Recombinant Salmonella schwarzengrund Cobalt transport protein CbiN (cbiN)

What is the structural composition of the CbiN protein?

The CbiN protein from Salmonella schwarzengrund is a membrane protein composed of two transmembrane helices connected by an extracytoplasmic loop of 37 amino acid residues. It spans 93 amino acids in total (1-93aa) and functions as an auxiliary component in the cobalt transport system . The full amino acid sequence is: MKKTLMLLAMVVALVILPFFINHGGEYGGSDGEAESQIQAIAPQYKPWFQPLYEPASGEI ESLLFTLQGSLGAAVIFYILGYCKGKQRRDDRA . When analyzing the protein's structure-function relationship, it's essential to note that any deletion in the CbiN loop abolishes transport activity, indicating the critical nature of this region for protein function .

How does CbiN function within the cobalt transport system?

CbiN functions as part of the energy-coupling factor (ECF) transporter system specifically designed for cobalt uptake in prokaryotic cells. Within this system, CbiN temporarily interacts with the CbiMQO2 components to facilitate cobalt transport . Experimental evidence shows that CbiN induces significant Co2+ transport activity even in the absence of CbiQO2 when co-expressed with the S component CbiM or as a Cbi(MN) fusion protein . This indicates that CbiN plays a crucial role in the transport mechanism beyond merely being a structural component. In the working model of the transporter, CbiN appears to function in coupling conformational changes between CbiQ and CbiM during the transport process .

What is the relationship between CbiN and other components of the ECF transporter complex?

The CbiN protein is part of a modular ECF transporter complex called CbiMNQO, where:

• CbiM functions as the substrate-binding component (corresponding to EcfS in other ECF transporters)

• CbiN serves as an auxiliary component unique to metal-specific systems

• CbiQ acts as the integral membrane scaffold component (corresponding to EcfT)

• CbiO represents the cytoplasmic ATP binding/hydrolysis component (corresponding to EcfA)

The interaction between these components creates a functional unit where:

Reconstitution experiments demonstrate that the substrate-binding subunit CbiM stimulates CbiQO's basal ATPase activity, highlighting the interconnected nature of these components . The transport process requires coordinated rotation or toppling of both CbiQ and CbiM, with CbiN serving as a coupling element for conformational changes between these components .

How do the interactions between CbiN and CbiM facilitate metal transport?

The interaction between CbiN and CbiM occurs primarily through protein-protein contacts between segments of the CbiN loop and loops in CbiM, as confirmed by cysteine-scanning mutagenesis and crosslinking experiments . These specific interactions are critical for proper function:

• The ordered structure of the CbiN loop, observed through electron paramagnetic resonance analysis after site-directed spin labeling, interacts with specific regions of CbiM .

• The N-terminal loop of CbiM, which contains three of the four metal ligands, becomes partially immobilized when interacting with wild-type CbiN but remains completely immobile in inactive variants with CbiN loop deletions .

• Decreased dynamics of inactive forms can be detected through solid-state nuclear magnetic resonance of isotope-labeled protein in proteoliposomes .

These interactions facilitate metal insertion into the binding pocket by creating the proper conformational arrangement for cobalt uptake. The dynamic nature of this interaction is essential, as reduced mobility in the N-terminal loop of CbiM correlates with loss of transport activity .

What conformational changes occur in the CbiMNQO complex during the transport cycle?

The CbiMNQO complex undergoes significant conformational changes during the transport cycle, driven by ATP binding and hydrolysis. Structural analysis reveals:

• The complex has been captured in an inward-open conformation (CbiMQO structure) .

• ATP binding induces conformational changes in CbiO, as demonstrated by the structure of CbiO in β, γ-methyleneadenosine 5′-triphosphate-bound closed conformation .

• The transport process likely involves rotation or toppling of both CbiQ and CbiM components, with CbiN functioning as a coupler for these conformational changes .

What is the functional significance of the L1 loop in CbiM?

When examining the structural elements involved in cobalt transport, researchers should pay special attention to:

• The positioning of the L1 loop relative to the metal binding site

• Conformational changes in the loop during different stages of transport

• Interactions between the L1 loop and other components, particularly CbiN

For experimental validation of L1 loop function, site-directed mutagenesis of key residues within this region, followed by transport assays, would provide valuable insights into its precise role in the gating mechanism .

What expression systems are optimal for recombinant production of CbiN?

E. coli has been successfully employed as an expression system for the recombinant production of CbiN, as demonstrated in multiple studies . When designing an expression protocol for CbiN, researchers should consider:

• Vector selection: Vectors allowing N-terminal His-tagging have proven effective for CbiN expression and subsequent purification .

• Expression conditions: The specific conditions (temperature, induction time, media composition) should be optimized based on the exact E. coli strain used.

• Protein extraction: As a membrane protein, CbiN requires appropriate detergent-based extraction methods.

A general expression protocol based on published methods would include:

• Amplification of the cbiN gene from Salmonella schwarzengrund

• Cloning into an appropriate expression vector with His-tag

• Transformation into E. coli expression strain

• Culture growth and protein expression induction

• Cell harvesting and membrane protein extraction

• Affinity purification using the His-tag

For the CbiMNQO complex studies, genes encoding all four subunits (CbiM, CbiN, CbiQ, and CbiO) were amplified and co-expressed to obtain functional complexes for structural and functional analyses .

What methods can be used to verify CbiN-CbiM interactions experimentally?

Several complementary techniques have been successfully employed to verify and characterize the interactions between CbiN and CbiM:

• Cysteine-scanning mutagenesis and crosslinking: This approach involves introducing cysteine residues at predicted interaction sites and then using crosslinking reagents to confirm proximity. This method has confirmed protein-protein contacts between segments of the CbiN loop and loops in CbiM .

• Electron paramagnetic resonance (EPR) analysis: Site-directed spin labeling followed by EPR has been used to observe the ordered structure of the CbiN loop and its interactions with CbiM .

• Solid-state nuclear magnetic resonance (NMR): This technique using isotope-labeled protein in proteoliposomes has detected decreased dynamics in inactive forms of the complex, providing insights into the functional significance of mobility .

• Reconstitution experiments: By reconstituting different combinations of CbiMNQO subunits and determining the resulting ATPase and transport activities, researchers have established functional interactions between components .

• In silico predictions: Computational models can predict protein-protein contacts between CbiN and CbiM loops, which can then be experimentally verified .

A comprehensive approach would combine multiple techniques to establish both structural proximity and functional relevance of the observed interactions.

How can transport activity of the CbiMNQO complex be measured?

Measuring the transport activity of the CbiMNQO complex requires specialized techniques that can detect the movement of cobalt ions across membranes. Established methodologies include:

• Cellular uptake assays: Utilizing cells expressing various combinations of CbiMNQO components to measure Co²⁺ uptake. This approach has demonstrated that CbiN induces significant Co²⁺ transport activity even in the absence of CbiQO₂ when co-expressed with CbiM .

• Reconstituted proteoliposome assays: Purified components can be reconstituted into proteoliposomes to measure transport activity in a controlled system. This approach allows for precise manipulation of the system's components and conditions .

• ATPase activity coupling: Since transport is coupled to ATP hydrolysis, measuring the ATPase activity can provide an indirect measure of transport. Studies have shown that the substrate-binding subunit CbiM stimulates CbiQO's basal ATPase activity .

What are the optimal storage conditions for recombinant CbiN protein?

Recombinant CbiN protein requires specific storage conditions to maintain stability and activity. Based on established protocols:

The protein should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles . For working aliquots, storage at 4°C for up to one week is recommended . The optimal storage buffer composition is Tris/PBS-based buffer with 6% Trehalose, pH 8.0 . For long-term storage, addition of glycerol to a final concentration of 50% is recommended .

It's important to note that repeated freezing and thawing is not recommended as it can lead to protein degradation and loss of activity . When planning experiments, researchers should consider:

• Preparing small aliquots during initial reconstitution to minimize freeze-thaw cycles

• Monitoring protein stability over time using appropriate quality control methods

• Using fresh preparations for critical experiments where maximal activity is required

How should recombinant CbiN be reconstituted after lyophilization?

For optimal reconstitution of lyophilized recombinant CbiN protein, follow these methodological steps:

• Briefly centrifuge the vial prior to opening to bring the contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation)

• Prepare appropriate aliquots for long-term storage at -20°C/-80°C

This reconstitution protocol ensures proper solubilization of the protein while maintaining its structural integrity and functional properties. The addition of glycerol serves as a cryoprotectant to prevent damage during freezing and thawing cycles.

When working with the reconstituted protein for functional assays or structural studies, researchers should verify protein integrity through analytical methods such as SDS-PAGE before proceeding with experiments.

What analytical methods can verify the quality of recombinant CbiN preparations?

Several analytical techniques can be employed to assess the quality and integrity of recombinant CbiN preparations:

• SDS-PAGE: The purity of recombinant CbiN should be greater than 90% as determined by SDS-PAGE . This technique allows visualization of protein size and purity.

• Western blotting: Using antibodies against the His-tag or CbiN itself can confirm the identity of the protein and detect any degradation products.

• Mass spectrometry: This technique can provide precise molecular weight determination and confirm the protein sequence through peptide mapping.

• Circular dichroism (CD) spectroscopy: CD can assess the secondary structure content of the protein, which is particularly relevant for membrane proteins like CbiN with defined transmembrane helices.

• Functional assays: Ultimately, the quality of CbiN preparations should be verified through functional assays, such as interaction studies with CbiM or transport activity measurements when combined with other components of the transport system .

For membrane proteins like CbiN, it's also important to verify proper folding and insertion into membranes or detergent micelles, which can be assessed through techniques such as tryptophan fluorescence or limited proteolysis assays.

How can mutagenesis studies identify critical functional regions of CbiN?

Mutagenesis approaches have been instrumental in identifying functionally important regions of CbiN, particularly the extracytoplasmic loop that connects its two transmembrane helices. Key methodological considerations include:

• Deletion mutagenesis: Studies have shown that any deletion in the 37-amino acid CbiN loop abolishes transport activity, demonstrating the critical nature of this region . Researchers can design systematic deletion series to map essential segments within the loop.

• Cysteine scanning mutagenesis: This approach involves replacing individual amino acids with cysteine residues, which can then be modified chemically or used for crosslinking studies. This method has successfully identified protein-protein contacts between segments of the CbiN loop and loops in CbiM .

• Alanine scanning: Systematic replacement of amino acids with alanine can identify residues that are critical for function without drastically altering protein structure.

• Site-directed mutagenesis of predicted interaction sites: Based on in silico predictions of protein-protein contacts, specific residues can be targeted for mutation to validate their role in interactions with CbiM .

For any mutagenesis strategy, functional validation through transport activity assays is essential to connect structural alterations to functional outcomes. Additionally, structural analyses of mutant proteins using techniques like EPR or solid-state NMR can reveal how mutations affect protein dynamics and interactions .

What role does CbiN play in bacterial cobalt homeostasis?

CbiN plays a critical role in bacterial cobalt homeostasis as an essential component of the ECF-type cobalt transport system. The significance of this system extends beyond simple nutrient acquisition:

• Nutrient acquisition in limited environments: As part of the CbiMNQO transporter, CbiN helps bacteria acquire cobalt from environments where this essential micronutrient may be limited .

• Supporting metabolic processes: Cobalt is required for various metabolic processes in bacteria, including the synthesis of vitamin B12 (cobalamin), which serves as a cofactor for several enzymes involved in central metabolism.

• Growth dependency: Research indicates that ABC-type cobalt transport systems like CbiMNQO are essential for bacterial growth under cobalt-limited conditions , highlighting their physiological importance.

• Potential antimicrobial target: Given its essential role in bacterial nutrition, the CbiMNQO system represents a potential target for developing new antimicrobial strategies that disrupt cobalt homeostasis.

Understanding the regulation of CbiN expression and activity in response to environmental cobalt levels could provide insights into bacterial adaptation mechanisms. Additionally, comparative studies across different bacterial species might reveal evolutionary adaptations in cobalt acquisition strategies.

How does the structure-function relationship in CbiN compare to other metal transport proteins?

The structure-function relationship of CbiN presents several unique features when compared to other metal transport proteins:

• Auxiliary component in ECF transporters: Unlike vitamin-specific ECF transporters, metal-specific systems like CbiMNQO rely on additional proteins such as CbiN with essential but specialized functions . This represents a distinct evolutionary adaptation for metal transport.

• Minimal structural organization: With just two transmembrane helices connected by an extracytoplasmic loop, CbiN has a relatively simple structure compared to many other metal transporters, yet plays a critical role in transport function .

• Dynamic interaction mechanism: CbiN functions through temporary interactions with other transport components rather than forming a static structural element of the transport pathway . This dynamic association represents a distinctive mechanistic approach to metal transport.

• Loop-mediated interactions: The functional importance of the CbiN loop in mediating interactions with CbiM highlights a unique structural feature that distinguishes this system from other metal transporters .

When examining metal transport systems across different organisms, researchers should consider:

• How the simplicity of CbiN's structure contributes to its functional specialization

• Whether similar auxiliary components exist in other metal transport systems

• How the dynamic interaction mechanism of CbiN compares to the more rigid structural arrangements in other transporters

This comparative approach can provide insights into the evolution of metal transport mechanisms and potentially inform the design of inhibitors targeting specific transport systems.

How can structural information about CbiN inform drug design targeting bacterial metal acquisition?

The structural details of CbiN and the CbiMNQO complex provide valuable insights for drug design strategies targeting bacterial metal acquisition systems:

• Targeting critical interfaces: The interaction interface between CbiN and CbiM represents a potential target for small molecule inhibitors. Disrupting this interaction could prevent proper cobalt transport without targeting highly conserved ATP-binding domains that might lead to off-target effects .

• Exploiting the essential loop structure: The extracytoplasmic loop of CbiN, which is essential for transport activity, presents a potential target for antibodies or peptide mimetics that could block its function. Since any deletion in this loop abolishes transport activity, it represents a vulnerable point in the system .

• Structure-based screening approaches: The available structural information on CbiMQO complex in its inward-open conformation provides a template for structure-based virtual screening of compound libraries to identify potential inhibitors .

• Allosteric inhibition strategies: Understanding the conformational changes induced by ATP binding and product release within the CbiMNQO transporter complex could inform the design of allosteric inhibitors that lock the transporter in an inactive conformation .

Development of such inhibitors could lead to novel antimicrobial agents that function by disrupting essential metal acquisition pathways, potentially addressing the growing challenge of antimicrobial resistance.

What methodological advances could improve structural studies of CbiN and related proteins?

Advancing our understanding of CbiN structure and function requires innovative methodological approaches:

• Cryo-electron microscopy (cryo-EM): This rapidly evolving technique has revolutionized structural biology of membrane proteins and could provide high-resolution structures of the complete CbiMNQO complex in different conformational states during the transport cycle .

• Advanced EPR techniques: Building on existing EPR studies , techniques such as double electron-electron resonance (DEER) could provide detailed information about distances between specific sites in CbiN and its binding partners during the transport process.

• Time-resolved structural methods: Techniques that capture transient structural states could reveal the dynamic aspects of CbiN function, particularly during its interactions with CbiM and other components of the transport system.

• Single-molecule approaches: Methods such as single-molecule FRET could track conformational changes in individual CbiMNQO complexes during transport cycles, providing insights into potential heterogeneity in transport mechanisms.

• Native mass spectrometry: This technique could help characterize the composition and stability of the CbiMNQO complex and subcomplexes under different conditions, providing insights into assembly and disassembly processes.

By combining these methodological advances, researchers could build a more comprehensive picture of CbiN function within the dynamic context of the complete transport system.

How can artificial intelligence tools support research on CbiN and related transport proteins?

Artificial intelligence (AI) tools offer significant potential to accelerate research on CbiN and related transport proteins:

• Literature analysis and knowledge extraction: AI tools like Consensus can help researchers quickly identify and synthesize information about CbiN from the scientific literature, identifying patterns and relationships that might not be immediately apparent .

• Structural prediction and modeling: While some structural information is available for the CbiMNQO complex , AI tools like AlphaFold or RoseTTAFold could predict structures of variants or related proteins, potentially revealing conservation patterns in structural elements across different organisms.

• Molecular dynamics simulations: AI-enhanced molecular dynamics approaches could model the dynamic interactions between CbiN and other components of the transport system, providing insights into conformational changes during the transport cycle.

• Drug discovery acceleration: AI-driven virtual screening approaches could identify potential inhibitors of CbiN function or CbiN-CbiM interactions, potentially leading to new antimicrobial strategies.

• Experimental design optimization: AI tools could help optimize expression conditions, purification protocols, and crystallization conditions for structural studies of CbiN and the complete CbiMNQO complex.

Researchers can leverage specialized AI research tools such as Elicit.org for literature-based brainstorming and Scite.ai for citation analysis and verification of research claims . These approaches could significantly accelerate the pace of discovery in CbiN research while ensuring scientific rigor.