COMMD9 Human

How does the COMMD9 structure compare to other COMMD family proteins?

COMMD9's HN domain shows structural similarity to equivalent domains in other COMMD proteins, particularly COMMD1, although with notable differences. Compared to COMMD1, COMMD9's HN domain contains an additional N-terminal α-helix that packs on top of the structure, providing greater stability . Additionally, COMMD9's central α4 helix forms a core element extending the length of the HN domain, whereas the equivalent helix in COMMD1 is bent and oriented differently .

Electrostatic surface analysis reveals two basic patches and a negatively charged region on the HN domain, while sequence conservation mapping shows highly conserved surface residues primarily in the α1 helix region . This structurally conserved but sequence-variable domain architecture is characteristic of the COMMD family, whose members share topological similarity despite sequence diversity.

What evolutionary insights can be derived from COMMD9's structure?

Intriguingly, COMMD9 shows remarkable structural similarity to bacterial proteins from Chlamydia species, specifically CT584 (C. trachomatis) and Cpn0803 (C. pneumoniae), with DALI Z-scores exceeding 7.5 . These bacterial orthologs possess modular structures analogous to COMMD9, with α-helical N-terminal domains and α/β C-terminal domains structurally similar to the HN and COMM domains respectively .

The primary structural differences include two additional C-terminal α-helices in the bacterial proteins and the absence of α1 and α5 helices in their N-terminal domains . This unexpected structural conservation between human COMMD9 and bacterial proteins suggests possible evolutionary significance and may provide insights into the fundamental cellular functions of these domains.

What are the primary cellular functions of human COMMD9?

COMMD9 exhibits multiple crucial cellular functions:

• Modulation of Cullin-RING E3 ubiquitin ligase (CRL) complexes: COMMD9 influences the activity of these essential complexes involved in protein ubiquitination and subsequent degradation .

• Regulation of NF-kappa-B activation: COMMD9 appears to down-regulate the activation of this key transcription factor, suggesting a role in inflammatory response and cell survival pathways .

• Ion transport regulation: COMMD9 modulates Na+ transport in epithelial cells by regulating the apical cell surface expression of amiloride-sensitive sodium channel (ENaC) subunits, indicating its importance in ion homeostasis .

• Membrane trafficking: As part of larger membrane-associated complexes, COMMD9 participates in endosomal protein trafficking and sorting mechanisms .

These diverse functions highlight COMMD9's significance in fundamental cellular processes related to protein degradation, transcriptional regulation, and ion channel trafficking.

What protein complexes does COMMD9 participate in?

COMMD9 functions as a component of several multiprotein complexes:

• CCC Complex: COMMD9 associates with coiled-coil domain-containing proteins CCDC22 and CCDC93 to form the CCC complex, which localizes to endosomes and regulates membrane protein trafficking .

• Commander Complex: The CCC complex further associates with a stable heterotrimeric assembly of VPS29, C16orf62, and DSCR3 to form the larger "Commander" or CCC/Retriever complex. Additional proteins including SNX17, RanBP1, SH3GLB1, and FAM45a are also thought to associate with this macromolecular assembly .

• COMMD-COMMD Interactions: COMMD9 can form both homodimers and heterodimers with other COMMD family proteins through its C-terminal COMM domain. Experimental evidence demonstrates that COMMD9 interacts directly with COMMD5, though with varying affinities for different family members (e.g., weaker interactions with COMMD6 and COMMD10) .

This complex network of interactions positions COMMD9 as a hub protein involved in coordinating membrane trafficking and cellular signaling pathways.

What expression systems are optimal for recombinant COMMD9 production?

Escherichia coli has been successfully employed for recombinant COMMD9 expression, as demonstrated in multiple studies . For structural and biochemical investigations, the following methodological considerations are important:

• Expression constructs: Full-length human COMMD9 (198 amino acids) can be expressed with purification tags (commonly His6 tags) for affinity purification .

• Domain-specific constructs: Separate expression of the HN domain and COMM domain may be beneficial for structural studies. The COMM domain alone has been successfully expressed and crystallized .

• Selenomethionine labeling: For crystallographic phase determination, selenomethionine labeling using the method described by Van Duyne et al. has proven effective for COMMD9 .

• Buffer optimization: For structural and biophysical studies, buffers containing 10 mM Tris (pH 8.0), 100 mM NaCl, and 2 mM DTT have been successfully used .

The resulting recombinant protein achieves >95% purity and is suitable for various analytical methods including SDS-PAGE and mass spectrometry .

What structural determination methods have been successful for COMMD9?

Multiple complementary structural biology techniques have been applied to characterize COMMD9:

Data processing utilized software including iMOSFLM for data integration, AIMLESS for scaling, AUTOSOL and PHENIX for phase calculation and refinement, and COOT for model building .

How can protein-protein interactions of COMMD9 be effectively studied?

Several complementary approaches have proven effective for investigating COMMD9's diverse protein interactions:

• Co-expression GST pull-down assays: Co-expression of GST-tagged COMMD proteins with His-tagged prey proteins in E. coli followed by affinity purification using glutathione sepharose beads effectively captures interaction partners. Western blotting can confirm these interactions, particularly when proteins have similar molecular weights .

• Cross-linking mass spectrometry (MS): Non-deuterated BS3 cross-linker has been successfully used to capture transient interactions between COMMD9 and binding partners such as the NN-CH domain of CCDC93. Analysis of the cross-linked peptides by MS enables identification of specific interaction surfaces .

• Domain-specific interaction studies: Isolated COMM domains can recapitulate many full-length protein interactions, demonstrating that this domain mediates most protein-protein interactions. In contrast, the HN domain of COMMD1 shows no detectable binding to other COMMD proteins .

An important observation is that COMMD-COMMD interactions appear to occur during co-translation and cannot be reconstituted by mixing pre-formed homodimeric proteins, suggesting that these interactions involve formation of specific dimeric structures analogous to the COMMD9 COMM domain crystal structure .

How does the COMMD9 COMM domain facilitate protein interactions?

The COMM domain plays a central role in mediating COMMD9's diverse protein interactions through several structural features:

• Dimerization interface: Crystal structures reveal that the COMM domain forms dimers, providing a structural basis for both homo- and heteromeric interactions with other COMMD proteins .

• Surface-exposed lysines: Cross-linking mass spectrometry identified three key lysine residues (K100, K133, and K152) that are solvent-accessible and involved in protein interactions. Notably, K133 and K152 are located on contiguous surfaces of the β-sheets, forming a likely binding surface for interaction partners such as CCDC93 .

• Promiscuity with specificity: While COMMD proteins can form various heterodimeric complexes through their COMM domains, there are preferences in binding affinities. For example, COMMD9 binds weakly to COMMD6, while COMMD10 binds most strongly to COMMD2 and COMMD5 but only weakly with COMMD9 .

• Co-translational assembly: COMMD-COMMD interactions appear to occur during protein synthesis and cannot be reconstituted by mixing pre-formed homodimers, suggesting that dimerization involves specific conformational states available only during protein folding .

These findings suggest that the COMM domain serves as a versatile protein-protein interaction module with both broad binding capacity and selective preferences that define COMMD9's functional specificity.

What is the significance of COMMD9's role in ion transport regulation?

COMMD9's role in regulating ion transport, particularly through modulation of the amiloride-sensitive sodium channel (ENaC), represents a critical function with potential physiological and pathological implications:

• Epithelial Na+ transport: COMMD9 regulates apical cell surface expression of ENaC subunits, directly influencing sodium homeostasis in epithelial tissues .

• Trafficking mechanism: As part of the CCC and Commander complexes, COMMD9 likely participates in the endosomal trafficking machinery that controls ENaC delivery to or retrieval from the plasma membrane .

• Potential disease relevance: Dysregulation of epithelial sodium transport is implicated in various pathological conditions including hypertension, cystic fibrosis, and certain kidney disorders. COMMD9's role in this process suggests potential involvement in these conditions .

• Integration with ubiquitination pathways: COMMD9's function in modulating cullin-RING E3 ubiquitin ligase complexes may connect protein ubiquitination with ion channel regulation, suggesting coordinated control of protein degradation and membrane trafficking .

This multifaceted role positions COMMD9 as a potential target for investigating ion transport disorders and developing therapeutic strategies for conditions involving epithelial sodium channel dysfunction.

What aspects of COMMD9 function remain poorly understood?

Despite significant advances in understanding COMMD9 structure and interactions, several important knowledge gaps remain:

• Tissue-specific functions: The expression pattern and tissue-specific roles of COMMD9 have not been fully characterized. Zebrafish expression data indicates specific expression patterns that may provide clues to tissue-specific functions .

• Regulatory mechanisms: How COMMD9 activity is itself regulated remains unclear. Potential post-translational modifications, subcellular localization signals, and expression control mechanisms warrant further investigation.

• Structural dynamics: While static structures of COMMD9 domains have been determined, the conformational dynamics during complex formation and function remain poorly understood.

• Evolutionary conservation: The striking structural similarity between COMMD9 and bacterial proteins from Chlamydia species suggests potential evolutionary relationships or convergent evolution that deserve deeper exploration .

• Pathological implications: The connection between COMMD9 dysfunction and specific human diseases requires further investigation, particularly given its roles in fundamental cellular processes like protein trafficking and ion transport.

Addressing these knowledge gaps will require integrated approaches combining structural biology, cell biology, and physiological studies to fully elucidate COMMD9's complex functional network.

What methodological approaches could advance COMMD9 research?

Several emerging methodological approaches could significantly enhance our understanding of COMMD9:

• Cryo-electron microscopy: While X-ray crystallography has provided high-resolution structures of individual domains, cryo-EM could reveal the architecture of larger COMMD9-containing complexes such as the complete Commander complex.

• Proximity labeling proteomics: Techniques like BioID or APEX could identify transient or context-dependent COMMD9 interaction partners in living cells, expanding our understanding beyond stable interactions identified through pull-down assays.

• Live-cell imaging: Advanced fluorescence microscopy approaches could track COMMD9 dynamics during endosomal trafficking and membrane protein regulation.

• CRISPR-based functional genomics: Systematic genetic screens could identify functional relationships and redundancies between COMMD9 and other cellular components.

• Integrative structural biology: Combining multiple structural techniques (X-ray crystallography, SAXS, cryo-EM, cross-linking MS) could provide comprehensive models of COMMD9 in its native functional complexes.

These approaches would complement the established methods (crystallography, SAXS, pull-downs) that have already yielded significant insights into COMMD9 structure and function.