Interesting Plant Protein & 3D Structures
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Structural basis for recognition of diverse transcriptional repressors by the TOPLESS family of corepressors

Structural basis for recognition of diverse transcriptional repressors by the TOPLESS family of corepressors | Interesting Plant Protein & 3D Structures | Scoop.it

TOPLESS (TPL) and TOPLESS-related (TPR) proteins comprise a conserved family of plant transcriptional corepressors that are related to Tup1, Groucho, and TLE (transducin-like enhancer of split) corepressors in yeast, insects, and mammals. In plants, TPL/TPR corepressors regulate development, stress responses, and hormone signaling through interaction with small ethylene response factor–associated amphiphilic repression (EAR) motifs found in diverse transcriptional repressors. How EAR motifs can interact with TPL/TPR proteins is unknown. We confirm the amino-terminal domain of the TPL family of corepressors, which we term TOPLESS domain (TPD), as the EAR motif–binding domain. To understand the structural basis of this interaction, we determined the crystal structures of the TPD of rice (Os) TPR2 in apo (apo protein) state and in complexes with the EAR motifs from Arabidopsis NINJA (novel interactor of JAZ), IAA1 (auxin-responsive protein 1), and IAA10, key transcriptional repressors involved in jasmonate and auxin signaling. The OsTPR2 TPD adopts a new fold of nine helices, followed by a zinc finger, which are arranged into a disc-like tetramer. The EAR motifs in the three different complexes adopt a similar extended conformation with the hydrophobic residues fitting into the same surface groove of each OsTPR2 monomer. Sequence alignments and structure-based mutagenesis indicate that this mode of corepressor binding is highly conserved in a large set of transcriptional repressors, thus providing a general mechanism for gene repression mediated by the TPL family of corepressors.

Dinesh Dhurvas's insight:

TOPLESS-RELATED (TPRs) proteins form tetramer and binds to EAR motif in proteins responsible for turning off genes (e.g AUX/IAA, a transcriptional repressor in auxin signalling)

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Structural basis for the auxin-induced transcriptional regulation by Aux/IAA17

Structural basis for the auxin-induced transcriptional regulation by Aux/IAA17 | Interesting Plant Protein & 3D Structures | Scoop.it

Auxin is the central hormone that regulates plant growth and organ development. Transcriptional regulation by auxin is mediated by the auxin response factor (ARF) and the repressor, AUX/IAA. Aux/IAA associates with ARF via domain III-IV for transcriptional repression that is reversed by auxin-induced Aux/IAA degradation. It has been known that Aux/IAA and ARF form homo- and hetero-oligomers for the transcriptional regulation, but what determines their association states is poorly understood. Here we report, to our knowledge, the first solution structure of domain III-IV of Aux/IAA17 (IAA17), and characterize molecular interactions underlying the homotypic and heterotypic oligomerization. The structure exhibits a compact β-grasp fold with a highly dynamic insert helix that is unique in Aux/IAA family proteins. IAA17 associates to form a heterogeneous ensemble of front-to-back oligomers in a concentration-dependent manner. IAA17 and ARF5 associate to form homo- or hetero-oligomers using a common scaffold and binding interfaces, but their affinities vary significantly. The equilibrium dissociation constants (KD) for homo-oligomerization are 6.6 μM and 0.87 μM for IAA17 and ARF5, respectively, whereas hetero-oligomerization reveals a ∼ 10- to ∼ 100-fold greater affinity (KD = 73 nM). Thus, individual homo-oligomers of IAA17 and ARF5 spontaneously exchange their subunits to form alternating hetero-oligomers for transcriptional repression. Oligomerization is mainly driven by electrostatic interactions, so that charge complementarity at the interface determines the binding affinity. Variable binding affinity by surface charge modulation may effectively regulate the complex interaction network between Aux/IAA and ARF family proteins required for the transcriptional control of auxin-response genes.

Dinesh Dhurvas's insight:

At AUX/IAA17 PB1 similar to ARF PB1 structure but with an addition alpha helix between DIII-IV !

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Structural basis for oligomerization of auxin transcriptional regulators

Structural basis for oligomerization of auxin transcriptional regulators | Interesting Plant Protein & 3D Structures | Scoop.it

The plant hormone auxin is a key morphogenetic regulator acting from embryogenesis onwards. Transcriptional events in response to auxin are mediated by the auxin response factor (ARF) transcription factors and the Aux/IAA (IAA) transcriptional repressors. At low auxin concentrations, IAA repressors associate with ARF proteins and recruit corepressors that prevent auxin-induced gene expression. At higher auxin concentrations, IAAs are degraded and ARFs become free to regulate auxin-responsive genes. The interaction between ARFs and IAAs is thus central to auxin signalling and occurs through the highly conserved domain III/IV present in both types of proteins. Here, we report the crystal structure of ARF5 domain III/IV and reveal the molecular determinants of ARF–IAA interactions. We further provide evidence that ARFs have the potential to oligomerize, a property that could be important for gene regulation in response to auxin.

Dinesh Dhurvas's insight:

ARF5 PB1 structure showing oligomerization mechanism !

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Nitrate-Regulated Auxin Transport by NRT1.1 Defines a Mechanism for Nutrient Sensing in Plants

Nitrate-Regulated Auxin Transport by NRT1.1 Defines a Mechanism for Nutrient Sensing in Plants | Interesting Plant Protein & 3D Structures | Scoop.it

Nitrate is a primary nutrient for plant growth, but its levels in soil can fluctuate by several orders of magnitude. Previous studies have identified Arabidopsis NRT1.1 as a dual-affinity nitrate transporter that can take up nitrate over a wide range of concentrations. The mode of action of NRT1.1 is controlled by phosphorylation of a key residue, Thr 101; however, how this post-translational modification switches the transporter between two affinity states remains unclear. Here we report the crystal structure of unphosphorylated NRT1.1, which reveals an unexpected homodimer in the inward-facing conformation. In this low-affinity state, the Thr 101 phosphorylation site is embedded in a pocket immediately adjacent to the dimer interface, linking the phosphorylation status of the transporter to its oligomeric state. Using a cell-based fluorescence resonance energy transfer assay, we show that functional NRT1.1 dimerizes in the cell membrane and that the phosphomimetic mutation of Thr 101 converts the protein into a monophasic high-affinity transporter by structurally decoupling the dimer. Together with analyses of the substrate transport tunnel, our results establish a phosphorylation-controlled dimerization switch that allows NRT1.1 to uptake nitrate with two distinct affinity modes.

Dinesh Dhurvas's insight:

Dual affinity nitrate transporters(NRT1.1) switch from low to high affinity state by phosphorylation ! Amazing nature !

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Cell Surface ABP1-TMK Auxin-Sensing Complex Activates ROP GTPase Signaling

Auxin-binding protein 1 (ABP1) was discovered nearly 40 years ago and was shown to be essential for plant development and morphogenesis, but its mode of action remains unclear. Here, we report that the plasma membrane–localized transmembrane kinase (TMK) receptor–like kinases interact with ABP1 and transduce auxin signal to activate plasma membrane–associated ROPs [Rho-like guanosine triphosphatases (GTPase) from plants], leading to changes in the cytoskeleton and the shape of leaf pavement cells in Arabidopsis. The interaction between ABP1 and TMK at the cell surface is induced by auxin and requires ABP1 sensing of auxin. These findings show that TMK proteins and ABP1 form a cell surface auxin perception complex that activates ROP signaling pathways, regulating nontranscriptional cytoplasmic responses and associated fundamental processes.


Via Jennifer Mach
Dinesh Dhurvas's insight:

ABP1+TMK structure yet to be solved !

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Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response

Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response | Interesting Plant Protein & 3D Structures | Scoop.it

The plant hormone auxin activates primary response genes by facilitating proteolytic removal of AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA)-inducible repressors, which directly bind to transcriptional AUXIN RESPONSE FACTORS (ARF). Most AUX/IAA and ARF proteins share highly conserved C-termini mediating homotypic and heterotypic interactions within and between both protein families. The high-resolution NMR structure of C-terminal domains III and IV of the AUX/IAA protein PsIAA4 from pea (Pisum sativum) revealed a globular ubiquitin-like β-grasp fold with homologies to the Phox and Bem1p (PB1) domain. The PB1 domain of wild-type PsIAA4 features two distinct surface patches of oppositely charged amino acid residues, mediating front-to-back multimerization via electrostatic interactions. Mutations of conserved basic or acidic residues on either face suppressed PsIAA4 PB1 homo-oligomerization in vitro and confirmed directional interaction of full-length PsIAA4 in vivo (yeast two-hybrid system). Mixing of oppositely mutated PsIAA4 PB1 monomers enabled NMR mapping of the negatively charged interface of the reconstituted PsIAA4 PB1 homodimer variant, whose stoichiometry (1:1) and equilibrium binding constant (KD ∼6.4 μM) were determined by isothermal titration calorimetry. In silico protein–protein docking studies based on NMR and yeast interaction data derived a model of the PsIAA4 PB1 homodimer, which is comparable with other PB1 domain dimers, but indicated considerable differences between the homodimeric interfaces of AUX/IAA and ARF PB1 domains. Our study provides an impetus for elucidating the molecular determinants that confer specificity to complex protein–protein interaction circuits between members of the two central families of transcription factors important to the regulation of auxin-responsive gene expression.

Dinesh Dhurvas's insight:

PsIAA4, a first AUX/IAA to be discovered in plant. Only PB1 structure to be solved as a monomer without mutations !

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Molecular basis for AUXIN RESPONSE FACTOR protein interaction and the control of auxin response repression

Molecular basis for AUXIN RESPONSE FACTOR protein interaction and the control of auxin response repression | Interesting Plant Protein & 3D Structures | Scoop.it

In plants, the AUXIN RESPONSE FACTOR (ARF) transcription factor family regulates gene expression in response to auxin. In the absence of auxin, ARF transcription factors are repressed by interaction with AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) proteins. Although the C termini of ARF and Aux/IAA proteins facilitate their homo- and heterooligomerization, the molecular basis for this interaction remained undefined. The crystal structure of the C-terminal interaction domain of Arabidopsis ARF7 reveals a Phox and Bem1p (PB1) domain that provides both positive and negative electrostatic interfaces for directional protein interaction. Mutation of interface residues in the ARF7 PB1 domain yields monomeric protein and abolishes interaction with both itself and IAA17. Expression of a stabilized Aux/IAA protein (i.e., IAA16) bearing PB1 mutations in Arabidopsis suggests a multimerization requirement for ARF protein repression, leading to a refined auxin-signaling model.

Dinesh Dhurvas's insight:

ARF7 PB1 structure.. Conserved acidic/basic patch mutants still form oligomers ! revisited @ http://www.jbc.org/content/290/20/12868.full

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Plant Physiology: Pseudomonas syringae type III effector AvrRpt2 promotes pathogen virulence via stimulating Arabidopsis Aux/IAA protein turnover (2013)

Plant Physiology: Pseudomonas syringae type III effector AvrRpt2 promotes pathogen virulence via stimulating Arabidopsis Aux/IAA protein turnover (2013) | Interesting Plant Protein & 3D Structures | Scoop.it

To accomplish successful infection, pathogens deploy complex strategies to interfere with host defense systems and subterfuge host physiology to favor pathogen survival and multiplication. Modulation of plant auxin physiology and signaling is emerging as a common virulence strategy for phytobacteria to cause diseases. However, the underlying mechanisms remain largely elusive. We have previously shown that the Pseudomonas syringae type III effector AvrRpt2 alters Arabidopsis auxin physiology. Here we report that AvrRpt2 promotes auxin response by stimulating the turnover of Aux/IAA proteins, the key negative regulators in auxin signaling. AvrRpt2 acts additively with auxin to stimulate Aux/IAA turnover, suggesting distinct, yet proteasome-dependent mechanisms operated by AvrRpt2 and auxin to control Aux/IAA stability. The cysteine protease activity is required for AvrRpt2-stimulated auxin signaling and Aux/IAA degradation. Importantly, the transgenic plants expressing the dominant axr2-1 mutation recalcitrant to AvrRpt2-mediated degradation ameliorated virulence functions of AvrRpt2, but did not alter the avirulent function mediated by the corresponding RPS2 resistance protein. Thus, promoting auxin response via modulating the stability of key transcription repressors Aux/IAA is a mechanism used by bacterial type III effector AvrRpt2 to promote pathogenicity.


Via Kamoun Lab @ TSL, Freddy Monteiro, tianxing84
Dinesh Dhurvas's insight:

Interestingly plant pathogen protein controls Aux/IAA turnover in plants..!

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An E4 Ligase Facilitates Polyubiquitination of Plant Immune Receptor Resistance Proteins in Arabidopsis

An E4 Ligase Facilitates Polyubiquitination of Plant Immune Receptor Resistance Proteins in Arabidopsis | Interesting Plant Protein & 3D Structures | Scoop.it

Proteins with nucleotide binding and leucine-rich repeat domains (NLRs) serve as immune receptors in animals and plants that recognize pathogens and activate downstream defense responses. As high accumulation of NLRs can result in unwarranted autoimmune responses, their cellular concentrations must be tightly regulated. However, the molecular mechanisms of this process are poorly detailed. The F-box protein Constitutive expressor of PR genes 1 (CPR1) was previously identified as a component of a Skp1, Cullin1, F-box protein E3 complex that targets NLRs, including Suppressor of NPR1, Constitutive 1 (SNC1) and Resistance to Pseudomonas syringae 2 (RPS2), for ubiquitination and further protein degradation. From a forward genetic screen, we identified Mutant, snc1-enhancing 3 (MUSE3), an E4 ubiquitin ligase involved in polyubiquitination of its protein targets. Knocking out MUSE3 in Arabidopsis thaliana results in increased levels of NLRs, including SNC1 and RPS2, whereas overexpressing MUSE3 together with CPR1 enhances polyubiquitination and protein degradation of these immune receptors. This report on the functional role of an E4 ligase in plants provides insight into the scarcely understood NLR degradation pathway.


Via Suayib Üstün, CP, Dr.donkey
Dinesh Dhurvas's insight:

E4 Ligase(MUSE3) adds up more Ub-Ub-Ub-Ub-Ub-Ubs to its target following E3 Ligase ! 

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Structural Basis for DNA Binding Specificity by the Auxin-Dependent ARF Transcription Factors

Structural Basis for DNA Binding Specificity by the Auxin-Dependent ARF Transcription Factors | Interesting Plant Protein & 3D Structures | Scoop.it

Auxin regulates numerous plant developmental processes by controlling gene expression via a family of functionally distinct DNA-binding auxin response factors (ARFs), yet the mechanistic basis for generating specificity in auxin response is unknown. Here, we address this question by solving high-resolution crystal structures of the pivotal Arabidopsis developmental regulator ARF5/MONOPTEROS (MP), its divergent paralog ARF1, and a complex of ARF1 and a generic auxin response DNA element (AuxRE). We show that ARF DNA-binding domains also homodimerize to generate cooperative DNA binding, which is critical for in vivo ARF5/MP function. Strikingly, DNA-contacting residues are conserved between ARFs, and we discover that monomers have the same intrinsic specificity. ARF1 and ARF5 homodimers, however, differ in spacing tolerated between binding sites. Our data identify the DNA-binding domain as an ARF dimerization domain, suggest that ARF dimers bind complex sites as molecular calipers with ARF-specific spacing preference, and provide an atomic-scale mechanistic model for specificity in auxin response.


Via Jennifer Mach
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