Biology subjects

Sluchanko, N. N.

Publications and source records attributed to Sluchanko, N. N..

At least 19 recordsLinked to original sources

bioRxiv2025

Structural and Functional Characterization of Encapsulin-Targeted Double Ferritin Fold Ferroxidases

Ferritins are a widespread family of proteins involved in iron homeostasis. While classic ferritins consist of four -helices and form 24-meric nanocages, related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity. Here, by analyzing the available genomic data, we identify a family of double ferritin-like proteins (DFLPs) composed of two four-helical domains, which arose by duplication of a ferritin fold protein. We characterize representative DFLPs from Thermocrinis minervae and Caldanaerovirga acetigignens, TmDFLP and CaDFLP, and show that they form homodimers and bind heme. We determine the X-ray structure of TmDFLP and demonstrate its ferroxidase activity. Furthermore, we show that some DFLPs, including TmDFLP and CaDFLP, are highly likely to be targeted into encapsulin shells. Our work expands the range of known iron metabolism systems and highlights the power of genome mining for discovery of new proteins. Graphical Abstract for Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/676036v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@72e6b0org.highwire.dtl.DTLVardef@18752fforg.highwire.dtl.DTLVardef@15a02e7org.highwire.dtl.DTLVardef@74cd02_HPS_FORMAT_FIGEXP M_FIG C_FIG A family of double ferritin-like proteins (DFLPs) composed of two four-helical domains is described and investigated. DFLPs are shown to form homodimers, bind heme, possess diiron sites and display ferroxidase activity. Some DFLPs are shown to be targeted to encapsulin shells as core or secondary cargo, thus representing a new type of iron metabolism systems.

biochemistry↗

bioRxiv2025

Architecture of the bundle-shaped phycobilisome from Gloeobacter violaceus

Cyanobacteria use soluble antenna megacomplexes, phycobilisomes (PBS), to maximize light-harvesting efficiency and small photoswitchable orange carotenoid proteins (OCPs) to down-regulate PBS in high light. Among known PBS morphologies, the one from the basal cyanobacterial genus Gloeobacter still lacks detailed structural characterization. Here, we present the cryo-EM structure of the G. violaceus PBS, a >10-MDa complex with a unique architecture consisting in 516 polypeptide chains totaling nearly 90,000 residues and harboring over 860 bilin chromophores. This unique PBS features diverging, conformationally mobile bundles of rods composed of stacked phycoerythrin and phycocyanin hexamers, stemming from a pentacylindrical allophycocyanin core belted by auxiliary phycocyanin hexamers. We show how two Gloeobacter-specific multidomain linker proteins, Glr1262 and Glr2806, maintain this bundle-shaped architecture and reveal its differential regulation via non-photochemical quenching by two OCP types of G. violaceus that recognize separate binding sites within the allophycocyanin core, including lateral cylinders absent in tricylindrical cores. Our findings provide the high-resolution structural insight into Gloeobacter PBS and its regulation, revealing divergent adaptations in early-branching cyanobacteria. The structure advances understanding of PBS diversity and evolution, offering a framework for bioengineering light-harvesting systems in synthetic biology and biotechnological applications.

biochemistry↗

bioRxiv2025

Target-induced Argonaute-HNH filaments confer bacterial immunity

Argonaute proteins provide innate immunity in all domains of life through guide-dependent recognition of invader nucleic acids. While eukaryotic Argonautes (eAgos) act on RNA during RNA interference, prokaryotic Argonautes (pAgos) mainly recognize DNA targets. Many eAgos and some pAgos are active nucleases that directly cleave their targets. In contrast, short pAgos lack the nuclease activity and are co-encoded with additional effectors. Diverse effector domains in short pAgo systems include NADases and tentative nucleases, but their mechanisms of activation remain largely unknown. Here, we characterize SPARHA systems (short prokaryotic argonautes, HNH-associated) encoding HNH nuclease effectors. We show that short pAgo activates the HNH effector after RNA-guided DNA recognition. Target recognition induces formation of filaments of SPARHA with double active sites formed at the interfaces of repetitive HNH domains, which results in indiscriminate collateral degradation of DNA and protects bacterial population from invaders. The results show that pAgos and associated effectors act as modular two-component systems that translate recognition of specific DNA into immune response through assembly of supramolecular complexes, deleterious for invaders and potentially useful for biotechnology.

molecular biology↗

bioRxiv2024

Multi-dimensional demarcation of phylogenetic groups of plant 14-3-3 isoforms using biochemical signatures

Interaction of dimeric 14-3-3 proteins with numerous phosphotargets regulates various physiological processes in plants, from flowering to transpiration and salt tolerance. Several genes express distinct 14-3-3 isoforms, particularly numerous in plants, but comparative studies of all 14-3-3 isoforms for a given organism have not been undertaken. Here we systematically investigated twelve 14-3-3 isoforms from the model plant Arabidopsis thaliana, uniformly capable of homodimerization at high protein concentration. We unexpectedly discovered that, at physiological protein concentrations, four isoforms representing a seemingly more ancestral, epsilon phylogenetic group (iota, mu, omicron, epsilon) demonstrate an outstanding monomerization propensity and enhanced surface hydrophobicity, which is uncharacteristic for eight non-epsilon isoforms (omega, phi, chi, psi, upsilon, nu, kappa, lambda). Further analysis revealed that dramatically lowered thermodynamic stabilities entail aggregation of the epsilon-group isoforms at near-physiological temperatures and provoke their proteolytic degradation. Structure-inspired single mutations in 14-3-3 iota could rescue non-epsilon behavior, thereby pinpointing key positions responsible for the phylogenetic demarcation. Combining two major demarcating positions (namely, 27th and 51st in omega) and multi-dimensional differences in biochemical properties identified here, we developed a predictor strongly supporting categorization of abundant 14-3-3 isoforms widely across plant groups, from Eudicots to Monocots, Gymnosperms and Lycophytes. In particular, our approach fully recapitulates the phylogenetic epsilon/non-epsilon demarcation in Eudicots and supports the presence of isoforms of both types in more primitive plant groups such as Selaginella, thereby refining solely sequence-based analysis in evolutionarily distant species and providing novel insights into the evolutionary history of the epsilon phylogenetic group. SignificanceDespite over 30 years of research, systematic comparative studies on the regulatory plant 14-3-3 proteins have not been undertaken, making phylogenetic classification of numerous plant 14-3-3 isoforms in different species unreliable. Working on twelve purified Arabidopsis 14-3-3 isoforms, we have discovered a set of biochemical signatures that can be used to robustly and widely categorize epsilon and non-epsilon plant 14-3-3 isoforms, also identifying at least two amino acid positions responsible for such multi-dimensional demarcation.

biochemistry↗

bioRxiv2024

Phosphorylation code of human nucleophosmin includes four cryptic sites for hierarchical binding of 14-3-3 proteins

Nucleophosmin (NPM1) is the 46th most abundant human protein with many functions whose dysregulation leads to various cancers. Pentameric NPM1 resides in the nucleolus but can also shuttle to the cytosol. NPM1 is regulated by multisite phosphorylation, yet molecular consequences of site-specific NPM1 phosphorylation remain elusive. Here we identify four 14-3-3 protein binding sites in NPM1 concealed within its oligomerization and -helical C-terminal domains that are found phosphorylated in vivo. By combining mutagenesis, in-cell phosphorylation and PermaPhos technology for site-directed incorporation of a non-hydrolyzable phosphoserine mimic, we show how phosphorylation promotes NPM1 monomerization and partial unfolding, to recruit 14-3-3 dimers with low-micromolar affinity. Using fluorescence anisotropy we quantified pairwise interactions of all seven human 14-3-3 isoforms with four recombinant NPM1 phosphopeptides and assessed their druggability by fusicoccin. This revealed a complex hierarchy of 14-3-3 affinities toward the primary (S48, S293) and secondary (S106, S260) sites, differentially modulated by the small molecule. As three of these 14-3-3 binding phospho-sites in NPM1 reside within signal sequences, this work highlights a key mechanism of NPM1 regulation by which NPM1 phosphorylation promotes 14-3-3 binding to control nucleocytoplasmic shuttling. It also provides further evidence that phosphorylation-induced structural rearrangements of globular proteins serve to expose otherwise cryptic 14-3-3-binding sites that are important for cellular function.

biochemistry↗

bioRxiv2023

Structural framework for the understanding spectroscopic and functional signatures of the cyanobacterial Orange Carotenoid Protein families

The Orange Carotenoid Protein (OCP) is a unique photoreceptor crucial for cyanobacterial photoprotection. Best studied Synechocystis sp. PCC 6803 OCP belongs to the large OCP1 family. Downregulated by the Fluorescence Recovery Protein (FRP) in low-light, high-light-activated OCP1 binds to the phycobilisomes and performs non-photochemical quenching. Recently discovered families OCP2 and OCP3 remain structurally and functionally underexplored, and no systematic comparative studies have ever been conducted. Here we present two first crystal structures of OCP2 from morphoecophysiologically different cyanobacteria and provide their comprehensive structural, spectroscopic and functional comparison with OCP1, the recently described OCP3 and all-OCP ancestor. Structures enable correlation of spectroscopic signatures with the effective number of hydrogen and discovered here chalcogen bonds anchoring the ketocarotenoid in OCP and rationalize the observed differences in OCP/FRP and OCP/phycobilisome functional interactions. These data are expected to foster OCP research and applications in optogenetics, targeted carotenoid delivery and cyanobacterial biomass engineering.

biochemistry↗

bioRxiv2023

DNA-targeting short Argonaute triggers effector nuclease to protect bacteria from invaders

Two prokaryotic defence systems, Argonautes (pAgos) and CRISPR-Cas, detect invader nucleic acids using complementary guides. Upon recognition, the target is cleaved through nuclease activities of pAgo or Cas proteins thus protecting the cell from invasion. However, not all pAgos are active nucleases. Members of a large clade of short pAgos bind nucleic acid guides but lack nuclease activity suggesting a different mechanism of action. Here, we have investigated short pAgo from Novosphingopyxis baekryungensis (NbaAgo). We have shown that NbaAgo forms a heterodimeric complex, SPARDA, with a co-encoded effector nuclease. RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA results in cell death during plasmid transformation or phage infection, thus protecting bacterial population from invaders. The collateral activity of SPARDA allows highly sensitive detection of specific DNA targets. SPARDA expands the list of prokaryotic immune systems that elicit suicidal cell response with a unique range of nuclease activities, creating additional opportunities for biotechnologies.

molecular biology↗

bioRxiv2023

luxA gene from Enhygromyxa salina encodes a functional homodimeric luciferase

Several clades of luminescent bacteria are known currently. They all contain similar lux operons, which include the genes luxA and luxB encoding a heterodimeric luciferase. The aldehyde oxygenation reaction is catalyzed by the subunit LuxA, while LuxB is inactive. Recently, genomic analysis identified a subset of bacterial species with rearranged lux operons lacking luxB. Here, we show that the product of the luxA gene from the reduced luxACDE operon of Enhygromyxa salina is luminescent upon addition of aldehydes both in vivo in Escherichia coli and in vitro. Overall, EsLuxA is less bright compared with luciferases from Aliivibrio fischeri (AfLuxAB) and Photorhabdus luminescens (PlLuxAB), and most active with medium-chain C4-C9 aldehydes. Crystal structure of EsLuxA determined at the resolution of 2.71 [A] reveals a classical monooxygenase fold, and the protein preferentially forms a dimer in solution. The mobile loop residues 264-293, which form a {beta}-hairpin or a coil in Vibrio harveyi LuxA, form -helices in EsLuxA. Phylogenetic analysis shows EsLuxA and related proteins may be bacterial protoluciferases that arose prior to duplication of the luxA gene and its speciation to luxA and luxB in the previously described luminescent bacteria. Our work paves the way for discovery of new luciferases that have an advantage of being encoded by a single gene.

biochemistry↗

bioRxiv2022

Structural basis for the ligand promiscuity of the neofunctionalized, carotenoid-binding fasciclin domain protein AstaP

Fasciclins (FAS1) are ancient adhesion protein domains found across different phyla from bacteria to humans, with no common small ligand binding function reported. A unique FAS1-containing astaxanthin-binding protein (AstaP) from green algae can efficiently bind an unusually broad repertoire of carotenoids (astaxanthin, zeaxanthin, canthaxanthin, {beta}-carotene), but the underlying mechanism is largely unknown. Here we dissect the structural basis for the ligand binding promiscuity of AstaP-orange1 (AstaPo1) by determining its solution NMR structure in complex with its natural ligand, astaxanthin (AXT), and validate this structure by SAXS, calorimetry, optical spectroscopy and mutagenesis data. While the unstructured tails of AstaPo1 are not essential for carotenoid binding, they enhance protein solubility. The a1-a2 helices of the AstaPo1 FAS1 domain embrace the carotenoid polyene like a jaw, organizing a conserved hydrophobic tunnel, too short to prevent the AXT {beta}-ionone rings from protruding on both sides of the tunnel, thereby not imposing specificity restrictions. The only specific protein-AXT interactions involve H-bonds between the oxygenated groups on AXT and a peripheral Gln56 residue. Remarkably, mapping of this and other AXT-contacting AstaPo1 residues revealed their different conservation in AstaP orthologs with the tentative carotenoid-binding function and in FAS1 proteins in general, supporting neofunctionalization of AstaPs within green algae. Correspondingly, a cyanobacterial homolog with a similar domain structure cannot bind carotenoids due to subtle differences in residues decorating the tunnel. These structure-activity relationships inform the sequence-based prediction of the carotenoid-binding FAS1 members. SIGNIFICANCEA water-soluble astaxanthin-binding protein (AstaP) is a photoprotective protein in green algae helping them to tolerate stress conditions. While belonging to a ubiquitous protein family sharing an ancient structural domain, fasciclin, involved in cell adhesion, AstaP possesses an outstanding ability to bind carotenoid pigments of a different type, which are potent antioxidants. To understand the molecular basis for such carotenoid-binding promiscuity of AstaP, here we determined its spatial structure - the first structure of a carotenoid-protein complex solved by nuclear magnetic resonance spectroscopy. Together with biochemical and sequence conservation analyses, our data illustrate a remarkable case of neofunctionalization of the ancient protein domain and pave the way for its bioengineering and practical use as antioxidant transporter for biomedical applications.

biochemistry↗

bioRxiv2022

Genetic encoding of 3-nitro-tyrosine reveals the impacts of 14-3-3 nitration on client binding and dephosphorylation

14-3-3 proteins are central hub regulators of hundreds of phosphorylated "client" proteins. They are subject to over 60 post-translational modifications (PTMs), yet little is known how these PTMs alter 14-3-3 function and its ability to regulate downstream signaling pathways. An often neglected, but well documented 14-3-3 PTM found under physiological and immune-stimulatory conditions is the conversion of tyrosine to 3-nitro-tyrosine at several Tyr sites, two of which are located at sites considered important for 14-3-3 function: Y130 ({beta}-isoform numbering) is located in the primary phospho-client peptide binding groove, while Y213 is found on a secondary binding site that engages with clients for full 14-3-3/client complex formation and client regulation. By genetically encoding 3-nitro-tyrosine, we sought to understand if nitration at Y130 and Y213 effectively modulated 14-3-3 structure, function, and client complexation. The 1.5 [A] resolution crystal structure of 14-3-3 nitrated at Y130 showed the nitro group altered the conformation of key residues in the primary binding site, while functional studies confirmed client proteins failed to bind this variant of 14-3-3. But, in contrast to other client-binding deficient variants, it did not localize to the nucleus. The 1.9 [A] resolution structure of 14-3-3 nitrated at Y213 revealed unusual flexibility of its C-terminal -helix resulting in domain swapping, suggesting additional structural plasticity though its relevance is not clear as this nitrated form retained its ability to bind clients. Collectively, our data suggest nitration of 14-3-3 will alter downstream signaling systems, and if uncontrolled could result in global dysregulation of the 14-3-3 interactome.

biochemistry↗

bioRxiv2022

The Tramtrack group BTB protein domains are Arthropoda-specific multimerization modules

BTB (Bric-a-brack, Tramtrack and Broad Complex) is a diverse group of protein-protein interaction domains found within metazoan proteins. Transcription factors contain a dimerizing BTB subtype with a characteristic N-terminal extension. The Tramtrack group (TTK) is a distinct type of BTB domain, which can multimerize. Single-particle cryo-EM microscopy revealed that the TTK-type BTB domains assemble into a hexameric structure consisting of three canonical BTB dimers connected through a previously uncharacterized interface. We demonstrated that the TTK-type BTB domains are found only in Arthropods and have undergone lineage-specific expansion in modern insects. The Drosophila genome encodes 24 transcription factors with TTK-type BTB domains, whereas only four have non-TTK-type BTB domains. Yeast two-hybrid analysis revealed that the TTK-type BTB domains have an unusually broad potential for heteromeric associations presumably through dimer-dimer interaction interface. Thus, the TTK-type BTB domains are a structurally and functionally distinct group of protein domains specific to Arthropodan transcription factors.

molecular biology↗

bioRxiv2022

Nanocontainer derived from silkworm carotenoprotein for carotenoid extraction and presentation in biotechnology and biomedical applications

Found in many organisms, soluble carotenoproteins are considered as antioxidant nanocarriers for biomedical applications, although the structural basis for their carotenoid transfer function, a prerequisite for rational bioengineering, is largely unknown. We report crystal structures of the Carotenoid-Binding Protein from Bombyx mori (BmCBP) in apo- and zeaxanthin (ZEA)-bound forms. We use spectroscopy and calorimetry to characterize how ZEA and BmCBP mutually affect each other in the complex, identify key carotenoid-binding residues, confirm their roles by crystallography and carotenoid-binding capacity of BmCBP mutants and reconstitute BmCBP complexes with biomedically-relevant xanthophylls lutein, zeaxanthin, canthaxanthin and astaxanthin. By cost-effectively and scalably solubilizing xanthophylls from various crude herbal extracts, His-tagged BmCBP remains monomeric and forms a dynamic nanocontainer delivering carotenoids to liposomes and to other carotenoid-binding proteins, which in particular makes the Orange Carotenoid Protein, a promising optogenetic tool, photoactive. Furthermore, BmCBP(ZEA) administration stimulates fibroblast growth, which paves the way for its biomedical applications.

biochemistry↗

bioRxiv2022

Crystal structure and functional peculiarities of a primordial Orange Carotenoid Protein (OCPX)

The two-domain photoactive Orange Carotenoid Protein (OCP) confers photoprotection in cyanobacteria and presumably stems from domain fusion. Yet, the primitive thylakoid-less cyanobacteria Gloeobacter encodes a complete OCP. Its photosynthesis regulation lacks the so-called Fluorescence Recovery Protein (FRP), which in Synechocystis inhibits OCP-mediated phycobilisome fluorescence quenching, and Gloeobacter OCP belongs to the recently defined, heterogeneous clade OCPX (GlOCPX), the least characterized compared to OCP2 and especially OCP1 clades. Here we describe the first crystal structure of OCPX and provide its detailed structural and functional comparison with OCP1 from Synechocystis. Monomeric GlOCPX quenches Synechocystis phycobilisomes but displays drastically accelerated, less temperature-dependent recovery after photoactivation, evades regulation by FRP from other species and reveals numerous structural features reflecting its functional peculiarities. Our detailed description of a primordial OCPX sheds light on the evolution of the OCP-dependent photoprotection mechanism, rationalizing subdivision of the OCPX clade into subclades.

biochemistry↗

bioRxiv2021

Potential coupling between SARS-CoV-2 replicative fitness and interactions of its nucleoprotein with human 14-3-3 proteins

Phosphorylated within its Ser/Arg-rich region, the SARS-CoV-2 nucleoprotein (N) recruits the phosphopeptide-binding human 14-3-3 proteins that play a well-recognized role in replication of many viruses. Here we use genetic code expansion to demonstrate that phosphorylation of SARS-CoV-2 N at either of two pseudo-repeats centered at Ser197 and Thr205 is sufficient for 14-3-3 binding. According to fluorescence anisotropy, the pT205-motif, present in SARS-CoV-2 but not in SARS-CoV, is preferred over the pS197-motif by all seven human 14-3-3 isoforms, which display unforeseen pT205/pS197 binding selectivity hierarchy. Crystal structures demonstrate that pS197 and pT205 are mutually exclusive 14-3-3-binding sites, whereas SAXS and biochemical data indicate 14-3-3 binding occludes the Ser/Arg-rich region, inhibiting its dephosphorylation. This Ser/Arg-rich region of N is highly prone to mutations, as exemplified by the Omicron and Delta variants, with our data suggesting how the strength of its 14-3-3 binding can be linked with the replicative fitness of the virus.

biochemistry↗

bioRxiv2021

Microalgal protein AstaP is a potent carotenoid solubilizer and delivery module with a broad carotenoid binding repertoire

Carotenoids are lipophilic substances with many biological functions, from coloration to photoprotection. Being potent antioxidants, carotenoids have multiple biomedical applications, including the treatment of neurodegenerative disorders and retina degeneration. Nevertheless, the delivery of carotenoids is substantially limited by their poor solubility in the aqueous phase. Natural water-soluble carotenoproteins can facilitate this task, necessitating studies on their ability to uptake and deliver carotenoids. One such promising carotenoprotein, AstaP (Astaxanthin-binding protein), was recently identified in eukaryotic microalgae, but its structure and functional properties remained largely uncharacterized. By using a correctly folded recombinant protein, here we show that AstaP is an efficient carotenoid solubilizer that can stably bind not only astaxanthin but also zeaxanthin, canthaxanthin, and, to a lesser extent, {beta}-carotene, i.e. carotenoids especially valuable to human health. AstaP accepts carotenoids provided as acetone solutions or embedded in membranes, forming carotenoid-protein complexes with an apparent stoichiometry of 1:1. We successfully produced AstaP holoproteins in specific carotenoid-producing strains of Escherichia coli, proving it is amenable to cost-efficient biotechnology processes. Regardless of the carotenoid type, AstaP remains monomeric in both apo- and holoforms, while its rather minimalistic mass ([~]20 kDa) makes it an especially attractive antioxidant delivery module. In vitro, AstaP transfers different carotenoids to the liposomes and to unrelated proteins from cyanobacteria, which can modulate their photoactivity and/or oligomerization. These findings expand the toolkit of the characterized carotenoid-binding proteins and outline the perspective of the use of AstaP as a unique monomeric antioxidant nanocarrier with an extensive carotenoid-binding repertoire.

biochemistry↗

bioRxiv2020

The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association can regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, while hijacking cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention. HighlightsSARS-CoV-2 nucleocapsid protein (N) binds to all seven human 14-3-3 isoforms. This association with 14-3-3 strictly depends on phosphorylation of N. The two proteins interact in 2:2 stoichiometry and with the Kd in a M range. Affinity of interaction depends on the specific 14-3-3 isoform. Conserved Ser197-phosphopeptide of N is critical for the interaction.

biochemistry↗

bioRxiv2020

Recognition of high-risk HPV E6 oncoproteins by 14-3-3 proteins studied by interactomics and crystallography

The seven human 14-3-3 isoforms, highly similar yet encoded by distinct genes, are among the top 1% highest-expressed human proteins. 14-3-3 proteins recognize phosphorylated motifs within numerous human or viral proteins. We analyzed by crystallography, fluorescence polarization, mutagenesis and fusicoccin-mediated modulation the structural basis and druggability of 14-3-3 binding to four E6 oncoproteins of tumorigenic HPV. The seven isoforms bound variant and mutated phospho-motifs of E6 and unrelated protein RSK1 with different affinities, albeit following an ordered ranking profile with conserved relative KD ratios. Remarkably, 14-3-3 isoforms obey the same hierarchy when binding to most of their established targets, nicely supported by a recent proteome-wide human complexome map. This knowledge allows predicting the proportions of 14-3-3 isoforms engaged with phosphoproteins in various tissues. Notwithstanding their individual functions, cellular concentrations of 14-3-3 may be collectively adjusted to buffer the strongest phosphorylation outbursts, explaining their expression variations in different tissues and tumors.

biochemistry↗

bioRxiv2020

Molecular basis for the recognition of steroidogenic acute regulatory protein by the 14-3-3 protein family

Steroidogenesis in adrenals and gonads starts from cholesterol transport to mitochondria by the steroidogenic acute regulatory protein STARD1, containing a mitochondrial import sequence followed by a cholesterol-binding START domain. Although mutations in this protein have been linked to lipoid congenital adrenal hyperplasia, the mechanism of steroidogenesis regulation by the STARD1 remains debatable, hypothetically involving a molten-globule structural transition and interaction with 14-3-3 proteins. We show that, while the isolated START domain does not interact with 14-3-3, interaction is enabled by STARD1 phosphorylation at Ser57, close to the mitochondrial peptide cleavage site. Biochemical analysis of the STARD1 affinity towards 14-3-3 and crystal structures of 14-3-3 complexes with Ser57 and Ser195 phosphopeptides, suggest distinct roles of site-specific phosphorylations in recruiting 14-3-3, to modulate STARD1 activity, processing and import to mitochondria. Phosphorylation at Ser195 creates a unique conditional site, that could only bind to 14-3-3 upon partial unfolding of the START domain.

biochemistry↗