Structural Basis Research Articles

Structural basis of human γ-secretase inhibition by anticancer clinical compounds.

Aberrant activation of Notch signaling, mediated by the Notch intracellular domain (NICD), is linked to certain types of cancer. The NICD is released through γ-secretase-mediated cleavage of the Notch receptor. Therefore, development of a γ-secretase inhibitor (GSI) represents an anticancer strategy. Here we report the cryo-electron microscopy structures of human γ-secretase bound individually to five clinically tested GSIs (RO4929097, crenigacestat, BMS906024, nirogacestat and MK-0752) at overall resolutions of 2.4-3.0 Å. Three of the five GSIs are in active anticancer clinical trials, while nirogacestat was recently approved. Each of these GSIs similarly occupies the substrate-binding site of presenilin 1 but shows characteristic differences in detailed recognition pattern. The size and shape of the binding pocket are induced by the bound GSI. Analysis of these structural features suggest strategies for modification of the GSI with improved inhibition potency.

Structural basis of the allosteric regulation of cyanobacterial glucose-6-phosphate dehydrogenase by the redox sensor OpcA

The oxidative pentose phosphate (OPP) pathway is a fundamental carbon catabolic route for generating reducing power and metabolic intermediates for biosynthetic processes. In addition, its first two reactions form the OPP shunt, which replenishes the Calvin-Benson cycle under certain conditions. Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-limiting reaction of this metabolic route. In photosynthetic organisms, G6PDH is redox-regulated to allow fine-tuning and to prevent futile cycles while carbon is being fixed. In cyanobacteria, regulation of G6PDH requires the redox protein OpcA, but the underlying molecular mechanisms behind this allosteric activation remain elusive. Here, we used enzymatic assays and in vivo interaction analyses to show that OpcA binds G6PDH under different environmental conditions. However, complex formation enhances G6PDH activity when OpcA is oxidized and inhibits it when OpcA is reduced. To understand the molecular basis of this regulation, we used cryogenic electron microscopy to determine the structure of Synechocystis G6PDH and the G6PDH-OpcA complex. OpcA binds the G6PDH tetramer and induces conformational changes in the active site of G6PDH. The redox sensitivity of OpcA is achieved by intramolecular disulfide bridge formation, which influences the allosteric regulation of G6PDH. In vitro assays reveal that the level of G6PDH activation depends on the number of bound OpcA molecules, which implies that this mechanism allows delicate fine-tuning. Our findings unveil a unique molecular mechanism governing the regulation of the OPP in Synechocystis.

A single mutation in bovine influenza H5N1 hemagglutinin switches specificity to human receptors.

In 2024, several human infections with highly pathogenic clade 2.3.4.4b bovine influenza H5N1 viruses in the United States raised concerns about their capability for bovine-to-human or even human-to-human transmission. In this study, analysis of the hemagglutinin (HA) from the first-reported human-infecting bovine H5N1 virus (A/Texas/37/2024, Texas) revealed avian-type receptor binding preference. Notably, a Gln226Leu substitution switched Texas HA binding specificity to human-type receptors, which was enhanced when combined with an Asn224Lys mutation. Crystal structures of the Texas HA with avian receptor analog LSTa and its Gln226Leu mutant with human receptor analog LSTc elucidated the structural basis for this preferential receptor recognition. These findings highlight the need for continuous surveillance of emerging mutations in avian and bovine clade 2.3.4.4b H5N1 viruses.

Molecular basis for the diversification of lincosamide biosynthesis by pyridoxal phosphate-dependent enzymes.

The biosynthesis of the lincosamide antibiotics lincomycin A and celesticetin involves the pyridoxal-5'-phosphate (PLP)-dependent enzymes LmbF and CcbF, which are responsible for bifurcation of the biosynthetic pathways. Despite recognizing the same S-glycosyl-L-cysteine structure of the substrates, LmbF catalyses thiol formation through β-elimination, whereas CcbF produces S-acetaldehyde through decarboxylation-coupled oxidative deamination. The structural basis for the diversification mechanism remains largely unexplored. Here we conduct structure-function analyses of LmbF and CcbF. X-ray crystal structures, docking and molecular dynamics simulations reveal that active-site aromatic residues play important roles in controlling the substrate binding mode and the reaction outcome. Furthermore, the reaction selectivity and oxygen-utilization of LmbF and CcbF were rationally engineered through structure- and calculation-based mutagenesis. Thus, the catalytic function of CcbF was switched to that of LmbF, and, remarkably, both LmbF and CcbF variants gained the oxidative-amidation activity to produce an unnatural S-acetamide derivative of lincosamide.

Molecular insights into the activation mechanism of GPR156 in maintaining auditory function

The class C orphan G-protein-coupled receptor (GPCR) GPR156, which lacks the large extracellular region, plays a pivotal role in auditory function through Gi2/3. Here, we firstly demonstrate that GPR156 with high constitutive activity is essential for maintaining auditory function, and further reveal the structural basis of the sustained role of GPR156. We present the cryo-EM structures of human apo GPR156 and the GPR156–Gi3 complex, unveiling a small extracellular region formed by extracellular loop 2 (ECL2) and the N-terminus. The GPR156 dimer in both apo state and Gi3 protein-coupled state adopt a transmembrane (TM)5/6-TM5/6 interface, indicating the high constitutive activity of GPR156 in the apo state. Furthermore, C-terminus in G-bound subunit of GPR156 plays a dual role in promoting G protein binding within G-bound subunit while preventing the G-free subunit from binding to additional G protein. Together, these results explain how GPR156 constitutive activity is maintained through dimerization and provide a mechanistic insight into the sustained role of GPR156 in maintaining auditory function.

Structural basis of deoxynucleotide addition by HIV-1 RT during reverse transcription

Reverse transcription of the retroviral RNA genome into DNA is an integral step during HIV-1 replication. Despite a wealth of structural information on reverse transcriptase (RT), we lack insight into the intermediate states of DNA synthesis. Using catalytically active substrates, and a blot/diffusion cryo-electron microscopy approach, we capture 11 structures encompassing reactant, intermediate and product states of dATP addition by RT at 2.2 to 3.0 Å resolution. In the reactant state, dATP binding to RT-template/primer involves a single Mg2+ (site B) inducing formation of a negatively charged pocket where a second floating Mg2+ can bind (site A). During the intermediate state, the α-phosphate oxygen from a previously unobserved dATP conformer aligns with site A Mg2+ and the primer 3′-OH for nucleophilic attack. The product state, comprises two substrate conformations including an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H-bonds. Moreover, K220 mutants significantly impact the rate of dNTP incorporation by RT and HIV-1 replication capacity. This work sheds light into the dynamic components of a reaction that is central to HIV-1 replication.

Structural and biochemical characterization of a widespread enterobacterial peroxidase encapsulin.

Encapsulins are self-assembling protein compartments found in prokaryotes and specifically encapsulate dedicated cargo enzymes. The most abundant encapsulin cargo class are Dye-decolorizing Peroxidases (DyPs). It has been previously suggested that DyP encapsulins are involved in oxidative stress resistance and bacterial pathogenicity due to DyPs' inherent ability to reduce and detoxify hydrogen peroxide while oxidizing a broad range of organic co-substrates. Here, we report the structural and biochemical analysis of a DyP encapsulin widely found across enterobacteria. Using bioinformatic approaches, we show that this DyP encapsulin is encoded by a conserved transposon-associated operon, enriched in enterobacterial pathogens. Through low pH and peroxide exposure experiments, we highlight the stability of this DyP encapsulin under harsh conditions and show that DyP catalytic activity is highest at low pH. We determine the structure of the DyP-loaded shell and free DyP via cryo-electron microscopy, revealing the structural basis for DyP cargo loading and peroxide preference. Our work lays the foundation to further explore the substrate range and physiological functions of enterobacterial DyP encapsulins.

Concept and Design of Cutting Tools for Osseodensification in Implant Dentistry

Osseodensification is an innovative surgical instrumentation technique based on additive (non-cutting) drilling using special burs. It is known from the literature, that the osseodensification burs should operate in a clockwise direction to drill holes and in a counterclockwise direction to compact the osteotomy walls. For these purposes, the burs have special design features, like conical contour shape, increased number of helical flutes, and negative rake angle on the peripheral part. However, although other parameters and features of the burs define their overall performance, they are not described sufficiently, and their influence on surgical quality is almost unknown both for clinicians and tool manufacturers. The purpose of the present research is to identify the key design features of burs for osseodensification and their functional relationship with the qualitative indices of the procedure based on an analytical review of research papers and patent documents. It will help to further improve the design of osseodensification burs and thereby enhance the surgical quality and, ultimately, patient satisfaction. Results: The most important design features and parameters of osseodensification burs are identified. Thereon, the structural model of osseodensification bur is first represented as a hypergraph. Based on the analysis of previous research, functional relationships between design parameters of osseodensification burs, osseodensification procedure conditions, and procedure performance data were established and, for the first time, described in the comprehensive form of a hypergraph. Conclusions: This study provides formal models that form the basis of database structure and its control interface, which will be used in the later developed computer-aided design module to create advanced types of burs under consideration. These models will also help to make good experimental designs used in studies aimed at improving the efficiency of the osseodensification procedure.

3D and 2D-QSAR Studies on Natural Flavonoids for Nitric Oxide Production Inhibitory Activity

Background: Nitric oxide (NO), an important second messenger molecule, regulates numerous physiological responses, while excessive NO generates negative effects on the circulatory, nervous and immune systems. Recently, some natural flavonoids were reported to possess the capability of inhibiting LPS-induced NO production. To fully understand the nature of their own NO inhibitory activity, it is necessary to address the structural requirements of flavonoids as NO inhibitors. Objective: The objective of this work was to develop efficient QSAR models for predicting the NOinhibitory activity of new flavonoids and improving insights into the critical properties of the chemical structures that were required for the ideal NO production inhibitory activities. Methods: To provide insights into the structural basis of flavonoids as NO inhibitors, 3D quantitative structure-activity relationship (3D-QSAR) and 2D-QSAR models were developed on a dataset of 55 flavonoids using comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA) and hologram quantitative structure-activity relationship (HQSAR) approaches. Results: The statistically significant models for CoMFA, CoMSIA and HQSAR resulted in crossvalidated coefficient (q2) values of 0.523, 0.572 and 0.639, non-cross-validated coefficient (r2) values of 0.793, 0.828 and 0.852, respectively. The robustness of these models was further affirmed using a test set of 18 compounds, which resulted in predictive correlation coefficients (r2 pred) of 0.968, 0.954 and 0.906. Furthermore, the models-derived contour maps were appraised for activity trends for the molecules analyzed. Conclusion: The 3D and 2D-QSAR models constructed in this paper were efficient in estimating the NO inhibitory activities of flavonoids and facilitating the design of flavonoid-derived NO production inhibitors.

Structural insights to the RRM-domain of the glycine-rich RNA-binding protein from Sorghum bicolor and its role in cold stress tolerance in E. coli

Sorghum bicolor Glycine-rich RNA-binding protein (SbGRBP), exhibit the ability to bind both single-stranded and double-stranded DNA. The expression of SbGRBP is regulated by heat stress, with the protein localizing to the nucleus and cytosol. The present study delves into the structure and ssDNA binding ability of its truncated version (SbGRBP1–119) which lacks glycine rich domain (GR). This protein has the ability to bind ssDNA Using Nuclear Magnetic Resonance (NMR) spectroscopy, we have revealed the secondary structure of SbGRBP1–119, highlighting the typical configuration of GRBPs with four β-sheets and two α-helices. Notably, we found two additional α-helices at the N-terminal region that seem to interact with ssDNA, a novel observation for GRBPs. Key residues crucial for ssDNA binding were identified, suggesting a specific interaction with the oligonucleotide sequence 5’-TTCTGG-3′. Preliminary assays hinted that SbGRBP1–119 might bolster E. coli resilience to cold stress, indicating a potential chaperone-like role under stress conditions. This study sheds light on the structural basis of SbGRBP1–119's interaction with nucleic acids, deepening our understanding about the role of GRBPs' in RNA metabolism and regulation.

Structural basis for the effects of Ser387 phosphorylation of MgcRacGAP on its GTPase-activating activities for CDC42 and RHOA

MgcRacGAP is a GTPase-activating protein (GAP) for the Rho family GTPases. During cytokinesis, MgcRacGAP localizes to the midbody, where it activates the GTPase activity of Rho family GTPases to facilitate cytokinesis. In the midbody, Aurora B phosphorylates Ser387 within the GAP domain of human MgcRacGAP, a modification that is suggested to influence GTPase preference. However, there are conflicting reports, with some studies indicating that Ser387 phosphorylation does not alter the GTPase preference of MgcRacGAP. This controversy highlights the need for a deeper understanding of the molecular interactions involved, which can be clarified through structural analyses. In the present study, we determined the crystal structures of the wild-type MgcRacGAP GAP domain complexed with CDC42•GDP•AlF4− and the S378D phosphomimetic mutant GAP domain fused with RHOA•GDP•AlF4−. Additionally, crystal structures of the GAP domains were determined for the S387D and S387A mutants. Our analysis revealed that neither GTPase binding nor S387D mutation affected the overall structure of the GAP domain. However, comparison of the CDC42•MgcRacGAP (wild-type) complex with the RHOA-MgcRacGAP(S378D) fusion protein structure indicated that the S387D mutation caused positional shifts in both CDC42 and RHOA relative to MgcRacGAP. These shifts reduced interactions with CDC42 more severely than those with RHOA. In fact, the S387D mutation decreased the GTPase-activating activity of MgcRacGAP toward CDC42, while its impact on RHOA was only moderate. This difference in the rate of activity reduction may play an important role in GTPase preference.

Evaluating the electronic and structural basis of carbon selenide-based quantum dots as photovoltaic design materials: A DFT and ML analysis

We present a new study on the design, discovery and space generation of carbon selenide based photovoltaic (PV) materials. By extending acceptors and leveraging density functional theory (DFT) and machine learning (ML) analysis, we discover new QDs with remarkable PV properties. We employ various ML models, to correlate the exciton binding energy (Eb) of 938 relevant compounds from literature with their molecular descriptors of structural features that influence their performance. Our study demonstrates the potential of ML approaches in streamlining the design and discovery of high-efficiency PV materials. Also the RDKit computed molecular descriptors correlates with PV parameters revealed maximum absorption (λmax) ranges of 509–531 nm, light harvesting efficiency (LHE) above 92 %, Open Circuit Voltage (Voc) of 0.22–0.45 V, and short Circuit (Jsc) currents of 37.92–42.75 mA/cm2. Their Predicted Power Conversion Efficiencies (PCE) using the Scharber method reaches upto 09–13 %. This study can pave the way for molecular descriptor-based design of new PV materials, promising a paradigm shift in the development of high-efficiency solar energy conversion technologies.

Receptor binding and structural basis of raccoon dog ACE2 binding to SARS-CoV-2 prototype and its variants.

Raccoon dog was proposed as a potential host of SARS-CoV-2, but no evidence support such a notion. In our study, we investigated the binding affinities of raccoon dog ACE2 (rdACE2) to the spike (S) protein receptor binding domain (RBD) of SARS-CoV-2 prototype (PT) and its variants. It revealed that the binding affinities of RBD from SARS-CoV-2 variants were generally lower than that of the PT RBD. Through structural and functional analyses, we found amino acids H34 and M82 play pivotal roles in maintaining the binding affinity of ACE2 to different SARS-CoV-2 sub-variants. These results suggest that raccoon dogs exhibit lower susceptibility to SARS-CoV-2 compared to those animal species with a high prevalence of SARS-CoV-2 transmission.

Conformational Plasticity of SpyCas9 Induced by AcrIIA4 and AcrIIA2: Insights from Molecular Dynamics Simulation

CRISPR-Cas9 systems constitute bacterial adaptive immune systems that protect against phage infections. Bacteriophages encode anti-CRISPR proteins (Acrs) that mitigate the bacterial immune response. However, the structural basis for their inhibitory actions from a molecular perspective remains elusive. In this study, through microsecond atomistic molecular dynamics simulations, we demonstrated the remarkable flexibility of Streptococcus pyogenes Cas9 (SpyCas9) and its conformational adaptability during interactions with AcrIIA4 and AcrIIA2. Specifically, we demonstrated that the binding of AcrIIA4 and AcrIIA2 to SpyCas9 induces a conformational rearrangement that causes spatial separation between the nuclease and cleavage sites, thus making the endonuclease inactive. This separation disrupts the transmission of signals between the protospacer adjacent motif recognition and nuclease domains, thereby impeding the efficient processing of double-stranded DNA. The simulation also reveals that AcrIIA4 and AcrIIA2 cause different structural variations of SpyCas9. Our research illuminates the precise mechanisms underlying the suppression of SpyCas9 by AcrIIA4 and AcrIIA2, thus presenting new possibilities for controlling genome editing with higher accuracy.

Structural Basis for the Pathogenicity of Parkin Catalytic Domain Mutants

Mutations in the E3 ubiquitin ligase parkin cause a familial form of Parkinson’s disease (PD). Parkin and the mitochondrial kinase PINK1 assure quality control of mitochondria through selective autophagy of mitochondria (mitophagy). Whereas numerous parkin mutations have been functionally and structurally characterized, several PD mutations found in the catalytic Rcat domain of parkin remain poorly understood. Here, we characterize two pathogenic Rcat mutants, T415N and P437L. We demonstrate that both mutants exhibit impaired activity using autoubiquitination and ubiquitin vinyl sulfone assays. We determine the minimal ubiquitin binding segment and show that both mutants display impaired binding of ubiquitin charged on the E2 enzyme. Finally, we use AlphaFold 3 to predict a model of the phospho-parkin:phospho-ubiquitin:ubiquitin-charged E2 complex. The model shows the repressor-element of parkin (REP) and the N-terminal residues of the catalytic domain form a helix to position ubiquitin for transfer from the E2 to parkin. Our results rationalize the pathogenicity of the parkin mutations and deepen our understanding of the active parkin-E2∼Ub complex.

Clustering of synaptic engram: Functional and structural basis of memory

Studies on memory engram have demonstrated how experience and learning can be allocated at a neuronal level for centuries. Recently emerging evidence narrowed down further to the synaptic connections and their patterned allocation on dendrites. Notably, groups of synapses within a specific range within dendrites known as ’synaptic clusters’ have been revealed in association with learning and memory. Previous investigations have shown that a variety of factors mediated by both presynaptic inputs and postsynaptic dendrites contribute to clustering. Here, we review the neural mechanism of synaptic clustering and its correlation with memory. We highlight the recent findings about the clustering of synaptic engrams and memory formation and discuss future directions.

Structural basis for full-length chemerin recognition and signaling through chemerin receptor 1

Chemerin, a chemotactic adipokine, plays essential roles in adipogenesis and inflammation. Serum chemerin concentration is closely associated with obesity and metabolism disorders. The mature form of chemerin (residues 21-157) acts primarily through chemerin receptor 1 (CMKLR1) for transmembrane signaling. As a result, CMKLR1 serves as a promising target for therapeutic intervention of immunometabolic diseases such as diabetes and multiple sclerosis. Here, we present a high-resolution cryo-EM structure of CMKLR1-Gi signaling complex bound to biologically active full-length chemerin. The mature chemerin shows binding features distinct from its C-terminal nonapeptide including interaction with both the extracellular loops (ECLs) and the N-terminus of CMKLR1. Combining results from functional assays, our studies demonstrate that chemerin interacts with CMKLR1 in a “two-site” mode similar to chemokine-chemokine receptor interactions, but acting as a “reverse chemokine” by inserting its C-terminus instead of the N-terminus as in the case of chemokines into the transmembrane binding pocket of CMKLR1. These structural insights are expected to help develop synthetic analogs with therapeutic potential.

High-resolution crystal structure of PD-1 in complex with retifanlimab, the FDA-approved immune checkpoint blocking antibody for treating Merkel cell carcinoma

Retifanlimab is a humanized monoclonal antibody that specifically targets programmed cell death protein 1 (PD-1), an essential immune checkpoint that modulates T-cell immune responses. Several anti-PD-1 antibodies have been market-approved, marking a significant advancement in the treatment of diverse tumor types by restoring the T-cell immune response. Recently, the US FDA approved retifanlimab for treating metastatic or recurrent locally advanced Merkel cell carcinoma. We present the crystal structure of PD-1 in complex with the retifanlimab Fab at a resolution of 1.54 Å to elucidate the structural basis for the mechanism of action of this antibody. This work clarifies the detailed interactions and conformational alterations that occur upon antibody binding. The epitope of retifanlimab partially overlaps with the ligand binding site, and its binding induced unique conformations of the flexible loops within PD-1, including BC, C'D, and FG loops, thereby optimizing interactions with the antibody. A thorough analysis of its interaction with PD-1 and other FDA-approved anti-PD-1 antibodies may provide valuable insights into the rational design of enhanced therapies to regulate immune responses in cancer treatment.

Structural basis of orientated asymmetry in a mGlu heterodimer

The structural basis for the allosteric interactions within G protein-coupled receptors (GPCRs) heterodimers remains largely unknown. The metabotropic glutamate (mGlu) receptors are complex dimeric GPCRs important for the fine tuning of many synapses. Heterodimeric mGlu receptors with specific allosteric properties have been identified in the brain. Here we report four cryo-electron microscopy structures of mGlu2-4 heterodimer in different states: an inactive state bound to antagonists, two intermediate states bound to either mGlu2 or mGlu4 agonist only and an active state bound to both glutamate and a mGlu4 positive allosteric modulator (PAM) in complex with Gi protein. In addition to revealing a unique PAM binding pocket among mGlu receptors, our data bring important information for the asymmetric activation of mGlu heterodimers. First, we show that agonist binding to a single subunit in the extracellular domain is not sufficient to stabilize an active dimer conformation. Single-molecule FRET data show that the monoliganded mGlu2-4 can be found in both intermediate states and an active one. Second, we provide a detailed view of the asymmetric interface in seven-transmembrane (7TM) domains and identified key residues within the mGlu2 7TM that limits its activation leaving mGlu4 as the only subunit activating G proteins.

Targeting histidinol-phosphate aminotransferase in Acinetobacter baumannii with salvianolic acid B: A structure-based approach to novel antibacterial strategies

Undoubtedly, Acinetobacter baumannii is a major ESKAPE pathogen that poses a significant threat to public health, causing severe nosocomial infections with high mortality rates in healthcare settings. Due to the rapid development of antibiotic resistance, only a limited number of antibiotics remain effective against infections caused by multidrug-resistant (MDR) Acinetobacter baumannii. The discovery of new class of antibiotic molecules still lags behind the rate of growing worldwide burden of antimicrobial resistance (AMR). To expedite the discovery of new therapeutic molecules, we have focused on HisC from A. baumannii (AbHisC), a crucial enzyme involved in the seventh step of histidine biosynthesis. This pathway is absent in humans. We have employed the advanced computational techniques to target this promising drug target. AbHisC was cloned, overexpressed, and purified. Three distinct sets of libraries containing ∼60,000 natural compounds from ZINC database were screened against AbHisC using Schrödinger's glide module software. Based on the docking score and glide energy, top 25 hits were further subjected to induced fit (IF) docking. Top four out of the twenty five compounds from IF docking were subjected to 100ns molecular dynamics simulations, and it was observed that salvianolic acid B (SA-B) (a naturally occurring compound) complex with AbHisC, was found to be extremely stable. The glide energy and docking score of SA-B were −88.59 kcal/mol and −10.4 kcal/mol. SA-B was also found to quench the intrinsic fluorescence of tyrosine indicating its binding to the target. The dissociation constant calculated using Surface Plasmon Resonance was found to be 3.4x10−9 M. Based on these results we can conclude that SA-B can serve as the potential inhibitor of AbHisC that can further form the basis of structure based drug design against this deadly pathogen.