Apostasy Research Articles

Conformational dynamics of the membrane enzyme LspA upon antibiotic and substrate binding

Lipoprotein signal peptidase (LspA) is an aspartyl protease that cleaves the transmembrane helix signal peptide of lipoproteins as part of the lipoprotein-processing pathway. Members of this pathway are excellent targets for the development of antibiotic therapeutics because they are essential in Gram-negative bacteria, are important for virulence in Gram-positive bacteria, and may not develop antibiotic resistance. Here, we report the conformational dynamics of LspA in the apo state and bound to the antibiotic globomycin determined using molecular dynamics simulations and electron paramagnetic resonance. The periplasmic helix fluctuates on the nanosecond timescale and samples unique conformations in the different states. In the apo state, the dominant conformation is the most closed and occludes the charged active site from the lipid bilayer. With antibiotic bound there are multiple binding modes with the dominant conformation of the periplasmic helix in a more open conformation. The different conformations observed in both bound and apo states indicate a flexible and adaptable active site, which explains how LspA accommodates and processes such a variety of substrates.

Assembly and Disassembly of PKA is Allosterically Controlled by Nucleotides and Metal Ions

Protein Kinase A (PKA) is a macromolecular assembly composed of a regulatory subunit (PKA‐R) that harbors cyclic‐nucleotide binding (CNB) domains, and a catalytic subunit (PKA‐C) that binds nucleotides and metal ions in its active site. During its phosphorylation cycle, PKA‐C experiences different conformations starting with a close conformation when bound to ATP‐Mg2+, to a dynamic equilibrium between close and open conformations with ADP‐Mg2+, and an open conformation in the absence of nucleotide and Mg2+. How the conformational changes in PKA‐C allosterically modulate interactions with the CNB domains of PKA‐R remain largely unknown. Here we use optical tweezers to selectively unfold and refold one CNB domain (termed CNB‐A) of PKA‐R in the presence of PKA‐C and different nucleotides. This approach allowed us to dissect the allosteric effects that the PKA‐C conformation exert over the folding energy landscape and conformation of CNB‐A. When PKA‐C is in an open conformation, we find that CNB‐A unfolds predominantly at low forces (9 pN) as in the apo state, indicating that CNB‐A interactions with PKA‐C require nucleotide occupancy. When PKA‐C is in a closed conformation bound to ATP‐Mg2+, CNB‐A predominately unfolds at high forces (19 pN), suggesting that CNB‐A and PKA‐C are in a single, tightly‐bound conformation. Interestingly, in the presence of ADP‐Mg2+, CNB‐A unfolds at low (9 pN), intermediate (13 pN) and high forces (19 pN), suggesting that CNB‐A can be in three conformations: apo, weakly‐ or tightly‐bound to PKA‐C. We further dissect the kinetics of interconversions between these three conformations by varying the PKA‐C concentration and the refolding and rebinding dwell time. We show that transition kinetics from the apo to the tightly‐bound conformation in the presence of ATP‐Mg2+ are much faster than in ADP‐Mg2+, which has consequences for the timing of assembly of the inactive PKA holoenzyme and dissociation of the PKA complex after cAMP‐mediate activation. Altogether, this study directly shows how nucleotides and metal ions at the active site of PKA‐C can allosterically modulate the strength and kinetics of interactions with the CNB domains, thereby highlighting the delicate regulation mechanisms for PKA to function as a proper switch in cell signaling processes.

Visualizing an orphan receptor GPR158 and it complex with neuronal regulator RGS7‐Gβ5 via cryoEM

G protein‐coupled receptors (GPCR) form the largest family of proteins (∼800 GPCRs) encoded in mammalian genomes that detect extracellular signals to program cellular response. They are essential to understanding physiology, disease, and drug development. More than 100 GPCRs are still awaits to identify its endogenous ligand and hence classified as orphan GPCRs. However, these orphan GPCRs have been implicated in several disorders from cancers to neurological diseases. Yet, in many cases, their mechanisms, ligands, and signaling reactions are poorly understood. One such orphan receptor in nervous system is GPR158 that is highly expressed in the brain where it controls synapse formation and function. GPR158 has also been implicated in depression, carcinogenesis, and cognition. However, the structural organization and signaling mechanism of GPR158 are largely unknown. We employed single‐particle cryogenic electron microscopy (cryoEM) to obtain structures of GPR158 in the apo state, and in complex with RGS7‐Gβ5, a regulator of GPCR signaling. The structure reveals highly unique domain organization of GPR158, not documented previously in any GPCRs structures. The homodimeric organization of protomers, phospholipids interaction and the presence of an extracellular Cache domain, an unusual ligand‐binding domain in GPCRs provide insights into the unusual biology of this orphan receptor and the formation of GPCR‐RGS complexes. These structures could help to deorphanize the receptor by serving the template for structure‐based discovery of its ligands that would leads to exploration of mechanistic of GPR158 mediated neuronal signaling.

Structural basis of lipopolysaccharide maturation by the O-antigen ligase.

The outer membrane of Gram-negative bacteria has an external leaflet that is largely composed of lipopolysaccharide, which provides a selective permeation barrier, particularly against antimicrobials1. The final and crucial step in the biosynthesis of lipopolysaccharide is the addition of a species-dependent O-antigen to the lipid A core oligosaccharide, which is catalysed by the O-antigen ligase WaaL2. Here we present structures of WaaL from Cupriavidus metallidurans, both in the apo state and in complex with its lipid carrier undecaprenyl pyrophosphate, determined by single-particle cryo-electron microscopy. The structures reveal that WaaL comprises 12 transmembrane helices and a predominantly α-helical periplasmic region, which we show contains many of the conserved residues that are required for catalysis. We observe a conserved fold within the GT-C family of glycosyltransferases and hypothesize that they have a common mechanism for shuttling the undecaprenyl-based carrier to and from the active site. The structures, combined with genetic, biochemical, bioinformatics and molecular dynamics simulation experiments, offermolecular details on how the ligands come in apposition, and allows us to propose a mechanistic model for catalysis. Together, our work provides a structural basis for lipopolysaccharide maturation in a member of the GT-C superfamily of glycosyltransferases.

Enhanced Conformational Sampling with an Adaptive Coarse-Grained Elastic Network Model Using Short-Time All-Atom Molecular Dynamics.

Compared to all-atom molecular dynamics (AA-MD) simulations, coarse-grained (CG) MD simulations can significantly reduce calculation costs. However, existing CG-MD methods are unsuitable for sampling structures that depart significantly from the initial structure without any biased force. In this study, we developed a new adaptive CG elastic network model (ENM), in which the dynamic cross-correlation coefficient based on short-time AA-MD of at most ns order is considered. By applying Bayesian optimization to search for a suitable parameter among the vast parameter space of adaptive CG-ENM, we succeeded in reducing the searching cost to approximately 10% of those for random sampling and exhaustive sampling. To evaluate the performance of adaptive CG-ENM, we applied the new methodology to adenylate kinase (ADK) and glutamine binding protein (GBP) in the apo state. The results showed that the structural ensembles explored by adaptive CG-ENM could be considerably more diverse than those by conventional ENMs with enhanced sampling such as temperature replica exchange MD and long-time AA-MD of 1 μs. In particular, some of the structures sampled by adaptive ENM are relatively close to the holo-type structures of ADK and GBP. Furthermore, as a challenging task, to demonstrate the advantages of the CG model with lower calculation cost, we applied our new methodology to a larger biomolecule, integrin (αV) in the inactive state. Then, we sampled various structural ensembles, including extended structures that are apparently different from inactive ones.

Long-range structural preformation in yes-associated protein precedes encounter complex formation with TEAD

SummaryYes-associated protein (YAP) is a partly intrinsically disordered protein (IDP) that plays a major role as the downstream element of the Hippo pathway. Although the structures of the complex between TEA domain transcription factors (TEADs) and the TEAD-binding domain of YAP are already well characterized, its apo state and the binding mechanism with TEADs are still not clearly defined. Here we characterize via a combination of different NMR approaches with site-directed mutagenesis and affinity measurements the intrinsically disordered solution state of apo YAP. Our results provide evidence that the apo state of YAP adopts several compact conformations that may facilitate the formation of the YAP:TEAD complex. The interplay between local secondary structure element preformation and long-range co-stabilization of these structured elements precedes the encounter complex formation with TEAD and we, therefore, propose that TEAD binding proceeds largely via conformational selection of the preformed compact substates displaying at least nanosecond lifetimes.

Structural Insights into the Catalytic Cycle of a Bacterial Multidrug ABC Efflux Pump

ABC (“ATP-Binding Cassette”) transporters of the type IV subfamily consist of exporters involved in the efflux of many compounds, notably those capable to confer multidrug resistance like the mammalian P-glycoprotein or the bacterial transporter BmrA. They function according to an alternating access mechanism between inward-facing (IF) and outward-facing (OF) conformations, but the extent of physical separation between the two nucleotide-binding domains (NBDs) in different states is still unsettled. Small Angle Neutron Scattering and hydrogen/deuterium exchange coupled to mass spectrometry were used to highlight different conformational states of BmrA during its ATPase cycle. In particular, mutation of the conserved Lysine residue of the Walker-A motif (K380A) captures BmrA in an ATP-bound IF conformation prior to NBD closure. While in the transition-like state induced by vanadate wild-type BmrA is mainly in an OF conformation, the transporter populates only IF conformations in either the apo state or in the presence of ADP/Mg. Importantly, in this post-hydrolytic step, distances between the two NBDs of BmrA seem to be more separated than in the apo state, but they remain shorter than the widest opening found in the related MsbA transporter. Overall, our results highlight the main steps of the catalytic cycle of a homodimeric bacterial multidrug transporter and underline structural and functional commonalities as well as oddities among the type IV subfamily of ABC transporters.

Activation Mechanism of Strigolactone Receptors and Its Impact on Ligand Selectivity between Host and Parasitic Plants.

Parasitic weeds such as Striga have led to significant losses in agricultural productivity worldwide. These weeds use the plant hormone strigolactone as a germination stimulant. Strigolactone signaling involves substrate hydrolysis followed by a conformational change of the receptor to a "closed" or "active" state that associates with a signaling partner, MAX2/D3. Crystal structures of active and inactive AtD14 receptors have helped elucidate the structural changes involved in activation. However, the mechanism by which the receptor activates remains unknown. The ligand dependence of AtD14 activation has been disputed by mutagenesis studies showing that enzymatically inactive receptors are able to associate with MAX2 proteins. Furthermore, activation differences between strigolactone receptor in Striga, ShHTL7, and AtD14 could contribute to the high sensitivity to strigolactones exhibited by parasitic plants. Using molecular dynamics simulations, we demonstrate that both AtD14 and ShHTL7 could adopt an active conformation in the absence of ligand. However, ShHTL7 exhibits a higher population in the inactive apo state as compared to the AtD14 receptor. We demonstrate that this difference in inactive state population is caused by sequence differences between their D-loops and interactions with the catalytic histidine that prevent full binding pocket closure in ShHTL7. These results indicate that ligand hydrolysis would enhance the active state population by destabilizing the inactive state in ShHTL7 as compared to AtD14. We also show that the mechanism of activation is more concerted in AtD14 than in ShHTL7 and that the main barrier to activation in ShHTL7 is closing of the binding pocket.

Structural and functional analyses explain Pea KAI2 receptor diversity and reveal stereoselective catalysis during signal perception

KAI2 proteins are plant α/β hydrolase receptors which perceive smoke-derived butenolide signals and endogenous, yet unidentified KAI2-ligands (KLs). The number of functional KAI2 receptors varies among species and KAI2 gene duplication and sub-functionalization likely plays an adaptative role by altering specificity towards different KLs. Legumes represent one of the largest families of flowering plants and contain many agronomic crops. Prior to their diversification, KAI2 underwent duplication resulting in KAI2A and KAI2B. Here we demonstrate that Pisum sativum KAI2A and KAI2B are active receptors and enzymes with divergent ligand stereoselectivity. KAI2B has a higher affinity for and hydrolyses a broader range of substrates including strigolactone-like stereoisomers. We determine the crystal structures of PsKAI2B in apo and butenolide-bound states. The biochemical, structural, and mass spectra analyses of KAI2s reveal a transient intermediate on the catalytic serine and a stable adduct on the catalytic histidine, confirming its role as a bona fide enzyme. Our work uncovers the stereoselectivity of ligand perception and catalysis by diverged KAI2 receptors and proposes adaptive sensitivity to KAR/KL and strigolactones by KAI2B.

Identification of the nucleotide-free state as a therapeutic vulnerability for inhibition of selected oncogenic RAS mutants.

SUMMARYRAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.

Subunit conformation modulates the concentration of CO2 in the active site of the most efficient carboxylase

Enoyl-CoA carboxylases/reductases (ECRs) belong to the most efficient CO2-fixing enzymes in Nature. X-Ray structures from Kitasatospora Setae at different steps of the catalytic cycle in conjunction with all-atoms molecular dynamics simulations of ECR, revealed that the enzyme modifies its conformation upon substrate binding, from a homotetramer, in its APO state, to a dimer of dimers with the bound substrate. Each dimer is composed of a catalytically active closed-form subunit and an inactive open-form subunit.

Structural basis for assembly of TRAPPII complex and specific activation of GTPase Ypt31/32.

Transport protein particle (TRAPP) complexes belong to the multiprotein tethering complex and exist in three forms—core TRAPP/TRAPPI, TRAPPII, and TRAPPIII. TRAPPII activates GTPase Ypt31/Ypt32 as the guanine nucleotide exchange factor in the trans-Golgi network to determine the maturation of Golgi cisternae into post-Golgi carriers in yeast. Here, we present cryo-EM structures of yeast TRAPPII in apo and Ypt32-bound states. All the structures show a dimeric architecture assembled by two triangle-shaped monomers, while the monomer in the apo state exhibits both open and closed conformations, and the monomer in the Ypt32-bound form only captures the closed conformation. Located in the interior of the monomer, Ypt32 binds with both core TRAPP/TRAPPI and Trs120 via its nucleotide-binding domain and binds with Trs31 via its hypervariable domain. Combined with functional analysis, the structures provide insights into the assembly of TRAPPII and the mechanism of the specific activation of Ypt31/Ypt32 by TRAPPII.

Sodium Binding Stabilizes the Outward-Open State of SERT by Limiting Bundle Domain Motions.

The human serotonin transporter (hSERT) removes the neurotransmitter serotonin from the synaptic cleft by reuptake into the presynaptic nerve terminal. A number of neurologic diseases are associated with dysfunction of the hSERT, and several medications for their treatment are hSERT blockers, including citalopram, fluoxetine, and paroxetine. The substrate transport is energized by the high concentration of external NaCl. We showed through molecular dynamics simulations that the binding of NaCl stabilized the hSERT in the substrate-binding competent conformation, which was characterized by an open access path to the substrate-binding site through the outer vestibule. Importantly, the binding of NaCl reduced the dynamics of the hSERT by decreasing the internal fluctuations of the bundle domain as well as the movement of the bundle domain relative to the scaffold domain. In contrast, the presence of only the bound chloride ion did not reduce the high domain mobility of the apo state.

Energy landscape of the SARS-CoV-2 reveals extensive conformational heterogeneity.

Cryo-electron microscopy (cryo-EM) has produced a number of structural models of the SARS-CoV-2 spike, already prompting biomedical outcomes. However, these reported models and their associated electrostatic potential maps represent an unknown admixture of conformations stemming from the underlying energy landscape of the spike protein. As with any protein, some of the spike's conformational motions are expected to be biophysically relevant, but cannot be interpreted only by static models. Using experimental cryo-EM images, we present the energy landscape of the glycosylated spike protein, and identify the diversity of low-energy conformations in the vicinity of its open (so called 1RBD-up) state. The resulting atomic refinement reveal global and local molecular rearrangements that cannot be inferred from an average 1RBD-up cryo-EM model. Here we report varied degrees of “openness” in global conformations of the 1RBD-up state, not revealed in the single-model interpretations of the density maps, together with conformations that overlap with the reported models. We discover how the glycan shield contributes to the stability of these low-energy conformations. Five out of six binding sites we analyzed, including those for engaging ACE2, therapeutic mini-proteins, linoleic acid, two different kinds of antibodies, switch conformations between their known apo- and holo-conformations, even when the global spike conformation is 1RBD-up. This apo-to-holo switching is reminiscent of a conformational preequilibrium. We found only one binding site, namely that of AB-C135 remains in apo state within all the sampled free energy-minimizing models, suggesting an induced fit mechanism for the docking of this antibody to the spike.

Deconversion and Identity Formation in Adolescents: The Role of Internal Dialogs and Religiousness of Parents

ABSTRACT Adolescence is a period of exceptional sensitivity to the ideals that are subject to verification. Therefore, a phenomenon of deconversion (i.e. abandonment of religion) is observed among adolescents. The paper aims to analyze deconversion and its connections with the identity formation as well as mediators and moderators of these relationships. Participants were 272 adolescents aged between 14 and 18. Three scales were used: the Circumplex Identity Modes Questionnaire, the Internal Dialogical Activity Scale-Revised, and the Adolescent Deconversion Scale. We found that identity modes such as moratorivity, diffusion, and defiance are positively related to deconversion, whereas socialization and normativity are negatively related to deconversion. The negative relationships between normativity and deconversion exist if the parent (especially mother) is assessed as religious or very religious. Moreover, internal dialogs moderate and mediate links between different identity modes and dimensions of deconversion.

Cryo-EM structure of mouse TRPML2 in lipid nanodiscs

In mammalians, transient receptor potential mucolipin ion channels (TRPMLs) exhibit variable permeability to cations such as Ca2+, Fe2+, Zn2+, and Na+ and can be activated by the phosphoinositide PI(3,5)P2 in the endolysosomal system. Loss or dysfunction of TRPMLs has been implicated in lysosomal storage disorders, infectious diseases, and metabolic diseases. TRPML2 has recently been identified as a mechanosensitive and hypotonicity-sensitive channel in endolysosomal organelles, which distinguishes it from TRPML1 and TRPML3. However, the molecular and gating mechanism of TRPML2 remains elusive. Here, we present the cryo-EM structure of the full-length mouse TRPML2 in lipid nanodiscs at 3.14 Å resolution. The TRPML2 homotetramer structure at pH 7.4 in the apo state reveals an inactive conformation and some unique features of the extracytosolic/luminal domain and voltage sensor-like domain that have implications for the ion-conducting pathway. This structure enables new comparisons between the different subgroups of TRPML channels with available structures and provides structural insights into the conservation and diversity of TRPML channels. These comparisons have broad implications for understanding a variety of molecular mechanisms of TRPMLs in different pH conditions, including with and without bound agonists and antagonists.

Communication pathways bridge local and global conformations in an IgG4 antibody

The affinity of an antibody for its antigen is primarily determined by the specific sequence and structural arrangement of the complementarity-determining regions (CDRs). Recent evidence, however, points toward a nontrivial relation between the CDR and distal sites: variations in the binding strengths have been observed upon mutating residues separated from the paratope by several nanometers, thus suggesting the existence of a communication network within antibodies, whose extension and relevance might be deeper than insofar expected. In this work, we test this hypothesis by means of molecular dynamics (MD) simulations of the IgG4 monoclonal antibody pembrolizumab, an approved drug that targets the programmed cell death protein 1 (PD-1). The molecule is simulated in both the apo and holo states, totalling 4 μs of MD trajectory. The analysis of these simulations shows that the bound antibody explores a restricted range of conformations with respect to the apo one, and that the global conformation of the molecule correlates with that of the CDR. These results support the hypothesis that pembrolizumab featues a multi-scale hierarchy of intertwined global and local conformational changes. The analysis pipeline developed in this work is general, and it can help shed further light on the mechanistic aspects of antibody function.

Binding-induced functional-domain motions in the Argonaute characterized by adaptive advanced sampling.

Argonaute proteins in combination with short microRNA (miRNAs) can target mRNA molecules for translation inhibition or degradation and play a key role in many regulatory processes. The miRNAs act as guide RNAs that associate with Argonaute and the complementary mRNA target region. The complex formation results in activation of Argonaute and specific cleavage of the target mRNA. Both the binding and activation processes involve essential domain rearrangements of functional importance. For the Thermus Thermophilus Argonaute (TtAgo) system guide-bound (binary) and guide/target-bound (ternary) complexes are known but how the binding of guide and target mediate domain movements is still not understood. We have studied the Argonaute domain motion in apo and guide/target bound states using Molecular Dynamics simulations and a Hamiltonian replica exchange (H-REMD) method that employs a specific biasing potential to accelerate domain motions. The H-REMD technique indicates sampling of a much broader distribution of domain arrangements both in the apo as well as binary and ternary complexes compared to regular MD simulations. In the apo state domain arrangements corresponding to more compact (closed) states are mainly sampled which undergo an opening upon guide and guide/target binding. Whereas only limited overlap in domain geometry between apo and bound states was found, a larger similarity in the domain distribution is observed for the simulations of binary and ternary complexes. Comparative simulations on ternary complexes with 15 or 16 base pairs (bp) formed between guide and target strands (instead of 14) resulted in dissociation of the 3'-guide strand from the PAZ domain and domain rearrangement. This agrees with the experimental observation that guide-target pairing beyond 14 bps is required for activation and gives a mechanistic explanation for the experimentally observed activation process.

Structural basis of cyclic oligoadenylate degradation by ancillary Type III CRISPR-Cas ring nucleases.

Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. These enzymes contain a CRISPR-associated Rossman-fold (CARF) domain, which binds and cleaves the cA molecule. Here, we present the structures of the standalone ring nuclease from Sulfolobus islandicus (Sis) 0811 in its apo and post-catalytic states. This enzyme is composed by a N-terminal CARF and a C-terminal wHTH domain. Sis0811 presents a phosphodiester hydrolysis metal-independent mechanism, which cleaves cA4 rings to generate linear adenylate species, thus reducing the levels of the second messenger and switching off the cell antiviral state. The structural and biochemical analysis revealed the coupling of a cork-screw conformational change with the positioning of key catalytic residues to proceed with cA4 phosphodiester hydrolysis in a non-concerted manner.

Structural Insight into the Binding of TGIF1 to SIN3A PAH2 Domain through a C-Terminal Amphipathic Helix.

TGIF1 is a transcriptional repressor playing crucial roles in human development and function and is associated with holoprosencephaly and various cancers. TGIF1-directed transcriptional repression of specific genes depends on the recruitment of corepressor SIN3A. However, to date, the exact region of TGIF1 binding to SIN3A was not clear, and the structural basis for the binding was unknown. Here, we demonstrate that TGIF1 utilizes a C-terminal domain (termed as SIN3A-interacting domain, SID) to bind with SIN3A PAH2. The TGIF1 SID adopts a disordered structure at the apo state but forms an amphipathic helix binding into the hydrophobic cleft of SIN3A PAH2 through the nonpolar side at the holo state. Residues F379, L382 and V383 of TGIF1 buried in the hydrophobic core of the complex are critical for the binding. Moreover, homodimerization of TGIF1 through the SID and key residues of F379, L382 and V383 was evidenced, which suggests a dual role of TGIF1 SID and a correlation between dimerization and SIN3A-PAH2 binding. This study provides a structural insight into the binding of TGIF1 with SIN3A, improves the knowledge of the structure–function relationship of TGIF1 and its homologs and will help in recognizing an undiscovered SIN3A-PAH2 binder and developing a peptide inhibitor for cancer treatment.