Heterotrimeric Complex Research Articles

Revisiting regulatory roles of replication protein A in plant DNA metabolism.

This review provides insight into the roles of heterotrimeric RPA protein complexes encompassing all aspects of DNA metabolism in plants along with specific function attributed by individual subunits. It highlights research gaps that need further attention. Replication protein A (RPA), a heterotrimeric protein complex partakes in almost every aspect of DNA metabolism in eukaryotes with its principle role being a single-stranded DNA-binding protein, thereby providing stability to single-stranded (ss) DNA. Although most of our knowledge of RPA structure and its role in DNA metabolism is based on studies in yeast and animal system, in recent years, plants have also been reported to have diverse repertoire of RPA complexes (formed by combination of different RPA subunit homologs arose during course of evolution), expected to be involved in plethora of DNA metabolic activities. Here, we have reviewed all studies regarding role of RPA in DNA metabolism in plants. As combination of plant RPA complexes may vary largely depending on number of homologs of each subunit, next step for plant biologists is to develop specific functional methods for detailed analysis of biological roles of these complexes, which we have tried to formulate in our review. Besides, complete absence of any study regarding regulatory role of posttranslational modification of RPA complexes in DNA metabolism in plants, prompts us to postulate a hypothetical model of same in light of information from animal system. With our review, we envisage to stimulate the RPA research in plants to shift its course from descriptive to functional studies, thereby bringing a new angle of studying dynamic DNA metabolism in plants.

CesL Regulates Type III Secretion Substrate Specificity of the Enteropathogenic E. coli Injectisome.

The type III secretion system (T3SS) is a complex molecular device used by several pathogenic bacteria to translocate effector proteins directly into eukaryotic host cells. One remarkable feature of the T3SS is its ability to secrete different categories of proteins in a hierarchical manner, to ensure proper assembly and timely delivery of effectors into target cells. In enteropathogenic Escherichia coli, the substrate specificity switch from translocator to effector secretion is regulated by a gatekeeper complex composed of SepL, SepD, and CesL proteins. Here, we report a characterization of the CesL protein using biochemical and genetic approaches. We investigated discrepancies in the phenotype among different cesL deletion mutants and showed that CesL is indeed essential for translocator secretion and to prevent premature effector secretion. We also demonstrated that CesL engages in pairwise interactions with both SepL and SepD. Furthermore, while association of SepL to the membrane does not depended on CesL, the absence of any of the proteins forming the heterotrimeric complex compromised the intracellular stability of each component. In addition, we found that CesL interacts with the cytoplasmic domains of the export gate components EscU and EscV. We propose a mechanism for substrate secretion regulation governed by the SepL/SepD/CesL complex.

Complex structure reveals CcmM and CcmN form a heterotrimeric adaptor in β-carboxysome.

Carboxysome is an icosahedral self-assembled microcompartment that sequesters RuBisCO and carbonic anhydrases within a selectively permeable protein shell. The scaffolding proteins, CcmM, and CcmN were proposed to act as adaptors that crosslink the enzymatic core to shell facets. However, the details of interaction pattern remain unknown. Here we obtained a stable heterotrimeric complex of CcmM γ-carbonic anhydrase domain (termed CcmMNT ) and CcmN, with a 1:2 stoichiometry, which interacts with the shell proteins CcmO and CcmL in vitro. The 2.9Å crystal structure of this heterotrimer revealed an asymmetric bundle composed of one CcmMNT and two CcmN subunits, all of which adopt a triangular left-handed β-helical barrel structure. The central CcmN subunit packs against CcmMNT and another CcmN subunit via a wall-to-edge or wall-to-wall pattern, respectively. Together with previous findings, we propose CcmMNT -CcmN functions as an adaptor to facilitate the recruitment of shell proteins and the assembly of intact β-carboxysome.

The translation initiation factor EIF4E5 from Leishmania: crystal structure and interacting partners

ABSTRACT The mRNA cap-binding protein, eIF4E, mediates the recognition of the mRNA 5ʹ end and, as part of the heterotrimeric eIF4F complex, facilitates the recruitment of the ribosomal subunits to initiate eukaryotic translation. Various regulatory events involving eIF4E and a second eIF4F subunit, eIF4G, are required for proper control of translation initiation. In pathogenic trypanosomatids, six eIF4Es and five eIF4Gs have been described, several forming different eIF4F-like complexes with yet unresolved roles. EIF4E5 is one of the least known of the trypanosomatid eIF4Es and has not been characterized in Leishmania species. Here, we used immunoprecipitation assays, combined with mass-spectrometry, to identify major EIF4E5 interacting proteins in L. infantum. A constitutively expressed, HA-tagged, EIF4E5 co-precipitated mainly with EIF4G1 and binding partners previously described in Trypanosoma brucei, EIF4G1-IP, RBP43 and the 14-3-3 proteins. In contrast, no clear co-precipitation with EIF4G2, also previously reported, was observed. EIF4E5 also co-precipitated with protein kinases, possibly associated with cell-cycle regulation, selected RNA binding proteins and histones. Phosphorylated residues were identified and mapped to the Leishmania-specific C-terminal end. Mutagenesis of the tryptophan residue (W53) postulated to mediate interactions with protein partners or of a neighbouring tryptophan conserved in Leishmania (W45) did not substantially impair the identified interactions. Finally, the crystal structure of Leishmania EIF4E5 evidences remarkable differences in the eIF4G interfacing region, when compared with human eIF4E-1 and with its Trypanosoma orthologue. Mapping of its C-terminal end near the cap-binding site also imply relevant differences in cap-binding function and/or regulation.

Underappreciation of the “Hook Effect” leading to Mismanagement of a Patient, a Tale of Preventable Disaster

Abstract Introduction: When evaluating pituitary adenomas, one needs to be cognizant of the “hook effect” - an assay artifact leading to under-reporting of extremely high prolactin levels. We report a case of a patient with a macroprolactinoma with an initial reported prolactin level of less than 1.0 ng/dl. Such an extreme case of the “hook effect” in a prolactinoma has not been reported yet. Case Presentation: A 53-year-old male with a medical history of type II DM initially presented in 2017 with blurred vision. Vital signs and labs, including pituitary hormone levels, were all within normal range. Prolactin level was reported as less than 1.0 ng/dl (normal 2-18 ng/dl). MRI brain showed a large pituitary mass and he was diagnosed with a non-secreting pituitary macroadenoma with mass effect. The patient subsequently underwent transsphenoidal resection of the pituitary adenoma. Prolactin level was noted to be elevated at 1608 ng/dl after the procedure. Unfortunately, the patient was lost to follow up. Two years later, the patient presented again with persistent headaches and worsening vision. MRI brain revealed a large suprasellar mass, measuring at 4.9 x 4 x 3 cm, with extension into the third ventricle and left cavernous sinus, indicating a recurrence of the pituitary adenoma. Prolactin levels measured were elevated 12,030 ng/dl. At this time, the patient was started on cabergoline with significant improvement in symptoms and reduction in prolactin levels to 1792 ng/dl within 4 weeks after initiation of therapy. He is currently still in remission with weekly cabergoline therapy. Conclusion: The most used prolactin assay typically reports the presence of detectable heterotrimeric immune complex formations made up of prolactin antigens sandwiched between ‘capture antibodies‘ at one end and ‘reported antibodies’ at the other end of the prolactin antigen. In prolactinomas, prolactin levels are usually present in relative excess to the two assay antibodies. This leads to the oversaturation of antibody sites with most of prolactin being complexed to only a single antibody instead of two antibodies, thus impairing adequate immune complex formation. Only a few remaining prolactin antigen are “sandwiched” to form the heterotrimeric complex and are therefore reported as “detectable.” As a result, true prolactin levels are substantially underreported, potentially leading to incorrect diagnosis, treatment delay, exposure to surgical risk and complications and increased economic burden to the patient and the healthcare system. The best method to avoid the “hook effect” and to correctly interpret prolactin values would be to obtain prolactin levels in appropriately diluted serum specimens (10-100-fold dilution of the sample). This will lead to appropriate immune complex formations and increase the sensitivity and specificity of the prolactin assay.

Structural Modeling and 3‐D Printing of the PTF1‐L Heterotrimeric Complex: Understanding the Structure and Function of a Ptf1a P191T Hypomorphic Mutation that Causes Pancreatic Aplasia in Children

Structural Modeling and 3‐D Printing of the PTF1‐L Heterotrimeric Complex: Understanding the Structure and Function of a Ptf1a P191T Hypomorphic Mutation that Causes Pancreatic Aplasia in Children

PPP2R1B is modulated by ubiquitination and is essential for spermatogenesis.

The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme complex that regulates many fundamental cellular processes. PP2A is involved in tumorigenesis because mutations in the scaffold subunit, PPP2R1B, were found in several types of cancers. However, the biological function of PPP2R1B remains largely unknown. We report here that homozygous deletion of Ppp2r1b in Mus musculus impairs meiotic recombination and causes meiotic arrest in spermatocytes. Consistently, male mice lacking Ppp2r1b are characterized with infertility. Furthermore, heterozygous missense mutations in the Homo sapiens PPP2R1B gene, which encodes PPP2R1B, are identified in azoospermia patients with meiotic arrest. We found that PPP2R1B mutants are susceptible to degradation by an E3 ligase CRL4ADCAF6 , and resistant to de-polyubiquitylation by ubiquitin-specific protease 5 (USP5). In addition, heterozygous mutations in PPP2R1B reduce stability of the wild-type PPP2R1B. Our results demonstrate an essential role of PPP2R1B in spermatogenesis and identify upstream regulators of PPP2R1B.

Biophysical Compatibility of a Heterotrimeric Tyrosinase-TYRP1-TYRP2 Metalloenzyme Complex.

Tyrosinase (TYR) is a copper-containing monooxygenase central to the function of melanocytes. Alterations in its expression or activity contribute to variations in skin, hair and eye color, and underlie a variety of pathogenic pigmentary phenotypes, including several forms of oculocutaneous albinism (OCA). Many of these phenotypes are linked to individual missense mutations causing single nucleotide variants and polymorphisms (SNVs) in TYR. We previously showed that two TYR homologues, TYRP1 and TYRP2, modulate TYR activity and stabilize the TYR protein. Accordingly, to investigate whether TYR, TYRP1, and TYRP2 are biophysically compatible with various heterocomplexes, we computationally docked a high-quality 3D model of TYR to the crystal structure of TYRP1 and to a high-quality 3D model of TYRP2. Remarkably, the resulting TYR-TYRP1 heterodimer was complementary in structure and energy with the TYR-TYRP2 heterodimer, with TYRP1 and TYRP2 docking to different adjacent surfaces on TYR that apposed a third realistic protein interface between TYRP1-TYRP2. Hence, the 3D models are compatible with a heterotrimeric TYR-TYRP1-TYRP2 complex. In addition, this heterotrimeric TYR-TYRP1-TYRP2 positioned the C-terminus of each folded enzymatic domain in an ideal position to allow their C-terminal transmembrane helices to form a putative membrane embedded three-helix bundle. Finally, pathogenic TYR mutations causing OCA1A, which also destabilize TYR biochemically, cluster on an unoccupied protein interface at the periphery of the heterotrimeric complex, suggesting that this may be a docking site for OCA2, an anion channel. Pathogenic OCA2 mutations result in similar phenotypes to those produced by OCA1A TYR mutations. While this complex may be difficult to detect in vitro, due to the complex environment of the vertebrate cellular membranous system, our results support the existence of a heterotrimeric complex in melanogenesis.

GTP binding by Arabidopsis extra-large G protein 2 is not essential for its functions.

The extra-large guanosine-5'-triphosphate (GTP)-binding protein 2, XLG2, is an unconventional Gα subunit of the Arabidopsis (Arabidopsis thaliana) heterotrimeric GTP-binding protein complex with a major role in plant defense. In vitro biochemical analyses and molecular dynamic simulations show that affinity of XLG2 for GTP is two orders of magnitude lower than that of the conventional Gα, AtGPA1. Here we tested the physiological relevance of GTP binding by XLG2. We generated an XLG2(T476N) variant with abolished GTP binding, as confirmed by in vitro GTPγS binding assay. Yeast three-hybrid, bimolecular fluorescence complementation, and split firefly-luciferase complementation assays revealed that the nucleotide-depleted XLG2(T476N) retained wild-type XLG2-like interactions with the Gβγ dimer and defense-related receptor-like kinases. Both wild-type and nucleotide-depleted XLG2(T476N) restored the defense responses against Fusarium oxysporum and Pseudomonas syringae compromised in the xlg2 xlg3 double mutant. Additionally, XLG2(T476N) was fully functional restoring stomatal density, root growth, and sensitivity to NaCl, but failed to complement impaired germination and vernalization-induced flowering. We conclude that XLG2 is able to function in a GTP-independent manner and discuss its possible mechanisms of action.

The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication.

The duplication and ninefold symmetry of the Drosophila centriole requires that the cartwheel molecule, Sas6, physically associates with Gorab, a trans-Golgi component. How Gorab achieves these disparate associations is unclear. Here, we use hydrogen-deuterium exchange mass spectrometry to define Gorab's interacting surfaces that mediate its subcellular localization. We identify a core stabilization sequence within Gorab's C-terminal coiled-coil domain that enables homodimerization, binding to Rab6, and thereby trans-Golgi localization. By contrast, part of the Gorab monomer's coiled-coil domain undergoes an antiparallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable heterotrimeric complex can be visualized by electron microscopy. Mutation of a single leucine residue in Sas6's Gorab-binding domain generates a Sas6 variant with a sixteenfold reduced binding affinity for Gorab that cannot support centriole duplication. Thus, Gorab dimers at the Golgi exist in equilibrium with Sas6-associated monomers at the centriole to balance Gorab's dual role.

Nuclear βArrestin1 regulates androgen receptor function in castration resistant prostate cancer.

Progression of prostate cancer (PC) to terminal castration-resistant PC (CRPC) involves a diverse set of intermediates, and androgen receptor (AR) is the key mediator of PC initiation and progression to CRPC. Hence, identification of factors involved in the regulation of AR expression and function is a necessary first-step to improve disease outcome. In this study, we identified ubiquitous βArrestin 1 (βArr1) as a regulator of AR function in CRPC. Unbiased gene expression analysis of public datasets revealed increased levels of ARRB1 (the gene encoding βArr1) in CRPC when compared to normal tissue. Further, βArr1 expression correlated with enhanced AR transcriptional function in these datasets. The βArr1 partitions to both nucleus and cytosol and mechanistic studies showed that nuclear, and not cytosolic, βArr1 formed a complex with AR and AR-coregulator βCatenin and that the heterotrimeric protein complex was recruited to androgen-response elements of AR-regulated genes. Functionally, we demonstrate that depletion of βArr1 attenuates PC cell and tumor growth and metastasis, and rescued expression of nuclear, but not cytosolic, βArr1 restores the PC colony growth and invasion of Matrigel in vitro and tumor growth and metastasis in mice. The targeting of βArr1-regulated AR transcriptional function may be used in the development of new drugs to treat lethal CRPC.

Deep transcriptome sequencing of subgenual anterior cingulate cortex reveals cross-diagnostic and diagnosis-specific RNA expression changes in major psychiatric disorders

Despite strong evidence of heritability and growing discovery of genetic markers for major mental illness, little is known about how gene expression in the brain differs across psychiatric diagnoses, or how known genetic risk factors shape these differences. Here we investigate expressed genes and gene transcripts in postmortem subgenual anterior cingulate cortex (sgACC), a key component of limbic circuits linked to mental illness. RNA obtained postmortem from 200 donors diagnosed with bipolar disorder, schizophrenia, major depression, or no psychiatric disorder was deeply sequenced to quantify expression of over 85,000 gene transcripts, many of which were rare. Case–control comparisons detected modest expression differences that were correlated across disorders. Case–case comparisons revealed greater expression differences, with some transcripts showing opposing patterns of expression between diagnostic groups, relative to controls. The ~250 rare transcripts that were differentially-expressed in one or more disorder groups were enriched for genes involved in synapse formation, cell junctions, and heterotrimeric G-protein complexes. Common genetic variants were associated with transcript expression (eQTL) or relative abundance of alternatively spliced transcripts (sQTL). Common genetic variants previously associated with disease risk were especially enriched for sQTLs, which together accounted for disproportionate fractions of diagnosis-specific heritability. Genetic risk factors that shape the brain transcriptome may contribute to diagnostic differences between broad classes of mental illness.

Eukaryotic Translation Initiation Factor 4AI: A Potential Novel Target in Neuroblastoma.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Children suffering from high-risk and/or metastatic NB often show no response to therapy, and new therapeutic approaches are urgently needed. Malignant tumor development has been shown to be driven by the dysregulation of eukaryotic initiation factors (eIFs) at the translation initiation. Especially the activity of the heterotrimeric eIF4F complex is often altered in malignant cells, since it is the direct connection to key oncogenic signaling pathways such as the PI3K/AKT/mTOR-pathway. A large body of literature exists that demonstrates targeting the translational machinery as a promising anti-neoplastic approach. The objective of this study was to determine whether eIF4F complex members are aberrantly expressed in NB and whether targeting parts of the complex may be a therapeutic strategy against NB. We show that eIF4AI is overexpressed in NB patient tissue using immunohistochemistry, immunoblotting, and RT-qPCR. NB cell lines exhibit decreased viability, increased apoptosis rates as well as changes in cell cycle distribution when treated with the synthetic rocaglate CR-1-31-B, which clamps eIF4A and eIF4F onto mRNA, resulting in a translational block. Additionally, this study reveals that CR-1-31-B is effective against NB cell lines at low nanomolar doses (≤20 nM), which have been shown to not affect non-malignant cells in previous studies. Thus, our study provides information of the expression status on eIF4AI in NB and offers initial promising insight into targeting translation initiation as an anti-tumorigenic approach for NB.

Single-Molecule Dynamics of eIF4F-Mediated mRNA Recognition

Recognition of the mRNA 5’ cap is by the heterotrimeric eIF4F complex is a critical step in eukaryotic translation initiation. As the final point of regulation prior to protein synthesis, regulation of steps of initiation is critical for maintaining cellular homeostasis. Despite efforts to characterize steps of translation initiation in real time, a detailed understanding of how cap recognition may vary between transcripts, and the contributions of individual initiation factors, is lacking due to an absence of appropriate methods. We recently developed a single molecule assay for examining this process on yeast mRNAs. Using single-molecule FRET, we in vitro reconstituted cap recognition by the mRNA cap binding protein eIF4E, characterizing this process in real-time. We apply this method to individual transcripts as well as heterogeneous pools of mRNA extracted from S.cerevisiae, characterizing the extent at which cap recognition by the cap binding subunit eIF4E varies throughout populations in the transcriptome. Additionally, we looked at the contributions of other eIF4F subunits and ATP to the rate of formation and stability of the eIF4E-cap complex, characterizing the kinetics of cap recognition. Our results suggest that mRNAs are inherently recognized at different rates by eIF4F, which can contribute to the wide range of translational efficiencies observed in vivo. Specifically, our findings highlight how factor composition and variations in mRNA features could tune the dynamics of protein synthesis by altering the rates of events during early initiation. Our work lays a foundation for isolating and studying the early steps of eukaryotic translation initiation, which is a multi-step, multi-factor process difficult to dissect in vivo due to its complexity.

Protein expression alteration in hippocampus upon genetic repression of AMPKα isoforms.

The AMP-activated protein kinase (AMPK) is a molecular sensor to help maintain cellular energy homeostasis. AMPK is a heterotrimeric complex and its enzymatic catalytic subunit includes two isoforms: α1 and α2. Dysregulation of AMPK signaling is linked to neuronal diseases characterized with cognitive impairments. Emerging evidence also suggest isoform-specific roles of AMPK in the brain. AMPK regulates protein synthesis, which is critical for memory formation and neuronal plasticity. However, the consequence of altering AMPK activity on the translation of specific proteins in the brain is unknown. Here, we use unbiased mass spectrometry-based proteomics approach to analyze protein profile alterations in hippocampus and prefrontal cortex of transgenic mice in which the genes for the two AMPKα isoforms are conditionally deleted. The study revealed identities of proteins whose expression is sensitive to suppression of AMPKα1 and/or α2 isoform. These data may serve as a basis for future in-depth study. Elucidation of the functional relevance of the alteration of specific proteins could provide insights into identification of novel therapeutic targets for neuronal disorders characterized with AMPK signaling dysregulation and impaired cellular energy metabolism.

Structure of a microtubule-bound axonemal dynein

Axonemal dyneins are tethered to doublet microtubules inside cilia to drive ciliary beating, a process critical for cellular motility and extracellular fluid flow. Axonemal dyneins are evolutionarily and biochemically distinct from cytoplasmic dyneins that transport cargo, and the mechanisms regulating their localization and function are poorly understood. Here, we report a single-particle cryo-EM reconstruction of a three-headed axonemal dynein natively bound to doublet microtubules isolated from cilia. The slanted conformation of the axonemal dynein causes interaction of its motor domains with the neighboring dynein complex. Our structure shows how a heterotrimeric docking complex specifically localizes the linear array of axonemal dyneins to the doublet microtubule by directly interacting with the heavy chains. Our structural analysis establishes the arrangement of conserved heavy, intermediate and light chain subunits, and provides a framework to understand the roles of individual subunits and the interactions between dyneins during ciliary waveform generation.

Lipid Signaling through G Proteins.

N-Acylethanolamine (NAE) signaling has received considerable attention in vertebrates as part of the endocannabinoid signaling system, where anandamide acts as a ligand for G protein-coupled cannabinoid receptors. Recent studies indicate that G proteins also are required for some types of NAE signaling in plants. The genetic ablation of the Gβγ dimer or loss of the full set of extra-large G proteins strongly attenuated NAE-induced chloroplast responses in seedlings. Intriguing parallels and distinct differences have emerged between plants and animals in NAE signaling, despite the conserved use of these lipid mediators to modulate cellular processes. Here we compare similarities and differences and identify open questions in a fundamental lipid signaling pathway in eukaryotes with components that are both conserved and diverged in plants.

A novel anti-human IL-1R7 antibody reduces IL-18-mediated inflammatory signaling

Unchecked inflammation can result in severe diseases with high mortality, such as macrophage activation syndrome (MAS). MAS and associated cytokine storms have been observed in COVID-19 patients exhibiting systemic hyperinflammation. Interleukin-18 (IL-18), a proinflammatory cytokine belonging to the IL-1 family, is elevated in both MAS and COVID-19 patients, and its level is known to correlate with the severity of COVID-19 symptoms. IL-18 binds its specific receptor IL-1 receptor 5 (IL-1R5, also known as IL-18 receptor alpha chain), leading to the recruitment of the coreceptor, IL-1 receptor 7 (IL-1R7, also known as IL-18 receptor beta chain). This heterotrimeric complex then initiates downstream signaling, resulting in systemic and local inflammation. Here, we developed a novel humanized monoclonal anti-IL-1R7 antibody to specifically block the activity of IL-18 and its inflammatory signaling. We characterized the function of this antibody in human cell lines, in freshly obtained peripheral blood mononuclear cells (PBMCs) and in human whole blood cultures. We found that the anti-IL-1R7 antibody significantly suppressed IL-18-mediated NFκB activation, reduced IL-18-stimulated IFNγ and IL-6 production in human cell lines, and reduced IL-18-induced IFNγ, IL-6, and TNFα production in PBMCs. Moreover, the anti-IL-1R7 antibody significantly inhibited LPS- and Candida albicans–induced IFNγ production in PBMCs, as well as LPS-induced IFNγ production in whole blood cultures. Our data suggest that blocking IL-1R7 could represent a potential therapeutic strategy to specifically modulate IL-18 signaling and may warrant further investigation into its clinical potential for treating IL-18-mediated diseases, including MAS and COVID-19.

A Reply to 'Cellular Toxicity of iHAP1 and DT-061 Does Not Occur Through PP2A-B56 Targeting'

While we appreciate the importance of independent validation of scientific discoveries and, thus, welcome all follow-up studies that evaluate the reported cytotoxic effects of DT-061 on cancer cells, we are somewhat flabbergasted by the claim by Nilsson and colleagues that they could not confirm effects of the compound depended on PP2A. This is particularly surprising as we presented a series of orthogonal assays directed at defining the mode of action of DT-061, especially binding and stabilization of specific PP2A heterotrimer complexes, but not a single one of these experiments was performed precisely as described (Leonard et al 2020). Programs like The Open Science initiative emphasize the need to exactly reproduce experiments when testing for reproducibility. As such, the title of the Matters Arising opinion submitted by Vit et al is misleading, as no direct experiments were undertaken to show that DT-061 is unable to interact with PP2A.

Novel perspectives of target-binding by the evolutionarily conserved PP4 phosphatase.

Protein phosphatase 4 (PP4) is an evolutionarily conserved and essential Ser/Thr phosphatase that regulates cell division, development and DNA repair in eukaryotes. The major form of PP4, present from yeast to human, is the PP4c-R2-R3 heterotrimeric complex. The R3 subunit is responsible for substrate-recognition via its EVH1 domain. In typical EVH1 domains, conserved phenylalanine, tyrosine and tryptophan residues form the specific recognition site for their target's proline-rich sequences. Here, we identify novel binding partners of the EVH1 domain of the Drosophila R3 subunit, Falafel, and demonstrate that instead of binding to proline-rich sequences this EVH1 variant specifically recognizes atypical ligands, namely the FxxP and MxPP short linear consensus motifs. This interaction is dependent on an exclusively conserved leucine that replaces the phenylalanine invariant of all canonical EVH1 domains. We propose that the EVH1 domain of PP4 represents a new class of the EVH1 family that can accommodate low proline content sequences, such as the FxxP motif. Finally, our data implicate the conserved Smk-1 domain of Falafel in target-binding. These findings greatly enhance our understanding of the substrate-recognition mechanisms and function of PP4.