Core Fold Research Articles

Turn-on RNA Mango Beacons for trans-acting fluorogenic nucleic acid detection.

The Mango I and II RNA aptamers have been widely used in vivo and in vitro as genetically encodable fluorogenic markers that undergo large increases in fluorescence upon binding to their ligand, TO1-Biotin. However, while studying nucleic acid sequences, it is often desirable to have trans-acting probes that induce fluorescence upon binding to a target sequence. Here, we rationally design three types of light-up RNA Mango Beacons based on a minimized Mango core that induces fluorescence upon binding to a target RNA strand. Our first design is bimolecular in nature and uses a DNA inhibition strand to prevent folding of the Mango aptamer core until binding to a target RNA. Our second design is unimolecular in nature, and features hybridization arms flanking the core that inhibit G-quadruplex folding until refolding is triggered by binding to a target RNA strand. Our third design builds upon this structure, and incorporates a self-inhibiting domain into one of the flanking arms that deliberately binds to, and precludes folding of, the aptamer core until a target is bound. This design separates G-quadruplex folding inhibition and RNA target hybridization into separate modules, enabling a more universal unimolecular beacon design. All three Mango Beacons feature high contrasts and low costs when compared to conventional molecular beacons, with excellent potential for in vitro and in vivo applications.

The GET insertase exhibits conformational plasticity and induces membrane thinning

The eukaryotic guided entry of tail-anchored proteins (GET) pathway mediates the biogenesis of tail-anchored (TA) membrane proteins at the endoplasmic reticulum. In the cytosol, the Get3 chaperone captures the TA protein substrate and delivers it to the Get1/Get2 membrane protein complex (GET insertase), which then inserts the substrate via a membrane-embedded hydrophilic groove. Here, we present structures, atomistic simulations and functional data of human and Chaetomium thermophilum Get1/Get2/Get3. The core fold of the GET insertase is conserved throughout eukaryotes, whilst thinning of the lipid bilayer occurs in the vicinity of the hydrophilic groove to presumably lower the energetic barrier of membrane insertion. We show that the gating interaction between Get2 helix α3’ and Get3 drives conformational changes in both Get3 and the Get1/Get2 membrane heterotetramer. Thus, we provide a framework to understand the conformational plasticity of the GET insertase and how it remodels its membrane environment to promote substrate insertion.

The insertase YidC chaperones the polytopic membrane protein MelB inserting and folding simultaneously from both termini

The insertion and folding of proteins into membranes is crucial for cell viability. Yet, the detailed contributions of insertases remain elusive. Here, we monitor how the insertase YidC guides the folding of the polytopic melibiose permease MelB into membranes. Invivo experiments using conditionally depleted E.coli strains show that MelB can insert in the absence of SecYEG if YidC resides in the cytoplasmic membrane. Invitro single-molecule force spectroscopy reveals that the MelB substrate itself forms two folding cores from which structural segments insert stepwise into the membrane. However, misfolding dominates, particularly in structural regions that interface the pseudo-symmetric α-helical domains of MelB. Here, YidC takes an important role in accelerating and chaperoning the stepwise insertion and folding process of both MelB folding cores. Our findings reveal a great flexibility of the chaperoning and insertase activity of YidC in the multifaceted folding processes of complex polytopic membrane proteins.

DNA rehybridization drives product release from Cas9 ribonucleoprotein to enable multiple-turnover cleavage.

The RNA-guided Cas9 endonuclease from Staphylococcus aureus (SauCas9) can catalyze multiple-turnover reactions whereas Cas9 from Streptococcus pyogenes (SpyCas9) is a single-turnover enzyme. Here we dissect the mechanism of multiple-turnover catalysis by SauCas9 and elucidate its molecular basis. We show that the multiple-turnover catalysis does not require more than stoichiometric RNA guides to Cas9 nuclease. Rather, the RNA-guide loaded ribonucleoprotein (RNP) is the reactive unity that is slowly released from product and recycled in the subsequent reaction. The mechanism that RNP is recycled for multiple-turnover reaction entails the unwinding of the RNA:DNA duplex in the R-loop. We argue that DNA rehybridization is required for RNP release by supplementing the energy cost in the process. Indeed, turnover is arrested when DNA rehybridization is suppressed. Further, under higher salt conditions, both SauCas9 and SpyCas9 showed increased turnover, and engineered SpyCas9 nucleases that form fewer direct or hydrogen bonding interactions with target DNA became multiple-turnover enzymes. Thus, these results indicate that for both SpyCas9 and SauCas9, turnover is determined by the energetic balance of the post-chemistry RNP-DNA interaction. Due to the conserved protein core folds, the mechanism underpinning turnover we establish here is likely operant in all Cas9 nucleases.

Structure of a RecT/Redβ family recombinase in complex with a duplex intermediate of DNA annealing

Some bacteriophage encode a recombinase that catalyzes single-stranded DNA annealing (SSA). These proteins are apparently related to RAD52, the primary human SSA protein. The best studied protein, Redβ from bacteriophage λ, binds weakly to ssDNA, not at all to dsDNA, but tightly to a duplex intermediate of annealing formed when two complementary DNA strands are added to the protein sequentially. We used single particle cryo-electron microscopy (cryo-EM) to determine a 3.4 Å structure of a Redβ homolog from a prophage of Listeria innocua in complex with two complementary 83mer oligonucleotides. The structure reveals a helical protein filament bound to a DNA duplex that is highly extended and unwound. Native mass spectrometry confirms that the complex seen by cryo-EM is the predominant species in solution. The protein shares a common core fold with RAD52 and a similar mode of ssDNA-binding. These data provide insights into the mechanism of protein-catalyzed SSA.

Structural visualization of the tubulin folding pathway directed by human chaperonin TRiC/CCT

The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of β-tubulin using human prefoldin and TRiC. We find unstructured β-tubulin is delivered by prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. Cryo-EM resolves four near-atomic-resolution structures containing progressively folded β-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Intrinsically disordered CCT C termini within the chamber promote subsequent folding of tubulin's core and middle domains and GTP-binding. Thus, TRiC's chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.

PSSNet—An Accurate Super-Secondary Structure for Protein Segmentation

A super-secondary structure (SSS) is a spatially unique ensemble of secondary structural elements that determine the three-dimensional shape of a protein and its function, rendering SSSs attractive as folding cores. Understanding known types of SSSs is important for developing a deeper understanding of the mechanisms of protein folding. Here, we propose a universal PSSNet machine-learning method for SSS recognition and segmentation. For various types of SSS segmentation, this method uses key characteristics of SSS geometry, including the lengths of secondary structural elements and the distances between them, torsion angles, spatial positions of Cα atoms, and primary sequences. Using four types of SSSs (βαβ-unit, α-hairpin, β-hairpin, αα-corner), we showed that extensive SSS sets could be reliably selected from the Protein Data Bank and AlphaFold 2.0 database of protein structures.

Crushing behaviours and energy absorption characteristics of square antiprisms structure against flatwise crushing

In this study, a type of thin-walled energy absorber named square antiprisms (SAP) is proposed, which can be used as a core of cladding “sandwich” panel design for improving crashworthiness. Three types of SAP structure, i.e. open SAP, half-enclosed SAP and all-enclosed SAP are studied. The purpose of this unique geometric structure is to increase the energy absorption capacity of the thin-walled structure while maintaining a uniform collapsing resistance under various crush loading rates as compared with other existing conventional thin-walled structures (i.e. cylindrical tube, square tube), and folded square dome shaped structure proposed recently [50]. A unit cell of standard Miura-origami folding core is numerically simulated and calibrated with the experimental results. Good agreement on the peak and average crushing force between numerical results and experimental data is achieved. The calibrated numerical model is then used to simulate the structural response of thin-walled energy-absorbing structures under various loading rates. The collapse modes and the energy absorption capacity including average and peak stress, energy absorption, crush force efficiency and densification strain are compared among these structures. The proposed open SAP structure shows good energy absorption characteristics under quasi-static loading and dynamic loading by yielding low initial peak stress, big crush force efficiency, large densification strain and more uniform collapsing. In addition, unlike the existing typical axial crashed thin-walled energy absorber, the SAP structure show shows insensitivity to the medium and low crushing speed (no more than 63 km/s) in terms of initial peak stress and uniformity ratio. Compared with the existing typical thin-walled structures, the proposed SAP structure demonstrates a superior performance to most of the axially loaded thin-walled components, indicating a potential application as an energy absorber.

Structural basis for shape-selective recognition and aminoacylation of a D-armless human mitochondrial tRNA

Human mitochondrial gene expression relies on the specific recognition and aminoacylation of mitochondrial tRNAs (mtRNAs) by nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs). Despite their essential role in cellular energy homeostasis, strong mutation pressure and genetic drift have led to an unparalleled sequence erosion of animal mtRNAs. The structural and functional consequences of this erosion are not understood. Here, we present cryo-EM structures of the human mitochondrial seryl-tRNA synthetase (mSerRS) in complex with mtRNASer(GCU). These structures reveal a unique mechanism of substrate recognition and aminoacylation. The mtRNASer(GCU) is highly degenerated, having lost the entire D-arm, tertiary core, and stable L-shaped fold that define canonical tRNAs. Instead, mtRNASer(GCU) evolved unique structural innovations, including a radically altered T-arm topology that serves as critical identity determinant in an unusual shape-selective readout mechanism by mSerRS. Our results provide a molecular framework to understand the principles of mito-nuclear co-evolution and specialized mechanisms of tRNA recognition in mammalian mitochondrial gene expression.

The Rysto immune receptor recognises a broadly conserved feature of potyviral coat proteins.

Summary Knowledge of the immune mechanisms responsible for viral recognition is critical for understanding durable disease resistance and successful crop protection. We determined how potato virus Y (PVY) coat protein (CP) is recognised by Rysto, a TNL immune receptor.We applied structural modelling, site‐directed mutagenesis, transient overexpression, co‐immunoprecipitation, infection assays and physiological cell death marker measurements to investigate the mechanism of Rysto–CP interaction.Rysto associates directly with PVY CP in planta that is conditioned by the presence of a CP central 149 amino acids domain. Each deletion that affects the CP core region impairs the ability of Rysto to trigger defence. Point mutations in the amino acid residues Ser125, Arg157, and Asp201 of the conserved RNA‐binding pocket of potyviral CP reduce or abolish Rysto binding and Rysto‐dependent responses, demonstrating that appropriate folding of the CP core is crucial for Rysto‐mediated recognition.Rysto recognises the CPs of at least 10 crop‐damaging viruses that share a similar core region. It confers immunity to plum pox virus and turnip mosaic virus in both Solanaceae and Brassicaceae systems, demonstrating potential utility in engineering virus resistance in various crops. Our findings shed new light on how R proteins detect different viruses by sensing conserved structural patterns.

The return of the rings: Evolutionary convergence of aromatic residues in the intrinsically disordered regions of RNA-binding proteins for liquid-liquid phase separation.

Aromatic residues appeared relatively late in the evolution of protein sequences to stabilize the globular proteins' folding core and are less in the intrinsically disordered regions (IDRs). Recent advances in protein liquid-liquid phase separation (LLPS) studies have also shown that aromatic residues in IDRs often act as "stickers" to promote multivalent interactions in forming higher-order oligomers. To study how general these structure-promoting residues are in IDRs, we compared levels of sequence disorder in RNA binding proteins (RBPs), which are often found to undergo LLPS, and the human proteome. We found that aromatic residues appear more frequently than expected in the IDRs of RBPs and, through multiple sequence alignment analysis, those aromatic residues are often conserved among chordates. Using TDP-43, FUS, and some other well-studied LLPS proteins as examples, the conserved aromatic residues are important to their LLPS-related functions. These analyses suggest that aromatic residues may have contributed twice to evolution: stabilizing structured proteins and assembling biomolecular condensates.

Hierarchical folding of the catalytic core during mitochondrial ribosome biogenesis.

Final maturation steps during ribosome biogenesis require the assistance of assembly and quality control factors to ensure the folding of rRNA and proteins into a functional translation machinery. Here we integrate several recent structural snapshots of native large ribosomal subunit intermediates into the complex pathway of mitochondrial ribosome assembly.

Localization of Energetic Frustration in Proteins.

We present a detailed heuristic method to quantify the degree of local energetic frustration manifested by protein molecules. Current applications are realized in computational experiments where a protein structure is visualized highlighting the energetic conflicts or the concordance of the local interactions in that structure. Minimally frustrated linkages highlight the stable folding core of the molecule. Sites of high local frustration, in contrast, often indicate functionally relevant regions such as binding, active, or allosteric sites.

Hepatitis C virus core protein uses triacylglycerols to fold onto the endoplasmic reticulum membrane.

Lipid droplets (LDs) are involved in viral infections, but exactly how remains unclear. Here, we study the hepatitis C virus (HCV) whose core capsid protein binds to LDs but is also involved in the assembly of virions at the endoplasmic reticulum (ER) bilayer. We found that the amphipathic helix-containing domain of core, D2, senses triglycerides (TGs) rather than LDs per se. In the absence of LDs, D2 can bind to the ER membrane but only if TG molecules are present in the bilayer. Accordingly, the pharmacological inhibition of the diacylglycerol O-acyltransferase enzymes, mediating TG synthesis in the ER, inhibits D2 association with the bilayer. We found that TG molecules enable D2 to fold into alpha helices. Sequence analysis reveals that D2 resembles the apoE lipid-binding region. Our data support that TG in LDs promotes the folding of core, which subsequently relocalizes to contiguous ER regions. During this motion, core may carry TG molecules to these regions where HCV lipoviroparticles likely assemble. Consistent with this model, the inhibition of Arf1/COPI, which decreases LD surface accessibility to proteins and ER-LD material exchange, severely impedes the assembly of virions. Altogether, our data uncover a critical function of TG in the folding of core and HCV replication and reveals, more broadly, how TG accumulation in the ER may provoke the binding of soluble amphipathic helix-containing proteins to the ER bilayer.

Seven Amino Acid Types Suffice to Create the Core Fold of RNA Polymerase.

The extant complex proteins must have evolved from ancient short and simple ancestors. The double-ψ β-barrel (DPBB) is one of the oldest protein folds and conserved in various fundamental enzymes, such as the core domain of RNA polymerase. Here, by reverse engineering a modern DPBB domain, we reconstructed its plausible evolutionary pathway started by "interlacing homodimerization" of a half-size peptide, followed by gene duplication and fusion. Furthermore, by simplifying the amino acid repertoire of the peptide, we successfully created the DPBB fold with only seven amino acid types (Ala, Asp, Glu, Gly, Lys, Arg, and Val), which can be coded by only GNN and ARR (R = A or G) codons in the modern translation system. Thus, the DPBB fold could have been materialized by the early translation system and genetic code.

Molecular basis for reduced cleavage activity and drug resistance in D30N HIV-1 protease

Nelfinavir is one of the FDA-approved HIV-1 protease inhibitors and a part of highly active anti-retroviral therapy (HAART) for the treatment of HIV-AIDS. Nelfinavir was the first HIV-1 protease inhibitor to be approved as a paediatric formulation. The application of HAART had resulted in significant improvement in the lives of AIDS patients. However, the emergence of drug resistance in HIV-1 protease has limited the use of many of these drugs including nelfinavir. A unique mutation observed frequently in patients treated with nelfinavir is D30N as it is selected exclusively by nelfinavir. The D30N mutation imparts very high resistance to nelfinavir but unlike other primary mutations does not give cross-resistance to the majority of other drugs. D30N mutation also significantly reduces cleavage activity of HIV-1 protease and affects viral fitness. Here, we have determined crystal structures of D30N HIV-1 protease in unliganded form and in complex with nelfinavir. These structures provide the rationale for reduced cleavage activity and the molecular basis of drug resistance induced by D30N mutation. The loss of coulombic interaction part of a crucial hydrogen bond between the drug and the protease is likely to play a major role in reduced affinity and resistance towards nelfinavir. The decreased catalytic activity of D30N HIV-1 protease due to altered interaction with the substrates and reduced stability of folding core may be the reason for the reduced replicative capacity of the virus harboring mutant HIV-1 protease. Communicated by Ramaswamy H. Sarma

Insight into Shared Properties and Differential Dynamics and Specificity of Secretory Phospholipase A2 Family Members

Understanding generic mechanisms of functions shared by the secretory phospholipase A2 (sPLA2) family involved in the lipid metabolism and cell signaling and the molecular basis of function specificity for family members is an intriguing but challenging problem for biologists. Here, we explore the issue through extensive analyses using a combination of structure-based methods and bioinformatics tools on130 sPLA2 family members. The principal component analysis of the structure ensemble reveals that the enzyme has an open-close motion which helps widen the substrate binding channel, facilitating its binding to phospholipid. Performing elastic network model and sequence analyses found that the residues critical for family functions, such as cysteine and catalytic residues, are highly conserved and undergo minimal movements, which is evolutionarily essential as their perturbation would impact the function, while the four residue regions involved in the association with the calcium ion/membrane are lowly conserved and of high mobility and large variations in low-to-intermediate frequency modes, which reflects the specificity of members. The analyses from perturbation response scanning also reveal that the above four regions with high sensitivity to an external perturbation are member-specific, suggesting their different roles in allosteric modulation, while the minimal sensitive residues are the shared characteristics across family members, which play an important role in maintaining structural stability as the folding core. This study is helpful for understanding how sequences, structures, and dynamics of sPLA2 family members evolve to ensure their common and specific functions and can provide a guide for accurate design of proteins with finely tuned activities.

Improved Peierls-Nabarro model for asymmetric nonplanar 1/2

The 1/2 < 11_0]{111} ordinary dislocations play an important role in the mechanical behaviors of L10 alloys. The core of this kind of dislocations has asymmetric four-fold nonplanar structure spreading onto two equivalent {111} planes, which makes the traditional planar core structure based Peierls-Nabarro (P–N) model inappropriate to describe the dislocation properties. Under this context, an improved Peierls-Nabarro (P–N) model is developed in the present work to investigate the dislocation width, stress field and Peierls stress of the ordinary 1/2 < 11_0]{111} screw dislocation in L10 alloys (TiAl, TiGa and CuAu). This extended P–N model considers the nonplanar core structure with physical information obtained by the density functional theory (DFT) simulations. By the variational method, the corresponding dislocation equation is solved. It is found that the dislocation width of the ordinary 1/2 < 11_0]{111} screw dislocation is as narrow as 0.5b. Compared to the results predicted for the planar core structure or the assumed Volterra dislocation, the long-range stress field induced by the real asymmetric four-fold core presents a clear rotation. When increasing the applied stress, the distribution of the Burgers vector along the four different core folds differs more and more from each other. When the applied stress attains a critical value (i.e., the Peierls stress), the dislocation core turns to be a planar structure and a fully discrete method is used to calculate the Peierls stress. Different from the original P–N model that underestimates the Peierls stress by several orders of magnitude, the Peierls stress predicted by the improved P–N model is well consistent with the previous DFT simulations. Apparently, when the long-range interaction between dislocations is taken into account for discrete dislocation simulations or theoretical analysis and when the Peierls stress is calculated for L10 alloys, the consideration of the asymmetric nonplanar core structure by the improved P–N model is necessary to have more accurate results.

Local extrema refinement‐based tensor product model transformation controller with problem independent sampling methods

AbstractSeveral convex hull manipulation methods have been proposed for tensor product (TP) model transformation, such as CNO, INO, IRNO, minimum volume simplex, and optimized CNO. Among the five convex hull manipulation methods, CNO, INO, and IRNO are the most often used convex hull manipulation methods; minimum volume simplex is an analysis method to shrink the tightness value of the TP model transformation, and optimized CNO convex hull manipulation method is used for convex hull rectification; it also uses core tensor unfolding and folding to obtain tight convex hull. However, all these convex hull manipulation methods are designed based on classical sampling method, while the local extrema are often omitted by the classical sampling method. To obtain a more precise sampling model, in this paper, most of the existing convex hull manipulation methods are extended with local extrema refinement strategy; a uniform local extrema refinement‐based convex hull manipulation framework is proposed for the existing convex hull manipulation methods. A single gantry system is used for simulation demonstration; results show that local extrema refinement strategy is needed in convex hull manipulation of TP model transformation; it might be a preferable way to enhance classical sampling method. Minimum volume simplex is more suitable than the optimized CNO in convex hull manipulation of TP model transformation with respect to computational efficiency.

O-glycans on death receptors in cells modulate their sensitivity to TRAIL-induced apoptosis through affecting on their stability and oligomerization.

The TNF-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in cells by signaling through the O-glycosylated death receptors (DR4 and DR5), but the sensitivity to TRAIL-induced apoptosis of cells varies, and the attributes of this phenomenon are complex. Human carcinoma cells often express truncated O-glycans, Tn (GalNAcα1-Ser/Thr), and Sialyl-Tn (Siaα2-6GalNAcα1-Ser/Thr, STn) on their surface glycoproteins, yet molecular mechanisms in terms of advantages for tumor cells to have these truncated O-glycans remain elusive. Normal extended O-glycan biosynthesis is regulated by a specific molecular chaperone Cosmc through assisting of the correct folding of Core 1 β3 Galactosyltransferase (T-synthase). Here, we use tumor cell lines harboring mutations in Cosmc, and therefore expressing Tn and STn antigens to study the role of O-glycans in TRAIL-induced apoptosis. Expression of Tn and STn in tumor cells attenuates their sensitivity to TRAIL treatment; when transfected with wild-type Cosmc, these tumor cells thus express normal extended O-glycans and become more sensitive to TRAIL treatment. Mechanistically, Tn/STn antigens impair homo-oligomerization and stability of DR4 and DR5. These results represent the first mechanistic insight into how O-glycan structures on cell surface modulate their sensitivity to apoptotic stimuli, suggesting expression of Tn/STn may offer tumor cell survival advantages through altering DR4 and/or DR5 activity.