HSCB Human
Description
Primary Structure
HSCB Human is a single polypeptide chain comprising 231 amino acids (residues 30–235) with a molecular mass of 26.7 kDa . A 25-amino-acid His-tag is fused at the N-terminus for purification purposes .
| Property | Detail |
|---|---|
| Expression Host | E. coli |
| Formulation | 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol |
| Purity | >90% (SDS-PAGE) |
| Storage | 4°C (short-term), -20°C (long-term with carrier protein) |
Tertiary Structure
The 3.0 Å crystal structure reveals an L-shaped conformation with two conserved domains:
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J-domain: Interacts with HSP70 chaperones (e.g., HscA in bacteria).
A unique N-terminal tetracysteine metal-binding domain (CXXC motifs) coordinates zinc or iron, structurally resembling rubredoxin . This domain is absent in bacterial homologs .
Gene Organization
The HSCB gene is located at chromosome 22q11-12, spanning six exons and five introns . Exon 1 encodes a mitochondrial presequence, while exons 2–6 cover the J- and C-domains .
Tissue-Specific Expression
HSCB is highly expressed in mitochondria-rich tissues:
This pattern mirrors frataxin, linking HSCB to mitochondrial energetics and potential roles in diseases like Friedreich’s ataxia .
Iron-Sulfur Cluster Biogenesis
HSCB acts as a co-chaperone with HSP70 to deliver nascent Fe-S clusters from scaffold proteins (e.g., ISCU) to client proteins . Key interactions include:
Beyond Fe-S Clusters
Recent studies highlight ISC-independent roles:
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FOG1 nuclear translocation: Required for erythropoiesis and megakaryopoiesis .
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Phosphorylation-dependent signaling: Modulates PI3K pathways .
Subcellular Localization
HSCB exists in two isoforms:
This dual localization suggests compartment-specific roles, though phosphorylation status does not alter distribution .
Disease Associations
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Mitochondrial disorders: Low CNS expression may predispose to neurodegeneration .
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Hematologic defects: HSCB knockdown impairs erythropoiesis and megakaryopoiesis .
Comparative Analysis
| Feature | Human HSCB | Bacterial HscB | Yeast Jac1p |
|---|---|---|---|
| N-terminal domain | Tetracysteine metal-binding | Absent | Absent |
| J-domain | ATPase activation of HSP70 | ATPase activation of HscA | ATPase activation of HSC70 |
| C-domain | Substrate binding | Scaffold protein binding | Scaffold protein binding |
| Localization | Mitochondria + cytosol | Cytosol | Mitochondria |
Product Specs
Q&A
What is the basic structure of human HscB and how does it differ from bacterial homologs?
Human HscB is an L-shaped protein that shares approximately 29% sequence identity with its Escherichia coli homolog. The most significant structural difference is that human HscB possesses a novel N-terminal domain (N-domain) capable of binding a metal ion, which is absent in the bacterial version. The human protein can be divided into three topologically distinct domains: the N-domain (residues 39-71), the J-domain (residues 72-145), and the C-domain (residues 156-235) .
The N-domain forms a small globular structure harboring a metal-binding site coordinated by four cysteine residues (Cys41, Cys44, Cys58, and Cys61) located on two apposed β-hairpins. This domain is stabilized by hydrogen bonds between specific residues, including Trp48, Gln65, and Ala63 .
What is the consensus motif in the N-domain of human HscB?
Multiple sequence alignment of HscB homologs has revealed a consensus motif in the N-domain: CWXCX9–13FCXXCXXXQ. The highly conserved residues Trp42 and Gln65 are crucial parts of this consensus motif. This tetracysteine motif coordinates a metal via the cysteine residues positioned on the two rubredoxin knuckles .
What is the biological role of human HscB in mitochondria?
Human HscB functions as a co-chaperone in the biogenesis of iron-sulfur proteins within mitochondria. It assists in the delivery of the iron-sulfur scaffold protein IscU to the molecular chaperone HscA (a member of the Hsp70 family) and enhances the intrinsic ATPase activity of the chaperone. This interaction facilitates the transfer of iron-sulfur clusters from the scaffold protein to acceptor apoproteins, likely by destabilizing the IscU·[FeS] complex .
What crystallization approaches have been successful for human HscB protein?
The full-length mature recombinant human co-chaperone Δ(1–21)HscB did not yield diffraction quality crystals in initial crystallization screens. Researchers found success by creating additional N-terminal truncation constructs. Specifically, the N-terminal truncated version Δ(1–29)HscB produced diffraction quality crystals that supported structure determination .
For structural studies, selenomethionine-labeled proteins were expressed following standard protocols for cloning, protein expression, and purification. The crystal structure was determined at 3.0Å resolution using single-wavelength anomalous diffraction method near the selenium K absorption edge .
How can normal mode analysis be applied to study HscB conformational dynamics?
Normal mode analysis can be applied to HscB to study its conformational flexibility and potential functional movements. Different models can be used to define the potential, ranging from complicated chemical force fields to simpler elastic network models.
A residue-level approach called the distance network model defines interactions between residues based on distances between their respective atoms. In this model, atomic contacts at different distances are given different weights (spring constants) that are added together to form the total Hessian matrix. This matrix can be diagonalized to analyze the normal modes, particularly focusing on the lowest-frequency modes that represent the largest, most global deformations of the protein structure .
What experimental considerations are important when studying the metal-binding properties of human HscB?
When investigating the metal-binding properties of human HscB's N-domain, researchers should consider:
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The tetracysteine motif (Cys41, Cys44, Cys58, and Cys61) coordinates the metal ion, so mutations of these residues would likely disrupt metal binding.
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The consensus sequence CWXCX9–13FCXXCXXXQ is conserved across eukaryotic HscB homologs, suggesting its functional importance .
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The identity of the coordinated metal in vivo should be determined using appropriate spectroscopic techniques.
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The structural stability of the N-domain is likely dependent on metal coordination, so metal chelation experiments might provide insights into the domain's stability and function.
What crystallographic methods are recommended for resolving difficult regions in HscB structures?
The crystal structure determination of human HscB revealed challenges with certain protein regions. For instance, two segments of molecule A (residues 48–53 and 148–159) showed markedly lower map quality, and residue Asp54 could not be modeled satisfactorily, remaining an outlier in the Ramachandran plot .
To address such challenges, researchers can employ:
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Iterative manual building in molecular visualization software (e.g., Coot) combined with refinement programs (e.g., REFMAC5).
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Model mask-guided density modification trials as implemented in density improvement strategies (e.g., autoSHARP).
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Use of noncrystallographic symmetry constraints during refinement when multiple molecules are present in the asymmetric unit.
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Multiple cycles of iterative building, refinement, and model mask constrained density modification to improve map quality .
How can researchers effectively analyze the interface between domains in human HscB?
Analysis of domain interfaces in HscB provides important insights into protein stability and function. The interface between the N- and J-domains buries a surface area of approximately 610 Ų, indicating significant interaction between these regions .
Researchers should:
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Calculate buried surface areas at domain interfaces using structural analysis software.
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Identify key residues participating in inter-domain interactions.
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Analyze the conservation of interface residues across homologs to infer functional importance.
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Consider using mutagenesis of interface residues to test the functional importance of domain interactions.
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Apply molecular dynamics simulations to study the dynamics of domain interfaces and potential conformational changes.
What are the key structural and functional differences between human and bacterial HscB proteins?
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Domain Organization: Human HscB possesses three domains (N-domain, J-domain, and C-domain), whereas E. coli HscB lacks the N-terminal metal-binding domain .
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Metal Binding: The N-domain of human HscB contains a tetracysteine motif that coordinates a metal ion, a feature absent in bacterial homologs .
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Sequence Identity: The two proteins share only 29% sequence identity in aligned regions, indicating substantial evolutionary divergence .
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Domain Orientation: The relative orientations of the J- and C-domains differ between the human and bacterial proteins, which may reflect functional adaptations .
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Size: Human HscB is larger due to the additional N-terminal domain, which may confer additional regulatory functions in the more complex eukaryotic cellular environment.
How might the differences between human and bacterial HscB impact experimental design?
When designing experiments to study human HscB, researchers should consider:
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Expression systems: Full-length human HscB may require eukaryotic expression systems for proper folding and metal incorporation.
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Truncation constructs: As seen in the structural studies, N-terminal truncations (e.g., Δ(1–29)HscB) may be needed for successful crystallization .
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Metal binding analysis: Experiments should assess the role of the N-domain in metal binding and its impact on protein function.
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Interaction studies: Investigations of protein-protein interactions should account for potential differences in binding partners between human and bacterial systems.
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Functional assays: Assays developed for bacterial HscB may need modification to account for the structural and functional differences in the human protein.
What techniques can be used to study the HPD motif function in human HscB?
The J-domain of human HscB contains the signature HPD motif (His102-Pro103-Asp104), which is implicated in interactions with molecular chaperones of the Hsp70 family . To study this functionally important motif, researchers can employ:
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Site-directed mutagenesis of the HPD residues followed by functional assays to assess the impact on chaperone interactions.
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Co-immunoprecipitation or pull-down assays to quantify the effect of HPD mutations on binding to Hsp70 chaperones.
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Surface plasmon resonance or isothermal titration calorimetry to determine binding kinetics and thermodynamics of wild-type versus mutant HscB with Hsp70 chaperones.
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Nuclear magnetic resonance (NMR) spectroscopy to investigate structural changes upon interaction with Hsp70 chaperones.
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Crosslinking studies combined with mass spectrometry to identify interaction interfaces.
What experimental controls should be included when studying human HscB in iron-sulfur protein biogenesis?
When investigating the role of human HscB in iron-sulfur protein biogenesis, researchers should include:
Product Science Overview
The HscB Iron-Sulfur Cluster Co-Chaperone, also known as Human Recombinant HscB, is a highly conserved mitochondrial protein that plays a crucial role in the assembly and maintenance of iron-sulfur (Fe-S) clusters. These clusters are essential cofactors involved in various biochemical processes, including electron transport, enzyme catalysis, and regulation of gene expression. HscB is a member of the heat shock cognate B (HscB) family of proteins and functions as a co-chaperone in the iron-sulfur cluster assembly machinery.
HscB primarily interacts with the scaffold protein IscU and the chaperone protein HSPA9 (also known as mortalin or GRP75). The interaction between HscB and IscU is critical for the successful assembly of Fe-S clusters. HscB facilitates the transfer of nascent Fe-S clusters from IscU to target apoproteins by stimulating the ATPase activity of HSPA9 . This process ensures the proper incorporation of Fe-S clusters into recipient proteins, which is vital for their functional activity.
The solution structure of HscB has been extensively studied using nuclear magnetic resonance (NMR) spectroscopy. These studies have revealed that HscB consists of a J-domain, a linker region, and a C-domain. The J-domain is responsible for binding to HSPA9, while the C-domain interacts with IscU . The conserved patch of residues in the C-domain is the principal binding site for IscU, and mutations in this region can significantly affect the binding affinity and functional activity of HscB .
HscB is essential for mitochondrial iron-sulfur cluster biogenesis, a process that is evolutionarily conserved across species. In humans, defects in the HscB gene can lead to various mitochondrial disorders, including congenital sideroblastic anemia (CSA). CSA is characterized by the presence of ringed sideroblasts in the bone marrow, resulting from impaired heme biosynthesis and mitochondrial iron metabolism . Mutations in HscB can disrupt the assembly of Fe-S clusters, leading to defective mitochondrial function and cellular iron homeostasis .
