negatively charged residue at position 8 represents a significant structural deviation that potentially confers novel pharmacological properties .

What is known about the genomic organization and expression of gamma-conopressin-vil?

Like other conopeptides, gamma-conopressin-vil is initially translated as a prepropeptide precursor. The canonical organization consists of a signal sequence at the NH2-terminal end (the "pre" region), followed by an intervening "pro" region, and the mature toxin region at the COOH-terminal end . Proteolytic cleavage of this precursor is required to generate the final functional toxin. The signal sequence of gamma-conopressin-vil would be expected to be conserved with other members of its gene superfamily, while the mature toxin region exhibits significant divergence, consistent with the hypermutation patterns observed in conopeptide evolution . The pro region likely contains recognition signals that recruit posttranslational modification enzymes, particularly those responsible for gamma-carboxylation of glutamate residues . Expression occurs in the venom duct of Conus villepinii, where the posttranslational modification enzymes are also expressed.

How is the gamma-carboxyglutamate residue formed in gamma-conopressin-vil?

The gamma-carboxyglutamate (Gla) residue in gamma-conopressin-vil is formed through posttranslational modification of a glutamate residue. This modification is catalyzed by a vitamin K-dependent gamma-glutamyl carboxylase expressed in the cone snail's venom duct . The enzyme recognizes specific signals in the pro region of the conopeptide precursor, which recruits the enzyme and directs it to modify specific glutamate residues in the mature toxin region . This posttranslational modification system is analogous to the medicinal chemistry optimization steps in drug development, representing a natural process for enhancing peptide functionality . The presence of this modification system in cone snails demonstrates their sophisticated biochemical adaptation for venom production and highlights the evolutionary significance of such modifications for conopeptide function.

What recombinant expression systems are most effective for producing functional gamma-conopressin-vil?

Recombinant expression of gamma-conopressin-vil presents unique challenges due to its posttranslational modifications, particularly the gamma-carboxylation of glutamate. Effective expression systems should account for:

• Disulfide Bond Formation: Expression hosts with oxidizing environments such as Pichia pastoris or specialized E. coli strains (e.g., SHuffle) that facilitate disulfide bond formation between the cysteine residues.

• Gamma-Carboxylation Capability: For authentic gamma-carboxyglutamate incorporation, expression systems must either:

  • Co-express vitamin K-dependent gamma-glutamyl carboxylase

  • Employ chemical synthesis followed by enzymatic modification

  • Utilize specialized mammalian cell lines with endogenous carboxylation machinery

• C-Terminal Amidation: Expression systems containing peptidylglycine alpha-amidating monooxygenase (PAM) or use of intein-based approaches for C-terminal modification.

A hybrid approach involving solid-phase peptide synthesis for the peptide backbone followed by enzymatic gamma-carboxylation may yield the most consistent results, as full recombinant expression with correct modifications presents significant technical hurdles . Alternatively, specialized mammalian expression systems engineered to overexpress the gamma-carboxylation machinery might prove viable for larger-scale production.

What analytical methods are most effective for confirming the structural integrity of recombinant gamma-conopressin-vil?

Multiple complementary analytical techniques should be employed to verify the structural integrity of recombinant gamma-conopressin-vil:

Primary Structure Confirmation:

• High-resolution mass spectrometry (HRMS) to confirm exact mass and isotopic distribution pattern characteristic of gamma-carboxyglutamate

• Tandem mass spectrometry (MS/MS) for sequence verification and posttranslational modification mapping

• Amino acid analysis to confirm composition

Secondary/Tertiary Structure Analysis:

• Nano-NMR spectroscopy, as used with the native peptide, to assess structural characteristics and calcium-induced conformational changes

• Circular dichroism (CD) spectroscopy to evaluate secondary structure elements

• Disulfide mapping using proteolytic digestion followed by MS analysis

Functional Verification:

• Calcium binding assays using isothermal titration calorimetry (ITC) or fluorescence-based methods

• Conformational change analysis in the presence vs. absence of calcium using small-angle X-ray scattering (SAXS)

• Receptor binding and activation assays compared against native peptide standards

The combination of these methods provides comprehensive validation of both structural and functional properties, ensuring that the recombinant peptide faithfully reproduces the characteristics of native gamma-conopressin-vil.

How can the calcium-binding properties of gamma-conopressin-vil be quantitatively characterized?

Quantitative characterization of calcium binding to gamma-conopressin-vil can be accomplished through multiple complementary approaches:

• Isothermal Titration Calorimetry (ITC):

  • Provides direct measurement of binding affinity (Kd), stoichiometry (n), and thermodynamic parameters (ΔH, ΔS)

  • Experiments should be conducted at physiologically relevant pH and ionic strength

  • Comparative analysis with gamma-carboxyglutamate-lacking mutants to isolate the contribution of this residue

• Fluorescence Spectroscopy:

  • If gamma-conopressin-vil contains aromatic residues sensitive to conformational changes

  • Alternatively, employing environmentally sensitive dyes that respond to calcium-induced conformational shifts

  • Titration curves can determine binding constants and cooperativity

• NMR Calcium Titration:

  • Building on the nano-NMR studies that first identified calcium-induced structural changes

  • Chemical shift perturbation analysis to map binding interface

  • Determination of dissociation constants and binding kinetics

• Calcium-Selective Electrodes:

  • Direct measurement of free calcium concentration changes upon peptide addition

  • Scatchard analysis to determine binding parameters

• Structural Studies Under Varying Calcium Concentrations:

  • X-ray crystallography or cryo-EM of peptide in calcium-free and calcium-bound states

  • Molecular dynamics simulations incorporating gamma-carboxyglutamate parameters

This multi-method approach would provide a comprehensive quantitative profile of the calcium-binding properties, which could be compared with other Gla-containing peptides to understand the novel binding mechanism suggested by previous research .

What structural changes occur in gamma-conopressin-vil upon calcium binding?

Nano-NMR spectroscopy of native gamma-conopressin-vil has revealed that the peptide undergoes significant structural changes when calcium is present . These conformational alterations are mediated by the gamma-carboxyglutamate (γ) residue at position 8. The specific structural transitions likely include:

These calcium-induced structural changes suggest gamma-conopressin-vil functions as a calcium-sensitive molecular switch, potentially enabling context-dependent bioactivity. This property distinguishes it from other vasopressin/oxytocin family members, which generally maintain consistent conformations independent of metal ion presence .

How does the binding mechanism of gamma-conopressin-vil differ from other calcium-binding peptides?

The calcium binding mechanism of gamma-conopressin-vil appears to employ a novel approach distinct from canonical calcium-binding peptides:

Comparison Table: Calcium Binding Mechanisms

The negatively charged residues in gamma-conopressin-vil, particularly the gamma-carboxyglutamate at position 8, create a unique calcium-binding pocket distinct from traditional calcium-binding motifs . This novel binding mechanism could potentially involve interactions between the gamma-carboxyglutamate and other residues in the peptide, creating a calcium-binding pocket that only forms in specific conformational states. This represents a variation from the typical calcium-binding mechanisms observed in other peptides, where multiple acidic residues create a pre-formed binding pocket .

What are the implications of calcium binding for the biological activity of gamma-conopressin-vil?

The calcium-dependent structural transitions of gamma-conopressin-vil suggest several potential implications for its biological activity:

• Environmental Sensing: The peptide may function as a calcium sensor, exhibiting different activities in environments with varying calcium concentrations. This could be particularly relevant in neuronal systems where calcium flux is a key signaling mechanism.

• Receptor Selectivity Modulation: The calcium-bound and calcium-free forms might preferentially interact with different receptor subtypes within the vasopressin/oxytocin receptor family, effectively functioning as two distinct ligands in a calcium-dependent manner.

• Synergistic Venom Action: Given that cone snail venoms function as "toxin cabals" with components acting synergistically , gamma-conopressin-vil's calcium sensitivity might coordinate its activity with other calcium-dependent processes during envenomation.

• Tissue-Specific Targeting: The peptide might exhibit specialized activity in calcium-rich microenvironments, potentially contributing to targeted prey immobilization.

• Evolutionary Adaptation: The unique calcium-binding property might represent an evolutionary adaptation to specific prey or defensive requirements of Conus villepinii.

Without detailed receptor binding and functional studies in calcium-variable conditions, the precise biological consequences of this structural switching remain speculative but represent a fascinating direction for future research .

How does gamma-conopressin-vil compare to other conopressins in terms of structure and receptor selectivity?

Gamma-conopressin-vil possesses unique structural features that distinguish it from other conopressins and likely influence its receptor selectivity profile:

Structural Comparison Table:

While most conopressins and vasopressin-like peptides contain a basic residue (Arg/Lys) at position 8, gamma-conopressin-vil's gamma-carboxyglutamate introduces a negative charge at this position . This substitution likely dramatically alters receptor interactions, as position 8 is critical for binding to vasopressin/oxytocin receptors. The other conopressins with acidic residues at position 8 (Conopressin-M1 and M2) show significantly reduced potency and altered selectivity profiles compared to traditional conopressins .

The calcium-binding capability introduces another dimension of potential receptor selectivity modulation not present in other conopressins, potentially allowing gamma-conopressin-vil to exhibit different receptor preferences in varying calcium concentrations .

What evolutionary insights can be gained from studying gamma-conopressin-vil?

Studying gamma-conopressin-vil provides several valuable evolutionary insights:

• Diversification Mechanisms: The presence of gamma-carboxyglutamate in gamma-conopressin-vil exemplifies the hypermutation mechanisms operating in conopeptide evolution. This represents an evolutionary equivalent to combinatorial library strategies in drug development .

• Posttranslational Innovation: The incorporation of gamma-carboxylation demonstrates how cone snails leverage posttranslational modifications to expand their venom peptide repertoire without requiring changes to the primary genetic sequence .

• Functional Repurposing: Conopressins likely evolved from pre-existing endogenous snail peptides involved in neurophysiological functions but were repurposed as venom components . Gamma-conopressin-vil represents a specialized adaptation of this repurposed system.

• Species-Specific Adaptations: The unique features of gamma-conopressin-vil may reflect adaptations specific to the ecological niche and prey preferences of Conus villepinii, a vermivorous species.

• Convergent Evolution: The calcium-binding capability might represent convergent evolution with other calcium-sensitive toxins, adapting similar functional principles through different structural innovations.

• Molecular Fossil Record: As a highly derived conopressin variant, gamma-conopressin-vil provides insights into the evolutionary history and plasticity of the vasopressin/oxytocin peptide family across metazoan lineages.

These evolutionary perspectives highlight how gamma-conopressin-vil exemplifies the remarkable molecular diversity generated through the specialized evolutionary processes operating in venomous animals .

What challenges exist in classifying gamma-conopressin-vil within traditional vasopressin/oxytocin peptide frameworks?

The classification of gamma-conopressin-vil within traditional vasopressin/oxytocin peptide frameworks presents several significant challenges:

• Ambiguous Sequence Classification: The eighth residue position is typically occupied by a basic residue (Arg/Lys) in vasopressins and a neutral residue (Leu/Ile) in oxytocins. Gamma-conopressin-vil's negatively charged gamma-carboxyglutamate at this position defies this classification criterion .

• Unique Posttranslational Modification: The presence of gamma-carboxyglutamate is unprecedented in the vasopressin/oxytocin family, introducing a structural and functional dimension not accounted for in traditional classification schemes .

• Calcium-Dependent Conformational Dynamics: The calcium-induced structural changes create essentially two conformational states, potentially with different classification profiles, challenging static classification approaches .

• Receptor Activity Profile Unknown: Without comprehensive receptor binding and activation data across the vasopressin/oxytocin receptor family, functional classification remains speculative.

• Evolutionary Placement: The significant sequence divergence complicates phylogenetic placement within the evolutionary tree of vasopressin/oxytocin peptides.

• Nomenclature Inconsistencies: The "conopressin" designation suggests vasopressin-like properties, but the structural features indicate a more complex identity that doesn't clearly align with either vasopressin or oxytocin subfamilies.

These classification challenges highlight the need for expanded frameworks that accommodate the structural and functional diversity exhibited by conopeptides like gamma-conopressin-vil that push the boundaries of traditional peptide hormone classification systems .

How might gamma-conopressin-vil's unique properties be leveraged for the development of novel research tools?

Gamma-conopressin-vil's distinctive properties offer several promising applications as research tools:

• Calcium-Sensitive Molecular Switch: The peptide could be developed as a calcium sensor or molecular switch for biological systems, potentially offering advantages over existing calcium indicators in specific contexts.

• Receptor Subtype Probes: If gamma-conopressin-vil shows calcium-dependent receptor selectivity, it could serve as a valuable probe for distinguishing between receptor subtypes or receptor states in complex tissues.

• Structure-Function Relationship Studies: The peptide provides a unique model for studying how gamma-carboxylation affects peptide conformation and function, informing broader questions about this posttranslational modification.

• Calcium Signaling Research: Modified versions could be developed as tools to manipulate calcium-dependent cellular processes with greater spatial or temporal precision than existing approaches.

• Peptide Engineering Templates: The calcium-binding mechanism could be incorporated into designer peptides to create novel calcium-dependent molecular switches for synthetic biology applications.

• Biosensor Development: The conformational change upon calcium binding could be coupled to reporter systems for developing new types of calcium biosensors with unique properties.

These applications leverage the natural evolutionary innovations found in gamma-conopressin-vil to create new capabilities for probing and manipulating biological systems in research contexts .

What structural modifications might enhance gamma-conopressin-vil's stability for research applications?

Several strategic modifications could enhance gamma-conopressin-vil's stability for research applications:

• Backbone Modifications:

  • N-methylation of specific amide bonds to reduce proteolytic susceptibility

  • Introduction of D-amino acids at proteolytically vulnerable positions

  • Cyclization beyond the disulfide bond (e.g., head-to-tail cyclization) to further constrain the structure

• Side Chain Alterations:

  • Substitution of non-essential residues with proteolytically resistant analogues

  • Introduction of non-natural amino acids with enhanced stability properties

  • PEGylation at specific sites to improve solubility and reduce proteolytic accessibility

• Disulfide Bond Engineering:

  • Replacement of the disulfide bond with selenocysteine pairs for enhanced redox stability

  • Addition of a second disulfide bond to further stabilize the active conformation

  • Using thioether linkages instead of disulfide bonds for redox-insensitive stability

• Gamma-Carboxyglutamate Preservation:

  • Protection strategies for the gamma-carboxyglutamate to prevent decarboxylation

  • Site-specific incorporation of non-natural calcium-binding moieties with enhanced stability

  • Engineering the local environment around the gamma-carboxyglutamate to enhance its stability

• Formulation Approaches:

  • Development of specialized buffer systems optimized for long-term stability

  • Lyophilization with appropriate excipients to maintain structure upon reconstitution

  • Encapsulation in nanoparticle or liposomal formulations for protection and controlled release

These modifications would need to be carefully balanced against preservation of the peptide's unique functional properties, particularly its calcium-binding capability and any bioactivity of interest .

What insights could gamma-conopressin-vil provide for the design of calcium-sensitive peptide therapeutics?

Gamma-conopressin-vil offers several valuable insights for designing calcium-sensitive peptide therapeutics:

• Minimal Calcium-Binding Motifs: The peptide demonstrates that a single gamma-carboxyglutamate residue, strategically positioned within a constrained peptide framework, can create effective calcium sensitivity. This minimalist approach could inform the design of smaller, more synthetically accessible calcium-responsive peptides .

• Conformational Switch Mechanisms: The calcium-induced structural transition provides a model for designing peptides with environmentally-triggered conformational changes, potentially enabling context-dependent therapeutic activity .

• Targeting Calcium-Rich Microenvironments: Gamma-conopressin-vil's properties suggest strategies for developing peptide therapeutics that selectively activate in calcium-rich microenvironments, such as:

  • Tumor microenvironments with dysregulated calcium

  • Sites of bone remodeling

  • Specific synaptic compartments

  • Regions of tissue damage with calcium influx

• Dual-Mode Therapeutics: The calcium-dependent conformational change suggests possibilities for dual-mode therapeutics that could exhibit different activities (e.g., receptor agonism vs. antagonism) depending on local calcium concentrations.

• Posttranslational Modification Strategy: The gamma-carboxylation demonstrates how specific posttranslational modifications can dramatically alter peptide properties, providing a template for incorporating similar modifications in designed therapeutics.

• Constrained Peptide Design: The integration of calcium-binding functionality within a disulfide-constrained framework illustrates strategies for maintaining peptide stability while incorporating responsive elements.

These insights could help address persistent challenges in peptide therapeutics, such as context-dependent activity, tissue-specific targeting, and controlled bioavailability .