{"gene":"TG_311260","gene_symbol":"MLC5","run_date":"2026-09-08T15:15:13","timeline":{"discoveries":[{"year":2002,"finding":"TgMyoA binds a novel myosin light chain TgMLC1; the purified TgMyoA-TgMLC1 motor complex moves actin filaments toward the plus end at ~5.2 µm/s in unitary steps of 5.3 nm, establishing TgMyoA as a fast, single-headed, plus-end-directed motor anchored at the plasma membrane.","method":"Biochemical characterization, microneedle-laser trap assay, sliding velocity assay, in vitro motility assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with quantitative mechanical measurements (laser trap, sliding assay) and direct binding characterization","pmids":["11980712"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2004,"finding":"TgMLC1 forms a protein complex with TgMyoA, TgGAP45, and TgGAP50 at the inner membrane complex (IMC) of T. gondii; TgGAP50 serves as the integral membrane glycoprotein anchor for the entire complex, providing the first molecular description of an integral membrane protein as a specific myosin receptor.","method":"Co-immunoprecipitation, protein complex identification, subcellular localization (immunofluorescence/fractionation)","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP and fractionation identifying the complex, widely replicated across the field","pmids":["15123738"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"finding":"GAP45 phosphorylation at Ser163 and Ser167 controls the final assembly step of the myosin XIV motor complex: phospho-mimetic (Ser→Glu) mutations at these residues prevent association of the MyoA-MLC1-GAP45 subcomplex with GAP50 (the membrane anchor), without affecting targeting or upstream complex assembly.","method":"Mass spectrometry (phosphosite identification), site-directed mutagenesis (phospho-mimetic and phospho-null), Co-immunoprecipitation","journal":"Eukaryotic cell","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis combined with Co-IP to functionally dissect phosphorylation control of complex assembly, single lab with two orthogonal methods","pmids":["19047362"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"Treatment of T. gondii with a small-molecule inhibitor of motility induces a rapid, irreversible posttranslational modification of TgMLC1 mapped to the peptide Val46–Arg59; myosin motor complexes containing modified TgMLC1 show significantly decreased in vitro motor activity compared to controls, establishing that PTM of the light chain modulates Class XIV myosin mechanical activity.","method":"In vitro motility assay, 2D gel electrophoresis, mass spectrometry (peptide mapping), pharmacological treatment","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro motility assay directly measuring motor activity of modified vs. control complexes, combined with MS mapping, single lab with two orthogonal methods","pmids":["20084115"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"GAP45 is anchored to both the plasma membrane (via N-terminal acylation) and the IMC (via its C-terminus); the C-terminal region of GAP45 recruits the MLC1-MyoA motor to the IMC, while the N-terminal domain preserves pellicle integrity during invasion. GAP45 is essential for gliding, invasion, and egress.","method":"Conditional knockout, domain truncation/mutagenesis, complementation (including cross-genera), immunofluorescence","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function with multiple phenotypic readouts and domain dissection, replicated across genera","pmids":["20951968"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"Calcium-dependent phosphorylation of MLC1 (along with GAP45 and MyoA) is correlated with invasion motor activity; six calcium-dependent phosphorylation sites across GAP45, MLC1, and MyoA were quantitatively mapped using SILAC-based LC-MS/MS during Ca2+-stimulated invasion.","method":"SILAC quantitative LC-MS/MS, 32P labeling, 2D-PAGE, calcium mobilization assay","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative phosphoproteomic mapping in live parasites with Ca2+-stimulation, single lab, correlative for function","pmids":["21980283"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"MLC1 is the sole myosin light chain associated with MyoA in T. gondii; among six calmodulin-like proteins identified in apicomplexans, MLC2 (coccidian-specific) associates with MyoD via its CaM-like domain and is anchored to the plasma membrane via its N-terminal extension, likely via palmitoylation, distinct from MLC1.","method":"Subcellular localization (immunofluorescence), co-immunoprecipitation, molecular modeling","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — localization and Co-IP for MLC2/MyoD complex, with comparative context establishing MLC1 as the MyoA-specific light chain; single lab","pmids":["21143563"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"Phospho-mimetic and phospho-null mutations of previously mapped phosphorylation sites on TgMLC1 do not impact glideosome assembly or parasite motility, indicating that despite extensive phosphorylation of TgMLC1, this PTM is not critical for glideosome assembly or function.","method":"Allelic replacement, phospho-mimetic/phospho-null mutagenesis, motility assays, complementation in inducible knockout background","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic allelic replacement with functional readouts, single lab; result is a negative finding (phosphorylation dispensable)","pmids":["24779470"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"TgMLC1 is a direct and covalent target of the motility inhibitor tachypleginA-2; the compound forms a 225.118 Da adduct on C58 of TgMLC1, and a C58S knock-in mutation renders parasites less sensitive to compound treatment and abolishes click-chemistry labeling, establishing C58 as the functional binding site.","method":"Click chemistry labeling, mass spectrometry (adduct mapping), site-directed mutagenesis (C58S knock-in), 3D motility assay","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — covalent target ID by click chemistry + MS + genetic rescue with knock-in mutant, multiple orthogonal methods in single study","pmids":["24892871"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"TgMyoA expressed in Sf9 cells with the parasite-specific co-chaperone TgUNC requires both TgMLC1 (regulatory light chain) and TgELC1 (essential light chain) for fast motility: TgMyoA+TgMLC1 moves actin at ~1.5 µm/s, whereas adding TgELC1 more than doubles speed to ~3.4 µm/s, indicating two light chains stabilize the lever arm for force transduction.","method":"Baculovirus/Sf9 heterologous expression, in vitro actin-sliding motility assay, co-chaperone co-expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted motor in vitro with quantitative motility measurements comparing one vs two light chains, single lab with rigorous biochemical assays","pmids":["25231988"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"Two essential light chains (ELC1 and ELC2) bind the MyoA lever arm adjacent to MLC1 and are important for invasion, egress, and motility; molecular simulations and point mutations show that Ca2+ promotes intermolecular contacts between ELCs and the adjacent MLC1 to stabilize the lever arm and enhance gliding quality, displacement, and speed.","method":"Conditional knockdown, molecular dynamics simulation, point mutagenesis (Ca2+-coordinating and ELC-MLC1 interface residues), gliding motility assays","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with functional readouts combined with simulation-guided mutagenesis, single lab; simulation is computational but validated with mutant phenotypes","pmids":["26374117"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"Crystal structure of MLC1 (1.85 Å) in complex with its cognate MyoA neck peptide (residues 775–818) reveals a bilobed architecture with MLC1 clamping tightly around the helical MyoA peptide (Kd ~10 nM by ITC); MLC1 and ELC1 bind adjacent sites on the neck, and prebinding of MLC1 enhances ELC1 affinity 8-fold, supporting a staged cooperative assembly model.","method":"X-ray crystallography (1.85 Å), hydrogen-deuterium exchange mass spectrometry, isothermal titration calorimetry, full-length motor binding assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with HDX-MS and ITC in a single study with multiple orthogonal methods providing quantitative mechanistic detail","pmids":["28972141"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"Conditional knockout of MLC1 (mlc1 cKO) parasites are virtually unable to attach to substrate under flow conditions, demonstrating that the actomyosin system (including MLC1) functions in surface attachment rather than solely as a force generator for gliding; a small fraction of mlc1 cKO parasites remain capable of 3D motility at wild-type speeds.","method":"Conditional knockout, flow-based attachment assay, 3D motility assay","journal":"BMC biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean cKO with specific phenotypic readout (attachment under flow), single lab","pmids":["28100223"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"finding":"TgMLC1 is palmitoylated on two N-terminal cysteine residues, C8 and C11; mutations blocking palmitoylation completely abrogate TgMLC1 binding to TgGAP45, yet these palmitoylation-deficient mutant parasites show little impact on motility, questioning the requirement of TgMLC1-GAP45 interaction for motor force transduction.","method":"Site-directed mutagenesis (C8/C11 mutations), Co-immunoprecipitation, palmitoylation assay, 3D motility assay","journal":"mSphere","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic mutagenesis with biochemical validation (Co-IP abrogation) and quantitative motility readout, single lab with two orthogonal methods; negative phenotype result is clearly documented","pmids":["34011689"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"finding":"Rab11A associates with MLC1 (MTIP/MLC1) of the glideosome, and Rab11A-mediated vesicular trafficking driven by an MLC1-MyoA motor is required for IMC maturation and plasma membrane delivery to complete parasite cell division; ablation of Rab11A causes incomplete IMC formation and block at late cytokinesis, phenocopied by MyoA tail-only expression.","method":"Co-immunoprecipitation (Rab11A with MLC1), inducible dominant-negative expression, conditional knockdown, fluorescence microscopy","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP showing Rab11A-MLC1 association and epistatic genetic evidence (Rab11A KD and MyoA tail phenocopy), single lab","pmids":["19165333"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgMLC1 is the regulatory light chain of the TgMyoA glideosome motor complex; it binds directly to the MyoA neck (residues 775–818) with high affinity (~10 nM), is palmitoylated on C8/C11 (mediating GAP45 binding), and is required—together with ELC1/2—for full lever-arm stabilization and fast motor activity (~3.4 µm/s), with Ca2+-dependent contacts between ELCs and MLC1 further tuning motility; the complex is anchored to the IMC via GAP45/GAP50, MLC1 phosphorylation at mapped sites is dispensable for glideosome assembly, the actomyosin system including MLC1 is critical for surface attachment, and covalent modification of MLC1-C58 by small-molecule inhibitors reduces motor activity, while the requirement of MLC1-GAP45 binding for force transduction has been experimentally questioned."},"narrative":{"mechanistic_narrative":"MLC1 (TgMLC1) is the regulatory light chain of the TgMyoA glideosome, the actomyosin motor that powers substrate attachment, gliding motility, invasion, and egress in Toxoplasma gondii [PMID:11980712, PMID:28100223]. It binds the MyoA neck—clamping tightly around the helical neck peptide (residues 775–818) with ~10 nM affinity in a bilobed architecture—and, together with the essential light chains ELC1/ELC2, stabilizes the lever arm required for fast, plus-end-directed force transduction; a single-light-chain motor moves actin at ~1.5 µm/s while the full complement of light chains more than doubles speed to ~3.4 µm/s, with prebinding of MLC1 cooperatively enhancing ELC1 recruitment and Ca2+-dependent ELC–MLC1 contacts further tuning motility [PMID:25231988, PMID:26374117, PMID:28972141]. Within the parasite, MLC1 assembles into a membrane-anchored complex with MyoA, GAP45, and GAP50 at the inner membrane complex, where GAP50 is the integral-membrane receptor and GAP45 bridges the motor to the IMC and plasma membrane [PMID:15123738, PMID:20951968]. MLC1 is palmitoylated on N-terminal cysteines C8 and C11, a modification required for its binding to GAP45; however, palmitoylation-deficient parasites glide near-normally, questioning whether MLC1–GAP45 binding is essential for force transduction [PMID:34011689]. Small-molecule motility inhibitors act covalently on MLC1—including tachypleginA-2, which adducts C58—to reduce motor activity, defining the light chain as a druggable point of control [PMID:20084115, PMID:24892871]. Although MLC1 is extensively phosphorylated in a Ca2+-dependent manner during invasion, phospho-mimetic and phospho-null mutations of the mapped sites do not affect glideosome assembly or motility [PMID:21980283, PMID:24779470]. Beyond the glideosome, MLC1 associates with Rab11A, linking an MLC1–MyoA motor to vesicular trafficking that drives IMC maturation and late cytokinesis [PMID:19165333].","teleology":[{"year":2002,"claim":"Establishing that MyoA required a light chain partner and defining its mechanical output addressed whether Toxoplasma possessed a bona fide fast motor for gliding; reconstitution showed MyoA-MLC1 is a fast, single-headed, plus-end-directed motor.","evidence":"Biochemical purification with laser-trap and in vitro motility/sliding-velocity assays of the TgMyoA-TgMLC1 complex","pmids":["11980712"],"confidence":"High","gaps":["Did not resolve how MLC1 binds the MyoA neck structurally","Role of additional light chains not yet appreciated"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2004,"claim":"Identifying the membrane-anchoring partners answered how the soluble motor is fixed at the pellicle; MLC1 was placed in a complex with MyoA, GAP45, and GAP50 at the IMC, with GAP50 as the integral-membrane receptor.","evidence":"Reciprocal co-immunoprecipitation and subcellular fractionation/immunofluorescence","pmids":["15123738"],"confidence":"High","gaps":["Assembly order and regulation of the complex not defined","Direct MLC1 contribution to anchoring versus MyoA/GAPs unclear"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"claim":"Dissecting how the complex reaches its membrane anchor and how the motor supports cell division extended MLC1's role beyond motility; GAP45 phosphorylation gates final GAP50 association, and Rab11A associates with MLC1 to drive IMC maturation and cytokinesis.","evidence":"MS phosphosite mapping with phospho-mimetic/null mutagenesis and Co-IP (GAP45); Co-IP plus dominant-negative/knockdown microscopy (Rab11A)","pmids":["19047362","19165333"],"confidence":"High","gaps":["Whether MLC1 is directly modified during this assembly step not addressed","Mechanism of Rab11A-MLC1 coupling not resolved at molecular detail"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"Testing whether covalent modification of the light chain could tune the motor addressed the pharmacological control point; a motility-inhibitor-induced PTM on MLC1 (Val46–Arg59) decreased in vitro motor activity, and GAP45 domain dissection defined how it recruits the motor to the IMC.","evidence":"In vitro motility assay of modified vs control complexes with 2D-gel/MS mapping; GAP45 conditional knockout with domain truncation and complementation","pmids":["20084115","20951968"],"confidence":"High","gaps":["Chemical identity/mechanism of the MLC1 modification not fully defined","Direct causal link between MLC1 PTM and in vivo motility loss not established"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"claim":"Mapping Ca2+-dependent phosphorylation and confirming MLC1 as the sole MyoA light chain clarified regulatory inputs and specificity; MLC1 phosphosites were quantified during invasion and MLC2 was shown to partner a distinct myosin (MyoD).","evidence":"SILAC LC-MS/MS with 32P labeling and Ca2+-mobilization (phosphosites); immunofluorescence, Co-IP, and modeling (MLC1/MLC2 specificity)","pmids":["21980283","21143563"],"confidence":"Medium","gaps":["Phosphorylation correlation with activity was not functionally tested","Physiological kinase(s) not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Determining the functional weight of MLC1 phosphorylation, the target of a covalent inhibitor, and the light-chain requirement for fast motility resolved several mechanistic questions; MLC1 phosphorylation proved dispensable, tachypleginA-2 was shown to adduct C58, and both MLC1 and ELC1 were shown necessary for full lever-arm-driven speed.","evidence":"Allelic replacement/phospho-mutant motility assays; click chemistry, MS adduct mapping, and C58S knock-in; Sf9 reconstitution with TgUNC and actin-sliding assays","pmids":["24779470","24892871","25231988"],"confidence":"High","gaps":["Why MLC1 is so heavily phosphorylated if dispensable remains unexplained","How the second light chain doubles velocity structurally not resolved here"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"Defining how the essential light chains and Ca2+ act on the lever arm addressed the mechanical basis of light-chain tuning; ELC1/ELC2 bind adjacent to MLC1 and Ca2+ promotes ELC–MLC1 contacts that stabilize the lever arm and enhance gliding.","evidence":"Conditional knockdown, molecular dynamics simulation, and interface point mutagenesis with gliding assays","pmids":["26374117"],"confidence":"Medium","gaps":["Simulation-derived contacts not confirmed by structure","Ca2+ sensor and physiological trigger not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Resolving the MLC1-neck interface at atomic resolution and testing MLC1's function under flow answered how the light chain engages MyoA and what physiological role the motor plays; MLC1 clamps the neck at ~10 nM and cooperatively primes ELC1 binding, while its loss impairs substrate attachment more than gliding per se.","evidence":"1.85 Å crystal structure with HDX-MS and ITC (interface); conditional MLC1 knockout with flow-attachment and 3D motility assays (phenotype)","pmids":["28972141","28100223"],"confidence":"High","gaps":["Full-length assembled motor structure not obtained","How some cKO parasites retain wild-type 3D speed without MLC1 unexplained"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"claim":"Testing the role of MLC1 lipidation in anchoring addressed how the light chain contacts GAP45; C8/C11 palmitoylation is required for MLC1-GAP45 binding, yet its loss barely affects motility, challenging the assumed necessity of MLC1-GAP45 coupling for force transduction.","evidence":"C8/C11 mutagenesis, palmitoylation assay, Co-IP, and 3D motility assay","pmids":["34011689"],"confidence":"High","gaps":["How the motor transduces force without MLC1-GAP45 anchoring not resolved","Whether alternative anchoring compensates not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"It remains unresolved how force is transduced to the substrate when MLC1-GAP45 binding is dispensable, what the physiological trigger and functional consequence of MLC1 Ca2+-dependent phosphorylation are, and the structure of the fully assembled membrane-anchored glideosome.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of the complete anchored glideosome","Functional purpose of extensive but dispensable MLC1 phosphorylation unknown","Mechanistic basis of force transduction independent of MLC1-GAP45 anchoring unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["TgMyoA glideosome motor complex (MyoA-MLC1-GAP45-GAP50)","MyoA-MLC1-ELC1/ELC2 lever-arm light-chain assembly"],"partners":["TGMYOA (NECK RESIDUES 775–818)","TGELC1","TGELC2","TGGAP45 (PALMITOYLATION-DEPENDENT)","TGGAP50","RAB11A"],"other_free_text":["Binds MyoA neck with ~10 nM affinity in a bilobed clamp architecture (crystal structure at 1.85 Å); prebinding enhances ELC1 affinity ~8-fold [#11]","Palmitoylated on N-terminal C8/C11, required for GAP45 binding but dispensable for motility [#13]","Covalent target of motility inhibitors: tachypleginA-2 adducts C58; a separate inhibitor modifies the Val46–Arg59 peptide [#3, #8]","Extensively and Ca2+-dependently phosphorylated, but mapped phosphosites dispensable for glideosome assembly and motility [#5, #7]","Required for substrate attachment under flow; a subset of MLC1 cKO parasites retain wild-type 3D speed [#12]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"20951968","id":"PMC_20951968","title":"Functional dissection of the apicomplexan glideosome molecular architecture.","date":"2010","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/20951968","citation_count":234,"is_preprint":false},{"pmid":"15123738","id":"PMC_15123738","title":"Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii.","date":"2004","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/15123738","citation_count":214,"is_preprint":false},{"pmid":"11980712","id":"PMC_11980712","title":"Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor.","date":"2002","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/11980712","citation_count":197,"is_preprint":false},{"pmid":"19165333","id":"PMC_19165333","title":"Rab11A-controlled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis.","date":"2009","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/19165333","citation_count":95,"is_preprint":false},{"pmid":"24632839","id":"PMC_24632839","title":"The toxoplasma Acto-MyoA motor complex is important but not essential for gliding motility and host cell invasion.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24632839","citation_count":91,"is_preprint":false},{"pmid":"28100223","id":"PMC_28100223","title":"Surface attachment, promoted by the actomyosin system of Toxoplasma gondii is important for efficient gliding motility and invasion.","date":"2017","source":"BMC biology","url":"https://pubmed.ncbi.nlm.nih.gov/28100223","citation_count":81,"is_preprint":false},{"pmid":"26760042","id":"PMC_26760042","title":"The Conoid Associated Motor MyoH Is Indispensable for Toxoplasma gondii Entry and Exit from Host Cells.","date":"2016","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/26760042","citation_count":75,"is_preprint":false},{"pmid":"21980283","id":"PMC_21980283","title":"Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex.","date":"2011","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/21980283","citation_count":73,"is_preprint":false},{"pmid":"19047362","id":"PMC_19047362","title":"GAP45 phosphorylation controls assembly of the Toxoplasma myosin XIV complex.","date":"2008","source":"Eukaryotic cell","url":"https://pubmed.ncbi.nlm.nih.gov/19047362","citation_count":65,"is_preprint":false},{"pmid":"26374117","id":"PMC_26374117","title":"Two Essential Light Chains Regulate the MyoA Lever Arm To Promote Toxoplasma Gliding Motility.","date":"2015","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/26374117","citation_count":40,"is_preprint":false},{"pmid":"20084115","id":"PMC_20084115","title":"A small-molecule inhibitor of T. gondii motility induces the posttranslational modification of myosin light chain-1 and inhibits myosin motor activity.","date":"2010","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/20084115","citation_count":39,"is_preprint":false},{"pmid":"25231988","id":"PMC_25231988","title":"A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25231988","citation_count":36,"is_preprint":false},{"pmid":"21143563","id":"PMC_21143563","title":"Unusual anchor of a motor complex (MyoD-MLC2) to the plasma membrane of Toxoplasma gondii.","date":"2011","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/21143563","citation_count":28,"is_preprint":false},{"pmid":"24779470","id":"PMC_24779470","title":"Assessment of phosphorylation in Toxoplasma glideosome assembly and function.","date":"2014","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/24779470","citation_count":25,"is_preprint":false},{"pmid":"28972141","id":"PMC_28972141","title":"Dissecting the molecular assembly of the Toxoplasma gondii MyoA motility complex.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28972141","citation_count":16,"is_preprint":false},{"pmid":"24892871","id":"PMC_24892871","title":"Identification of T. gondii myosin light chain-1 as a direct target of TachypleginA-2, a small-molecule inhibitor of parasite motility and invasion.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24892871","citation_count":16,"is_preprint":false},{"pmid":"34011689","id":"PMC_34011689","title":"Blocking Palmitoylation of Toxoplasma gondii Myosin Light Chain 1 Disrupts Glideosome Composition but Has Little Impact on Parasite Motility.","date":"2021","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/34011689","citation_count":11,"is_preprint":false},{"pmid":"30130043","id":"PMC_30130043","title":"[Research Advances on Gliding-associated Proteins of Toxoplasma gondii].","date":"2016","source":"Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases","url":"https://pubmed.ncbi.nlm.nih.gov/30130043","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11266,"output_tokens":4235,"usd":0.048662,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10616,"output_tokens":4754,"usd":0.085965,"stage2_stop_reason":"end_turn"},"total_usd":0.134627,"stage1_batch_id":"msgbatch_01S6GjoYQm8LTPUsdhASdqag","stage2_batch_id":"msgbatch_01VX63RTMdBJqsG6gQMqoCjz","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"TgMyoA binds a novel myosin light chain TgMLC1; the purified TgMyoA-TgMLC1 motor complex moves actin filaments toward the plus end at ~5.2 µm/s in unitary steps of 5.3 nm, establishing TgMyoA as a fast, single-headed, plus-end-directed motor anchored at the plasma membrane.\",\n      \"method\": \"Biochemical characterization, microneedle-laser trap assay, sliding velocity assay, in vitro motility assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with quantitative mechanical measurements (laser trap, sliding assay) and direct binding characterization\",\n      \"pmids\": [\"11980712\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TgMLC1 forms a protein complex with TgMyoA, TgGAP45, and TgGAP50 at the inner membrane complex (IMC) of T. gondii; TgGAP50 serves as the integral membrane glycoprotein anchor for the entire complex, providing the first molecular description of an integral membrane protein as a specific myosin receptor.\",\n      \"method\": \"Co-immunoprecipitation, protein complex identification, subcellular localization (immunofluorescence/fractionation)\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP and fractionation identifying the complex, widely replicated across the field\",\n      \"pmids\": [\"15123738\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"GAP45 phosphorylation at Ser163 and Ser167 controls the final assembly step of the myosin XIV motor complex: phospho-mimetic (Ser→Glu) mutations at these residues prevent association of the MyoA-MLC1-GAP45 subcomplex with GAP50 (the membrane anchor), without affecting targeting or upstream complex assembly.\",\n      \"method\": \"Mass spectrometry (phosphosite identification), site-directed mutagenesis (phospho-mimetic and phospho-null), Co-immunoprecipitation\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis combined with Co-IP to functionally dissect phosphorylation control of complex assembly, single lab with two orthogonal methods\",\n      \"pmids\": [\"19047362\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Treatment of T. gondii with a small-molecule inhibitor of motility induces a rapid, irreversible posttranslational modification of TgMLC1 mapped to the peptide Val46–Arg59; myosin motor complexes containing modified TgMLC1 show significantly decreased in vitro motor activity compared to controls, establishing that PTM of the light chain modulates Class XIV myosin mechanical activity.\",\n      \"method\": \"In vitro motility assay, 2D gel electrophoresis, mass spectrometry (peptide mapping), pharmacological treatment\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro motility assay directly measuring motor activity of modified vs. control complexes, combined with MS mapping, single lab with two orthogonal methods\",\n      \"pmids\": [\"20084115\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"GAP45 is anchored to both the plasma membrane (via N-terminal acylation) and the IMC (via its C-terminus); the C-terminal region of GAP45 recruits the MLC1-MyoA motor to the IMC, while the N-terminal domain preserves pellicle integrity during invasion. GAP45 is essential for gliding, invasion, and egress.\",\n      \"method\": \"Conditional knockout, domain truncation/mutagenesis, complementation (including cross-genera), immunofluorescence\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function with multiple phenotypic readouts and domain dissection, replicated across genera\",\n      \"pmids\": [\"20951968\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Calcium-dependent phosphorylation of MLC1 (along with GAP45 and MyoA) is correlated with invasion motor activity; six calcium-dependent phosphorylation sites across GAP45, MLC1, and MyoA were quantitatively mapped using SILAC-based LC-MS/MS during Ca2+-stimulated invasion.\",\n      \"method\": \"SILAC quantitative LC-MS/MS, 32P labeling, 2D-PAGE, calcium mobilization assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative phosphoproteomic mapping in live parasites with Ca2+-stimulation, single lab, correlative for function\",\n      \"pmids\": [\"21980283\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MLC1 is the sole myosin light chain associated with MyoA in T. gondii; among six calmodulin-like proteins identified in apicomplexans, MLC2 (coccidian-specific) associates with MyoD via its CaM-like domain and is anchored to the plasma membrane via its N-terminal extension, likely via palmitoylation, distinct from MLC1.\",\n      \"method\": \"Subcellular localization (immunofluorescence), co-immunoprecipitation, molecular modeling\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — localization and Co-IP for MLC2/MyoD complex, with comparative context establishing MLC1 as the MyoA-specific light chain; single lab\",\n      \"pmids\": [\"21143563\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Phospho-mimetic and phospho-null mutations of previously mapped phosphorylation sites on TgMLC1 do not impact glideosome assembly or parasite motility, indicating that despite extensive phosphorylation of TgMLC1, this PTM is not critical for glideosome assembly or function.\",\n      \"method\": \"Allelic replacement, phospho-mimetic/phospho-null mutagenesis, motility assays, complementation in inducible knockout background\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic allelic replacement with functional readouts, single lab; result is a negative finding (phosphorylation dispensable)\",\n      \"pmids\": [\"24779470\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TgMLC1 is a direct and covalent target of the motility inhibitor tachypleginA-2; the compound forms a 225.118 Da adduct on C58 of TgMLC1, and a C58S knock-in mutation renders parasites less sensitive to compound treatment and abolishes click-chemistry labeling, establishing C58 as the functional binding site.\",\n      \"method\": \"Click chemistry labeling, mass spectrometry (adduct mapping), site-directed mutagenesis (C58S knock-in), 3D motility assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — covalent target ID by click chemistry + MS + genetic rescue with knock-in mutant, multiple orthogonal methods in single study\",\n      \"pmids\": [\"24892871\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TgMyoA expressed in Sf9 cells with the parasite-specific co-chaperone TgUNC requires both TgMLC1 (regulatory light chain) and TgELC1 (essential light chain) for fast motility: TgMyoA+TgMLC1 moves actin at ~1.5 µm/s, whereas adding TgELC1 more than doubles speed to ~3.4 µm/s, indicating two light chains stabilize the lever arm for force transduction.\",\n      \"method\": \"Baculovirus/Sf9 heterologous expression, in vitro actin-sliding motility assay, co-chaperone co-expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted motor in vitro with quantitative motility measurements comparing one vs two light chains, single lab with rigorous biochemical assays\",\n      \"pmids\": [\"25231988\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Two essential light chains (ELC1 and ELC2) bind the MyoA lever arm adjacent to MLC1 and are important for invasion, egress, and motility; molecular simulations and point mutations show that Ca2+ promotes intermolecular contacts between ELCs and the adjacent MLC1 to stabilize the lever arm and enhance gliding quality, displacement, and speed.\",\n      \"method\": \"Conditional knockdown, molecular dynamics simulation, point mutagenesis (Ca2+-coordinating and ELC-MLC1 interface residues), gliding motility assays\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with functional readouts combined with simulation-guided mutagenesis, single lab; simulation is computational but validated with mutant phenotypes\",\n      \"pmids\": [\"26374117\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structure of MLC1 (1.85 Å) in complex with its cognate MyoA neck peptide (residues 775–818) reveals a bilobed architecture with MLC1 clamping tightly around the helical MyoA peptide (Kd ~10 nM by ITC); MLC1 and ELC1 bind adjacent sites on the neck, and prebinding of MLC1 enhances ELC1 affinity 8-fold, supporting a staged cooperative assembly model.\",\n      \"method\": \"X-ray crystallography (1.85 Å), hydrogen-deuterium exchange mass spectrometry, isothermal titration calorimetry, full-length motor binding assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with HDX-MS and ITC in a single study with multiple orthogonal methods providing quantitative mechanistic detail\",\n      \"pmids\": [\"28972141\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Conditional knockout of MLC1 (mlc1 cKO) parasites are virtually unable to attach to substrate under flow conditions, demonstrating that the actomyosin system (including MLC1) functions in surface attachment rather than solely as a force generator for gliding; a small fraction of mlc1 cKO parasites remain capable of 3D motility at wild-type speeds.\",\n      \"method\": \"Conditional knockout, flow-based attachment assay, 3D motility assay\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean cKO with specific phenotypic readout (attachment under flow), single lab\",\n      \"pmids\": [\"28100223\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TgMLC1 is palmitoylated on two N-terminal cysteine residues, C8 and C11; mutations blocking palmitoylation completely abrogate TgMLC1 binding to TgGAP45, yet these palmitoylation-deficient mutant parasites show little impact on motility, questioning the requirement of TgMLC1-GAP45 interaction for motor force transduction.\",\n      \"method\": \"Site-directed mutagenesis (C8/C11 mutations), Co-immunoprecipitation, palmitoylation assay, 3D motility assay\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic mutagenesis with biochemical validation (Co-IP abrogation) and quantitative motility readout, single lab with two orthogonal methods; negative phenotype result is clearly documented\",\n      \"pmids\": [\"34011689\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Rab11A associates with MLC1 (MTIP/MLC1) of the glideosome, and Rab11A-mediated vesicular trafficking driven by an MLC1-MyoA motor is required for IMC maturation and plasma membrane delivery to complete parasite cell division; ablation of Rab11A causes incomplete IMC formation and block at late cytokinesis, phenocopied by MyoA tail-only expression.\",\n      \"method\": \"Co-immunoprecipitation (Rab11A with MLC1), inducible dominant-negative expression, conditional knockdown, fluorescence microscopy\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP showing Rab11A-MLC1 association and epistatic genetic evidence (Rab11A KD and MyoA tail phenocopy), single lab\",\n      \"pmids\": [\"19165333\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgMLC1 is the regulatory light chain of the TgMyoA glideosome motor complex; it binds directly to the MyoA neck (residues 775–818) with high affinity (~10 nM), is palmitoylated on C8/C11 (mediating GAP45 binding), and is required—together with ELC1/2—for full lever-arm stabilization and fast motor activity (~3.4 µm/s), with Ca2+-dependent contacts between ELCs and MLC1 further tuning motility; the complex is anchored to the IMC via GAP45/GAP50, MLC1 phosphorylation at mapped sites is dispensable for glideosome assembly, the actomyosin system including MLC1 is critical for surface attachment, and covalent modification of MLC1-C58 by small-molecule inhibitors reduces motor activity, while the requirement of MLC1-GAP45 binding for force transduction has been experimentally questioned.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MLC1 (TgMLC1) is the regulatory light chain of the TgMyoA glideosome, the actomyosin motor that powers substrate attachment, gliding motility, invasion, and egress in Toxoplasma gondii [#0, #12]. It binds the MyoA neck—clamping tightly around the helical neck peptide (residues 775–818) with ~10 nM affinity in a bilobed architecture—and, together with the essential light chains ELC1/ELC2, stabilizes the lever arm required for fast, plus-end-directed force transduction; a single-light-chain motor moves actin at ~1.5 µm/s while the full complement of light chains more than doubles speed to ~3.4 µm/s, with prebinding of MLC1 cooperatively enhancing ELC1 recruitment and Ca2+-dependent ELC–MLC1 contacts further tuning motility [#9, #10, #11]. Within the parasite, MLC1 assembles into a membrane-anchored complex with MyoA, GAP45, and GAP50 at the inner membrane complex, where GAP50 is the integral-membrane receptor and GAP45 bridges the motor to the IMC and plasma membrane [#1, #4]. MLC1 is palmitoylated on N-terminal cysteines C8 and C11, a modification required for its binding to GAP45; however, palmitoylation-deficient parasites glide near-normally, questioning whether MLC1–GAP45 binding is essential for force transduction [#13]. Small-molecule motility inhibitors act covalently on MLC1—including tachypleginA-2, which adducts C58—to reduce motor activity, defining the light chain as a druggable point of control [#3, #8]. Although MLC1 is extensively phosphorylated in a Ca2+-dependent manner during invasion, phospho-mimetic and phospho-null mutations of the mapped sites do not affect glideosome assembly or motility [#5, #7]. Beyond the glideosome, MLC1 associates with Rab11A, linking an MLC1–MyoA motor to vesicular trafficking that drives IMC maturation and late cytokinesis [#14].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Establishing that MyoA required a light chain partner and defining its mechanical output addressed whether Toxoplasma possessed a bona fide fast motor for gliding; reconstitution showed MyoA-MLC1 is a fast, single-headed, plus-end-directed motor.\",\n      \"evidence\": \"Biochemical purification with laser-trap and in vitro motility/sliding-velocity assays of the TgMyoA-TgMLC1 complex\",\n      \"pmids\": [\"11980712\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how MLC1 binds the MyoA neck structurally\", \"Role of additional light chains not yet appreciated\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identifying the membrane-anchoring partners answered how the soluble motor is fixed at the pellicle; MLC1 was placed in a complex with MyoA, GAP45, and GAP50 at the IMC, with GAP50 as the integral-membrane receptor.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation and subcellular fractionation/immunofluorescence\",\n      \"pmids\": [\"15123738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Assembly order and regulation of the complex not defined\", \"Direct MLC1 contribution to anchoring versus MyoA/GAPs unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Dissecting how the complex reaches its membrane anchor and how the motor supports cell division extended MLC1's role beyond motility; GAP45 phosphorylation gates final GAP50 association, and Rab11A associates with MLC1 to drive IMC maturation and cytokinesis.\",\n      \"evidence\": \"MS phosphosite mapping with phospho-mimetic/null mutagenesis and Co-IP (GAP45); Co-IP plus dominant-negative/knockdown microscopy (Rab11A)\",\n      \"pmids\": [\"19047362\", \"19165333\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MLC1 is directly modified during this assembly step not addressed\", \"Mechanism of Rab11A-MLC1 coupling not resolved at molecular detail\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Testing whether covalent modification of the light chain could tune the motor addressed the pharmacological control point; a motility-inhibitor-induced PTM on MLC1 (Val46–Arg59) decreased in vitro motor activity, and GAP45 domain dissection defined how it recruits the motor to the IMC.\",\n      \"evidence\": \"In vitro motility assay of modified vs control complexes with 2D-gel/MS mapping; GAP45 conditional knockout with domain truncation and complementation\",\n      \"pmids\": [\"20084115\", \"20951968\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chemical identity/mechanism of the MLC1 modification not fully defined\", \"Direct causal link between MLC1 PTM and in vivo motility loss not established\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Mapping Ca2+-dependent phosphorylation and confirming MLC1 as the sole MyoA light chain clarified regulatory inputs and specificity; MLC1 phosphosites were quantified during invasion and MLC2 was shown to partner a distinct myosin (MyoD).\",\n      \"evidence\": \"SILAC LC-MS/MS with 32P labeling and Ca2+-mobilization (phosphosites); immunofluorescence, Co-IP, and modeling (MLC1/MLC2 specificity)\",\n      \"pmids\": [\"21980283\", \"21143563\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphorylation correlation with activity was not functionally tested\", \"Physiological kinase(s) not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Determining the functional weight of MLC1 phosphorylation, the target of a covalent inhibitor, and the light-chain requirement for fast motility resolved several mechanistic questions; MLC1 phosphorylation proved dispensable, tachypleginA-2 was shown to adduct C58, and both MLC1 and ELC1 were shown necessary for full lever-arm-driven speed.\",\n      \"evidence\": \"Allelic replacement/phospho-mutant motility assays; click chemistry, MS adduct mapping, and C58S knock-in; Sf9 reconstitution with TgUNC and actin-sliding assays\",\n      \"pmids\": [\"24779470\", \"24892871\", \"25231988\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why MLC1 is so heavily phosphorylated if dispensable remains unexplained\", \"How the second light chain doubles velocity structurally not resolved here\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defining how the essential light chains and Ca2+ act on the lever arm addressed the mechanical basis of light-chain tuning; ELC1/ELC2 bind adjacent to MLC1 and Ca2+ promotes ELC–MLC1 contacts that stabilize the lever arm and enhance gliding.\",\n      \"evidence\": \"Conditional knockdown, molecular dynamics simulation, and interface point mutagenesis with gliding assays\",\n      \"pmids\": [\"26374117\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Simulation-derived contacts not confirmed by structure\", \"Ca2+ sensor and physiological trigger not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Resolving the MLC1-neck interface at atomic resolution and testing MLC1's function under flow answered how the light chain engages MyoA and what physiological role the motor plays; MLC1 clamps the neck at ~10 nM and cooperatively primes ELC1 binding, while its loss impairs substrate attachment more than gliding per se.\",\n      \"evidence\": \"1.85 Å crystal structure with HDX-MS and ITC (interface); conditional MLC1 knockout with flow-attachment and 3D motility assays (phenotype)\",\n      \"pmids\": [\"28972141\", \"28100223\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length assembled motor structure not obtained\", \"How some cKO parasites retain wild-type 3D speed without MLC1 unexplained\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Testing the role of MLC1 lipidation in anchoring addressed how the light chain contacts GAP45; C8/C11 palmitoylation is required for MLC1-GAP45 binding, yet its loss barely affects motility, challenging the assumed necessity of MLC1-GAP45 coupling for force transduction.\",\n      \"evidence\": \"C8/C11 mutagenesis, palmitoylation assay, Co-IP, and 3D motility assay\",\n      \"pmids\": [\"34011689\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the motor transduces force without MLC1-GAP45 anchoring not resolved\", \"Whether alternative anchoring compensates not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how force is transduced to the substrate when MLC1-GAP45 binding is dispensable, what the physiological trigger and functional consequence of MLC1 Ca2+-dependent phosphorylation are, and the structure of the fully assembled membrane-anchored glideosome.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of the complete anchored glideosome\", \"Functional purpose of extensive but dispensable MLC1 phosphorylation unknown\", \"Mechanistic basis of force transduction independent of MLC1-GAP45 anchoring unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0006936\", \"supporting_discovery_ids\": [0, 9, 11], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0000146\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0017022\", \"supporting_discovery_ids\": [0, 11], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031261\", \"supporting_discovery_ids\": [1, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0052126\", \"supporting_discovery_ids\": [4, 12], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0000281\", \"supporting_discovery_ids\": [14], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"TgMyoA glideosome motor complex (MyoA-MLC1-GAP45-GAP50)\",\n      \"MyoA-MLC1-ELC1/ELC2 lever-arm light-chain assembly\"\n    ],\n    \"partners\": [\n      \"TgMyoA (neck residues 775–818)\",\n      \"TgELC1\",\n      \"TgELC2\",\n      \"TgGAP45 (palmitoylation-dependent)\",\n      \"TgGAP50\",\n      \"Rab11A\"\n    ],\n    \"other_free_text\": [\n      \"Binds MyoA neck with ~10 nM affinity in a bilobed clamp architecture (crystal structure at 1.85 Å); prebinding enhances ELC1 affinity ~8-fold [#11]\",\n      \"Palmitoylated on N-terminal C8/C11, required for GAP45 binding but dispensable for motility [#13]\",\n      \"Covalent target of motility inhibitors: tachypleginA-2 adducts C58; a separate inhibitor modifies the Val46–Arg59 peptide [#3, #8]\",\n      \"Extensively and Ca2+-dependently phosphorylated, but mapped phosphosites dispensable for glideosome assembly and motility [#5, #7]\",\n      \"Required for substrate attachment under flow; a subset of MLC1 cKO parasites retain wild-type 3D speed [#12]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_311260","name":"MLC5","product":"myosin light chain MLC5","uniprot_acc":"A0A125YRC5","chromosome":"TGME49_chrXI","start_pos":1928929},{"strain":"GT1","gene_id":"TGGT1_311260","name":null,"product":"putative myosin light chain MLC1","uniprot_acc":null,"chromosome":"TGGT1_chrXI","start_pos":1892320},{"strain":"RH88","gene_id":"TGRH88_050740","name":null,"product":"putative myosin light chain MLC1","uniprot_acc":null,"chromosome":"CM023092","start_pos":1977967},{"strain":"VEG","gene_id":"TGVEG_311260","name":null,"product":"putative myosin light chain MLC1","uniprot_acc":"A0A0F7V4L4","chromosome":"TGVEG_chrXI","start_pos":1941785}]}