Native Structure Research Articles

Super Enhanced Purification of Denatured-Refolded Ubiquitinated Proteins by ThUBD Revealed Ubiquitinome Dysfunction in Liver Fibrosis

Ubiquitination is crucial for maintaining protein homeostasis and plays a vital role in diverse biological processes. Ubiquitinome profiling and quantification are of great scientific significance. Artificial ubiquitin-binding domains (UBDs) have been widely employed to capture ubiquitinated proteins. The success of this enrichment relies on recognizing native spatial structures of ubiquitin and ubiquitin chains by UBDs under native conditions. However, the use of native lysis conditions presents significant challenges, including insufficient protein extraction, heightened activity of deubiquitinating enzymes (DUBs) and proteasomes in removing the ubiquitin signal, and purification of a substantial number of contaminant proteins, all of which undermine the robustness and reproducibility of ubiquitinomics. In this study, we introduced a novel approach that combines denatured-refolded ubiquitinated sample preparation (DRUSP) with a tandem hybrid UBD (ThUBD) for ubiquitinomic analysis. The samples were effectively extracted using strongly denatured buffers and subsequently refolded using filters. DRUSP yielded a significantly stronger ubiquitin signal, nearly 3 times greater than that of the Control method. Then, 8 types of ubiquitin chains were quickly and accurately restored; therefore, they were recognized and enriched by ThUBD with high efficiency and no biases. Compared with the Control method, DRUSP showed extremely high efficiency in enriching ubiquitinated proteins, improving overall ubiquitin signal enrichment by approximately 10-fold. Moreover, when combined with ubiquitin chain-specific UBDs, DRUSP had also been proven to be a versatile approach. This new method significantly enhanced the stability and reproducibility of ubiquitinomics research. Finally, DRUSP was successfully applied to deep ubiquitinome profiling of early mouse liver fibrosis with increased accuracy, revealing novel insights for liver fibrosis research.

Toward Diverse Plant Proteins for Food Innovation.

This review highlights the development of plant proteins from a wide variety of sources, as most of the research and development efforts to date have been limited to a few sources including soy, chickpea, wheat, and pea. The native structure of plant proteins during production and their impact on food colloids including emulsions, foams, and gels are considered in relation to their fundamental properties, while highlighting the recent developments in the production and processing technologies with regard to their impacts on the molecular properties and aggregation of the proteins. The ability to quantify structural, morphological, and rheological properties can provide a better understanding of the roles of plant proteins in food systems. The applications of plant proteins as dairy and meat alternatives are discussed from the perspective of food structure formation. Future directions on the processing of plant proteins and potential applications are outlined to encourage the generation of more diverse plant-based products.

Comparative analysis of polysaccharide and cell wall structure in Aspergillus nidulans and Aspergillus fumigatus by solid-state NMR

Invasive aspergillosis poses a significant threat to immunocompromised patients, leading to high mortality rates associated with these infections. Targeting the biosynthesis of cell wall carbohydrates is a promising strategy for antifungal drug development and will be advanced by a molecular-level understanding of the native structures of polysaccharides within their cellular context. Solid-state NMR spectroscopy has recently provided detailed insights into the cell wall organization of Aspergillus fumigatus, but genetic and biochemical evidence highlights species-specific differences among Aspergillus species. In this study, we employed a combination of 13C, 15N, and 1H-detection solid-state NMR, supplemented by Dynamic Nuclear Polarization (DNP), to compare the structural organization of cell wall polymers and their assembly in the cell walls of A. fumigatus and A. nidulans, both of which are key model organisms and human pathogens. The two species exhibited a similar rigid core architecture, consisting of chitin, α-glucan, and β-glucan, which contributed to comparable cell wall properties, including polymer dynamics, water retention, and supramolecular organization. However, differences were observed in the chitin, galactosaminogalactan, protein, and lipid content, as well as in the dynamics of galactomannan and the structure of the glucan matrix.

Tyrosinase-Mediated Conjugation for Antigen Display on Ferritin Nanoparticles.

Ferritin (Ft) nanoparticles have become versatile platforms for displaying antigens, being a promising technology for vaccine development. While genetic fusion has traditionally been the preferred method for antigen display, concerns about improper folding and steric hindrance that may compromise vaccine efficacy or stability have prompted alternative approaches. Bioconjugation offers the advantage of preserving native protein structure and function, with recent advancements improving efficiency and specificity. In this study, we used tyrosinase (TYR) to bioconjugate the receptor binding domain of the SARS-CoV-2 spike protein, tagged with a tyrosine (RBD-Y), to native cysteines on Ft, resulting in RBD-Y-Ft nanoparticles. We quantified available cysteines on ferritin using Ellman's assay and monitored their reduction during the reactions. Denaturing analytics (via SDS-PAGE, Western blot, and LC-TOF-MS) confirmed the formation of RBD-Y-Ft monomers with an expected molecular weight of 46 kDa. Mass photometry and HPLC estimated a molecular weight of RBD-Y-Ft nanoparticles of 680 kDa, which was higher than that of nonfunctionalized ferritin (480 kDa), indicating successful binding of up to eight RBD-Y antigens per 24-mer Ft nanoparticle. This work enhances our understanding of how Ft nanoparticles can be engineered to present antigens, leveraging them as a robust scaffold for producing tailored-made candidate vaccines in a timely manner.

Molecular architecture of the mammalian 2-oxoglutarate dehydrogenase complex

The 2-oxoglutarate dehydrogenase complex (OGDHc) orchestrates a critical reaction regulating the TCA cycle. Although the structure of each OGDHc subunit has been solved, the architecture of the intact complex and inter-subunit interactions still remain unknown. Here we report the assembly of native, intact OGDHc from Sus scrofa heart tissue using cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET), and subtomogram averaging (STA) to discern native structures of the whole complex and each subunit. Our cryo-EM analyses revealed the E2o cubic core structure comprising eight homotrimers at 3.3-Å resolution. More importantly, the numbers, positions and orientations of each OGDHc subunit were determined by cryo-ET and the STA structures of the core were resolved at 7.9-Å with the peripheral subunits reaching nanometer resolution. Although the distribution of the peripheral subunits E1o and E3 vary among complexes, they demonstrate a certain regularity within the position and orientation. Moreover, we analyzed and validated the interactions between each subunit, and determined the flexible binding mode for E1o, E2o and E3, resulting in a proposed model of Sus scrofa OGDHc. Together, our results reveal distinctive factors driving the architecture of the intact, native OGDHc.

The Dsc complex and its role in Golgi quality control.

Membrane proteins play crucial roles in cellular functions. However, processes such as the insertion of membrane proteins into the endoplasmic reticulum (ER), their folding into native structures, the assembly of multi-subunit membrane protein complexes, and their targeting from the ER to specific organelles are prone to errors and have a relatively high failure rate. To prevent the accumulation of defective or orphaned membrane proteins, quality control mechanisms assess folding, quantity, and localization of these proteins. This quality control is vital for preserving organelle integrity and maintaining cellular health. In this mini-review, we will focus on how selective membrane protein quality control at the Golgi apparatus, particularly through the defective for SREBP cleavage (Dsc) ubiquitin ligase complex, detects orphaned proteins and prevents their mis-localization to other organelles.

Resveratrol Effect on α-Lactalbumin Thermal Stability.

The effect of resveratrol (RESV) on α-lactalbumin (α-LA) thermal stability was evaluated using differential scanning calorimetry (DSC), circular dichroism (CD) and dynamic light scattering (DLS) measurements. Complementary information offered by molecular docking served to identify the binding site of the ligand on the native structure of protein and the type of interacting forces. DSC thermograms revealed a double-endotherm pattern with partial overlapping of the two components. The most relevant effect of RESV is manifested in the narrowing of the protein thermal fingerprint: the first process (peak temperature T1) is shifted to higher temperatures while the second one (peak temperature T2) to lower values. The CD data indicated partial conformational changes in the protein non-α-helix domain at T1, resulting in a β-sheet richer intermediate (BSRI) with an unaffected, native-like α-helix backbone. The RESV influence on this process may be defined as slightly demoting, at least within DSC conditions (linear heating rate of 1 K min-1). On further heating, unfolding of the α-helix domain takes place at T2, with RESV acting as a promoter of the process. Long time incubation at 333 K produced the same type of BSRI: no significant effect of RESV on the secondary structure content was detected by CD spectroscopy. Nevertheless, the size distribution of the protein population obtained from DLS measurements revealed the free (non-bound) RESV action manifested in the developing of larger size aggregates.

The role of microwave absorption capacity and water mobility in the microwave treatment of grain vs. flour: Impact on the treated flour characteristics

Four alternative crops (amaranth, buckwheat, quinoa, and sorghum) were subjected to microwave-assisted heat-moisture treatment (MWT) under identical conditions (100 °C for 30 min at 25% moisture content) in two different forms (grain and flour). The effects of MWT on the microstructure, thermal properties, and techno-functional properties of the resulting flours were evaluated. The microwave absorption capacity and water mobility in flours and grains during heating were also determined to explain the different impacts of MWT depending on the matrix and its form. The results revealed that the microwave absorption capacity was comparable for all the samples, with water being the primary microwave-absorbing component. However, a different mobility and distribution of water during heating were observed for grains and flour and among matrices, leading to variations in the properties measured. The matrix, the form of treatment, and their interaction significantly influenced most of the analysed properties. MWT resulted in disruption of the native structure with partial fusion and loss of integrity of constituents, reduction in gelatinisation enthalpy, increase in the extent of amylopectin retrogradation, increase in water absorption capacity, and decrease in emulsifying capacity. The water absorption index and water solubility index showed increments or decrements depending on the matrix and the form of treatment. Moreover, the MWT of grain proved effective in limiting the colour change observed in the MWT of flour while enhancing the water absorption capacity. These findings highlight the importance of controlling not only the MWT conditions, but also the form of the matrix, as this influences the mobility of water and the final flour properties.

Production of Nanofibrillar Patterned Collagen for Tissue Engineering.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

Interpenetrating networks of fibrillar and amorphous collagen promote cell spreading and hydrogel stability.

Collagen hydrogels are widely used as scaffolds for tissue engineering due to their support of cellular activity. However, collagen hydrogels often undergo undesired changes in size and shape due to cell-generated forces, and conventional strategies to mitigate this deformation typically compromise either the fibrillar microstructure or cytocompatibility of the collagen. In this study, we introduce an innovative interpenetrating network (IPN) that combines physically self-assembled, fibrillar collagen-ideal for promoting cell adhesion and spreading-with covalently crosslinked, amorphous collagen-ideal for enhancing bulk hydrogel stability. Our IPN design maintains the native fibrillar structure of collagen while significantly improving resistance against cell-induced contraction, providing a promising solution to enhance the performance and reliability of collagen hydrogels for tissue engineering applications.

Protein Microarrays for High Throughput Hydrogen/Deuterium Exchange Monitored by FTIR Imaging.

Proteins form the fastest-growing therapeutic class. Due to their intrinsic instability, loss of native structure is common. Structure alteration must be carefully evaluated as structural changes may jeopardize the efficiency and safety of the protein-based drugs. Hydrogen deuterium exchange (HDX) has long been used to evaluate protein structure and dynamics. The rate of exchange constitutes a sensitive marker of the conformational state of the protein and of its stability. It is often monitored by mass spectrometry. Fourier transform infrared (FTIR) spectroscopy is another method with very promising capabilities. Combining protein microarrays with FTIR imaging resulted in high throughput HDX FTIR measurements. BaF2 slides bearing the protein microarrays were covered by another slide separated by a spacer, allowing us to flush the cell continuously with a flow of N2 gas saturated with 2H2O. Exchange occurred simultaneously for all proteins and single images covering ca. 96 spots of proteins that could be recorded on-line at selected time points. Each protein spot contained ca. 5 ng protein, and the entire array covered 2.5 × 2.5 mm2. Furthermore, HDX could be monitored in real time, and the experiment was therefore not subject to back-exchange problems. Analysis of HDX curves by inverse Laplace transform and by fitting exponential curves indicated that quantitative comparison of the samples is feasible. The paper also demonstrates how the whole process of analysis can be automatized to yield fast analyses.

Abstract A085: Engineered novel bioactive Nectin-4 dimer protein significantly enhances the binding of TIGIT

Abstract The goals of this study are to: (a) create a novel soluble bioactive Nectin-4 dimer molecule mimicking its native dimer quaternary structure on the cell surface and (b) evaluate the recombinant Nectin-4 dimer binding potency to its receptor vs the monomer form, so that the dimer can be used as an immunogen and an antigen to increase the potential of cancer therapeutic antibody and vaccine discovery when targeting quaternary structural epitopes. Nectin-4 is a cell adhesion molecule belonging to the nectin family. It's involved in cell-cell junctions and plays a role in cancer progression by influencing cell adhesion, migration, and proliferation. Nectin-4 is overexpressed on various types of tumor cells including breast, lung, ovarian and pancreatic cancers. Nectin-4 has been recently identified as a novel ligand of T cell immunoglobulin and ITIM domain (TIGIT) and interaction between Nectin-4 and TIGIT inhibits NK cell cytotoxicity thus suppressing immune responses, allowing cancer cells to evade immune surveillance. Nectin-4 and Nectin-1 interact in a heterophilic manner, however in the context of cancer, altered expression of Nectin-4 and Nectin-1 can contribute to tumor progression. Nectin-4 is a type 1 single transmembrane protein, and can form homodimers on the cell surface. While therapeutics such as Enfortumab Vedotin, an antibody-drug conjugate, binds Nectin-4 on the surface of cancer cells, mimicking the Nectin-4 dimer quaternary structure can present many more critical opportunities for therapeutic antibody and vaccine discovery. To better mimic the native dimer conformation and quaternary structure, we designed a novel soluble Nectin-4 dimer with 3 extracellular domains fused to a dimer motif at the C-terminus. The Nectin-4 dimer protein was expressed and purified from HEK293 cells and confirmed by SDS-PAGE and Western blot analyses. We characterized the Nectin-4 dimer protein binding potency to TIGIT and Nectin-1 by ELISA. Very interestingly, the results demonstrated that the Nectin-4 dimer binding potency to TIGIT increased dramatically as measured by ELISA, compared to the Nectin-4 monomer protein. Conversely, the Nectin-4 dimer binding to Nectin-1 monomer was significantly decreased, compared to Nectin-4 monomer, as expected. We also studied Nectin-4 dimer immunogenicity using a Nectin-4 dimer DNA immunization to induce potent antibody responses in mice. In summary, these findings imply that the Nectin-4 dimer protein is bioactive and in the desirable conformation, resulting in an increased binding potency to its receptor TIGIT, reduced binding to Nectin-1 (as expected), and eliciting strong antibody responses in mice. This novel Nectin-4 dimer protein can be a very useful molecule for cancer research, therapeutic antibody and vaccine discovery. The Nectin-4 dimer protein can be used to: (i) elicit antibody responses against the dimer quaternary structure, (ii) screen antibodies targeting more conformational epitopes, and (iii) study the Nectin-4 and TIGIT ligand/receptor interactions. Citation Format: Christofer Walsh, Timothy Smith, Mikhail Rashkovskii, Richard Parent, Jean Qiu, Shixia Wang. Engineered novel bioactive Nectin-4 dimer protein significantly enhances the binding of TIGIT [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A085.

Unraveling the Interplay between Stability and Flexibility in the Design of Polyethylene Terephthalate (PET) Hydrolases.

The accumulation of polyethylene terephthalate (PET), a widely used polyester plastic in packaging and textiles, has led to a global environmental crisis. Biodegradation presents a promising strategy for PET recycling, with PET hydrolases (PETase) undertaking the task at the molecular level. Unfortunately, PETase operates only at ambient temperatures with low efficiency, limiting its industrial application. Current engineering efforts focus on enhancing the thermostability of PETase, but increased stability can reduce the structural dynamics needed for substrate binding, potentially slowing enzymatic activity. To elucidate the balance between stability and flexibility in optimizing PETase catalytic activity, we performed theoretical investigations on both wild-type PETase (WT-PETase) and a thermophilic variant (Thermo-PETase) using molecular dynamics simulations and frustration analysis. Despite being initially designed to stabilize the native structure of the enzyme, our findings reveal that Thermo-PETase exhibits an unprecedented increase in structural flexibility at the PET-binding and catalytic sites, beneficial for substrate recruitment and product release, compared to WT-PETase. Upon PET binding, we observed that the structural dynamics of Thermo-PETase is largely quenched, favoring the proximity between the catalytic residues and the carbonyl of the PET substrate. This may potentially contribute to a higher probability of a catalytic reaction occurring in Thermo-PETase compared to WT-PETase. We suggest that Thermo-PETase can exhibit higher PET-degradation performance than WT-PETase across a broad temperature range by leveraging stability and flexibility at high and low temperatures, respectively. Our findings provide valuable insights into how PETase optimizes its enzymatic performance by balancing stability and flexibility, which may contribute to future PETase design strategies.

Target-Specific De Novo Peptide Binder Design with DiffPepBuilder.

Despite the exciting progress in target-specific de novo protein binder design, peptide binder design remains challenging due to the flexibility of peptide structures and the scarcity of protein-peptide complex structure data. In this study, we curated a large synthetic data set, referred to as PepPC-F, from the abundant protein-protein interface data and developed DiffPepBuilder, a de novo target-specific peptide binder generation method that utilizes an SE(3)-equivariant diffusion model trained on PepPC-F to codesign peptide sequences and structures. DiffPepBuilder also introduces disulfide bonds to stabilize the generated peptide structures. We tested DiffPepBuilder on 30 experimentally verified strong peptide binders with available protein-peptide complex structures. DiffPepBuilder was able to effectively recall the native structures and sequences of the peptide ligands and to generate novel peptide binders with improved binding free energy. We subsequently conducted de novo generation case studies on three targets. In both the regeneration test and case studies, DiffPepBuilder outperformed AfDesign and RFdiffusion coupled with ProteinMPNN, in terms of sequence and structure recall, interface quality, and structural diversity. Molecular dynamics simulations confirmed that the introduction of disulfide bonds enhanced the structural rigidity and binding performance of the generated peptides. As a general peptide binder de novo design tool, DiffPepBuilder can be used to design peptide binders for given protein targets with three-dimensional and binding site information.

Mild three-stage alkali-oxygen treatment preserving the native macromolecular structure of lignin for effective disassembling of tobacco stalk

Tobacco stalks, as one of the annual economic crops rich in biomacromolecules such as cellulose and hemicellulose, are more difficult to decompose into cellulose fibers due to their high degree of lignification compared to other ordinary straw feedstocks, resulting in their underutilization. In this study, we developed a mild three-stage alkali‑oxygen (AO) process to efficiently deconstruct the tobacco stalk cell walls. The process, involving alkaline dosages of 15 %, 10 %, and 3 % at each stage, effectively dissociated the cell walls and yielded cellulose fibers with high brightness (42.0 % ISO). The organics in the spent liquor, including lignin, hemicellulose, and small-molecular extracts, were isolated through acid/ethanol precipitation and organic solvent extraction. Lignin characterization by 2D HSQC NMR indicated that the majority of native β-aryl ether linkages were preserved after AO treatment, making it suitable for producing chemicals or biofuels via depolymerization. Additionally, the small-molecular extracts contained numerous depolymerized products from lignin and carbohydrates, as well as bioactive compounds derived from the tobacco stalk. Overall, this mild, efficient, and eco-friendly process offers a promising approach for the valorization of tobacco stalks and similar biomass resources.

Identification and biophysical characterization of epitope atlas of Porcine Reproductive and Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) have been a critical threat to swine health since 1987 due to its high mutation rate and substantial economic loss over half a billion dollar in USA. The rapid mutation rate of PRRSV presents a significant challenge in developing an effective vaccine. Even though surveillance and intervention studies have recently (2019) unveiled utilization of PRRSV glycoprotein 5 (GP5; encoded by ORF5 gene) to induce immunogenic reaction and production of neutralizing antibodies in porcine populations, the future viral generations can accrue escape mutations. In this study we identify 63 porcine-PRRSV protein-protein interactions which play primary or ancillary roles in viral entry and infection. Using genome–proteome annotation, protein structure prediction, multiple docking experiments, and binding energy calculations, we identified a list of 75 epitope locations on PRRSV proteins crucial for infection. Additionally, using machine learning-based diffusion model, we designed 56 stable immunogen peptides that contain one or more of these epitopes with their native tertiary structures stabilized through optimized N– and C–terminus flank sequences and interspersed with appropriate linker regions. Our workflow successfully identified numerous known interactions and predicted several novel PRRSV-porcine interactions. By leveraging the structural and sequence insights, this study paves the way for more effective, high-avidity, multi-valent PRRSV vaccines, and leveraging neural networks for immunogen design.

Hemoglobin-PEG Interactions Probed by Small-Angle X-ray Scattering: Insights for Crystallization and Diagnostics Applications.

Protein-protein interactions, controlling protein aggregation in the solution phase, are crucial for the formulation of protein therapeutics and the use of proteins in diagnostic applications. Additives in the solution phase are factors that may enhance the protein's conformational stability or induce crystallization. Protein-PEG interactions do not always stabilize the native protein structure. Structural information is needed to validate excipients for protein stabilization in the development of protein therapeutics or use proteins in diagnostic assays. The present study investigates the impact of polyethylene glycol (PEG) molecular weight and concentration on the spatial structure of human hemoglobin (Hb) at neutral pH. Small-angle X-ray scattering (SAXS) in combination with size-exclusion chromatography is employed to characterize the Hb structure in solution without and with the addition of PEG. Our results evidence that human hemoglobin maintains a tetrameric conformation at neutral pH. The dummy atom model, reconstructed from the SAXS data, aligns closely with the known crystallographic structure of methemoglobin (metHb) from the Protein Data Bank. We established that the addition of short-chain PEG600, at concentrations of up to 10% (w/v), acts as a stabilizer for hemoglobin, preserving its spatial structure without significant alterations. By contrast, 5% (w/v) PEG with higher molecular weights of 2000 and 4000 leads to a slight reduction in the maximum particle dimension (Dmax), while the radius of gyration (Rg) remains essentially unchanged. This implies a reduced hydration shell around the protein due to the dehydrating effect of longer PEG chains. At a concentration of 10% (w/v), PEG2000 interacts with Hb to form a complex that does not distort the protein's spatial configuration. The obtained results provide a deeper understanding of PEG's influence on the Hb structure in solution and broader knowledge regarding protein-PEG interactions.

Osteochondral regeneration with a tri-layered biomimetic resorbable scaffold: In vivo study in a sheep model up to 12 months of follow-up

The treatment of osteochondral joint lesions requires the regeneration of both articular cartilage and subchondral bone tissue. Scaffold-based strategies aimed at mimicking the native osteochondral structure have been explored with mixed results. The aim of this study was to evaluate the regenerative potential of a tri-layered osteochondral cell-free scaffold in a large animal model at both 6 and 12 months of follow-up. Bilateral critical-sized osteochondral defects were created in 22 sheep. One defect was filled with the scaffold, whereas the contralateral was left empty. The repair tissue quality was evaluated at 6 and 12 months of follow-up in terms of macroscopic appearance, histology, trabecular bone formation, and inflammation grade. The mean global ICRS II score in the scaffold and control groups was 41 ± 11 vs 30 ± 6 at 6 months (p = 0.004) and 54 ± 13 vs 37 ± 11 at 12 months (p = 0.002), respectively. A higher percentage of bone was found in the treatment group compared to controls both at 6 (BV/TV 48.8 ± 8.6 % vs 37.4 ± 9.5 %, respectively; p < 0.001) and 12 months (BV/TV 51.8 ± 8.8 % vs 42.1 ± 12.6 %, respectively; p = 0.023). No significant levels of inflammation were seen. These results demonstrated the scaffold safety and potential to regenerate both cartilage and subchondral tissues in a large animal model of knee osteochondral lesions.

Empowering AlphaFold2 for protein conformation selective drug discovery with AlphaFold2-RAVE.

Small-molecule drug design hinges on obtaining co-crystallized ligand-protein structures. Despite AlphaFold2's strides in protein native structure prediction, its focus on apo structures overlooks ligands and associated holo structures. Moreover, designing selective drugs often benefits from the targeting of diverse metastable conformations. Therefore, direct application of AlphaFold2 models in virtual screening and drug discovery remains tentative. Here, we demonstrate an AlphaFold2-based framework combined with all-atom enhanced sampling molecular dynamics and Induced Fit docking, named AF2RAVE-Glide, to conduct computational model-based small-molecule binding of metastable protein kinase conformations, initiated from protein sequences. We demonstrate the AF2RAVE-Glide workflow on three different mammalian protein kinases and their type I and II inhibitors, with special emphasis on binding of known type II kinase inhibitors which target the metastable classical DFG-out state. These states are not easy to sample from AlphaFold2. Here, we demonstrate how with AF2RAVE these metastable conformations can be sampled for different kinases with high enough accuracy to enable subsequent docking of known type II kinase inhibitors with more than 50% success rates across docking calculations. We believe the protocol should be deployable for other kinases and more proteins generally.

Contact-Map-Driven Exploration of Heterogeneous Protein-Folding Paths.

We have recently shown how physically realizable protein-folding pathways can be generated using directed walks in the space of inter-residue contact-maps; combined with a back-transformation to move from protein contact-maps to Cartesian coordinates, we have demonstrated how this approach can generate protein-folding trajectory ensembles without recourse to molecular dynamics. In this article, we demonstrate that this framework can be used to study a challenging protein-folding problem that is known to exhibit two different folding paths which were previously identified through molecular dynamics simulation at several different temperatures. From the viewpoint of protein-folding mechanism prediction, this particular problem is extremely challenging to address, specifically involving folding to an identical nontrivial compact native structure along distinct pathways defined by heterogeneous secondary structural elements. Here, we show how our previously reported contact-map-based protein-folding strategy can be significantly enhanced to enable accurate and robust prediction of heterogeneous folding paths by (i) introducing a novel topologically informed metric for comparing two protein contact maps, (ii) reformulating our graph-represented folding path generation, and (iii) introducing a new and more reliable structural back-mapping algorithm. These changes improve the reliability of generating structurally sound folding intermediates and dramatically decrease the number of physically irrelevant folding intermediates generated by our previous simulation strategy. Most importantly, we demonstrate how our enhanced folding algorithm can successfully identify the alternative folding mechanisms of a multifolding-pathway protein, in line with direct molecular dynamics simulations.