Open Conformation Research Articles

Insights into structure and activity of a UDP-GlcNAc 2-epimerase involved in secondary cell wall polymer biosynthesis in Paenibacillus alvei.

S-layer anchoring in Paenibacillus alvei is enabled by a non-covalent interaction between an S-layer homology domain trimer and a secondary cell wall polymer (SCWP), ensuring the structural integrity of the bacterial cell wall. Within the SCWP repeat, pyruvylated ManNAc serves as the ligand and the UDP-GlcNAc-2-epimerase MnaA supplies UDP-ManNAc to SCWP biosynthesis. To better understand SCWP biosynthesis and identify strategies for inhibiting pathogens with comparable cell wall architecture, like Bacillus anthracis, MnaA and rational variants were produced in E. coli and their kinetic constants determined. The effect of UDP-GlcNAc as a predicted allosteric activator and tunicamycin as a potential inhibitor of MnaA was tested in vitro supported by molecular docking experiments. Additionally, wild-type MnaA was crystallized. We present the crystal structure of unliganded P. alvei MnaA resolved at 2.20 Å. It adopts a GT-B fold consistent with other bacterial non-hydrolyzing UDP-GlcNAc 2-epimerases. A comparison of amino acid sequences reveals conservation of putative and known catalytic and allosteric-site residues in MnaA, which was confirmed through analysis of Q42A, Q69A, E135A and H241A MnaA variants. The kinetic parameters K M and k cat of MnaA were determined to be 3.91 mM and 33.44 s-1 for the forward, and 2.41 mM and 6.02 s-1 for the reverse reaction. While allosteric regulation by UDP-GlcNAc has been proposed as a mechanism for enzyme activation, UDP-GlcNAc was not found to be essential for UDP-ManNAc epimerization by P. alvei MnaA. However, the reaction rate doubled upon addition of 5% UDP-GlcNAc. Unexpectedly, the UDP-GlcNAc analog tunicamycin did not inhibit MnaA. Molecular docking experiments comparing tunicamycin binding of P. alvei MnaA and Staphylococcus aureus MnaA, which is inhibited by tunicamycin, revealed different residues exposed to the antibiotic excluding, those at the predicted allosteric site of P. alvei MnaA, corroborating tunicamycin resistance. The unliganded crystal structure of P. alvei MnaA reveals an open conformation characterized by an accessible cleft between the N- and C-terminal domains. Despite the conservation of residues involved in binding the allosteric activator UDP-GlcNAc, the enzyme is not strictly regulated by the substrate. Unlike S. aureus MnaA, the activity of P. alvei MnaA remains unaffected by tunicamycin.

Exploring the Mechanism of Activation of CFTR by Curcuminoids: An Ensemble Docking Study.

Curcumin, a major constituent of turmeric (Curcuma longa L.), has beneficial effects against several diseases. In cystic fibrosis (CF), this compound improves patients' symptoms by recovering the activity of a number of mutants of the cystic fibrosis transmembrane conductance regulator (CFTR). Despite holding promise in the treatment of CF, the curcumin binding site in CFTR and the molecular mechanism of activation of this channel are still unknown. The results of this study, based on docking and molecular dynamics (MD) simulations, allow us to propose that curcumin binds the closed ATP-free CFTR near the nucleotide-binding domain 1 (NBD1)/ICl1/ICl4 interface. The bound ligand, once approached by the nucleotide-binding domain 2 (NBD2) during transient channel opening, lays at a multiple interdomain cross point. Thereafter, curcumin can bridge NBD1 and NBD2, and also ICL1/ICL4 and ICL2/ICL3, finally tightening the same interdomain interactions that normally uphold the open conformation in the wild-type ATP-bound CFTR. The proposed binding site is compatible with biochemical observations made in previous CFTR-curcumin interaction studies. These findings provide a framework for the design of novel drugs that activate CFTR mutants characterized by defects in ATP binding and/or NBD dimerization or even lacking NBD2.

ADAMTS-13 conformation influences autoimmune recognition in immune thrombotic thrombocytopenic purpura

BackgroundPatients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) have anti–ADAMTS-13 immunoglobulin G (IgG) autoantibodies that enhance ADAMTS-13 clearance and/or inhibit its function. ADAMTS-13 normally circulates in a closed conformation, which is manifested by the interaction of the CUB domains with the central spacer domain. Disruption of the spacer–CUB interaction opens ADAMTS-13, which augments its proteolytic function but may also expose cryptic autoimmune epitopes that promote further autoantibody recognition. ObjectivesTo explore differences in autoantibody binding to ADAMTS-13 in its closed or open conformations in patients with iTTP and to correlate these differences with disease-related parameters. MethodsWe developed a novel assay to measure autoantibodies binding to closed and open ADAMTS-13. Autoantibody titer and IgG subclass binding to open or closed ADAMTS-13 were measured in 70 iTTP first presentation samples and correlated with clinical data, remission, and relapse. ResultsIn 70 patients with iTTP, the mean autoantibody titer against open ADAMTS-13 was, on average, approximately 2-fold greater than that against closed ADAMTS-13, suggesting that ADAMTS-13 opening increases epitope exposure and immune complex formation. Autoantibody titer against closed/open ADAMTS-13 and IgG subclass did not correlate with ADAMTS-13 antigen at presentation. Two patients with iTTP and persistent autoantibodies lost specificity for closed ADAMTS-13 in remission. Recognition of closed/open ADAMTS-13 and autoantibody IgG subclass between the first and second iTTP episodes were very similar. ConclusionADAMTS-13 autoantibody binding is highly influenced by ADAMTS-13 conformation. Although this does not appear to modify the pathogenicity of autoantibodies, the autoantibody signature at relapse suggests that relapse represents re-emergence of the original autoimmune response rather than de novo presentation.

Mechanism of hydrophobic gating in the acetylcholine receptor channel pore.

Neuromuscular acetylcholine receptors (AChRs) are hetero-pentameric, ligand-gated ion channels. The binding of the neurotransmitter acetylcholine (ACh) to two target sites promotes a global conformational change of the receptor that opens the channel and allows ion conduction through the channel pore. Here, by measuring free-energy changes from single-channel current recordings and using molecular dynamics simulations, we elucidate how a constricted hydrophobic region acts as a "gate" to regulate the channel opening in the pore of AChRs. Mutations of gate residues, including those implicated in congenital myasthenia syndrome, lower the permeation barrier of the channel substantially and increase the unliganded gating equilibrium constant (constitutive channel openings). Correlations between hydrophobicity and the observed free-energy changes, supported by calculations of water densities in the wild-type versus mutant channel pores, provide evidence for hydrophobic wetting-dewetting transition at the gate. The analysis of a coupled interaction network provides insight into the molecular mechanism of closed- versus open-state conformational changes at the gate. Studies of the transition state by "phi"(φ)-value analysis indicate that agonist binding serves to stabilize both the transition and the open state. Intersubunit interaction energy measurements and molecular dynamics simulations suggest that channel opening involves tilting of the pore-lining M2 helices, asymmetric outward rotation of amino acid side chains, and wetting transition of the gate region that lowers the barrier to ion permeation and stabilizes the channel open conformation. Our work provides new insight into the hydrophobic gate opening and shows why the gate mutations result in constitutive AChR channel activity.

Cation Chloride Cotransporter NKCC1 Operates through a Rocking-Bundle Mechanism.

The sodium, potassium, and chloride cotransporter 1 (NKCC1) plays a key role in tightly regulating ion shuttling across cell membranes. Lately, its aberrant expression and function have been linked to numerous neurological disorders and cancers, making it a novel and highly promising pharmacological target for therapeutic interventions. A better understanding of how NKCC1 dynamically operates would therefore have broad implications for ongoing efforts toward its exploitation as a therapeutic target through its modulation. Based on recent structural data on NKCC1, we reveal conformational motions that are key to its function. Using extensive deep-learning-guided atomistic simulations of NKCC1 models embedded into the membrane, we captured complex dynamical transitions between alternate open conformations of the inner and outer vestibules of the cotransporter and demonstrated that NKCC1 has water-permeable states. We found that these previously undefined conformational transitions occur via a rocking-bundle mechanism characterized by the cooperative angular motion of transmembrane helices (TM) 4 and 9, with the contribution of the extracellular tip of TM 10. We found these motions to be critical in modulating ion transportation and in regulating NKCC1's water transporting capabilities. Specifically, we identified interhelical dynamical contacts between TM 10 and TM 6, which we functionally validated through mutagenesis experiments of 4 new targeted NKCC1 mutants. We conclude showing that those 4 residues are highly conserved in most Na+-dependent cation chloride cotransporters (CCCs), which highlights their critical mechanistic implications, opening the way to new strategies for NKCC1's function modulation and thus to potential drug action on selected CCCs.

Analysis of Telomerase RNA Structure in Physcomitrium patens Indicates Functionally Relevant Transitions Between OPEN and CLOSED Conformations

Telomerase RNA (TR) conformation determines its function as a template for telomere synthesis and as a scaffold for the assembly of the telomerase nucleoprotein complex. Experimental analyses of TR secondary structure using DMS-Map Seq and SHAPE-Map Seq techniques show its CLOSED conformation as the consensus structure where the template region cannot perform its function. Our data show that the apparent discrepancy between experimental results and predicted TR functional conformation, mostly ignored in published studies, can be explained using data analysis based on single-molecule structure prediction from individual sequencing reads by the recently established DaVinci method. This method results in several clusters of secondary structures reflecting the structural dynamics of TR, possibly related to its multiple functional states. Interestingly, the presumed active (OPEN) conformation of TR corresponds to a minor fraction of TR under in vivo conditions. Therefore, structural polymorphism and dynamic TR transitions between CLOSED and OPEN conformations may be involved in telomerase activity regulation as a switch that functions independently of total TR transcript levels.

Cryo-EM structure of bovine chaperonin TRiC/CCT in open conformation

In this work, conditions were selected for obtaining a sample of eukaryotic chaperonin TRiC suitable for studying by cryo-electron microscopy. Using the method of differential scanning (time-resolved) fluorimetry, the temperature stability of protein samples at different concentrations of salt and glycerol was compared, and then the selected conditions were used to prepare the sample for microscopy. As a result, the structure of bovine TRiC in an open conformation was obtained at 4.42 Å resolution.

Comprehending conformational changes in EmrE, multidrug transporter at different pH: insights from molecular dynamics simulations

EmrE is a small multidrug resistance (SMR) pump of antiparallel topology that confers resistance to a broad range of polyaromatic cations in Escherichia coli. Atomic-level understanding of conformational changes for the selectivity of substrate and transport of a diverse array of drugs through the smallest known efflux pumps is crucial to multi-drug resistance. Therefore, the present study aims to provide insights into conformational changes during the transport through EmrE transporter at different pH. Molecular dynamics simulations have been carried out on the complete structure of EmrE in the absence of substrate. Computational analyses such as secondary structure, principal component, dynamic cross-correlation matrix, and hydrogen bond calculations have been performed. Analysis of MD trajectories in this study revealed pH-dependent interactions that influenced the structural dynamics of EmrE. Notably, at high pH, Glu14 and Tyr60 in both monomers formed electrostatic interactions, while these interactions decreased significantly at a low pH. Interestingly, a kink at helix 3 (H3) and dual open conformation of EmrE at low pH were also observed in contrast to a closed state discerned towards the periplasmic side at high pH. Significant interactions between C-terminal residues and residues at the edge of H1 & Loop1 and H3 & Loop3 were identified, suggesting their role in stabilizing the closed conformation of EmrE at the periplasmic end under high pH conditions. The present study enhances our understanding of EmrE’s conformational changes, shedding light on the pH-dependent mechanisms that are likely to impact its function in multi-drug resistance. Communicated by Ramaswamy H. Sarma

Structural dynamics of the CROPs domain control stability and toxicity of Paeniclostridium sordellii lethal toxin

Paeniclostridium sordellii lethal toxin (TcsL) is a potent exotoxin that causes lethal toxic shock syndrome associated with fulminant bacterial infections. TcsL belongs to the large clostridial toxin (LCT) family. Here, we report that TcsL with varied lengths of combined repetitive oligopeptides (CROPs) deleted show increased autoproteolysis as well as higher cytotoxicity. We next present cryo-EM structures of full-length TcsL, at neutral (pH 7.4) and acidic (pH 5.0) conditions. The TcsL at neutral pH exhibits in the open conformation, which resembles reported TcdB structures. Low pH induces the conformational change of partial TcsL to the closed form. Two intracellular interfaces are observed in the closed conformation, which possibly locks the cysteine protease domain and hinders the binding of the host receptor. Our findings provide insights into the structure and function of TcsL and reveal mechanisms for CROPs-mediated modulation of autoproteolysis and cytotoxicity, which could be common across the LCT family.

New insights into the structure and function of the complex between the Escherichia coli Hsp70, DnaK, and its nucleotide-exchange factor, GrpE

The 70 kDa heat shock proteins (Hsp70s) play a pivotal role in many cellular functions using allosteric communication between their nucleotide-binding domain (NBD) and substrate-binding domain, mediated by an interdomain linker, to modulate their affinity for protein clients. Critical to modulation of the Hsp70 allosteric cycle, nucleotide-exchange factors (NEFs) act by a conserved mechanism involving binding to the ADP-bound NBD and opening of the nucleotide-binding cleft to accelerate the release of ADP and binding of ATP. The crystal structure of the complex between the NBD of the Escherichia coli Hsp70, DnaK, and its NEF, GrpE, was reported previously, but the GrpE in the complex carried a point mutation (G122D). Both the functional impact of this mutation and its location on the NEF led us to revisit the DnaK NBD/GrpE complex structurally using AlphaFold modeling and validation by solution methods that report on protein conformation and mutagenesis. This work resulted in a new model for the DnaK NBD in complex with GrpE in which subdomain IIB of the NBD rotates more than in the crystal structure, resulting in an open conformation of the nucleotide-binding cleft, which now resembles more closely what is seen in other Hsp/NEF complexes. Moreover, the new model is consistent with the increased ADP off-rate accompanying GrpE binding. Excitingly, our findings point to an interdomain allosteric signal in DnaK triggered by GrpE binding.

Modeling Substrate Entry into the P-Glycoprotein Efflux Pump at the Blood-Brain Barrier.

We report molecular dynamics simulations of rhodamine entry into the central binding cavity of P-gp in the inward open conformation. Rhodamine can enter the inner volume via passive transport across the luminal membrane or lateral diffusion in the lipid bilayer. Entry into the inner volume is determined by the aperture angle at the apex of the protein, with a critical angle of 27° for rhodamine. The central binding cavity has an aqueous phase with a few lipids, which significantly reduces substrate diffusion. Within the central binding cavity, we identified regions with relatively weak binding, suggesting that the combination of reduced mobility and weak substrate binding confines rhodamine to enable the completion of the efflux cycle. Tariquidar, a P-gp inhibitor, aggregates at the lower arms of the P-gp, suggesting that inhibition involves steric hindrance of entry into the inner volume and/or steric hindrance of access of ATP to the nucleotide-binding domains.

Hemophagocytic lymphohistiocytosis is associated with deficiency and closed conformation of ADAMTS-13

BackgroundA disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13 (ADAMTS-13) is the specific von Willebrand factor–cleaving protease and circulates in a closed and latent conformation due to a spacer/CUB1 domain interaction. ADAMTS-13 is allosterically activated after binding of its substrate or antibodies, inducing an open conformation. Recently, we suggested a potential role of plasmin (fibrinolysin) in hemostasis disorders reported in most patients with hemophagocytic lymphohistiocytosis (HLH), a rare and life-threatening condition related to a severe systemic inflammatory state. Most patients with HLH had a partial ADAMTS-13 deficiency, and plasmin could induce a truncation of the C-terminal part of ADAMTS-13 and thus an open conformation. ObjectivesTo understand the effect of plasmin on ADAMTS-13, our study aimed to investigate ADAMTS-13 conformation in patients with HLH. MethodsForty-five critically ill patients with HLH were prospectively enrolled between April 2015 and December 2018. ADAMTS-13 activity was measured by fluorescent resonance energy transfer-VWF73 assay, ADAMTS-13 antigen, and conformation with our homemade 3H9-enzyme-linked immunosorbent assay and 1C4-enzyme-linked immunosorbent assay. ResultsADAMTS-13 activity ranged from <10 to 65 IU/dL, and 41 of the 45 patients had a quantitative deficiency in ADAMTS-13 (activity <50 IU/dL). Twenty patients had a severe ADAMTS-13 deficiency (activity <20 IU/dL). ADAMTS-13 conformation was folded in all patients under normal conditions. Surprisingly, the switch of ADAMTS-13 conformation expected with the monoclonal antibody 17G2 (anti-CUB1) was disturbed in 6 patients (activity <20 IU/dL). ConclusionOur study reported that ADAMTS-13 conformation is closed in HLH and provides an indirect proof that plasmin is not able to massively degrade ADAMTS-13. Further studies on glycosylation and citrullination profiles of ADAMTS-13 are needed to understand their role in HLH.

Conformational Dynamics of the Most Efficient Carboxylase Contributes to Efficient CO2 Fixation.

Crotonyl-CoA carboxylase/reductase (Ccr) is one of the fastest CO2 fixing enzymes and has become part of efficient artificial CO2-fixation pathways in vitro, paving the way for future applications. The underlying mechanism of its efficiency, however, is not yet completely understood. X-ray structures of different intermediates in the catalytic cycle reveal tetramers in a dimer of dimers configuration with two open and two closed active sites. Upon binding a substrate, this active site changes its conformation from the open state to the closed state. It is challenging to predict how these coupled conformational changes will alter the CO2 binding affinity to the reaction's active site. To determine whether the open or closed conformations of Ccr affect binding of CO2 to the active site, we performed all-atom molecular simulations of the various conformations of Ccr. The open conformation without a substrate showed the highest binding affinity. The CO2 binding sites are located near the catalytic relevant Asn81 and His365 residues and in an optimal position for CO2 fixation. Furthermore, they are unaffected by substrate binding, and CO2 molecules stay in these binding sites for a longer time. Longer times at these reactive binding sites facilitate CO2 fixation through the nucleophilic attack of the reactive enolate in the closed conformation. We previously demonstrated that the Asn81Leu variant cannot fix CO2. Simulations of the Asn81Leu variant explain the loss of activity through the removal of the Asn81 and His365 binding sites. Overall, our findings show that the conformational dynamics of the enzyme controls CO2 binding. Conformational changes in Ccr increase the level of CO2 in the open subunit before the substrate is bound, the active site closes, and the reaction starts. The full catalytic Ccr cycle alternates among CO2 addition, conformational change, and chemical reaction in the four subunits of the tetramer coordinated by communication between the two dimers.

Probing the pH Sensitivity of OprM: Insights into Metastable States and Semi-Open Conformation.

Efflux pumps are specialized transport proteins that play a key role in the bacterial defense against a wide spectrum of antibiotics. Hence, understanding the biophysical mechanism associated with this complex system of drug expulsion becomes crucial. This work deals with some vital aspects of the outer membrane factor (OMF) of MexAB-OprM. After being passed through MexB and MexA, efflux substrates have to go through OprM for their final judgment. Thus, it is very important to understand the periplasmic pore opening mechanism and the associated biophysical changes during this process. Our study captures a detailed analysis of the pore opening mechanism involving OprM. With powerful molecular dynamics (MD) techniques such as well-tempered metadynamics, the presence of metastable states in between open and closed states was confirmed. Also, upon mutating R376, the energy barrier for the conversion of the close to open conformation decreases, indicating an important role played by the residue. Further, constant pH MD was performed to capture the effect of pH in both conformations. OprM exhibits distinct conformational states at pH values greater than 5.5 and lower than 5.5, suggesting its pH-responsive characteristics. Overall, our study elucidates a crucial undertaking toward discovering potential inhibitors for MexAB-OprM efflux pumps.

Management and follow-up of pregnancy-onset thrombotic thrombocytopenic purpura: the French experience

AbstractPregnancy-onset thrombotic thrombocytopenic purpura (TTP) is a rare and life-threatening disease of which diagnosis and management requires experienced multidisciplinary teams. The mechanisms responsible for a deficiency in the disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13 (ADAMTS13) leading to pregnancy-onset TTP may be congenital or acquired, and studying ADAMTS13 conformation could be of interest. The differential diagnosis between TTP and other pregnancy-associated thrombotic microangiopathies (TMA) is often challenging. Our retrospective multicenter study highlights the significance and the challenges associated with pregnancy-onset TTP and childbirth in terms of diagnosis, obstetric management, and follow-up aspects. Among 1174 pregnancy-onset TMA enrolled in the French Registry for TMA from 2000 to 2020, we identified 108 pregnancy-onset TTP: 52 immune-mediated TTP (iTTP, 48.1%), 27 acquired TTP of unidentified mechanism (uTTP, 25%), and 29 congenital TTP (cTTP, 26.9%). Data show that maternal outcome is good (survival rate: 95%) and fetal outcome is linked to the gestational age at the onset of the disease (survival rate: 75.5%). Three distinct entities with different natural histories emerged: pregnancy-onset iTTP appears similar to idiopathic iTTP, with an open ADAMTS13 conformation, and is marked by a relapse risk independent of subsequent pregnancies; pregnancy-onset uTTP appears to have a different pathophysiology with an unexpected open ADAMTS13 conformation and a very low relapse risk independent of subsequent pregnancies; finally, pregnancy-onset cTTP is characterized by the necessity of pregnancy as a systematic and specific trigger and a need for prophylactic plasmatherapy for subsequent pregnancies. This trial was registered at www.clinicaltrials.gov as #NCT00426686, and at the Health Authority and the French Ministry of Health (P051064/PHRC AOM05012).

The AI‐based AlphaFold 2 algorithm predicts an open structure for tauC3

AbstractBackgroundTauC3 is an especially noxious C‐terminal tau fragment produced by caspase‐3 cleavage at Asp421. Two decades of accumulating evidence support its importance in the pathogenesis of AD and other tauopathies, notably FTD and PSP. TauC3 promotes aggregation and filament formation, recruits full length tau (FLT) to incipient fibrils, and despite low concentration in AD brain tissue relative to total tau, has been reported to account for substantially all the seeding activity in AD brain extracts. TauC3 has also been shown to slow fast axonal transport (FAT), with deleterious consequences. Although tau’s largely disordered structure prevents analysis by X‐ray crystallography, FRET analysis suggested the existence of a hairpin conformation in which the N‐terminus folds over the C‐terminus close to the MTBR. In this model, Ser432 is the point of closest contact with Pro301. Opening of the hairpin has been hypothesized to 1) make FLT prone to aggregation by exposing the MTBR, and 2) impair FAT by overexposing the phosphatase activating domain near the N‐terminus. We postulated that tauC3 cannot form the hairpin because it lacks Ser432 and thus has an “open” conformation. This would explain tauC3’s substantially increased pathogenicity compared to FLT. The AI‐based tool AlphaFold 2 (AF2) is being used to predict the conformation of many proteins with great accuracy. Applying it to FLT and tauC3 could provide valuable data about the hairpin and whether caspase cleavage alters it.MethodWe tested AF2’s ability to predict the conformation of 4‐repeat FLT and tauC3 from FASTA sequences, aligning the models produced with the MTBR.ResultAF2 modeling revealed a dramatically different conformation for tauC3 compared to FLT, specifically, an open conformation with the MTBR and N‐terminus far apart (Figure). Interestingly, it also revealed that the entire C‐terminus region of FLT starting at Gln423 is composed of a single α‐helix.ConclusionAF2 predictions support the existence of the hairpin conformation for tau and underscore the critical importance of residue 432 in maintaining it. The extended C‐terminus α‐helix may have functional implications. TauC3’s open conformation is consistent with its slowing of FAT and its ability to seed aggregation even at low concentrations.

Cryo-EM Structure of Bovine Chaperonin TRiC/CCT in Open Conformation

Cryo-EM Structure of Bovine Chaperonin TRiC/CCT in Open Conformation

AlphaFold 2 predicts opening of the tau hairpin conformation by Mapt mutations and the extent of opening reflects their location

AbstractBackgroundDozens of Mapt mutations can cause FTD, but the molecular mechanisms involved are poorly understood. We recently reported that Pro301 mutations increase the susceptibility of full‐length tau (FLT) to caspase‐3 cleavage at Asp421, which produces the highly noxious C‐terminal fragment tauC3. We speculated that the increased susceptibility could be caused by exposing the cleavage site through impact on critical hairpin interactions between Ser432 and P301. In another study, we used the AI‐based AlphaFold 2 (AF2) algorithm to predict and compare structural models of FLT and tauC3. This revealed dramatic differences in their conformations: tauC3 has an open conformation while FLT is closed by a hairpin. We postulated that other Mapt mutations may also open the conformation of tau in the same way.MethodWe performed AF2 predictions on FASTA sequences of several 4‐repeat FLT variants: R5H, P301S, P301L, and R406W. We also obtained the predicted intramolecular distances between Ser432 and the MTBR to assess the effect of the mutations on the conformation of tau quantitatively.ResultThere were notable differences in the magnitude of opening across the variants. Rank order for openness (Figure) was tauC3 &gt; P301L &gt; P301S &gt; R406W &gt; R5H ∼ non‐mutated FLT. P301L had a strong effect that approximated the opening in tauC3. P301S and R06W had moderate effects, while R5H has a minimal effect.ConclusionThe finding that P301L’s effect was greater than that of P301S may be explained by leucine’s greater hydrophobicity. The relatively strong effect of both Pro301 mutations reflect this residue being closest to Ser432. The R406W mutation’s intermediate but lesser effect is consistent with its relative proximity to Ser432, while the lack of effect for R5H suggests that the extreme N‐terminus is minimally involved. Collectively, our data shows the power of AF2 to predict effects of sequence alterations. The findings also have important implications for understanding tau biology and the development of therapeutics for tauopathies.

Abstract B090: Partially open conformation of the G323E mutated HIF-2α PASB domain captured by X-ray crystallography

Abstract Under hypoxic conditions, vital cell processes such as angiogenesis, erythropoiesis, cell proliferation and metabolism are regulated by a group of transcription factors known as hypoxia-inducible factors (HIFs). These proteins belong to the helix-loop-helix family of transcription factors and require heterodimerization to function. Constitutively expressed HIF-1β protein forms a heterodimer with one of the oxygen-labile counterparts: HIF-1α, HIF-2α, or HIF-3α. The HIF-αs are degraded under normoxic conditions via ubiquitination by the von Hippel Lindau (VHL) E3 ubiquitin ligase and become available for dimerization under either hypoxia or VHL dysfunction. Certain cancer types take advantage of this HIF-regulated transcription for survival and growth, as exemplified by VHL-deficient clear cell renal cell carcinoma (ccRCC). Inhibitors aimed at disruption of the HIF-1β:HIF-2α heterodimer have been advanced into clinical development, including belzutifan (approved by the Food and Drug Administration for adult patients with VHL disease who require therapy for RCC among other indications) and AB521 (Arcus Biosciences). This class of small molecules functions by binding to and causing a conformational change in the internal cavity of the PasB domain of HIF-2α. A mutation within this internal cavity, G323E, was reported in a patient undergoing treatment with HIF-2α inhibitor. This mutation restored the ability of HIF-1β to heterodimerize with HIF-2α in presence of the inhibitor. To better understand this potential resistance mechanism and how the G323E mutation affects inhibitor binding, we used X-ray crystallography to examine the structure of the mutant HIF-2α PasB domain in complex with a novel small molecule inhibitor active in vitro against the G323E mutant form. We report a co-crystal structure that reveals a partially disordered open conformation of the HIF-2α PasB domain. This structure strengthens existing hypotheses regarding the route of compound entry into the HIF-2α PasB cavity and lays the foundation for structure-based design of HIF-2α PasB G323E-active inhibitors. Citation Format: Steven Shia, Ian Malgapo, Jeffrey Chen, Amber Pham, Cesar A Meleza, Lunda Shen, Mariya Morar, Kenneth V Lawson, Kelsey E Sivick Gauthier, Hyock J Kwon. Partially open conformation of the G323E mutated HIF-2α PASB domain captured by X-ray crystallography [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B090.

Design and Synthesis of Novel Near-Infrared Fluorescence Probes Based on an Open Conformation of a Cytochrome P450 1B1 Complex for Molecular Imaging of Colorectal Tumors.

Cytochrome P450 1B1 (CYP1B1) is induced during the early stage of cancer and is universally overexpressed in tumors. Thus, it was considered as a potential biomarker for the monitoring of cancer. In this study, we designed and synthesized CYP1B1-targeted near-infrared (NIR) fluorescence molecular probes based on the latest reported open conformation of the CYP1B1-α-naphthoflavone (ANF) complex. According to the architecture of the open channel, we introduced linkers and a Cy5.5 fragment at the 5' position of ANF derivatives with strong CYP1B1 inhibitory activity to obtain probes 19-21. Then, in vitro cell-based studies showed that the probes could be enriched in tumor cells by binding to CYP1B1. During in vivo and ex vivo imaging in a xenograft mouse model, probe 19 with the best binding affinity was proven to be able to identify tumor sites in both fluorescence imaging and photoacoustic imaging modes.