Characterization of<i>Arabidopsis</i>aldolases AtFBA4 and AtFBA5; inhibition by morin and interaction with calmodulin

Symonds, Kyle; Smith, Milena A.; Esme, Oona; Plaxton, William C.; Snedden, Wayne A.

doi:10.1101/2024.03.29.587371

2024

DOI: 10.1101/2024.03.29.587371

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Characterization ofArabidopsisaldolases AtFBA4 and AtFBA5; inhibition by morin and interaction with calmodulin

Kyle Symonds,

Milena A. Smith,

Oona Esme

et al.

Abstract: Fructose bisphosphate aldolases (FBAs) catalyze the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. We analyzed two previously uncharacterized cytosolic Arabidopsis FBAs, AtFBA4 and AtFBA5. Based on a recent report, we examined the interaction of AtFBA4 with calmodulin (CaM)-like protein 11 (AtCML11). AtFBA4 did not bind AtCML11, however, we found that CaM bound AtFBA5 in a Ca2+-dependent manner with high specificity and affinity (KD ~ 190 nM) an… Show more

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Cited by 2 publications

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Characterization of the calmodulin-like protein family inChara brauniiand their conserved interaction with the calmodulin-binding transcription activator family

Symonds,

Wali,

Duff

et al. 2024

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Calcium sensor proteins play important roles by detecting changes in intracellular calcium and relaying that information onto downstream targets through protein-protein interaction. Very little is known about calcium sensors from plant species that predate land colonization and the evolution of embryophytes. Here, we examined the genome of the multicellular algae,Chara braunii, for orthologs to the evolutionarily-conserved calcium sensor calmodulin (CaM), and for CaM-like proteins (CMLs). We identified one CaM and eight CML isoforms which rang in size from 16.4 to 21.3 kDa and are predicted to have between two to four calcium-binding (EF-hand) domains. Using recombinant protein, we tested whether CbCaM and CbCMLs1-7 possess biochemical properties of typical calcium sensors. CbCaM and the CbCMLs all displayed high-affinity calcium binding with estimated globalKDvalues in the physiological µM range. In response to calcium binding, CbCaM and the CbCMLs exhibited varying degrees of increase in exposed hydrophobicity, suggesting different calcium-induced conformational changes occur among isoforms. We found many examples of putative CaM targets encoded in theC. brauniigenome and explored the ability of CbCaM and CbCMLs to interactin plantawith a representative putative target, aC. brauniiCaM-binding transcription factor (CbCAMTA1). CbCaM, CbCML2, and CbCML4 associated with the C-terminal region of CbCAMTA1. Collectively, our data support the hypothesis that complex calcium signaling and sensing networks involving CaM and CMLs evolved early in the green lineage. Similarly, it seems likely that calcium-mediated regulation of transcription occurs inC. brauniivia CAMTAs and is an ancient trait predating embryophytic emergence.HighlightsAlthough calmodulin (CaM) and calmodulin-like (CML) proteins are well studied in vascular plants, little is known about their orthologs in ancient lineages. We characterized CaM and CMLs fromChara braunii, and assessed their ability to bind a representative target protein, a calmodulin-binding transcription factor, CbCAMTA1.

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Characterization of the calmodulin-like protein family inChara brauniiand their conserved interaction with the calmodulin-binding transcription activator family

Symonds,

Wali,

Duff

et al. 2024

Preprint

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The Calmodulin-like proteins, CML13 and CML14 Function as Myosin Light Chains for the Class XI Myosins inArabidopsis

Symonds,

Duff,

Dwivedi

et al. 2024

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Myosins are a crucial motor protein associated with the actin cytoskeleton in eukaryotic cells. Structurally, myosins form heteromeric complexes, with smaller light chains such as calmodulin (CaM) bound to isoleucine–glutamine (IQ) domains in the neck region. These interactions facilitate mechano-enzymatic activity. Recently, we identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors with proteins containing multiple IQ domains, that function as the myosin VIII light chains. This study demonstrates that CaM, CML13, and CML14 specifically bind to the neck region of all 13 Arabidopsis myosin XI isoforms, with some preference among the CaM/CML-IQ domains. Additionally, we observed distinct residue preferences within the IQ domains for CML13, CML14, and CaM.In vitroexperiments revealed that recombinant CaM, CML13, and CML14 exhibit calcium-independent binding to the IQ domains of myosin XIs. Furthermore, when co-expressed with MAP65-1–myosin fusion proteins containing the IQ domains of myosin XIs, CaM, CML13, and CML14 co-localize to microtubules.In vitroactin motility assays demonstrated that recombinant CML13, CML14, and CaM function as myosin XI light chains. Acml13T-DNA mutant exhibited a shortened primary root phenotype that was complemented by the wild-type CML13 and was similar to that observed in a triple myosin XI mutant (xi3KO). Overall, our data indicate that Arabidopsis CML13 and CML14 are novel myosin XI light chains that likely participate in a breadth of myosin XI functions.HighlightMyosin XI proteins play a crucial role in the plant cytoskeleton, but their associated light chains have remained unidentified. Here, we show that calmodulin-like proteins, CML13 and CML14, serve as light chains for myosin XI, similar to their role for myosin VIII proteins

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Characterization ofArabidopsisaldolases AtFBA4 and AtFBA5; inhibition by morin and interaction with calmodulin

Cited by 2 publications

References 53 publications

Characterization of the calmodulin-like protein family inChara brauniiand their conserved interaction with the calmodulin-binding transcription activator family

Characterization of the calmodulin-like protein family inChara brauniiand their conserved interaction with the calmodulin-binding transcription activator family

The Calmodulin-like proteins, CML13 and CML14 Function as Myosin Light Chains for the Class XI Myosins inArabidopsis

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