ABSTRACT
Abiotic D-proteins that selectively bind to natural L-proteins have gained significant biotechnological interest. However, the underlying structural principles governing such heterochiral protein-protein interactions remain largely unknown. In this study, we present the de novo design of D-proteins consisting of 50-65 residues, aiming to target specific surface regions of L-proteins or L-peptides. Our designer D-protein binders exhibit nanomolar affinity toward an artificial L-peptide, as well as two naturally occurring proteins of therapeutic significance: the D5 domain of human tropomyosin receptor kinase A (TrkA) and human interleukin-6 (IL-6). Notably, these D-protein binders demonstrate high enantiomeric specificity and target specificity. In cell-based experiments, designer D-protein binders effectively inhibited the downstream signaling of TrkA and IL-6 with high potency. Moreover, these binders exhibited remarkable thermal stability and resistance to protease degradation. Crystal structure of the designed heterochiral D-protein-L-peptide complex, obtained at a resolution of 2.0 Å, closely resembled the design model, indicating that the computational method employed is highly accurate. Furthermore, the crystal structure provides valuable information regarding the interactions between helical L-peptides and D-proteins, particularly elucidating a novel mode of heterochiral helix-helix interactions. Leveraging the design of D-proteins specifically targeting L-peptides or L-proteins opens up avenues for systematic exploration of the mirror-image protein universe, paving the way for a diverse range of applications.
ABSTRACT
Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo.
Subject(s)
Extracellular Vesicles , Nanoparticles , Extracellular Vesicles/metabolism , Humans , Nanoparticles/chemistry , Protein Engineering/methods , Membrane Microdomains/metabolism , Lipids/chemistry , Cell Membrane/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , Animals , Drug Delivery Systems , Protein Transport , HEK293 Cells , LiposomesABSTRACT
Latent bioreactive unnatural amino acids (Uaas) have been widely used in the development of covalent drugs and identification of protein interactors, such as proteins, DNA, RNA and carbohydrates. However, it is challenging to perform high-throughput identification of Uaa cross-linking products due to the complexities of protein samples and the data analysis processes. Enrichable Uaas can effectively reduce the complexities of protein samples and simplify data analysis, but few cross-linked peptides were identified from mammalian cell samples with these Uaas. Here we develop an enrichable and multiple amino acids reactive Uaa, eFSY, and demonstrate that eFSY is MS cleavable when eFSY-Lys and eFSY-His are the cross-linking products. An identification software, AixUaa is developed to decipher eFSY mass cleavable data. We systematically identify direct interactomes of Thioredoxin 1 (Trx1) and Selenoprotein M (SELM) with eFSY and AixUaa.
Subject(s)
Amino Acids , Thioredoxins , Amino Acids/metabolism , Amino Acids/chemistry , Humans , Thioredoxins/metabolism , Thioredoxins/genetics , Thioredoxins/chemistry , Cross-Linking Reagents/chemistry , Protein Binding , Peptides/metabolism , Peptides/chemistry , Selenoproteins/metabolism , Selenoproteins/genetics , Selenoproteins/chemistry , Software , Proteins/metabolism , Proteins/chemistry , HEK293 CellsABSTRACT
The organization of membrane proteins between and within membrane-bound compartments is critical to cellular function. Yet we lack approaches to regulate this organization in a range of membrane-based materials, such as engineered cells, exosomes, and liposomes. Uncovering and leveraging biophysical drivers of membrane protein organization to design membrane systems could greatly enhance the functionality of these materials. Towards this goal, we use de novo protein design, molecular dynamic simulations, and cell-free systems to explore how membrane-protein hydrophobic mismatch could be used to tune protein cotranslational integration and organization in synthetic lipid membranes. We find that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We use this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to design self-organizing membrane-based materials with applications such as artificial cells, biosensors, and therapeutic nanoparticles.
Subject(s)
Artificial Cells , Membrane Proteins , Cell Membrane/metabolism , Membranes/metabolism , Membrane Proteins/metabolism , Liposomes , Lipid Bilayers/chemistryABSTRACT
In recent decades, major progress has been made in the design of water-soluble proteins, yet the design of transmembrane proteins has lagged considerably. Despite their biological and pharmaceutical importance, only a limited number of transmembrane proteins have been successfully designed owing to the complexity of the membrane environment and difficulties in experimental characterization. Here, we introduce principles for transmembrane protein design in general and discuss design examples, including scaffold proteins and functional proteins. We also discuss how developments in design methods have advanced the field and what we may achieve with recent breakthroughs in structural biology.
Subject(s)
Computational Biology , Membrane Proteins , Membrane Proteins/chemistryABSTRACT
Membrane transport proteins, which include transporters and channels, are delicate protein machineries that mediate the exchange of a variety of substances across biomembranes. Accumulated structural and functional knowledge allows for the de novo design of transport proteins with new structures that do not exist in nature. Analysis based on these novel proteins provides new insights into the principles that govern protein assembly, conformational change, and substrate recognition. Here, we review the advances in the de novo design of transporters and channels over recent years and highlight the challenges and opportunities in this field.
Subject(s)
Carrier Proteins , Membrane Transport Proteins , Biological Transport , Carrier Proteins/chemistry , Membrane Transport Proteins/chemistryABSTRACT
Wntless (WLS), an evolutionarily conserved multi-pass transmembrane protein, is essential for secretion of Wnt proteins. Wnt-triggered signaling pathways control many crucial life events, whereas aberrant Wnt signaling is tightly associated with many human diseases including cancers. Here, we report the cryo-EM structure of human WLS in complex with Wnt3a, the most widely studied Wnt, at 2.2 Å resolution. The transmembrane domain of WLS bears a GPCR fold, with a conserved core cavity and a lateral opening. Wnt3a interacts with WLS at multiple interfaces, with the lipid moiety on Wnt3a traversing a hydrophobic tunnel of WLS transmembrane domain and inserting into membrane. A ß-hairpin of Wnt3a containing the conserved palmitoleoylation site interacts with WLS extensively, which is crucial for WLS-mediated Wnt secretion. The flexibility of the Wnt3a loop/hairpin regions involved in the multiple binding sites indicates induced fit might happen when Wnts are bound to different binding partners. Our findings provide important insights into the molecular mechanism of Wnt palmitoleoylation, secretion and signaling.
Subject(s)
Cryoelectron Microscopy , Receptors, G-Protein-Coupled/ultrastructure , Wnt3A Protein/ultrastructure , Frizzled Receptors/metabolism , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Models, Molecular , Protein Conformation , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Wnt3A Protein/chemistry , Wnt3A Protein/metabolismABSTRACT
Despite recent success in computational design of structured cyclic peptides, de novo design of cyclic peptides that bind to any protein functional site remains difficult. To address this challenge, we develop a computational "anchor extension" methodology for targeting protein interfaces by extending a peptide chain around a non-canonical amino acid residue anchor. To test our approach using a well characterized model system, we design cyclic peptides that inhibit histone deacetylases 2 and 6 (HDAC2 and HDAC6) with enhanced potency compared to the original anchor (IC50 values of 9.1 and 4.4 nM for the best binders compared to 5.4 and 0.6 µM for the anchor, respectively). The HDAC6 inhibitor is among the most potent reported so far. These results highlight the potential for de novo design of high-affinity protein-peptide interfaces, as well as the challenges that remain.
Subject(s)
Drug Design , Histone Deacetylase Inhibitors/pharmacology , Peptides, Cyclic/pharmacology , Structure-Activity Relationship , Catalytic Domain/drug effects , Crystallography, X-Ray , Enzyme Assays , Histone Deacetylase 2/antagonists & inhibitors , Histone Deacetylase 2/isolation & purification , Histone Deacetylase 2/metabolism , Histone Deacetylase 2/ultrastructure , Histone Deacetylase 6/antagonists & inhibitors , Histone Deacetylase 6/genetics , Histone Deacetylase 6/isolation & purification , Histone Deacetylase 6/ultrastructure , Histone Deacetylase Inhibitors/chemistry , Inhibitory Concentration 50 , Molecular Docking Simulation , Nuclear Magnetic Resonance, Biomolecular , Peptide Library , Peptides, Cyclic/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Zebrafish Proteins/genetics , Zebrafish Proteins/ultrastructureABSTRACT
An atomically dispersed structure is attractive for electrochemically converting carbon dioxide (CO2) to fuels and feedstock due to its unique properties and activity. Most single-atom electrocatalysts are reported to reduce CO2 to carbon monoxide (CO). Herein, we develop atomically dispersed indium (In) on a nitrogen-doped carbon skeleton (In-N-C) as an efficient catalyst to produce formic acid/formate in aqueous media, reaching a turnover frequency as high as 26771 h-1 at -0.99 V relative to a reversible hydrogen electrode (RHE). Electrochemical measurements show that trace amounts of In loaded on the carbon matrix significantly improve the electrocatalytic behavior for the CO2 reduction reaction, outperforming conventional metallic In catalysts. Further experiments and density functional theory (DFT) calculations reveal that the formation of intermediate *OCHO on isolated In sites plays a pivotal role in the efficiency of the CO2-to-formate process, which has a lower energy barrier than that on metallic In.
Subject(s)
Receptors, Chimeric Antigen , Antigens, Surface , Immunotherapy, Adoptive , Logic , ProteinsABSTRACT
Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.
Subject(s)
Computer Simulation , Genes, Synthetic/genetics , Ion Channels/chemistry , Ion Channels/genetics , Models, Molecular , Synthetic Biology , Cell Line , Cryoelectron Microscopy , Crystallography, X-Ray , Electric Conductivity , Escherichia coli/genetics , Escherichia coli/metabolism , Hydrazines , Ion Channels/metabolism , Ion Transport , Liposomes/metabolism , Patch-Clamp Techniques , Porins/chemistry , Porins/genetics , Porins/metabolism , Protein Engineering , Protein Structure, Secondary , Solubility , Water/chemistryABSTRACT
Global demand for green and clean energy is increasing day by day owing to ongoing developments by the human race that are changing the face of the earth at a rate faster than ever. Exploring alternative sources of energy to replace fossil fuel consumption has become even more vital to control the growing concentration of CO2, and reduction of CO2 into CO or other useful hydrocarbons (e.g., C1 and C≥2 products), as well as reduction of N2 into ammonia, can greatly help in this regard. Various materials have been developed for the reduction of CO2 and N2. The introduction of pores in these materials by porosity engineering has been demonstrated to be highly effective in increasing the efficiency of the involved redox reactions, over 40% increment for CO2 reduction to date, by providing an increased number of exposed facets, kinks, edges, and catalytically active sites of catalysts. By shaping the surface porous structure, the selectivity of the redox reaction can also be enhanced. In order to better understand this area benefiting rational design for future solutions, this review systematically summarizes and constructively discusses the porosity engineering in catalytic materials, including various synthesis methods, characterization of porous materials, and the effects of porosity on performance of CO2 reduction and N2 reduction.
ABSTRACT
A novel and efficient method to synthesize rigid bis-coumarins based on the dimerization of coumarinyl aldehydes was developed. This procedure is additive- and column-free, providing a facile and environment-friendly way to prepare fluorophores. The prepared novel fluorescent bis-coumarins exhibit favorable photophysical properties with good sensitivity and selectivity towards G-quadruplexes (G4s).
ABSTRACT
Electrochemically converting carbon dioxide (CO2) to formate offers a promising approach for energy conversion and storage. Bismuth is believed to be one of the promising candidates for CO2 electroreduction, but the poor selectivity and complexity of synthesis limit its real application on a large scale. In this work, a facile one-step-reduction method was developed to prepare a bismuth nanostructure in aqueous solution. Owing to its enhanced reactive sites and exposed crystal plane, the prepared Bi nanostructure exhibits excellent performance for CO2 electroreduction, which reaches the maximum faradaic efficiency for formate as high as 92% at a potential of -0.9 V versus a reversible hydrogen electrode. Additionally, the large current density and remarkable durability also reveal its high intrinsic CO2 electroreduction activity. The density functional theory calculation confirms that the formation of intermediate *OCHO that finally converts to formate is thermodynamically favorable on Bi high-index planes. We anticipate that such a facile synthesis strategy and excellent electrocatalytic performance of the Bi nanostructure will be easy to scale up, realizing its industrialization applications in CO2 electrochemical conversion.
ABSTRACT
The top-down fabrication of catalytically active molecular metal oxide anions, or polyoxometalates, is virtually unexplored, although these materials offer unique possibilities, for catalysis, energy conversion and storage. Here, we report a novel top-down route, which enables the scalable synthesis and deposition of sub-nanometer molybdenum-oxo clusters on electrically conductive mesoporous carbon. The new approach uses a unique redox-cycling process to convert crystalline MoIVO2 particles into sub-nanometer molecular molybdenum-oxo clusters with a nuclearity of â¼1-20. The resulting molybdenum-oxo cluster/carbon composite shows outstanding, stable electrocatalytic performance for the oxygen reduction reaction with catalyst characteristics comparable to those of commercial Pt/C. This new material design could give access to a new class of highly reactive polyoxometalate-like metal oxo clusters as high-performance, earth abundant (electro-)catalysts.
ABSTRACT
The electrochemical oxygen evolution reaction (OER) is sparked extensive interest in efficient energy storage and conversion. Cobalt Selenide (CoSe2) is believed to be one of the promising candidates for OER based on Yang Shao-Horn's principle. However, owing to low exposure of active sites and/or low efficiency of electron transfer, the electrocatalytic activity of CoSe2 is far less than expected. In this work, a novel carbon nanotubes (CNT) grafted 3D core-shell structured CoSe2@C-CNT nanohybrid is developed by a general hydrothermal-calcination strategy. Zeolite imidazole frameworks (ZIF) was used as the precursor to synthesis of the materials. It is found that both the calcination temperature and the selenium content can significantly regulate the catalytic performance of the hybrids. The obtained best catalysts requires the overpotential of only 306â¯mV and 345â¯mV to reach a current density of 10â¯mAâ¯cm-2 and 50â¯mAâ¯cm-2 in 1.0â¯MKOH medium, respectively. It also exhibits a small Tafel slope of 46â¯mVâ¯dec-1 and excellent durability, which is superior to most of recently reported CoSe2-based and Co-based materials. These superior performances can be ascribed to synergistic effects of the highly active CoSe2 nanostructure, defect carbon species and the carbon nanotubes exist in the catalyst. Besides, the unique morphology leads to large electrochemical surface area of the catalyst, which is in favor of the exposure of active sites for OER. Due to high efficiency, low cost and excellent durability for OER, the prepared catalysts showed can be potentially used to substitute noble metals utilized in related energy storage and conversion devices.
ABSTRACT
Specificity of interactions between two DNA strands, or between protein and DNA, is often achieved by varying bases or side chains coming off the DNA or protein backbone-for example, the bases participating in Watson-Crick pairing in the double helix, or the side chains contacting DNA in TALEN-DNA complexes. By contrast, specificity of protein-protein interactions usually involves backbone shape complementarity1, which is less modular and hence harder to generalize. Coiled-coil heterodimers are an exception, but the restricted geometry of interactions across the heterodimer interface (primarily at the heptad a and d positions2) limits the number of orthogonal pairs that can be created simply by varying side-chain interactions3,4. Here we show that protein-protein interaction specificity can be achieved using extensive and modular side-chain hydrogen-bond networks. We used the Crick generating equations5 to produce millions of four-helix backbones with varying degrees of supercoiling around a central axis, identified those accommodating extensive hydrogen-bond networks, and used Rosetta to connect pairs of helices with short loops and to optimize the remainder of the sequence. Of 97 such designs expressed in Escherichia coli, 65 formed constitutive heterodimers, and the crystal structures of four designs were in close agreement with the computational models and confirmed the designed hydrogen-bond networks. In cells, six heterodimers were fully orthogonal, and in vitro-following mixing of 32 chains from 16 heterodimer designs, denaturation in 5 M guanidine hydrochloride and reannealing-almost all of the interactions observed by native mass spectrometry were between the designed cognate pairs. The ability to design orthogonal protein heterodimers should enable sophisticated protein-based control logic for synthetic biology, and illustrates that nature has not fully explored the possibilities for programmable biomolecular interaction modalities.
Subject(s)
Computer Simulation , Protein Engineering , Protein Interaction Domains and Motifs , Protein Multimerization , Proteins/chemistry , Proteins/metabolism , DNA/chemistry , DNA/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Guanidine/pharmacology , Hydrogen Bonding , Models, Molecular , Protein Binding , Protein Denaturation/drug effects , Protein Structure, Secondary , Proteins/geneticsABSTRACT
Membrane-bound O-acyltransferases (MBOATs) are a superfamily of integral transmembrane enzymes that are found in all kingdoms of life1. In bacteria, MBOATs modify protective cell-surface polymers. In vertebrates, some MBOAT enzymes-such as acyl-coenzyme A:cholesterol acyltransferase and diacylglycerol acyltransferase 1-are responsible for lipid biosynthesis or phospholipid remodelling2,3. Other MBOATs, including porcupine, hedgehog acyltransferase and ghrelin acyltransferase, catalyse essential lipid modifications of secreted proteins such as Wnt, hedgehog and ghrelin, respectively4-10. Although many MBOAT proteins are important drug targets, little is known about their molecular architecture and functional mechanisms. Here we present crystal structures of DltB, an MBOAT responsible for the D-alanylation of cell-wall teichoic acid in Gram-positive bacteria11-16, both alone and in complex with the D-alanyl donor protein DltC. DltB contains a ring of 11 peripheral transmembrane helices, which shield a highly conserved extracellular structural funnel extending into the middle of the lipid bilayer. The conserved catalytic histidine residue is located at the bottom of this funnel and is connected to the intracellular DltC through a narrow tunnel. Mutation of either the catalytic histidine or the DltC-binding site of DltB abolishes the D-alanylation of lipoteichoic acid and sensitizes the Gram-positive bacterium Bacillus subtilis to cell-wall stress, which suggests cross-membrane catalysis involving the tunnel. Structure-guided sequence comparison among DltB and vertebrate MBOATs reveals a conserved structural core and suggests that MBOATs from different organisms have similar catalytic mechanisms. Our structures provide a template for understanding structure-function relationships in MBOATs and for developing therapeutic MBOAT inhibitors.