Apostasy Research Articles

Allosteric activation of VCP, an AAA unfoldase, by small molecule mimicry

The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme function, activator binding must be permissive to different conformational states needed for mechanochemistry. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, an AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP ATPase Activator 1 (VAA1), a compound that dose-dependently stimulates VCP ATPase activity up to ~threefold. Our cryo-EM studies resulted in structures (ranging from ~2.9 to 3.7 Å-resolution) of VCP in apo and ADP-bound states and revealed that VAA1 binds an allosteric pocket near the C-terminus in both states. Engineered mutations in the VAA1-binding site confer resistance to VAA1, and furthermore, modulate VCP activity. Mutation of a phenylalanine residue in the VCP C-terminal tail that can occupy the VAA1 binding site also stimulates ATPase activity, suggesting that VAA1 acts by mimicking this interaction. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry.

NMR chemical shift assignment of Drosophila odorant binding protein 44a in complex with 8(Z)-eicosenoic acid

The odorant binding protein, OBP44a is one of the most abundant proteins expressed in the brain of the developing fruit fly Drosophila melanogaster. Its cellular function has not yet been determined. The OBP family of proteins is well established to recognize hydrophobic molecules. In this study, NMR is employed to structurally characterize OBP44a. NMR chemical shift perturbation measurements confirm that OBP44a binds to fatty acids. Complete assignments of the backbone chemical shifts and secondary chemical shift analysis demonstrate that the apo state of OBP44a is comprised of six α-helices. Upon binding 8(Z)-eicosenoic acid (8(Z)-C20:1), the OBP44a C-terminal region undergoes a conformational change, from unstructured to α-helical. In addition to C-terminal restructuring upon ligand binding, some hydrophobic residues show dramatic chemical shift changes. Surprisingly, several charged residues are also strongly affected by lipid binding. Some of these residues could represent key structural features that OBP44a relies on to perform its cellular function. The NMR chemical shift assignment is the first step towards characterizing the structure of OBP44a and how specific residues might play a role in lipid binding and release. This information will be important in deciphering the biological function of OBP44a during fly brain development.

Cryo-EM structure of cadmium-bound human ABCB6

ATP-binding cassette transporter B6 (ABCB6), a protein essential for heme biosynthesis in mitochondria, also functions as a heavy metal efflux pump. Here, we present cryo-electron microscopy structures of human ABCB6 bound to a cadmium Cd(II) ion in the presence of antioxidant thiol peptides glutathione (GSH) and phytochelatin 2 (PC2) at resolutions of 3.2 and 3.1 Å, respectively. The overall folding of the two structures resembles the inward-facing apo state but with less separation between the two halves of the transporter. Two GSH molecules are symmetrically bound to the Cd(II) ion in a bent conformation, with the central cysteine protruding towards the metal. The N-terminal glutamate and C-terminal glycine of GSH do not directly interact with Cd(II) but contribute to neutralizing positive charges of the binding cavity by forming hydrogen bonds and van der Waals interactions with nearby residues. In the presence of PC2, Cd(II) binding to ABCB6 is similar to that observed with GSH, except that two cysteine residues of each PC2 molecule participate in Cd(II) coordination to form a tetrathiolate. Structural comparison of human ABCB6 and its homologous Atm-type transporters indicate that their distinct substrate specificity might be attributed to variations in the capping residues situated at the top of the substrate-binding cavity.

Integrating cryo-OrbiSIMS with computational modelling and metadynamics simulations enhances RNA structure prediction at atomic resolution

The 3D architecture of RNAs governs their molecular interactions, chemical reactions, and biological functions. However, a large number of RNAs and their protein complexes remain poorly understood due to the limitations of conventional structural biology techniques in deciphering their complex structures and dynamic interactions. To address this limitation, we have benchmarked an integrated approach that combines cryogenic OrbiSIMS, a state-of-the-art solid-state mass spectrometry technique, with computational methods for modelling RNA structures at atomic resolution with enhanced precision. Furthermore, using 7SK RNP as a test case, we have successfully determined the full 3D structure of a native RNA in its apo, native and disease-remodelled states, which offers insights into the structural interactions and plasticity of the 7SK complex within these states. Overall, our study establishes cryo-OrbiSIMS as a valuable tool in the field of RNA structural biology as it enables the study of challenging, native RNA systems.

Molecular basis of bacterial DSR2 anti-phage defense and viral immune evasion

Defense-associated sirtuin 2 (DSR2) systems are widely distributed across prokaryotic genomes, providing robust protection against phage infection. DSR2 recognizes phage tail tube proteins and induces abortive infection by depleting intracellular NAD+, a process that is counteracted by another phage-encoded protein, DSR Anti Defense 1 (DSAD1). Here, we present cryo-EM structures of Bacillus subtilis DSR2 in its apo, Tube-bound, and DSAD1-bound states. DSR2 assembles into an elongated tetramer, with four NADase catalytic modules clustered in the center and the regulatory-sensing modules distributed at four distal corners. Interestingly, monomeric Tube protein, rather than its oligomeric states, docks at each corner of the DSR2 tetramer to form a 4:4 DSR2-Tube assembly, which is essential for DSR2 NADase activity. DSAD1 competes with Tube for binding to DSR2 by occupying an overlapping region, thereby inhibiting DSR2 immunity. Thus, our results provide important insights into the assembly, activation and inhibition of the DSR2 anti-phage defense system.

Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment.

Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation.

Functional and structural characterization of an IclR family transcription factor for the development of dicarboxylic acid biosensors.

Prokaryotic transcription factors (TFs) regulate gene expression in response to small molecules, thus representing promising candidates as versatile small molecule-detecting biosensors valuable for synthetic biology applications. The engineering of such biosensors requires thorough in vitro and in vivo characterization of TF ligand response as well as detailed molecular structure information. In this work, we functionally and structurally characterize the Pca regulon regulatory protein (PcaR) transcription factor belonging to the IclR transcription factor family. Here, we present in vitro functional analysis of the ligand profile of PcaR and the construction of genetic circuits for the characterization of PcaR as an in vivo biosensor in the model eukaryote Saccharomyces cerevisiae. We report the crystal structures of PcaR in the apo state and in complex with one of its ligands, succinate, which suggests the mechanism of dicarboxylic acid recognition by this transcription factor. This work contributes key structural and functional insights enabling the engineering of PcaR for dicarboxylic acid biosensors, in addition to providing more insights into the IclR family of regulators.

Structure and activity of the septal peptidoglycan hydrolysis machinery crucial for bacterial cell division.

The peptidoglycan (PG) layer is a critical component of the bacterial cell wall and serves as an important target for antibiotics in both gram-negative and gram-positive bacteria. The hydrolysis of septal PG (sPG) is a crucial step of bacterial cell division, facilitated by FtsEX through an amidase activation system. In this study, we present the cryo-EM structures of Escherichia coli FtsEX and FtsEX-EnvC in the ATP-bound state at resolutions of 3.05 Å and 3.11 Å, respectively. Our PG degradation assays in E. coli reveal that the ATP-bound conformation of FtsEX activates sPG hydrolysis of EnvC-AmiB, whereas EnvC-AmiB alone exhibits autoinhibition. Structural analyses indicate that ATP binding induces conformational changes in FtsEX-EnvC, leading to significant differences from the apo state. Furthermore, PG degradation assays of AmiB mutants confirm that the regulation of AmiB by FtsEX-EnvC is achieved through the interaction between EnvC-AmiB. These findings not only provide structural insight into the mechanism of sPG hydrolysis and bacterial cell division, but also have implications for the development of novel therapeutics targeting drug-resistant bacteria.

X-ray crystal structure of a designed rigidified imaging scaffold in the ligand-free conformation.

Imaging scaffolds composed of designed protein cages fused to designed ankyrin repeat proteins (DARPins) have enabled the structure determination of small proteins by cryogenic electron microscopy (cryo-EM). One particularly well characterized scaffold type is a symmetric tetrahedral assembly composed of 24 subunits, 12 A and 12 B, which has three cargo-binding DARPins positioned on each vertex. Here, the X-ray crystal structure of a representative tetrahedral scaffold in the apo state is reported at 3.8 Å resolution. The X-ray crystal structure complements recent cryo-EM findings on a closely related scaffold, while also suggesting potential utility for crystallographic investigations. As observed in this crystal structure, one of the three DARPins, which serve as modular adaptors for binding diverse `cargo' proteins, present on each of the vertices is oriented towards a large solvent channel. The crystal lattice is unusually porous, suggesting that it may be possible to soak crystals of the scaffold with small (≤30 kDa) protein cargo ligands and subsequently determine cage-cargo structures via X-ray crystallography. The results suggest the possibility that cryo-EM scaffolds may be repurposed for structure determination by X-ray crystallography, thus extending the utility of electron-microscopy scaffold designs for alternative structural biology applications.

Genetic Encoding of Fluoro-l-tryptophans for Site-Specific Detection of Conformational Heterogeneity in Proteins by NMR Spectroscopy.

The substitution of a single hydrogen atom in a protein by fluorine yields a site-specific probe for sensitive detection by 19F nuclear magnetic resonance (NMR) spectroscopy, where the absence of background signal from the protein facilitates the detection of minor conformational species. We developed genetic encoding systems for the site-selective incorporation of 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, and 7-fluorotryptophan in response to an amber stop codon and used them to investigate conformational heterogeneity in a designed amino acid binding protein and in flaviviral NS2B-NS3 proteases. These proteases have been shown to present variable conformations in X-ray crystal structures, including flips of the indole side chains of tryptophan residues. The 19F NMR spectra of different fluorotryptophan isomers installed at the conserved site of Trp83 indicate that the indole ring flip is common in flaviviral NS2B-NS3 proteases in the apo state and suppressed by an active-site inhibitor.

Leveraging Bidirectional Nature of Allostery To Inhibit Protein-Protein Interactions (PPIs): A Case Study of PCSK9-LDLR Interaction.

The protein PCSK9 (proprotein convertase subtilisin/Kexin type 9) negatively regulates the recycling of LDLR (low-density lipoprotein receptor), leading to an elevated plasma level of LDL. Inhibition of PCSK9-LDLR interaction has emerged as a promising therapeutic strategy to manage hypercholesterolemia. However, the large interaction surface area between PCSK9 and LDLR makes it challenging to identify a small molecule competitive inhibitor. An alternative strategy would be to identify distal cryptic sites as targets for allosteric inhibitors that can remotely modulate PCSK9-LDLR interaction. Using several microseconds long molecular dynamics (MD) simulations, we demonstrate that on binding with LDLR, there is a significant conformational change (population shift) in a distal loop (residues 211-222) region of PCSK9. Consistent with the bidirectional nature of allostery, we establish a clear correlation between the loop conformation and the binding affinity with LDLR. Using a thermodynamic argument, we establish that the loop conformations predominantly present in the apo state of PCSK9 would have lower LDLR binding affinity, and they would be potential targets for designing allosteric inhibitors. We elucidate the molecular origin of the allosteric coupling between this loop and the LDLR binding interface in terms of the population shift in a set of salt bridges and hydrogen bonds. Overall, our work provides a general strategy toward identifying allosteric hotspots: compare the conformational ensemble of the receptor between the apo and bound states of the protein and identify distal conformational changes, if any. The inhibitors should be designed to bind and stabilize the apo-specific conformations.

Cryo-EM structure of the human Asc-1 transporter complex

The Alanine-Serine-Cysteine transporter 1 (Asc-1 or SLC7A10) forms a crucial heterodimeric transporter complex with 4F2hc (SLC3A2) through a covalent disulfide bridge. This complex enables the sodium-independent transport of small neutral amino acids, including L-Alanine (L-Ala), Glycine (Gly), and D-Serine (D-Ser), within the central nervous system (CNS). D-Ser and Gly are two key endogenous glutamate co-agonists that activate N-methyl-d-aspartate (NMDA) receptors by binding to the allosteric site. Mice deficient in Asc-1 display severe symptoms such as tremors, ataxia, and seizures, leading to early postnatal death. Despite its physiological importance, the functional mechanism of the Asc-1-4F2hc complex has remained elusive. Here, we present cryo-electron microscopy (cryo-EM) structures of the human Asc-1-4F2hc complex in its apo state, D-Ser bound state, and L-Ala bound state, resolved at 3.6 Å, 3.5 Å, and 3.4 Å, respectively. Through detailed structural analysis and transport assays, we uncover a comprehensive alternating access mechanism that underlies conformational changes in the complex. In summary, our findings reveal the architecture of the Asc-1 and 4F2hc complex and provide valuable insights into substrate recognition and the functional cycle of this essential transporter complex.

Cryo-EM structures of prokaryotic ligand-gated ion channel GLIC provide insights into gating in a lipid environment

GLIC, a proton-activated prokaryotic ligand-gated ion channel, served as a model system for understanding the eukaryotic counterparts due to their structural and functional similarities. Despite extensive studies conducted on GLIC, the molecular mechanism of channel gating in the lipid environment requires further investigation. Here, we present the cryo-EM structures of nanodisc-reconstituted GLIC at neutral and acidic pH in the resolution range of 2.6 – 3.4 Å. In our apo state at pH 7.5, the extracellular domain (ECD) displays conformational variations compared to the existing apo structures. At pH 4.0, three distinct conformational states (C1, C2 and O states) are identified. The protonated structures exhibit a compacted and counter-clockwise rotated ECD compared with our apo state. A gradual widening of the pore in the TMD is observed upon reducing the pH, with the widest pore in O state, accompanied by several layers of water pentagons. The pore radius and molecular dynamics (MD) simulations suggest that the O state represents an open conductive state. We also observe state-dependent interactions between several lipids and proteins that may be involved in the regulation of channel gating. Our results provide comprehensive insights into the importance of lipids impact on gating.

Bifurcated assembly pathway and dual function of a Lon-like protease revealed by cryo-EM Analysis

The Lon proteases are evolutionarily conserved oligomeric hydrolases characterized by a built-in ATPase module. Here, we report the structures of LonC from Meiothermus taiwanensis (MtaLonC), a unique member in the Lon protein family with a dual chaperone-protease function but lacking ATPase activity, determined by cryo-electron microscopy (cryo-EM). In its apo state, LonC forms phosphate-bound open-ring pentamers, open-ring hexamers, and close-ring heptamers. However, upon interaction with ATPγS, inhibitor, or substrate, MtaLonC assembles into close-ring hexamers, wherein the protease domain's substrate-binding loop adopts an extended active conformation. We show that proteolytic and chaperone activities may be carried out by the close-ring hexameric and heptameric forms, respectively. We further show that MtaLonC forms exclusively close-ring heptamers at elevated temperatures. This study sheds light on the bifurcated assembly pathway of MtaLonC, leading to two distinct oligomeric close-ring complexes that carry out a dual function.

Using Protein Design and Directed Evolution to Monomerize a Bright Near-Infrared Fluorescent Protein.

The small ultrared fluorescent protein (smURFP) is a bright near-infrared (NIR) fluorescent protein (FP) that forms a dimer and binds its fluorescence chromophore, biliverdin, at its dimer interface. To engineer a monomeric NIR FP based on smURFP potentially more suitable for bioimaging, we employed protein design to extend the protein backbone with a new segment of two helices that shield the original dimer interface while covering the biliverdin binding pocket in place of the second chain in the original dimer. We experimentally characterized 13 designs and obtained a monomeric protein with a weak fluorescence. We enhanced the fluorescence of this designed protein through two rounds of directed evolution and obtained designed monomeric smURFP (DMsmURFP), a bright, stable, and monomeric NIR FP with a molecular weight of 19.6 kDa. We determined the crystal structures of DMsmURFP both in the apo state and in complex with biliverdin, which confirmed the designed structure. The use of DMsmURFP in in vivo imaging of mammalian systems was demonstrated. The backbone design-based strategy used here can also be applied to monomerize other naturally multimeric proteins with intersubunit functional sites.

Cryo-EM structures of the human NaS1 and NaDC1 transporters revealed the elevator transport and allosteric regulation mechanism.

The solute carrier 13 (SLC13) family comprises electrogenic sodium ion-coupled anion cotransporters, segregating into sodium ion-sulfate cotransporters (NaSs) and sodium ion-di- and-tricarboxylate cotransporters (NaDCs). NaS1 and NaDC1 regulate sulfate homeostasis and oxidative metabolism, respectively. NaS1 deficiency affects murine growth and fertility, while NaDC1 affects urinary citrate and calcium nephrolithiasis. Despite their importance, the mechanisms of substrate recognition and transport remain insufficiently characterized. In this study, we determined the cryo-electron microscopy structures of human NaS1, capturing inward-facing and combined inward-facing/outward-facing conformations within a dimer both in apo and sulfate-bound states. In addition, we elucidated NaDC1's outward-facing conformation, encompassing apo, citrate-bound, and N-(p-amylcinnamoyl) anthranilic acid (ACA) inhibitor-bound states. Structural scrutiny illuminates a detailed elevator mechanism driving conformational changes. Notably, the ACA inhibitor unexpectedly binds primarily anchored by transmembrane 2 (TM2), Loop 10, TM11, and TM6a proximate to the cytosolic membrane. Our findings provide crucial insights into SLC13 transport mechanisms, paving the way for future drug design.

Ligand-induced protein transition state stabilization switches the binding pathway from conformational selection to induced fit

Protein-ligand complex formation is fundamental to biological function. A central question is whether proteins spontaneously adopt binding-competent conformations to which ligands bind conformational selection (CS) or whether ligands induce the binding-competent conformation induced fit (IF). Here, we resolve the CS and IF binding pathways by characterizing protein conformational dynamics over a wide range of ligand concentrations using NMR relaxation dispersion. We determined the relative flux through the two pathways using a four-state binding model that includes both CS and IF. Experiments conducted without ligand show that galectin-3 exchanges between the ground-state conformation and a high-energy conformation similar to the ligand-bound conformation, demonstrating that CS is a plausible pathway. Near-identical crystal structures of the apo and ligand-bound states suggest that the high-energy conformation in solution corresponds to the apo crystal structure. Stepwise additions of the ligand lactose induce progressive changes in the relaxation dispersions that we fit collectively to the four-state model, yielding all microscopic rate constants and binding affinities. The ligand affinity is higher for the bound-like conformation than for the ground state, as expected for CS. Nonetheless, the IF pathway contributes greater than 70% of the total flux even at low ligand concentrations. The higher flux through the IF pathway is explained by considerably higher rates of exchange between the two protein conformations in the ligand-associated state. Thus, the ligand acts to decrease the activation barrier between protein conformations in a manner reciprocal to enzymatic transition-state stabilization of reactions involving ligand transformation.

Molecular basis for substrate transport of Mycobacterium tuberculosis ABC importer DppABCD.

The type I adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter DppABCD is believed to be responsible for the import of exogenous heme as an iron source into the cytoplasm of the human pathogen Mycobacterium tuberculosis (Mtb). Additionally, this system is also known to be involved in the acquisition of tri- or tetra-peptides. Here, we report the cryo-electron microscopy structures of the dual-function Mtb DppABCD transporter in three forms, namely, the apo, substrate-bound, and ATP-bound states. The apo structure reveals an unexpected and previously uncharacterized assembly mode for ABC importers, where the lipoprotein DppA, a cluster C substrate-binding protein (SBP), stands upright on the translocator DppBCD primarily through its hinge region and N-lobe. These structural data, along with biochemical studies, reveal the assembly of DppABCD complex and the detailed mechanism of DppABCD-mediated transport. Together, these findings provide a molecular roadmap for understanding the transport mechanism of a cluster C SBP and its translocator.

Design of amyloidogenic peptide traps.

Segments of proteins with high β-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in β-strand and β-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein-peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid β1-42 (Aβ42). The Aβ binders block the assembly of Aβ fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aβ42 species.

Equilibrium landscape of ingress/egress channels and gating residues of the Cytochrome P450 3A4.

The Cytochrome P450 (CYP) enzymes metabolize a variety of drugs, which may potentially lead to toxicity or reduced efficacy when drugs are co-administered. These drug-drug interactions are often manifested by CYP3A4, the most prevalent of all CYP isozymes. We carried out multiple MD simulations employing CAVER to quantify the channels, and Hidden Markov Models (HMM) to characterize the behavior of the gating residues. We discuss channel properties, bottleneck residues with respect to their likelihood to deem the respective channel ingress or egress, gating residues regarding their open or closed states, and channel location relative to the membrane. Channels do not display coordinated motion and randomly transition between different conformations. Gateway residues also behave in a random fashion. Our findings shed light on the equilibrium behavior of the gating residues and channels in the apo state.