DVU_3336 is a KdpD-like sensory protein located in the kdp locus of D. vulgaris Hildenborough, positioned between kdpD and the structural kdpFABC genes. The protein contains an N-terminal KdpD sensor domain (IPR003852) and a C-terminal UspA stress-response domain (IPR006016), along with a P-loop NTPase fold. Critically, the catalytic histidine kinase domains (HisKA and HATPase_c) required for phosphotransfer activity are located on the adjacent DVU3335 gene, not on DVU_3336. This protein likely functions as a sensory/adapter component that participates in osmotic and K+ stress sensing, feeding into the KdpD/KdpE two-component regulatory system that controls expression of the high-affinity K+ transporter KdpFABC. The UspA domain suggests involvement in stress sensing, while the P-loop NTPase domain may enable nucleotide binding for signal modulation.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005886 plasma membrane | IEA GO_REF:0000118 | ACCEPT | Summary: KdpD-like proteins are typically membrane-associated sensors in the two-component KdpD/KdpE system. The N-terminal KdpD domain (IPR003852) described in UniProt as "Signal transduction histidine kinase osmosensitive K+ channel sensor N-terminal" is typically membrane-associated. However, DVU_3336 lacks predicted transmembrane helices in its UniProt entry, and the membrane association is inferred by analogy rather than direct evidence (Freeman 2013). Reason: Plasma membrane localization is consistent with the role of KdpD-like sensors, which function at the membrane interface to sense osmotic/K+ changes. While direct localization evidence for DVU_3336 is lacking, the TreeGrafter annotation based on phylogenetic inference (PANTHER:PTN008137371) supports membrane association. The KdpD domain in canonical systems requires membrane proximity for signal integration (Freeman 2013). Supporting Evidence: DOI:10.1371/journal.ppat.1003201 There appear to be no reports of KdpE or KdpD being located anywhere other than the cytoplasm and cytoplasmic membrane |
| GO:0000155 phosphorelay sensor kinase activity | IEA GO_REF:0000120 | REMOVE | Summary: This annotation is problematic. While DVU_3336 contains a KdpD N-terminal sensor domain (IPR003852), it LACKS the catalytic histidine kinase domains (HisKA/HATPase_c) required for actual kinase activity. Domain analysis shows DVU_3336 has only the sensory portion (aa 13-218) and a UspA domain (aa 243-357). The adjacent DVU3335 contains the conserved histidine kinase catalytic domains. The IEA annotation was generated from InterPro:IPR003852 combined with PANTHER, but this represents an over-annotation - having a sensor domain associated with kinases does not confer kinase activity itself. Reason: DVU_3336 lacks the conserved histidine kinase catalytic domains (HisKA containing the phosphorylatable His residue, and HATPase_c for ATP binding/hydrolysis). Domain architecture shows only the N-terminal KdpD sensor domain (Pfam PF02702, aa 13-218) and UspA domain (Pfam PF00582, aa 243-357). The actual histidine kinase domains are on adjacent DVU3335 in DvH. Assigning kinase activity based on a sensor domain that is often found fused with kinase domains (in other organisms) but is separate in this operon organization is an over-annotation. Supporting Evidence: DOI:10.1371/journal.ppat.1003201 Mutation analysis suggests that an activating stimulus causes the inhibition of the phospho-KdpE-specific phosphatase activity of KdpD, leading to an accumulation of phospho-KdpE. file:DESVH/Q725T9/Q725T9-deep-research-falcon.md In DvH, the conserved histidine kinase catalytic domains are explicitly assigned to the adjacent DVU3335 gene |
| GO:0000160 phosphorelay signal transduction system | IEA GO_REF:0000120 | ACCEPT | Summary: DVU_3336 is located within the kdp locus and contains domains consistent with participation in the KdpD/KdpE phosphorelay signal transduction system. While it does not possess catalytic kinase activity itself, the KdpD sensor domain and UspA stress-sensing domain suggest it participates in sensing environmental signals (K+ limitation, osmotic stress) that are transduced through this system. Reason: Involvement in phosphorelay signal transduction is appropriate even for non-catalytic components. The protein's location in the kdp operon, its KdpD sensor domain, and UspA domain all indicate participation in sensing and signal relay within the KdpD/KdpE two-component system. The term describes involvement in the process, not necessarily catalytic function. Supporting Evidence: DOI:10.1371/journal.ppat.1003201 The genes kdpD and kdpE together encode the KdpD/KdpE TCS, which is well-studied for its regulation of the Kdp-ATPase potassium ion (K +) pump operon kdpFABC. file:DESVH/Q725T9/Q725T9-deep-research-falcon.md DVU_3336 is located within the kdp locus of DvH. Operon maps in DvH position DVU_3336 between kdpD and the structural kdpFABC genes |
| GO:0004673 protein histidine kinase activity | IEA GO_REF:0000002 | REMOVE | Summary: This annotation is incorrect. It was assigned via InterPro:IPR003852 mapping, but IPR003852 is "Signal transduction histidine kinase KdpD N-terminal" - a SENSOR domain, not a catalytic kinase domain. The InterPro-to-GO mapping conflates the sensor domain with kinase activity because in many organisms these domains are fused on a single protein. In DVU_3336, only the sensor domain is present; the catalytic domains are on the adjacent DVU3335. Reason: DVU_3336 lacks the HisKA domain containing the conserved phosphorylatable histidine residue and lacks the HATPase_c domain required for ATP hydrolysis and phosphotransfer. The protein is 379 aa with domains at positions 13-218 (KdpD N-terminal sensor) and 243-357 (UspA). There is no sequence region corresponding to histidine kinase catalytic function. This is a clear case of over-annotation where a sensory domain associated with kinases was incorrectly annotated as having kinase activity. Supporting Evidence: file:DESVH/Q725T9/Q725T9-deep-research-falcon.md the conserved histidine kinase catalytic domains are explicitly assigned to the adjacent DVU3335 gene UniProt:Q725T9 Signal transduction histidine kinase osmosensitive K+ channel sensor N-terminal |
| GO:0005737 cytoplasm | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA annotation for cytoplasm localization. KdpD-like sensors are membrane-associated but their cytoplasmic portions (including the UspA domain) extend into the cytoplasm. The UspA domain and P-loop NTPase region would be cytoplasmic. This is consistent with the dual annotation of plasma membrane and cytoplasm. Reason: For membrane-associated signaling proteins, the cytoplasmic annotation captures the localization of the functional domains (KdpD sensor C-terminal region, UspA domain) that interact with cytoplasmic partners and nucleotides. The annotation is not contradictory with plasma membrane localization but rather complementary. |
| GO:0016020 membrane | IEA GO_REF:0000002 | ACCEPT | Summary: Generic membrane annotation from InterPro:IPR003852 (KdpD N-terminal domain). This is consistent with the more specific plasma membrane annotation. KdpD-like proteins are membrane-associated sensors. Reason: Membrane localization is consistent with KdpD-like sensors. While more specific than "plasma membrane" would be ideal, this general term is acceptable as a broader annotation alongside the more specific plasma membrane annotation. Both annotations capture the membrane association of this sensory protein. |
| GO:0016301 kinase activity | IEA GO_REF:0000043 | REMOVE | Summary: Annotation derived from UniProtKB keyword KW-0418 (Kinase). This keyword was likely assigned based on the protein name containing "histidine kinase domain" and the presence of KdpD-related domains. However, DVU_3336 lacks catalytic kinase domains - it has only sensory/regulatory domains. Reason: DVU_3336 does not possess kinase activity. The "kinase" in the protein name refers to the type of signaling system (histidine kinase two-component system) it participates in, not its own enzymatic function. Domain analysis confirms absence of HisKA and HATPase_c catalytic domains. The P-loop NTPase fold present in the protein may bind ATP but does not confer kinase activity - it is a distinct nucleotide-binding fold found in many non-kinase proteins. Supporting Evidence: UniProt:Q725T9 Potassium channel histidine kinase domain protein/universal stress protein |
| GO:0016740 transferase activity | IEA GO_REF:0000043 | REMOVE | Summary: Annotation derived from UniProtKB keyword KW-0808 (Transferase). This is a parent term of kinase activity and was assigned for the same incorrect reason - the protein name and associated keywords suggest kinase function that the protein does not actually possess. Reason: DVU_3336 is not a transferase. It lacks the catalytic domains required for phosphotransfer activity. This annotation cascades from the incorrect kinase-related keywords. The protein functions as a sensor/adapter in signal transduction, not as an enzyme catalyzing group transfer. |
| GO:0034220 monoatomic ion transmembrane transport | IEA GO_REF:0000043 | REMOVE | Summary: Annotation derived from UniProtKB keyword KW-0407 (Ion transport). This is incorrect - DVU_3336 is a sensor protein that participates in REGULATING ion transport (via the KdpD/KdpE system controlling KdpFABC), but it is NOT itself an ion transporter. The protein lacks transmembrane transporter domains. The "potassium channel" in the protein name refers to its role in sensing/ regulating potassium channel/transporter expression, not to being a channel itself. Reason: DVU_3336 is NOT an ion transporter. It is a sensory protein that participates in the regulatory system controlling the high-affinity K+ transporter KdpFABC. The KdpFABC complex (encoded by separate genes) performs the actual ion transport. The keywords "Ion channel" and "Ion transport" in UniProt are misleading - they reflect the pathway context (kdp locus) rather than the protein's direct function. This is a clear over-annotation where regulatory components were annotated with the function they regulate. Supporting Evidence: DOI:10.1371/journal.ppat.1003201 This in turn binds to an operator sequence in the promoter DNA to activate transcription of kdpFABC [9]. file:DESVH/Q725T9/Q725T9-deep-research-falcon.md DVU_3336 likely participates in sensing and signaling that controls high-affinity K+ uptake via KdpFABC |
| GO:0005524 ATP binding | ISS file:DESVH/Q725T9/Q725T9-deep-research-falcon.md | NEW | Summary: DVU_3336 contains a P-loop NTPase domain (IPR027417) which is associated with nucleotide binding. This domain is distinct from the HATPase_c domain found in histidine kinases. The P-loop (Walker A motif) is a conserved nucleotide- binding element found in many ATP/GTP-binding proteins. Reason: The presence of the P-loop NTPase domain (IPR027417) and Rossmann-like fold (IPR014729) strongly suggests ATP binding capability. This is a structural prediction based on conserved domain architecture. ATP binding by UspA-domain proteins and P-loop containing proteins is well documented. This annotation captures a likely molecular function that is not currently annotated. Supporting Evidence: UniProt:Q725T9 InterPro; IPR027417; P-loop_NTPase |
| GO:0006970 response to osmotic stress | ISS file:DESVH/Q725T9/Q725T9-deep-research-falcon.md | NEW | Summary: The KdpD/KdpE system responds to osmotic stress and K+ limitation. DVU_3336 contains both a KdpD N-terminal sensor domain (osmosensitive K+ channel sensor) and a UspA domain. UspA domains are characteristic of universal stress proteins that respond to various stresses including osmotic stress. Reason: The protein's domain architecture (osmosensitive KdpD sensor domain plus UspA stress domain) and operon context (kdp locus controlling K+ homeostasis under stress) strongly support involvement in osmotic stress response. The KdpD/KdpE system is a well-characterized osmotic stress response pathway, and DVU_3336 is positioned within this regulatory network. Supporting Evidence: DOI:10.1371/journal.ppat.1003201 Expression of the operon is triggered by three stimuli perceived and integrated by the HK KdpD: K + concentration, osmolarity, and ATP concen- tration [8]. UniProt:Q725T9 Signal transduction histidine kinase osmosensitive K+ channel sensor N-terminal |
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Download this section (compressed HTML)Q: What is the precise mechanism by which DVU_3336 and DVU3335 interact in D. vulgaris?
Q: Does DVU_3336 have any autonomous regulatory function, or does it strictly depend on DVU3335 for signal relay?
Q: What specific signals (K+ concentration, osmolarity) does the UspA domain of DVU_3336 sense?
Experiment: Deletion mutant of DVU_3336 to assess phenotype under K+ limitation and osmotic stress
Hypothesis: DVU_3336 deletion will impair osmotic stress response and K+ homeostasis
Experiment: Co-immunoprecipitation or bacterial two-hybrid to determine protein-protein interactions with DVU3335 and KdpE
Hypothesis: DVU_3336 physically interacts with DVU3335 to modulate histidine kinase activity
Experiment: ATP binding assays to confirm P-loop NTPase domain function
Hypothesis: The P-loop NTPase domain of DVU_3336 binds ATP
Experiment: Transcriptomic analysis of kdpFABC expression in DVU_3336 mutant background
Hypothesis: DVU_3336 is required for proper induction of kdpFABC under K+ limitation
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