Crystal structure reveals canonical recognition of the phosphorylated cytoplasmic loop of human alpha7 nicotinic acetylcholine receptor by 14-3-3 protein.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels composed of five homologous subunits. The homopentameric α7-nAChR, abundantly expressed in the brain, is involved in the regulation of the neuronal plasticity and memory and undergoes phosphorylation by protein kinase A (PKA). Here, we extracted native α7-nAChR from murine brain, validated its assembly by cryo-EM and showed that phosphorylation by PKA in vitro enables its interaction with the abundant human brain protein 14-3-3ζ. Bioinformatic analysis narrowed the putative 14-3-3-binding site down to the fragment of the intracellular loop (ICL) containing Ser365 (Q361RRCSLASVEMS372), known to be phosphorylated in vivo. We reconstructed the 14-3-3ζ/ICL peptide complex and determined its structure by X-ray crystallography, which confirmed the Ser365 phosphorylation-dependent canonical recognition of the ICL by 14-3-3. A common mechanism of nAChRs' regulation by ICL phosphorylation and 14-3-3 binding that potentially affects nAChR activity, stoichiometry, and surface expression is suggested.

Structural basis for the recognition by 14-3-3 proteins of a conditional binding site within the oligomerization domain of human nucleophosmin.

Nucleophosmin 1 (NPM1) is a multifunctional protein regulating ribosome biogenesis, centrosome duplication and chromatin remodeling. Being a major nucleolar protein, NPM1 can migrate to the nucleus and the cytoplasm, which is controlled by changes of NPM1 oligomerization and interaction with other cell factors. NPM1 forms a stable pentamer with its N-terminal structured domain, where two nuclear export signals and several phosphorylation sites reside. This domain undergoes dissociation and disordering upon Ser48 phosphorylation in the subunit interface. Recent studies indicated that Ser48 is important for NPM1 interaction with other proteins including 14-3-3, the well-known phosphoserine/phosphothreonine binders, but the structural basis for 14-3-3/NPM1 interaction remained unaddressed. By fusing human 14-3-3ζ with an NPM1 segment surrounding Ser48, which was phosphorylated inside Escherichia coli cells by co-expressed protein kinase A, here we obtained the desired protein/phosphopeptide complex and determined its crystal structure. While biochemical data indicated that the interaction is driven by Ser48 phosphorylation, the crystallographic 14-3-3/phosphopeptide interface reveals an NPM1 conformation distinctly different from that in the NPM1 pentamer. Given the canonical phosphopeptide-binding mode observed in our crystal structure, Ser48 emerges as a conditional binding site whose recognition by 14-3-3 proteins is enabled by NPM1 phosphorylation, disassembly and disordering under physiological circumstances.

Effect of Trehalose on Oligomeric State and Anti-Aggregation Activity of αB-Crystallin.

αB-Crystallin (αB-Cr), one of the main crystalline lens proteins, along with other crystallins maintains lens transparency suppressing protein aggregation and thus preventing cataractogenesis. αB-Cr belongs to the class of molecular chaperones; being expressed in many tissues it has a dynamic quaternary structure, which is essential for its chaperone-like activity. Shift in the equilibrium between ensembles of oligomers of different size allows regulating the chaperone activity. Trehalose is known to inhibit protein aggregation in vivo and in vitro, and it is widely used in biotechnology. The results of studying the effect of trehalose on the chaperone-like activity of crystallins can serve as a basis for the design of drugs delaying cataractogenesis. We have studied the trehalose effect on the quaternary structure and anti-aggregation activity of αB-Cr using muscle glycogen phosphorylase b (Phb) as a target protein. According to the dynamic light scattering data, trehalose affects the nucleation stage of Phb thermal aggregation at 48°C, and an increase in the αB-Cr adsorption capacity (AC0) is the main effect of trehalose on the aggregation process in the presence of the protein chaperone (AC0 increases 1.5-fold in the presence of 66 mM trehalose). According to the sedimentation analysis data, trehalose stabilizes the dimeric form of Phb at the stages of denaturation and dissociation and enhances the interaction of αB-Cr with the target protein. Moreover, trehalose shifts the equilibrium between the αB-Cr oligomers towards the smaller forms. Thus, trehalose affects the quaternary structure of αB-Cr and increases its anti-aggregation activity at the nucleation stage.

Effect of Betaine and Arginine on Interaction of αB-Crystallin with Glycogen Phosphorylase b.

Protein-protein interactions (PPIs) play an important role in many biological processes in a living cell. Among them chaperone-client interactions are the most important. In this work PPIs of αB-crystallin and glycogen phosphorylase b (Phb) in the presence of betaine (Bet) and arginine (Arg) at 48 °C and ionic strength of 0.15 M were studied using methods of dynamic light scattering, differential scanning calorimetry, and analytical ultracentrifugation. It was shown that Bet enhanced, while Arg reduced both the stability of αB-crystallin and its adsorption capacity (AC0) to the target protein at the stage of aggregate growth. Thus, the anti-aggregation activity of αB-crystallin increased in the presence of Bet and decreased under the influence of Arg, which resulted in inhibition or acceleration of Phb aggregation, respectively. Our data show that chemical chaperones can influence the tertiary and quaternary structure of both the target protein and the protein chaperone. The presence of the substrate protein also affects the quaternary structure of αB-crystallin, causing its disassembly. This is inextricably linked to the anti-aggregation activity of αB-crystallin, which in turn affects its PPI with the target protein. Thus, our studies contribute to understanding the mechanism of interaction between chaperones and proteins.

Crystal structure of human 14-3-3ζ complexed with the noncanonical phosphopeptide from proapoptotic BAD.

Several signaling pathways control phosphorylation of the proapoptotic protein BAD and its phosphorylation-dependent association with 14-3-3 proteins in the cytoplasm. The stability of the 14-3-3/BAD complex determines the cell fate: unphosphorylated BAD escapes from 14-3-3, migrates to the mitochondria and initiates apoptosis. While the 14-3-3/BAD interaction represents a promising drug target, it lacks structural characterization. Among several phosphosites identified in vivo, Ser75 and Ser99 of human BAD match the consensus sequence RXXpSXP recognized by 14-3-3 and, therefore, represent canonical 14-3-3-binding sites. Yet, BAD contains other serines phosphorylatable in vivo, whose role is less understood. Here, we report a 2.36 Å crystal structure of 14-3-3ζ complexed with a BAD fragment which includes residues Ser74 and Ser75, both being substrates for protein kinases. While the BAD peptide is anchored to 14-3-3 by phosphoserine as expected, the BAD peptide was unexpectedly phosphorylated at Ser74 instead of Ser75, revealing noncanonical binding within the amphipathic groove and leading to a one-step positional shift and reorganization of the interface. This observation exemplifies plasticity of the amphipathic 14-3-3 groove in accommodating various peptides and suggests the redundancy of Ser74 and Ser75 phosphosites with respect to binding of BAD to 14-3-3.

Design, expression, purification and crystallization of human 14-3-3ζ protein chimera with phosphopeptide from proapoptotic protein BAD.

14-3-3 protein isoforms regulate multiple processes in eukaryotes, including apoptosis and cell division. 14-3-3 proteins preferentially recognize phosphorylated unstructured motifs, justifying the protein-peptide binding approach to study 14-3-3/phosphotarget complexes. Tethering of human 14-3-3σ with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides, simultaneously facilitating purification and crystallization. Nevertheless, the broader applicability to other 14-3-3 isoforms and phosphopeptides was unclear. Here, we designed a novel 14-3-3ζ chimera with a conserved phosphopeptide from BAD, whose complex with 14-3-3 is a gatekeeper of apoptosis regulation. The chimera could be bacterially expressed and purified without affinity tags. Co-expressed PKA efficiently phosphorylates BAD within the chimera and blocks its interaction with a known 14-3-3 phosphotarget, suggesting occupation of the 14-3-3 grooves by the tethered BAD phosphopeptide. Efficient crystallization of the engineered protein suggests suitability of the "chimeric" approach for studies of other relevant 14-3-3 complexes.

Effect of the NBD-group position on interaction of fluorescently-labeled cholesterol analogues with human steroidogenic acute regulatory protein STARD1.

Steroidogenic acute regulatory protein (StAR, STARD1) is a key factor of intracellular cholesterol transfer to mitochondria, necessary for adrenal and gonadal steroidogenesis, and is an archetypal member of the START protein family. Despite the common overall structural fold, START members differ in their binding selectivity toward various lipid ligands, but the lack of direct structural information hinders complete understanding of the binding process and cholesterol orientation in the STARD1 complex in particular. Cholesterol binding has been widely studied by commercially available fluorescent steroids, but the effect of the fluorescent group position on binding remained underexplored. Here, we dissect STARD1 interaction with cholesterol-like steroids bearing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group in different positions, namely, with 22-NBD-cholesterol (22NC), 25-NBD-cholesterol (25NC), 20-((NBDamino)-pregn-5-en-3-ol (20NP) and 3-(NBDamino)-cholestane (3NC). While being able to stoichiometrically bind 22NC and 20NP with high fluorescence yield and quantitative exhaustion of fluorescence of some protein tryptophans, STARD1 binds 25NC and 3NC with much lower affinity and poor fluorescence response. In contrast to 3NC, binding of 20NP leads to STARD1 stabilization and substantially increases the NBD fluorescence lifetime. Remarkably, in terms of fluorescence response, 20NP slightly outperforms commonly used 22NC and can thus be used for screening of various potential ligands by a competition mechanism in the future.

Solution structure of human steroidogenic acute regulatory protein STARD1 studied by small-angle X-ray scattering.

Intracellular cholesterol transfer to mitochondria, a bottleneck of adrenal and gonadal steroidogenesis, relies on the functioning of the steroidogenic acute regulatory protein (StAR, STARD1), for which many disease-associated mutations have been described. Despite significant progress in the field, the exact mechanism of cholesterol binding and transfer by STARD1 still remains debatable and often considers significant structural rearrangements to achieve ligand binding. The crystal structure of STARD1, obtained recently at medium resolution, suggests that this protein has the same fold as other members of the START family. However, hydrodynamic properties and solution conformation of STARD1 are insufficiently characterized, partially due to poor solubility of this protein. Here, we used our recent protocol to obtain stable and soluble STARD1 and analyzed its hydrodynamic properties and solution conformation using a previously inapplicable small-angle X-ray scattering (SAXS). The SAXS data obtained exclusively from a monodisperse fraction of the monomeric protein suggest that, apart from movements of the flexible Ω1-loop, STARD1 unlikely undergoes significant spontaneous rearrangements proposed earlier as a gating mechanism for cholesterol binding. The consistency with the previously reported solution NMR structure of STARD6 suggests similarity of hydrodynamic behavior of other STARD-containing proteins.

High-yield soluble expression, purification and characterization of human steroidogenic acute regulatory protein (StAR) fused to a cleavable Maltose-Binding Protein (MBP).

Steroidogenic acute regulatory protein (StAR) is responsible for the rapid delivery of cholesterol to mitochondria where the lipid serves as a source for steroid hormones biosynthesis in adrenals and gonads. Despite many successful investigations, current understanding of the mechanism of StAR action is far from being completely clear. StAR was mostly obtained using denaturation/renaturation or in minor quantities in a soluble form at decreased temperatures that, presumably, limited the possibilities for its consequent detailed exploration. In our hands, existing StAR expression constructs could be bacterially expressed almost exclusively as insoluble forms, even upon decreased expression temperatures and in specific strains of Escherichia coli, and isolated protein tended to aggregate and was difficult to handle. To maximize the yield of soluble protein, optimized StAR sequence encompassing functional domain STARD1 (residues 66-285) was fused to the C-terminus of His-tagged Maltose-Binding Protein (MBP) with the possibility to cleave off the whole tag by 3C protease. The developed protocol of expression and purification comprising of a combination of subtractive immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography allowed us to obtain up to 25 mg/1 L culture of completely soluble StAR protein, which was (i) homogenous according to SDS-PAGE, (ii) gave a single symmetrical peak on a gel-filtration, (iii) showed the characteristic CD spectrum and (iv) pH-dependent ability to bind a fluorescently-labeled cholesterol analogue. We conclude that our strategy provides fully soluble and native StAR protein which in future could be efficiently used for biotechnology and drug discovery aimed at modulation of steroids production.

DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity.

Two prokaryotic defence systems, prokaryotic Argonautes (pAgos) and CRISPR-Cas, detect and cleave invader nucleic acids using complementary guides and the nuclease activities of pAgo or Cas proteins. However, not all pAgos are active nucleases. A large clade of short pAgos bind nucleic acid guides but lack nuclease activity, suggesting a different mechanism of action. Here we investigate short pAgos associated with a putative effector nuclease, NbaAgo from Novosphingopyxis baekryungensis and CmeAgo from Cupriavidus metallidurans. We show that these pAgos form a heterodimeric complex with co-encoded effector nucleases (short prokaryotic Argonaute, DNase and RNase associated (SPARDA)). RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA by plasmids or phages results in degradation of cellular DNA and cell death or dormancy, conferring target-specific population protection and expanding the range of known prokaryotic immune systems.

Human 14-3-3 Proteins Site-selectively Bind the Mutational Hotspot Region of SARS-CoV-2 Nucleoprotein Modulating its Phosphoregulation.

Phosphorylation of SARS-CoV-2 nucleoprotein recruits human cytosolic 14-3-3 proteins playing a well-recognized role in replication of many viruses. Here we use genetic code expansion to demonstrate that 14-3-3 binding is triggered by phosphorylation of SARS-CoV-2 nucleoprotein at either of two pseudo-repeats centered at Ser197 and Thr205. According to fluorescence anisotropy measurements, the pT205-motif,presentin SARS-CoV-2 but not in SARS-CoV, is preferred over the pS197-motif by all seven human 14-3-3 isoforms, which collectively display an unforeseen pT205/pS197 peptide binding selectivity hierarchy. Crystal structures demonstrate that pS197 and pT205 are mutually exclusive 14-3-3-binding sites, whereas SAXS and biochemical data obtained on the full protein-protein complex indicate that 14-3-3 binding occludes the Ser/Arg-rich region of the nucleoprotein, inhibiting its dephosphorylation. This Ser/Arg-rich region is highly prone to mutations, as exemplified by the Omicron and Delta variants, with our data suggesting that the strength of 14-3-3/nucleoprotein interaction can be linked with the replicative fitness of the virus.

Hierarchized phosphotarget binding by the seven human 14-3-3 isoforms.

The seven 14-3-3 isoforms are highly abundant human proteins encoded by similar yet distinct genes. 14-3-3 proteins recognize phosphorylated motifs within numerous human and viral proteins. Here, we analyze by X-ray crystallography, fluorescence polarization, mutagenesis and fusicoccin-mediated modulation the structural basis and druggability of 14-3-3 binding to four E6 oncoproteins of tumorigenic human papillomaviruses. 14-3-3 isoforms bind variant and mutated phospho-motifs of E6 and unrelated protein RSK1 with different affinities, albeit following an ordered affinity ranking with conserved relative KD ratios. Remarkably, 14-3-3 isoforms obey the same hierarchy when binding to most of their established targets, as supported by literature and a recent human complexome map. This knowledge allows predicting 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.

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 could regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, and could also hijack cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.

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. This is mediated 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 (LCAH), the mechanism of steroidogenesis regulation by STARD1 remains debatable. It has been hypothesized to involve a molten-globule structural transition and interaction with 14-3-3 proteins. In this study, we aimed to address the structural basis for the 14-3-3-STARD1 interaction. We show that, while the isolated START domain does not interact with 14-3-3, this interaction is enabled by STARD1 phosphorylation at Ser57, close to the mitochondrial peptide cleavage site. Biochemical analysis of the STARD1 affinity toward 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 the mitochondria. Phosphorylation at Ser195 creates a unique conditional site that could only bind to 14-3-3 upon partial unfolding of the START domain. Overall, our findings on the interaction between 14-3-3 and STARD1 may have potential clinical implications for patients with LCAH.

New 20-hydroxycholesterol-like compounds with fluorescent NBD or alkyne labels: Synthesis, in silico interactions with proteins and uptake by yeast cells.

20-hydroxycholesterol is a signaling oxysterol with immunomodulating functions and, thus, structural analogues with reporter capabilities could be useful for studying and modulating the cellular processes concerned. We have synthesized three new 20-hydroxycholesterol-like pregn-5-en-3ß-ol derivatives with fluorescent 7-nitrobenzofurazan (NBD) or Raman-sensitive alkyne labels in their side-chains. In silico computations demonstrated the compounds possess good membrane permeability and can bind within active sites of known 20-hydroxycholesterol targets (e.g. Smoothened and yeast Osh4) and some other sterol-binding proteins (human LXRß and STARD1; yeast START-kins Lam4S2 and Lam2S2). Having found good predicted membrane permeability and binding to some yeast proteins, we tested the compounds on microorganisms. Fluorescent microscopy indicated the uptake of the steroids by both Saccharomyces cerevisiae and Yarrowia lipolytica, whereas only S. cerevisiae demonstrated conversion of the compounds into 3-O-acetates, likely because 3-O-acetyltransferase Atf2p is present only in its genome. The new compounds provide new options to study the uptake, intracellular distribution and metabolism of sterols in yeast cells as well as might be used as ligands for sterol-binding proteins.

Concatenation of 14-3-3 with partner phosphoproteins as a tool to study their interaction.

Regulatory 14-3-3 proteins interact with a plethora of phosphorylated partner proteins, however 14-3-3 complexes feature intrinsically disordered regions and often a transient type of interactions making structural studies difficult. Here we engineer and examine a chimera of human 14-3-3 tethered to a nearly complete partner HSPB6 which is phosphorylated by protein kinase A (PKA). HSPB6 includes a long disordered N-terminal domain (NTD), a phosphorylation motif around Ser16, and a core α-crystallin domain (ACD) responsible for dimerisation. The chosen design enables an unstrained binding of pSer16 in each 1433 subunit and secures the correct 2:2 stoichiometry. Differential scanning calorimetry, limited proteolysis and small-angle X-ray scattering (SAXS) support the proper folding of both the 14-3-3 and ACD dimers within the chimera, and indicate that the chimera retains the overall architecture of the native complex of 14-3-3 and phosphorylated HSPB6 that has recently been resolved using crystallography. At the same time, the SAXS data highlight the weakness of the secondary interface between the ACD dimer and the C-terminal lobe of 14-3-3 observed in the crystal structure. Applied to other 14-3-3 complexes, the chimeric approach may help probe the stability and specificity of secondary interfaces for targeting them with small molecules in the future.

Bacterial co-expression of human Tau protein with protein kinase A and 14-3-3 for studies of 14-3-3/phospho-Tau interaction.

Abundant regulatory 14-3-3 proteins have an extremely wide interactome and coordinate multiple cellular events via interaction with specifically phosphorylated partner proteins. Notwithstanding the key role of 14-3-3/phosphotarget interactions in many physiological and pathological processes, they are dramatically underexplored. Here, we focused on the 14-3-3 interaction with human Tau protein associated with the development of several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Among many known phosphorylation sites within Tau, protein kinase A (PKA) phosphorylates several key residues of Tau and induces its tight interaction with 14-3-3 proteins. However, the stoichiometry and mechanism of 14-3-3 interaction with phosphorylated Tau (pTau) are not clearly elucidated. In this work, we describe a simple bacterial co-expression system aimed to facilitate biochemical and structural studies on the 14-3-3/pTau interaction. We show that dual co-expression of human fetal Tau with PKA in Escherichia coli results in multisite Tau phosphorylation including also naturally occurring sites which were not previously considered in the context of 14-3-3 binding. Tau protein co-expressed with PKA displays tight functional interaction with 14-3-3 isoforms of a different type. Upon triple co-expression with 14-3-3 and PKA, Tau protein could be co-purified with 14-3-3 and demonstrates complex which is similar to that formed in vitro between individual 14-3-3 and pTau obtained from dual co-expression. Although used in this study for the specific case of the previously known 14-3-3/pTau interaction, our co-expression system may be useful to study of other selected 14-3-3/phosphotarget interactions and for validations of 14-3-3 complexes identified by other methods.

Chimeric 14-3-3 proteins for unraveling interactions with intrinsically disordered partners.

In eukaryotes, several "hub" proteins integrate signals from different interacting partners that bind through intrinsically disordered regions. The 14-3-3 protein hub, which plays wide-ranging roles in cellular processes, has been linked to numerous human disorders and is a promising target for therapeutic intervention. Partner proteins usually bind via insertion of a phosphopeptide into an amphipathic groove of 14-3-3. Structural plasticity in the groove generates promiscuity allowing accommodation of hundreds of different partners. So far, accurate structural information has been derived for only a few 14-3-3 complexes with phosphopeptide-containing proteins and a variety of complexes with short synthetic peptides. To further advance structural studies, here we propose a novel approach based on fusing 14-3-3 proteins with the target partner peptide sequences. Such chimeric proteins are easy to design, express, purify and crystallize. Peptide attachment to the C terminus of 14-3-3 via an optimal linker allows its phosphorylation by protein kinase A during bacterial co-expression and subsequent binding at the amphipathic groove. Crystal structures of 14-3-3 chimeras with three different peptides provide detailed structural information on peptide-14-3-3 interactions. This simple but powerful approach, employing chimeric proteins, can reinvigorate studies of 14-3-3/phosphoprotein assemblies, including those with challenging low-affinity partners, and may facilitate the design of novel biosensors.