mlcD

UniProt ID: Q7Z2B8
Organism: Dictyostelium discoideum
Review Status: COMPLETE
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Gene Description

MlcD is a calmodulin-like myosin light chain that serves as the dedicated light chain for the class I myosin MyoD (Myo1D) in Dictyostelium discoideum. It contains four EF-hand motifs but only retains low-affinity calcium binding (Kd ~52 uM) due to mutations in key Ca2+-coordinating residues of EF-hands 2-4, and thus cannot function as a physiological calcium sensor. Two copies of MlcD bind the MyoD IQ motifs in a calcium-insensitive manner, forming the regulatory light chain component of the Myo1D complex. Through its association with MyoD, MlcD participates in membrane dynamics at macropinocytic cups and actin waves.

Existing Annotations Review

GO Term Evidence Action Reason
GO:0005509 calcium ion binding
IBA
GO_REF:0000033 		KEEP AS NON CORE
Summary: Phylogenetic inference of calcium ion binding is partially supported but misleading. MlcD retains one functional EF-hand with low-affinity calcium binding (Kd 52 uM), well above physiological levels. The protein cannot function as a sensor of physiological calcium, so this annotation overstates the functional relevance.
Reason: MlcD does bind calcium but with very low affinity that is not physiologically relevant. The IBA inference from calmodulin family members with high-affinity calcium binding is misleading for this divergent paralog. The annotation is technically correct but not representative of the core function.
Supporting Evidence:
PMID:12826013
yielding apparent dissociation constants ( K'(d)) of 52 microM for Ca2+ and 450 microM for Mg2+
PMID:12826013
The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+
GO:0005737 cytoplasm
IBA
GO_REF:0000033 		ACCEPT
Summary: Cytoplasmic localization is correct and well-supported by direct evidence from the same gene product.
Reason: MlcD is a cytoplasmic protein as shown by direct experimental evidence, consistent with its role as a myosin light chain that operates in the cytoplasm.
Supporting Evidence:
PMID:12826013
FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD
GO:0030234 enzyme regulator activity
IBA
GO_REF:0000033 		REMOVE
Summary: This IBA annotation from the calmodulin family is questionable for MlcD. While calmodulin regulates numerous enzymes, MlcD has diverged substantially and specifically associates only with MyoD. There is no direct evidence that MlcD regulates enzyme activity per se; it functions as a structural light chain of a motor complex rather than as an enzyme regulator.
Reason: MlcD is a dedicated structural light chain for MyoD, not a general enzyme regulator. Unlike calmodulin, which activates kinases and phosphatases, MlcD binds only MyoD in a calcium-insensitive manner. The IBA inference from calmodulin family members does not transfer to this divergent paralog that has lost both high-affinity calcium binding and promiscuous target engagement.
Supporting Evidence:
PMID:12826013
FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC
PMID:12826013
The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+
GO:0000226 microtubule cytoskeleton organization
IBA
GO_REF:0000033 		REMOVE
Summary: This IBA annotation transfers microtubule-related functions from calmodulin orthologs. There is no evidence that MlcD or its partner MyoD are involved in microtubule cytoskeleton organization. MyoD/Myo1D is an actin-based motor that functions at membranes and actin waves.
Reason: MlcD is a dedicated myosin I light chain with no known connection to microtubule biology. All experimental evidence places MlcD/MyoD in the context of actin-based structures (macropinocytic cups, actin waves). The IBA inference from calmodulin, which does participate in microtubule biology through different targets, does not transfer to this divergent paralog.
Supporting Evidence:
PMID:31774725
Myo1D localized, like short-tail Myo1E and Myo1F, in the PIP3-rich region encircled by the actin wave
PMID:12826013
FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC
GO:0005509 calcium ion binding
IEA
GO_REF:0000002 		KEEP AS NON CORE
Summary: InterPro2GO annotation from the EF-hand domain (IPR002048). While MlcD does contain EF-hand domains and retains low-affinity calcium binding, this annotation overstates functional importance since the binding is not physiologically relevant.
Reason: The InterPro2GO inference from EF-hand domains is technically correct (MlcD does bind calcium) but functionally misleading. Three of four EF-hands have degenerate calcium-coordinating residues, and the measured Kd of 52 uM is far above physiological calcium concentrations. This is not a core function.
Supporting Evidence:
PMID:12826013
EF-hands 2-4 contain mutations in key Ca2+-co-ordinating residues that would be predicted to impair Ca2+ binding
PMID:12826013
The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+
GO:0005737 cytoplasm
IEA
GO_REF:0000044 		ACCEPT
Summary: Correct annotation based on UniProt subcellular location. MlcD is a cytoplasmic protein.
Reason: Cytoplasmic localization is well established for MlcD from direct experimental evidence.
Supporting Evidence:
PMID:12826013
FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD
GO:0005509 calcium ion binding
IDA
PMID:12826013
The Dictyostelium class I myosin, MyoD, contains a novel lig... 		KEEP AS NON CORE
Summary: Direct experimental evidence demonstrates calcium binding by MlcD via electrospray ionization MS and tryptophan fluorescence quenching. However, the binding is low affinity (Kd 52 uM), so while the annotation is experimentally supported, it does not represent the core evolved function.
Reason: The IDA evidence from De La Roche et al. 2003 is solid - MlcD does bind calcium as demonstrated by ESI-MS and fluorescence spectroscopy. However, the authors themselves conclude the binding is not physiologically relevant. This is a vestigial capacity from calmodulin ancestry, not a core function.
Supporting Evidence:
PMID:12826013
Electrospray ionization MS of MlcD in the presence of Ca2+ and La3+ showed the presence of one major and one minor metal-binding site
PMID:12826013
yielding apparent dissociation constants ( K'(d)) of 52 microM for Ca2+ and 450 microM for Mg2+
PMID:12826013
The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+
GO:0016459 myosin complex
IDA
PMID:12826013
The Dictyostelium class I myosin, MyoD, contains a novel lig... 		MODIFY
Summary: MlcD is part of the MyoD (Myo1D) myosin complex. This is a core annotation. The more specific term GO:0045160 myosin I complex would be more precise since MyoD is a class I myosin.
Reason: MlcD co-purifies with MyoD as part of a myosin I complex (two copies of MlcD per MyoD heavy chain). The annotation is correct but the more specific child term myosin I complex is more appropriate since MyoD is definitively a class I myosin.
Proposed replacements: myosin I complex
Supporting Evidence:
PMID:12826013
MyoD, a long-tailed class I myosin, co-purified with two copies of a 16 kDa light chain
PMID:21671662
Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD)
GO:0032036 myosin heavy chain binding
IPI
PMID:12826013
The Dictyostelium class I myosin, MyoD, contains a novel lig... 		MODIFY
Summary: MlcD specifically binds the MyoD heavy chain at its IQ motifs. Since MyoD is a class I myosin, the more specific term GO:0032037 myosin I heavy chain binding is more appropriate.
Reason: MlcD binds specifically to MyoD (a class I myosin) heavy chain IQ motifs. The general term myosin heavy chain binding is correct but the child term myosin I heavy chain binding more precisely captures the specificity of this interaction.
Proposed replacements: myosin I heavy chain binding
Supporting Evidence:
PMID:12826013
Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs
PMID:12826013
FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC
GO:0062201 actin wave
IDA
PMID:31774725
Basic-hydrophobic sites are localized in conserved positions... 		ACCEPT
Summary: MlcD localizes to actin waves as part of the Myo1D complex. Specifically, Myo1D localizes to the PIP3-rich region encircled by the actin wave, not the actin wave crest itself. This is a valid localization annotation reflecting the behavior of the Myo1D complex.
Reason: Brzeska et al. 2019 directly demonstrated Myo1D (and hence its light chain MlcD) localizing to actin wave structures, specifically in the PIP3-rich region. This is a core localization for the Myo1D motor complex.
Supporting Evidence:
PMID:31774725
Myo1D localized, like short-tail Myo1E and Myo1F, in the PIP3-rich region encircled by the actin wave
GO:0070687 macropinocytic cup cytoskeleton
IDA
PMID:31774725
Basic-hydrophobic sites are localized in conserved positions... 		ACCEPT
Summary: Myo1D localizes to macropinocytic cups, and MlcD as its light chain would be present there. The annotation to macropinocytic cup cytoskeleton is well supported by the localization data.
Reason: Brzeska et al. 2019 showed that Myo1D localizes to macropinocytic protrusions, cups, and freshly internalized vesicles. As the obligate light chain, MlcD is present at these sites.
Supporting Evidence:
PMID:31774725
Myo1D is also found in pinocytic protrusions and cups, and freshly internalized vesicles
file:DICDI/mlcD/mlcD-deep-research-bioreason-sft.md
Its recruitment to large endocytic structures is consistent with GO:0070685 macropinocytic cup

Core Functions

MlcD serves as the dedicated regulatory light chain subunit of the Myo1D (MyoD) class I myosin complex. Two copies of MlcD bind the MyoD heavy chain IQ motifs in a calcium-insensitive manner, providing structural stabilization of the motor complex neck region.

Molecular Function:
myosin I heavy chain binding
Cellular Locations:
cytoplasm actin wave macropinocytic cup cytoskeleton
In Complex:
myosin I complex
Supporting Evidence:
  • PMID:12826013
    MyoD, a long-tailed class I myosin, co-purified with two copies of a 16 kDa light chain
  • PMID:12826013
    Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs
  • PMID:21671662
    Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD)

References

GO_REF:0000002
Gene Ontology annotation through association of InterPro records with GO terms
GO_REF:0000033
Annotation inferences using phylogenetic trees
GO_REF:0000044
Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt
PMID:12826013
The Dictyostelium class I myosin, MyoD, contains a novel light chain that lacks high-affinity calcium-binding sites.
  • MlcD is a 16 kDa calmodulin-like protein that co-purifies with MyoD as two copies per heavy chain.
    "MyoD, a long-tailed class I myosin, co-purified with two copies of a 16 kDa light chain"
  • MlcD has four EF-hands but only retains low-affinity calcium binding due to degenerate calcium-coordinating residues in EF-hands 2-4.
    "MlcD comprises four EF-hands; however, EF-hands 2-4 contain mutations in key Ca2+-co-ordinating residues that would be predicted to impair Ca2+ binding"
  • MlcD binds calcium with a Kd of 52 uM, far above physiological calcium concentrations.
    "yielding apparent dissociation constants ( K'(d)) of 52 microM for Ca2+ and 450 microM for Mg2+"
  • MlcD cannot function as a physiological calcium sensor.
    "The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+"
  • MlcD binding to MyoD is calcium-insensitive.
    "Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs"
  • MlcD specifically associates with MyoD and not with MyoB or MyoC.
    "FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC"
PMID:16415352
Identification and characterization of an 8-kDa light chain associated with Dictyostelium discoideum MyoB, a class I myosin.
  • MlcB is a distinct light chain for MyoB, confirming that each long-tailed myosin I has a unique light chain partner.
    "FLAG-MlcB expressed in Dictyostelium co-immunoprecipitated with MyoB but not with the related class myosins and MyoD"
PMID:21671662
Identification of calmodulin and MlcC as light chains for Dictyostelium myosin-I isozymes.
  • Each long-tailed Dictyostelium myosin I has a unique light chain, while short-tailed ones use calmodulin.
    "Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD), whereas the short-tailed myosins-I, MyoA and MyoE, have the multifunctional calmodulin as a light chain"
  • Light chain diversity contributes to functional specialization of myosin I isoforms.
    "The diversity in light chain composition is likely to contribute to the distinct cellular functions of each myosin-I isozyme"
PMID:31774725
Basic-hydrophobic sites are localized in conserved positions inside and outside of PH domains and affect localization of Dictyostelium myosin 1s.
  • Myo1D localizes to the PIP3-rich region inside actin waves, distinct from Myo1B which colocalizes with the actin wave itself.
    "Myo1D localized, like short-tail Myo1E and Myo1F, in the PIP3-rich region encircled by the actin wave"
  • Myo1D localizes to macropinocytic cups and freshly internalized vesicles.
    "Myo1D is also found in pinocytic protrusions and cups, and freshly internalized vesicles"

📚 Additional Documentation

Deep Research Bioreason Sft

(mlcD-deep-research-bioreason-sft.md) 
---
provider: bioreason-pro
model: SFT
source: huggingface.co/datasets/wanglab/protein_catalogue
date: '2026-04-04'
uniprot_id: Q7Z2B8
gene: mlcD
organism: Dictyostelium discoideum (Social amoeba)
---

## Thinking Trace

The architecture begins with IPR050230 (Calmodulin/Myosin light chain/Troponin C-like family, residues 2–146), which defines a compact, helical EF-hand calcium sensor specialized for binding target peptides such as IQ motifs on myosins. This family scaffold is refined by IPR011992 (EF-hand domain pair homologous superfamily, residues 5–144), indicating two EF-hand pairs arranged N- and C-terminally. The N-terminal pair is marked by IPR002048 EF-hand domains at residues 8–43, 12–40, and 14–60, with IPR018247 specifying the canonical Ca2+-coordinating loop at residues 21–33 within this first pair. The C-terminal pair is demarcated by IPR002048 domains at residues 79–114 and 81–144, with overlapping confidence windows (83–111 and 83–144) that emphasize a functional Ca2+-binding site in the second lobe. This ordered layout—two Ca2+-binding pairs within a calmodulin-like fold—causes Ca2+-dependent conformational switching that exposes hydrophobic pockets for target engagement.

This EF-hand architecture directly encodes GO:0005509 calcium ion binding as the primary molecular function. The calmodulin/myosin light chain-like family context predicts high-affinity recognition of IQ motifs on myosin heavy chains, supporting GO:0032036 myosin heavy chain binding. In this protein, the paired EF-hands act as a Ca2+ sensor that toggles between low- and high-affinity states for myosin II regulatory segments, thereby modulating motor activity.

Calcium-gated control of myosin II provides a mechanistic route to multiple cellular processes. By binding the myosin II heavy chain, the protein can tune filament assembly and spacing at the cleavage furrow, accounting for GO:0043520 regulation of myosin II filament assembly and GO:0000281 mitotic cytokinesis. Targeting to the division plane is consistent with GO:0071976 protein localization to cell division site. The same Ca2+-dependent exposure of target pockets can regulate interactions with actin regulators, explaining GO:0030041 actin filament polymerization and GO:0030837 negative regulation of actin filament polymerization, as different target states promote or restrain filament growth depending on Ca2+ levels. In endocytic and phagocytic remodeling, Ca2+ spikes recruit this sensor to the cortex to coordinate myosin II contractility and actin assembly, aligning with GO:0006911 phagocytosis, engulfment and GO:0006907 pinocytosis. Rapid Ca2+-triggered assembly at membrane lesions leverages the same mechanism for GO:0001778 plasma membrane repair. By modulating the activity of myosin II and associated enzymes, the protein exerts GO:0043462 regulation of ATP-dependent activity. Its Ca2+-dependent binding to kinases, phosphatases, and small GTPase regulators provides a route to GO:0043087 regulation of GTPase activity and GO:0032092 positive regulation of protein binding. In Dictyostelium development, these cytoskeletal and signaling effects influence multicellular aggregation dynamics (GO:0031152 aggregation involved in sorocarp development) and can restrain proliferative cycles, supporting GO:1903665 negative regulation of asexual reproduction. Nuclear actomyosin and envelope mechanics suggest a role in GO:0006997 nucleus organization.

The EF-hand sensor lacks transmembrane segments and is optimized for dynamic association with actomyosin assemblies, placing it in the GO:0016459 myosin complex and at the GO:0030864 cortical actin cytoskeleton. Its recruitment to large endocytic structures is consistent with GO:0070685 macropinocytic cup. These localizations follow directly from Ca2+-induced exposure of myosin-binding pockets and affinity for IQ motifs on myosin II at the cortex and division site.

A coherent mechanistic model emerges: Ca2+ binding to the N-terminal EF-hands primes the sensor to engage myosin II heavy chain IQ motifs, while the C-terminal EF-hands stabilize the complex and tune affinity. This engagement alters myosin II filament assembly and spacing, thereby controlling contractility and actin polymerization at the cleavage furrow, during macropinocytosis, phagocytosis, and membrane repair. Through Ca2+-dependent docking, the protein likely associates with ROCO-family kinases (roco11, roco6, roco5, roco8) to couple cytoskeletal state to kinase signaling; with the serine/threonine-protein phosphatase 2B catalytic subunit to reset phosphorylation states in a Ca2+-dependent manner; with an inositol 1,4,5-trisphosphate receptor-like protein A to integrate Ca2+ release dynamics; and with the Ras GTPase-activating-like protein gapA to coordinate small GTPase cycles. Together, these interactions position the protein as a Ca2+-sensor that orchestrates myosin II–driven mechanics and signaling at the cortex and division site.

## Functional Summary

A calmodulin-like calcium sensor in Dictyostelium that binds calcium to expose target-specific pockets and engage the myosin II heavy chain. By tuning myosin II filament assembly and contractility at the cortex and cleavage furrow, it coordinates actin polymerization dynamics for cytokinesis, macropinocytosis, phagocytic engulfment, and rapid plasma membrane repair. Its calcium-dependent interactions with kinases, phosphatases, and small GTPase regulators integrate cytoskeletal mechanics with signaling, influencing aggregation during development and restraining asexual proliferation. The protein operates within myosin-containing assemblies at the cortical actin cytoskeleton and macropinocytic cups.

## UniProt Summary

Binds calcium. Binds to the myosin II heavy chain.

## InterPro Domains

    - IPR050230: Calmodulin/Myosin light chain/Troponin C-like (family) [2-146]
- IPR011992: EF-hand domain pair (homologous_superfamily) [5-144]
- IPR002048: EF-hand domain (domain) [8-43]
- IPR002048: EF-hand domain (domain) [12-40]
- IPR002048: EF-hand domain (domain) [14-60]
- IPR018247: EF-Hand 1, calcium-binding site (binding_site) [21-33]
- IPR002048: EF-hand domain (domain) [79-114]
- IPR002048: EF-hand domain (domain) [81-144]
- IPR002048: EF-hand domain (domain) [83-111]
- IPR002048: EF-hand domain (domain) [83-144]

## GO Term Predictions

### Molecular Function


### Biological Process


### Cellular Component

Notes

(mlcD-notes.md)

mlcD (Dictyostelium discoideum) - Research Notes

Gene identity

mlcD encodes the myosin-ID light chain (UniProt Q7Z2B8), a 147 amino acid calmodulin-like protein that serves as the dedicated light chain for the class I myosin MyoD (Myo1D) in Dictyostelium discoideum.

Key findings from literature

Discovery and biochemical characterization (De La Roche et al. 2003)

MlcD was discovered as a novel 16 kDa light chain that co-purifies with MyoD. Two copies of MlcD associate with each MyoD heavy chain PMID:12826013. The protein has four EF-hand motifs but only weak calcium binding PMID:12826013. Critically, MlcD binds calcium only at low affinity PMID:12826013. This low affinity means it is not a physiological calcium sensor PMID:12826013. MlcD binding to MyoD is calcium-insensitive PMID:12826013. MlcD is specific to MyoD and does not interact with MyoB or MyoC PMID:12826013. The protein is localized to the cytoplasm PMID:12826013.

Comparison with MlcB (Crawley et al. 2006)

The related light chain MlcB for MyoB was characterized and compared with MlcD. MlcD was confirmed as specifically associated with MyoD PMID:16415352. This work established that each long-tailed class I myosin in Dictyostelium has a distinct light chain.

Diversity of myosin I light chains (Crawley et al. 2011)

A comprehensive study established the complete light chain complement: MyoB-MlcB, MyoC-MlcC, MyoD-MlcD for the long-tailed myosin I isoforms, while short-tailed MyoA and MyoE use calmodulin PMID:21671662. The diversity in light chain composition contributes to distinct cellular functions PMID:21671662.

MyoD/Myo1D localization and membrane targeting (Brzeska et al. 2019)

MlcD localizes with MyoD (Myo1D) to macropinocytic structures and actin waves. Myo1D localizes to macropinocytic cups and the PIP3-enriched region inside actin waves PMID:31774725. This is distinct from Myo1B, which localizes to the actin wave itself PMID:31774725. Myo1D is found in pinocytic protrusions and cups, and freshly internalized vesicles PMID:31774725.

Summary of core biology

MlcD is a structural/regulatory light chain subunit of the myosin I complex containing MyoD (Myo1D). It is a calmodulin-related protein with degenerate EF-hands that retains only low-affinity calcium binding. Its primary molecular function is binding the MyoD heavy chain at IQ motifs in a calcium-insensitive manner, serving as an essential structural subunit of the Myo1D motor complex. Through this association, MlcD participates in macropinocytosis-related membrane dynamics and actin wave organization.

Bioreason Sft Review

(mlcD-bioreason-sft-review.md)

BioReason-Pro SFT Review: mlcD (Dictyostelium discoideum)

Source: mlcD-deep-research-bioreason-sft.md

  • Correctness: 2/5
  • Completeness: 3/5

Functional Summary Review

The BioReason SFT functional summary describes mlcD as:

A calmodulin-like calcium sensor in Dictyostelium that binds calcium to expose target-specific pockets and engage the myosin II heavy chain.

This contains a fundamental error: MlcD does not bind the myosin II heavy chain. MlcD is the dedicated light chain for MyoD, which is a class I myosin. The paper that originally characterized MlcD explicitly identifies it as associated with "MyoD, a long-tailed class I myosin" (PMID:12826013). The summary also incorrectly describes MlcD as a "calcium sensor," when the original characterization explicitly states that "the low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+" (PMID:12826013, Kd = 52 uM).

The summary further claims:

By tuning myosin II filament assembly and contractility at the cortex and cleavage furrow, it coordinates actin polymerization dynamics for cytokinesis, macropinocytosis, phagocytic engulfment, and rapid plasma membrane repair.

This is largely fabricated. There is no evidence that MlcD regulates myosin II filament assembly, participates in cytokinesis, controls actin filament polymerization, mediates phagocytic engulfment, or is involved in plasma membrane repair. The thinking trace arrives at these functions by assuming MlcD acts as a calcium-dependent regulator of myosin II, which is incorrect on both counts (wrong myosin class, and calcium-insensitive binding).

The summary correctly notes localization to macropinocytic cups, which is supported by Brzeska et al. 2019 (PMID:31774725), though this localization derives from the Myo1D heavy chain rather than from MlcD acting independently.

Its calcium-dependent interactions with kinases, phosphatases, and small GTPase regulators integrate cytoskeletal mechanics with signaling

There is no evidence for any of these interactions. MlcD specifically and exclusively associates with the MyoD heavy chain. Unlike calmodulin, which promiscuously regulates many targets, MlcD has diverged to become a dedicated structural subunit.

Comparison with interpro2go

The InterPro2GO annotation (GO_REF:0000002) for MlcD predicts GO:0005509 calcium ion binding based on the EF-hand domain (IPR002048). This is technically correct (MlcD does bind calcium, albeit at low affinity) but functionally misleading.

The BioReason SFT trace starts from the same InterPro domain architecture and arrives at the same calcium binding prediction but then makes far more egregious errors. It interprets the calmodulin/myosin light chain family membership (IPR050230) as evidence for calcium-dependent conformational switching and promiscuous target engagement, which is the calmodulin paradigm but not what MlcD actually does. The thinking trace explicitly states: "This EF-hand architecture directly encodes GO:0005509 calcium ion binding as the primary molecular function. The calmodulin/myosin light chain-like family context predicts high-affinity recognition of IQ motifs on myosin heavy chains, supporting GO:0032036 myosin heavy chain binding." The first part is essentially InterPro2GO. The second part (myosin heavy chain binding) is actually correct, but then the trace veers off into myosin II biology, which is wrong.

BioReason adds substantial speculative biology beyond InterPro2GO -- cytokinesis, GTPase regulation, actin polymerization control, phagocytosis, nucleus organization, plasma membrane repair -- none of which is supported by evidence for this specific protein. This represents a hallucination pattern where the model fills in biology from the calmodulin literature and applies it indiscriminately to a divergent family member.

In summary, InterPro2GO is more honest in its limited prediction (calcium binding from EF-hand domains), while BioReason generates a more detailed narrative that is largely incorrect because it fails to account for the functional divergence of MlcD from calmodulin.

Notes on thinking trace

The thinking trace correctly identifies the InterPro domain architecture with multiple EF-hand domains. However, it makes a critical error early on by treating MlcD as a canonical "Ca2+-dependent conformational switch" rather than recognizing the degenerate calcium-binding sites. The trace states "Ca2+ binding to the N-terminal EF-hands primes the sensor to engage myosin II heavy chain IQ motifs" -- this is wrong on two counts: MlcD binding to MyoD is calcium-insensitive, and the target is myosin I, not myosin II.

The trace then cascades this error into an elaborate but unsupported functional network involving myosin II filament assembly regulation, cytokinesis, ROCO-family kinases, protein phosphatase 2B, IP3 receptors, and Ras GTPase-activating proteins. None of these interactions have been demonstrated for MlcD. This is a clear failure mode where the model treats domain-family membership as sufficient to predict specific protein-protein interactions and biological processes.

The GO term predictions section in the BioReason output is empty (no MF, BP, or CC predictions listed), so the scoring here is based entirely on the functional summary and thinking trace narrative.

📄 View Raw YAML

 
id: Q7Z2B8
gene_symbol: mlcD
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:44689
  label: Dictyostelium discoideum
description: MlcD is a calmodulin-like myosin light chain that serves as the 
  dedicated light chain for the class I myosin MyoD (Myo1D) in Dictyostelium 
  discoideum. It contains four EF-hand motifs but only retains low-affinity 
  calcium binding (Kd ~52 uM) due to mutations in key Ca2+-coordinating residues
  of EF-hands 2-4, and thus cannot function as a physiological calcium sensor. 
  Two copies of MlcD bind the MyoD IQ motifs in a calcium-insensitive manner, 
  forming the regulatory light chain component of the Myo1D complex. Through its
  association with MyoD, MlcD participates in membrane dynamics at 
  macropinocytic cups and actin waves.
existing_annotations:
- term:
    id: GO:0005509
    label: calcium ion binding
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: Phylogenetic inference of calcium ion binding is partially 
      supported but misleading. MlcD retains one functional EF-hand with 
      low-affinity calcium binding (Kd 52 uM), well above physiological levels. 
      The protein cannot function as a sensor of physiological calcium, so this 
      annotation overstates the functional relevance.
    action: KEEP_AS_NON_CORE
    reason: MlcD does bind calcium but with very low affinity that is not 
      physiologically relevant. The IBA inference from calmodulin family members
      with high-affinity calcium binding is misleading for this divergent 
      paralog. The annotation is technically correct but not representative of 
      the core function.
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: yielding apparent dissociation constants ( K'(d)) of 52 
        microM for Ca2+ and 450 microM for Mg2+
    - reference_id: PMID:12826013
      supporting_text: The low affinity of MlcD for Ca2+ indicates that it 
        cannot function as a sensor of physiological Ca2+
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: Cytoplasmic localization is correct and well-supported by direct 
      evidence from the same gene product.
    action: ACCEPT
    reason: MlcD is a cytoplasmic protein as shown by direct experimental 
      evidence, consistent with its role as a myosin light chain that operates 
      in the cytoplasm.
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: FLAG-MlcD expressed in Dictyostelium formed a complex 
        with MyoD
- term:
    id: GO:0030234
    label: enzyme regulator activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: This IBA annotation from the calmodulin family is questionable for 
      MlcD. While calmodulin regulates numerous enzymes, MlcD has diverged 
      substantially and specifically associates only with MyoD. There is no 
      direct evidence that MlcD regulates enzyme activity per se; it functions 
      as a structural light chain of a motor complex rather than as an enzyme 
      regulator.
    action: REMOVE
    reason: MlcD is a dedicated structural light chain for MyoD, not a general 
      enzyme regulator. Unlike calmodulin, which activates kinases and 
      phosphatases, MlcD binds only MyoD in a calcium-insensitive manner. The 
      IBA inference from calmodulin family members does not transfer to this 
      divergent paralog that has lost both high-affinity calcium binding and 
      promiscuous target engagement.
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: FLAG-MlcD expressed in Dictyostelium formed a complex 
        with MyoD, but not with the two other long-tailed Dictyostelium myosin I
        isoenzymes, MyoB and MyoC
    - reference_id: PMID:12826013
      supporting_text: The low affinity of MlcD for Ca2+ indicates that it 
        cannot function as a sensor of physiological Ca2+
- term:
    id: GO:0000226
    label: microtubule cytoskeleton organization
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: This IBA annotation transfers microtubule-related functions from 
      calmodulin orthologs. There is no evidence that MlcD or its partner MyoD 
      are involved in microtubule cytoskeleton organization. MyoD/Myo1D is an 
      actin-based motor that functions at membranes and actin waves.
    action: REMOVE
    reason: MlcD is a dedicated myosin I light chain with no known connection to
      microtubule biology. All experimental evidence places MlcD/MyoD in the 
      context of actin-based structures (macropinocytic cups, actin waves). The 
      IBA inference from calmodulin, which does participate in microtubule 
      biology through different targets, does not transfer to this divergent 
      paralog.
    supported_by:
    - reference_id: PMID:31774725
      supporting_text: Myo1D localized, like short-tail Myo1E and Myo1F, in the 
        PIP3-rich region encircled by the actin wave
    - reference_id: PMID:12826013
      supporting_text: FLAG-MlcD expressed in Dictyostelium formed a complex 
        with MyoD, but not with the two other long-tailed Dictyostelium myosin I
        isoenzymes, MyoB and MyoC
- term:
    id: GO:0005509
    label: calcium ion binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  review:
    summary: InterPro2GO annotation from the EF-hand domain (IPR002048). While 
      MlcD does contain EF-hand domains and retains low-affinity calcium 
      binding, this annotation overstates functional importance since the 
      binding is not physiologically relevant.
    action: KEEP_AS_NON_CORE
    reason: The InterPro2GO inference from EF-hand domains is technically 
      correct (MlcD does bind calcium) but functionally misleading. Three of 
      four EF-hands have degenerate calcium-coordinating residues, and the 
      measured Kd of 52 uM is far above physiological calcium concentrations. 
      This is not a core function.
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: EF-hands 2-4 contain mutations in key Ca2+-co-ordinating 
        residues that would be predicted to impair Ca2+ binding
    - reference_id: PMID:12826013
      supporting_text: The low affinity of MlcD for Ca2+ indicates that it 
        cannot function as a sensor of physiological Ca2+
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  review:
    summary: Correct annotation based on UniProt subcellular location. MlcD is a
      cytoplasmic protein.
    action: ACCEPT
    reason: Cytoplasmic localization is well established for MlcD from direct 
      experimental evidence.
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: FLAG-MlcD expressed in Dictyostelium formed a complex 
        with MyoD
- term:
    id: GO:0005509
    label: calcium ion binding
  evidence_type: IDA
  original_reference_id: PMID:12826013
  review:
    summary: Direct experimental evidence demonstrates calcium binding by MlcD 
      via electrospray ionization MS and tryptophan fluorescence quenching. 
      However, the binding is low affinity (Kd 52 uM), so while the annotation 
      is experimentally supported, it does not represent the core evolved 
      function.
    action: KEEP_AS_NON_CORE
    reason: The IDA evidence from De La Roche et al. 2003 is solid - MlcD does 
      bind calcium as demonstrated by ESI-MS and fluorescence spectroscopy. 
      However, the authors themselves conclude the binding is not 
      physiologically relevant. This is a vestigial capacity from calmodulin 
      ancestry, not a core function.
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: Electrospray ionization MS of MlcD in the presence of 
        Ca2+ and La3+ showed the presence of one major and one minor 
        metal-binding site
    - reference_id: PMID:12826013
      supporting_text: yielding apparent dissociation constants ( K'(d)) of 52 
        microM for Ca2+ and 450 microM for Mg2+
    - reference_id: PMID:12826013
      supporting_text: The low affinity of MlcD for Ca2+ indicates that it 
        cannot function as a sensor of physiological Ca2+
- term:
    id: GO:0016459
    label: myosin complex
  evidence_type: IDA
  original_reference_id: PMID:12826013
  review:
    summary: MlcD is part of the MyoD (Myo1D) myosin complex. This is a core 
      annotation. The more specific term GO:0045160 myosin I complex would be 
      more precise since MyoD is a class I myosin.
    action: MODIFY
    reason: MlcD co-purifies with MyoD as part of a myosin I complex (two copies
      of MlcD per MyoD heavy chain). The annotation is correct but the more 
      specific child term myosin I complex is more appropriate since MyoD is 
      definitively a class I myosin.
    proposed_replacement_terms:
    - id: GO:0045160
      label: myosin I complex
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: MyoD, a long-tailed class I myosin, co-purified with two 
        copies of a 16 kDa light chain
    - reference_id: PMID:21671662
      supporting_text: Each long-tailed Dictyostelium myosin-I has now been 
        shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD)
- term:
    id: GO:0032036
    label: myosin heavy chain binding
  evidence_type: IPI
  original_reference_id: PMID:12826013
  review:
    summary: MlcD specifically binds the MyoD heavy chain at its IQ motifs. 
      Since MyoD is a class I myosin, the more specific term GO:0032037 myosin I
      heavy chain binding is more appropriate.
    action: MODIFY
    reason: MlcD binds specifically to MyoD (a class I myosin) heavy chain IQ 
      motifs. The general term myosin heavy chain binding is correct but the 
      child term myosin I heavy chain binding more precisely captures the 
      specificity of this interaction.
    proposed_replacement_terms:
    - id: GO:0032037
      label: myosin I heavy chain binding
    supported_by:
    - reference_id: PMID:12826013
      supporting_text: Ca2+ did not affect the binding of MlcD to MyoD or to 
        either of the two MyoD IQ (Ile-Gln) motifs
    - reference_id: PMID:12826013
      supporting_text: FLAG-MlcD expressed in Dictyostelium formed a complex 
        with MyoD, but not with the two other long-tailed Dictyostelium myosin I
        isoenzymes, MyoB and MyoC
- term:
    id: GO:0062201
    label: actin wave
  evidence_type: IDA
  original_reference_id: PMID:31774725
  review:
    summary: MlcD localizes to actin waves as part of the Myo1D complex. 
      Specifically, Myo1D localizes to the PIP3-rich region encircled by the 
      actin wave, not the actin wave crest itself. This is a valid localization 
      annotation reflecting the behavior of the Myo1D complex.
    action: ACCEPT
    reason: Brzeska et al. 2019 directly demonstrated Myo1D (and hence its light
      chain MlcD) localizing to actin wave structures, specifically in the 
      PIP3-rich region. This is a core localization for the Myo1D motor complex.
    supported_by:
    - reference_id: PMID:31774725
      supporting_text: Myo1D localized, like short-tail Myo1E and Myo1F, in the 
        PIP3-rich region encircled by the actin wave
- term:
    id: GO:0070687
    label: macropinocytic cup cytoskeleton
  evidence_type: IDA
  original_reference_id: PMID:31774725
  review:
    summary: Myo1D localizes to macropinocytic cups, and MlcD as its light chain
      would be present there. The annotation to macropinocytic cup cytoskeleton 
      is well supported by the localization data.
    action: ACCEPT
    reason: Brzeska et al. 2019 showed that Myo1D localizes to macropinocytic 
      protrusions, cups, and freshly internalized vesicles. As the obligate 
      light chain, MlcD is present at these sites.
    supported_by:
    - reference_id: PMID:31774725
      supporting_text: Myo1D is also found in pinocytic protrusions and cups, 
        and freshly internalized vesicles
    - reference_id: file:DICDI/mlcD/mlcD-deep-research-bioreason-sft.md
      supporting_text: Its recruitment to large endocytic structures is 
        consistent with GO:0070685 macropinocytic cup
references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with 
    GO terms
  findings: []
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  findings: []
- id: GO_REF:0000044
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular 
    Location vocabulary mapping, accompanied by conservative changes to GO terms
    applied by UniProt
  findings: []
- id: PMID:12826013
  title: The Dictyostelium class I myosin, MyoD, contains a novel light chain 
    that lacks high-affinity calcium-binding sites.
  findings:
  - statement: MlcD is a 16 kDa calmodulin-like protein that co-purifies with 
      MyoD as two copies per heavy chain.
    supporting_text: MyoD, a long-tailed class I myosin, co-purified with two 
      copies of a 16 kDa light chain
  - statement: MlcD has four EF-hands but only retains low-affinity calcium 
      binding due to degenerate calcium-coordinating residues in EF-hands 2-4.
    supporting_text: MlcD comprises four EF-hands; however, EF-hands 2-4 contain
      mutations in key Ca2+-co-ordinating residues that would be predicted to 
      impair Ca2+ binding
  - statement: MlcD binds calcium with a Kd of 52 uM, far above physiological 
      calcium concentrations.
    supporting_text: yielding apparent dissociation constants ( K'(d)) of 52 
      microM for Ca2+ and 450 microM for Mg2+
  - statement: MlcD cannot function as a physiological calcium sensor.
    supporting_text: The low affinity of MlcD for Ca2+ indicates that it cannot 
      function as a sensor of physiological Ca2+
  - statement: MlcD binding to MyoD is calcium-insensitive.
    supporting_text: Ca2+ did not affect the binding of MlcD to MyoD or to 
      either of the two MyoD IQ (Ile-Gln) motifs
  - statement: MlcD specifically associates with MyoD and not with MyoB or MyoC.
    supporting_text: FLAG-MlcD expressed in Dictyostelium formed a complex with 
      MyoD, but not with the two other long-tailed Dictyostelium myosin I 
      isoenzymes, MyoB and MyoC
- id: PMID:16415352
  title: Identification and characterization of an 8-kDa light chain associated 
    with Dictyostelium discoideum MyoB, a class I myosin.
  findings:
  - statement: MlcB is a distinct light chain for MyoB, confirming that each 
      long-tailed myosin I has a unique light chain partner.
    supporting_text: FLAG-MlcB expressed in Dictyostelium co-immunoprecipitated 
      with MyoB but not with the related class myosins and MyoD
- id: PMID:21671662
  title: Identification of calmodulin and MlcC as light chains for Dictyostelium
    myosin-I isozymes.
  findings:
  - statement: Each long-tailed Dictyostelium myosin I has a unique light chain,
      while short-tailed ones use calmodulin.
    supporting_text: Each long-tailed Dictyostelium myosin-I has now been shown 
      to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD), 
      whereas the short-tailed myosins-I, MyoA and MyoE, have the 
      multifunctional calmodulin as a light chain
  - statement: Light chain diversity contributes to functional specialization of
      myosin I isoforms.
    supporting_text: The diversity in light chain composition is likely to 
      contribute to the distinct cellular functions of each myosin-I isozyme
- id: PMID:31774725
  title: Basic-hydrophobic sites are localized in conserved positions inside and
    outside of PH domains and affect localization of Dictyostelium myosin 1s.
  findings:
  - statement: Myo1D localizes to the PIP3-rich region inside actin waves, 
      distinct from Myo1B which colocalizes with the actin wave itself.
    supporting_text: Myo1D localized, like short-tail Myo1E and Myo1F, in the 
      PIP3-rich region encircled by the actin wave
  - statement: Myo1D localizes to macropinocytic cups and freshly internalized 
      vesicles.
    supporting_text: Myo1D is also found in pinocytic protrusions and cups, and 
      freshly internalized vesicles
core_functions:
- description: MlcD serves as the dedicated regulatory light chain subunit of 
    the Myo1D (MyoD) class I myosin complex. Two copies of MlcD bind the MyoD 
    heavy chain IQ motifs in a calcium-insensitive manner, providing structural 
    stabilization of the motor complex neck region.
  molecular_function:
    id: GO:0032037
    label: myosin I heavy chain binding
  locations:
  - id: GO:0005737
    label: cytoplasm
  - id: GO:0062201
    label: actin wave
  - id: GO:0070687
    label: macropinocytic cup cytoskeleton
  supported_by:
  - reference_id: PMID:12826013
    supporting_text: MyoD, a long-tailed class I myosin, co-purified with two 
      copies of a 16 kDa light chain
  - reference_id: PMID:12826013
    supporting_text: Ca2+ did not affect the binding of MlcD to MyoD or to 
      either of the two MyoD IQ (Ile-Gln) motifs
  - reference_id: PMID:21671662
    supporting_text: Each long-tailed Dictyostelium myosin-I has now been shown 
      to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD)
  in_complex:
    id: GO:0045160
    label: myosin I complex