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. 2018 Aug 24;293(34):13134-13150.
doi: 10.1074/jbc.RA118.002073. Epub 2018 Jun 26.

The FCS-like zinc finger scaffold of the kinase SnRK1 is formed by the coordinated actions of the FLZ domain and intrinsically disordered regions

Muhammed Jamsheer K  1 , Brihaspati N Shukla  1 , Sunita Jindal  1 , Nandu Gopan  2 , Chanchal Thomas Mannully  1 , Ashverya Laxmi  3
Affiliations

The FCS-like zinc finger scaffold of the kinase SnRK1 is formed by the coordinated actions of the FLZ domain and intrinsically disordered regions

Muhammed Jamsheer K et al. J Biol Chem. .

Abstract

The SNF1-related protein kinase 1 (SnRK1) is a heterotrimeric eukaryotic kinase that interacts with diverse proteins and regulates their activity in response to starvation and stress signals. Recently, the FCS-like zinc finger (FLZ) proteins were identified as a potential scaffold for SnRK1 in plants. However, the evolutionary and mechanistic aspect of this complex formation is currently unknown. Here, in silico analyses predicted that FLZ proteins possess conserved intrinsically disordered regions (IDRs) with a propensity for protein binding in the N and C termini across the plant lineage. We observed that the Arabidopsis FLZ proteins promiscuously interact with SnRK1 subunits, which formed different isoenzyme complexes. The FLZ domain was essential for mediating the interaction with SnRK1α subunits, whereas the IDRs in the N termini facilitated interactions with the β and βγ subunits of SnRK1. Furthermore, the IDRs in the N termini were important for mediating dimerization of different FLZ proteins. Of note, the interaction of FLZ with SnRK1 was confined to cytoplasmic foci, which colocalized with the endoplasmic reticulum. An evolutionary analysis revealed that in general, the IDR-rich regions are under more relaxed selection than the FLZ domain. In summary, the findings in our study reveal the structural details, origin, and evolution of a land plant-specific scaffold of SnRK1 formed by the coordinated actions of IDRs and structured regions in the FLZ proteins. We propose that the FLZ protein complex might be involved in providing flexibility, thus enhancing the binding repertoire of the SnRK1 hub in land plants.

Keywords: FCS-like zinc finger; SnRK1; energy signaling; intrinsically disordered protein; plant; protein complex; protein evolution; protein–protein interaction; scaffold protein; zinc finger.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Disorder in FLZ protein family across the plant lineage. A, schematic representation of domain structure of FLZ proteins. The domain structure of Arabidopsis FLZ1 is shown here as an example. B, average disorder of different regions of FLZ proteins from Arabidopsis thaliana (Ath). C, average disorder of different regions of FLZ proteins from K. flaccidum (Kfa), Marchantia polymorpha (Mpo), Physcomitrella patens (Ppa), Sphagnum fallax (Sfa), and Selaginella moellendorffii (Smo). D, average disorder of different regions of FLZ proteins from 28 Spermatophyta proteomes. Each species is shown in different background colors. E, relationship between IDR length and frequency of occurrence in FLZ protein family. The bars indicate the relationship between IDR length and frequency after categorizing IDRs into groups based on length. The IDRs with a length difference of up to five amino acids were grouped together. The red dots indicate the relationship between IDR length and frequency without any grouping. F, average distribution of short (SIDR; 10–29 residues) and long (LIDR; 30 or more residues) IDRs in different regions of FLZ protein family. The detailed table of IDRs in FLZ protein family is given in Table S3.
Figure 2.
Figure 2.
Binding propensity and post-translational modification sites of FLZ proteins. A, average RNA, DNA, and protein-binding propensity of N and C termini of FLZ proteins. B, predicted phosphorylation sites in different regions of FLZ proteins. C, predicted arginine methylation sites in different regions of FLZ proteins. D, predicted lysine acetylation sites in different regions of FLZ proteins. The detailed table of PTM sites in the FLZ protein family is given in Table S4. The p value obtained in the paired t test indicating the statistically significant difference in the number of PTM sites between N or C termini and FLZ domain is given in the graphs.
Figure 3.
Figure 3.
Interaction of FLZ proteins with SnRK1 subunits. The CDS of SnRK1 and FLZ genes were cloned in AD and BD vectors respectively. The constructs were cotransformed, and interaction was screened on DDO (upper row in each group) and QDO plates supplemented with X-α-Gal and AbA (lower row in each group). Simultaneously, a negative control experiment with BD vector and AD construct was carried out to identify false interactions.
Figure 4.
Figure 4.
Role of FLZ domain in mediating the interaction of FLZ proteins with SnRK1 subunits. A, composition of FLZ proteins. The FLZ domain is represented by red box. Number indicates the position of the FLZ domain and other regions in the protein. B, interaction of FLZ domain of FLZ1, FLZ2, FLZ3, FLZ8, and FLZ15 with SnRK1α subunits. C, interaction of N and C termini of FLZ1 with SnRK1α subunits. D, FLZ domain of FLZ8 indicating the position of Δ1, Δ2, and Δ3 mutations. The cysteine residues that form the CX2CX18FCSX2C signature zinc finger motif are indicated by red, and the nonconserved cysteine residues are indicated by blue. E, interaction of FLZ8 and mutated FLZ8 (Δ1, Δ2, Δ1-Δ2, and Δ1-Δ3) with SnRK1α2 subunit. F, interaction of FLZ domain of FLZ1, FLZ2, and FLZ8 with SnRK1β1 subunit. G, interaction of FLZ domain of FLZ1, FLZ2, and FLZ8 with SnRK1β2 subunit. H, interaction of FLZ domain of FLZ1, FLZ2, FLZ3, FLZ8, and FLZ15 with SnRK1β3 subunit. I, interaction of FLZ domain of FLZ2 with SnRK1βγ subunit. The SnRK1 and FLZ constructs were prepared in AD and BD vectors, respectively. The cotransformed colonies of Y2H Gold were screened on DDO (upper row in each group) and QDO plates supplemented with X-α-Gal and AbA (lower row in each group).
Figure 5.
Figure 5.
Role of N terminus in mediating the interaction of FLZ proteins with SnRK1β and βγ subunits. A, full-length and partial construct of FLZ1 and their interaction with SnRK1β subunits with negative control experiment. B, full-length and partial construct of FLZ2 and their interaction with SnRK1β and βγ subunits with negative control experiment. C, interaction of FLZ8 and mutated FLZ8 (Δ1, Δ2, and Δ1-Δ2) with SnRK1β1. D, interaction of FLZ8 and mutated FLZ8 (Δ1, Δ2, and Δ1-Δ2) with SnRK1β2. E, interaction of FLZ8 and mutated FLZ8 (Δ1, Δ2, and Δ1-Δ2) with SnRK1β3. The SnRK1 and FLZ1 constructs were prepared in AD and BD vector, respectively. The cotransformed colonies of Y2H Gold were screened on DDO (1st column in each group) and QDO plates supplemented with X-α-Gal and AbA (2nd column in each group).
Figure 6.
Figure 6.
In planta interaction of FLZ proteins with SnRK1α kinase subunits. A, BiFC analysis of the interaction of FLZ proteins with SnRK1α1 and -α2. The whole cell representation is given in Fig. S6. FLZ proteins were fused to (YFP(1–174)) N terminus of YFP, and SnRK1α1 and -α2 were fused with (YFP(175-end)) C terminus of YFP. The interactions were co-localized with ER marker construct fused with mCherry. B, colocalization of FLZ15–SnRK1α1 interaction with ER–Tracker Red. Wavelengths used for activation/detection (absorption/emission) of the fluorophores: YFP(514/530 nm); DAPI (351/450 nm); mCherry (575/610 nm); ER–Tracker Red (587/615 nm).
Figure 7.
Figure 7.
Homo- and heterodimerization of FLZ proteins. The FLZ genes were cloned in AD and BD vectors; constructs were cotransformed; and interaction was screened on DDO (upper row in each group) and QDO plates supplemented with X-α-Gal and AbA (lower row in each group). Simultaneously, a negative control experiment with BD vector and AD construct was carried out to identify false interactions.
Figure 8.
Figure 8.
Role of N terminus in mediating the heterodimerization of FLZ proteins. A, interaction of FLZ7 and FLZ15 with full-length and partial constructs of FLZ1. B, interaction of FLZ7, FLZ10, FLZ11, FLZ12, and FLZ15 with full-length and partial constructs of FLZ2. C, composition of FLZ7 and FLZ15. D, interaction analysis of N terminus of FLZ1 and FLZ2 with the N terminus of FLZ7 and FLZ15. The constructs prepared in AD and BD were cotransformed in Y2H Gold strain and screened on DDO (1st column in each group) and QDO plates supplemented with X-α-Gal and AbA (2nd column in each group).
Figure 9.
Figure 9.
Role of IDRs in N terminus in mediating heterodimerization of FLZ proteins and interaction with SnRK1 subunits. A, sequence alignment of the N terminus of FLZ1 and FLZ2 showing the position of IDRs. The conserved residues are marked by violet color. B, interaction analysis of FLZ2NIDR1 and FLZ2NIDR2 with FLZ7 and FLZ10. C, interaction analysis of FLZ2NIDR1 and FLZ2NIDR2 with SnRK1β and βγ subunits. D, interaction analysis of FLZ1NIDR1 (1–48) and FLZ1NIDR1 (49–81) with FLZ7 and FLZ15. E, interaction analysis of FLZ1NIDR1 (1–48) and FLZ1NIDR1 (49–81) with SnRK1β subunits. F, interaction analysis of FLZ1N and FLZ1N-FLZ2NIDR2 with FLZ10. The constructs prepared in AD and BD were cotransformed in Y2H Gold strain and screened on DDO (1st column in each group) and QDO plates supplemented with X-α-Gal and AbA (2nd column in each group).
Figure 10.
Figure 10.
Estimation of sequence divergence rate in different regions of FLZ proteins. The Ka/Ks ratio between putative orthologous FLZ genes from 12 different angiosperms was calculated. The cumulative ratio of each orthologous gene pair is represented by single bar. The Ka/Ks ratio of each region of the gene pair is marked by different color. The detailed table of Ka/Ks estimation is given in Table S5. The species used for this analysis were abbreviated in the graph as follows: A. thaliana (Ath); Brassica rapa (Bra); Fragaria vesca (Fve); Malus domestica (Mdo); Medicago truncatula (Mdo); Glycine max (Gma); Zostera marina (Zmar); Spirodela polyrhiza (Spo); Oryza sativa (Osa); Brachypodium distachyon (Bdi); Sorghum bicolor (Sbi); and Zea mays (Zma).
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