Chemistry/structural biology of psychedelic drugs and their receptor(s).

This brief review highlights some of the structure-activity relationships of classic serotonergic psychedelics. In particular, we discuss structural features of three chemotypes: phenethylamines, ergolines and certain tryptamines, which possess psychedelic activity in humans. Where they are known, we point out the underlying molecular mechanisms utilized by each of the three chemotypes of psychedelic molecules. With a focus on the 5-HT2A receptor subtype, a G-protein coupled receptor known to be the primary target of psychedelics, we refer to several X-ray and cryoEM structures, with a variety of ligands bound, to illustrate the underlying atomistic basis for some of the known pharmacological observations of psychedelic drug actions.

Coherent X-ray diffraction imaging of single particles: background impact on 3D reconstruction.

Coherent diffractive imaging with X-ray free-electron lasers could enable structural studies of macromolecules at room temperature. This type of experiment could provide a means to study structural dynamics on the femtosecond timescale. However, the diffraction from a single protein is weak compared with the incoherent scattering from background sources, which negatively affects the reconstruction analysis. This work evaluates the effects of the presence of background on the analysis pipeline. Background measurements from the European X-ray Free-Electron Laser were combined with simulated diffraction patterns and treated by a standard reconstruction procedure, including orientation recovery with the expand, maximize and compress algorithm and 3D phase retrieval. Background scattering did have an adverse effect on the estimated resolution of the reconstructed density maps. Still, the reconstructions generally worked when the signal-to-background ratio was 0.6 or better, in the momentum transfer shell of the highest reconstructed resolution. The results also suggest that the signal-to-background requirement increases at higher resolution. This study gives an indication of what is possible at current setups at X-ray free-electron lasers with regards to expected background strength and establishes a target for experimental optimization of the background.

Recent advances in expression screening and sample evaluation for structural studies of membrane proteins.

Membrane proteins are involved in numerous biological processes and represent more than half of all drug targets; thus, structural information on these proteins is invaluable. However, the low expression level of membrane proteins, as well as their poor stability in solution and tendency to precipitate and aggregate, are major bottlenecks in the preparation of purified membrane proteins for structural studies. Traditionally, the evaluation of membrane protein constructs for structural studies has been quite time consuming and expensive since it is necessary to express and purify the proteins on a large scale, particularly for X-ray crystallography. The emergence of fluorescence detection size exclusion chromatography (FSEC) has drastically changed this situation, as this method can be used to rapidly evaluate the expression and behavior of membrane proteins on a small scale without the need for purification. FSEC has become the most widely used method for the screening of expression conditions and sample evaluation for membrane proteins, leading to the successful determination of numerous structures. Even in the era of cryo-EM, FSEC and the new generation of FSEC derivative methods are being widely used in various manners to facilitate structural analysis. In addition, the application of FSEC is not limited to structural analysis; this method is also widely used for functional analysis of membrane proteins, including for analysis of oligomerization state, screening of antibodies and ligands, and affinity profiling. This review presents the latest advances and applications in membrane protein expression screening and sample evaluation, with a particular focus on FSEC methods.

Activation of polycystin-1 signaling by binding of stalk-derived peptide agonists.

Polycystin-1 (PC1) is the protein product of the PKD1 gene whose mutation causes autosomal dominant Polycystic Kidney Disease (ADPKD). PC1 is an atypical G protein-coupled receptor (GPCR) with an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal and membrane-embedded C-terminal (CTF) fragments. Recently, activation of PC1 CTF signaling was shown to be regulated by a stalk tethered agonist (TA), resembling the mechanism observed for adhesion GPCRs. Here, synthetic peptides of the first 9- (p9), 17- (p17), and 21-residues (p21) of the PC1 stalk TA were shown to re-activate signaling by a stalkless CTF mutant in human cell culture assays. Novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) simulations elucidated binding conformations of p9, p17, and p21 and revealed multiple specific binding regions to the stalkless CTF. Peptide agonists binding to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of stalk TA-mediated PC1 CTF activation. Additional sequence coevolution analyses showed the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. These insights into the structural dynamic mechanism of PC1 activation by TA peptide agonists provide an in-depth understanding that will facilitate the development of therapeutics targeting PC1 for ADPKD treatment.

Dynamic basis of lipopolysaccharide export by LptB2FGC.

Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB2FG forms an ATP-binding cassette transporter that transfers LPS to LptC. How LptB2FG couples ATP binding and hydrolysis with LPS transport to LptC remains unclear. We observed the conformational heterogeneity of LptB2FG and LptB2FGC in micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored LPS binding and release using laser-induced liquid bead ion desorption mass spectrometry. The ß-jellyroll domain of LptF stably interacts with the LptG and LptC ß-jellyrolls in both the apo and vanadate-trapped states. ATP binding at the cytoplasmic side is allosterically coupled to the selective opening of the periplasmic LptF ß-jellyroll domain. In LptB2FG, ATP binding closes the nucleotide binding domains, causing a collapse of the first lateral gate as observed in structures. However, the second lateral gate, which forms the putative entry site for LPS, exhibits a heterogeneous conformation. LptC binding limits the flexibility of this gate to two conformations, likely representing the helix of LptC as either released from or inserted into the transmembrane domains. Our results reveal the regulation of the LPS entry gate through the dynamic behavior of the LptC transmembrane helix, while its ß-jellyroll domain is anchored in the periplasm. This, combined with long-range ATP-dependent allosteric gating of the LptF ß-jellyroll domain, may ensure efficient and unidirectional transport of LPS across the periplasm.

Hybrid Exb/Mot stators require substitutions distant from the chimeric pore to power flagellar rotation.

Powered by ion transport across the cell membrane, conserved ion-powered rotary motors (IRMs) drive bacterial motility by generating torque on the rotor of the bacterial flagellar motor. Homologous heteroheptameric IRMs have been structurally characterized in ion channels such as Tol/Ton/Exb/Gld, and most recently in phage defense systems such as Zor. Functional stator complexes synthesized from chimeras of PomB/MotB (PotB) have been used to study flagellar rotation at low ion-motive force achieved via reduced external sodium concentration. The function of such chimeras is highly sensitive to the location of the fusion site, and these hybrid proteins have thus far been arbitrarily designed. To date, no chimeras have been constructed using interchange of components from Tol/Ton/Exb/Gld and other ion-powered motors with more distant homology. Here, we synthesized chimeras of MotAB, PomAPotB, and ExbBD to assess their capacity for cross-compatibility. We generated motile strains powered by stator complexes with B-subunit chimeras. This motility was further optimized by directed evolution. Whole-genome sequencing of these strains revealed that motility-enhancing residue changes occurred in the A-subunit and at the peptidoglycan binding domain of the B-unit, which could improve motility. Overall, our work highlights the complexity of stator architecture and identifies the challenges associated with the rational design of chimeric IRMs. IMPORTANCE: Ion-powered rotary motors (IRMs) underpin the rotation of one of nature's oldest wheels, the flagellar motor. Recent structures show that this complex appears to be a fundamental molecular module with diverse biological utility where electrical energy is coupled to torque. Here, we attempted to rationally design chimeric IRMs to explore the cross-compatibility of these ancient motors. We succeeded in making one working chimera of a flagellar motor and a non-flagellar transport system protein. This had only a short hybrid stretch in the ion-conducting channel, and function was subsequently improved through additional substitutions at sites distant from this hybrid pore region. Our goal was to test the cross-compatibility of these homologous systems and highlight challenges arising when engineering new rotary motors.

Selective deuteration of an RNA:RNA complex for structural analysis using small-angle scattering.

The structures of RNA:RNA complexes regulate many biological processes. Despite their importance, protein-free RNA:RNA complexes represent a tiny fraction of experimentally-determined structures. Here, we describe a joint small-angle X-ray and neutron scattering (SAXS/SANS) approach to structurally interrogate conformational changes in a model RNA:RNA complex. Using SAXS, we measured the solution structures of the individual RNAs in their free state and of the overall RNA:RNA complex. With SANS, we demonstrate, as a proof-of-principle, that isotope labeling and contrast matching (CM) can be combined to probe the bound state structure of an RNA within a selectively deuterated RNA:RNA complex. Furthermore, we show that experimental scattering data can validate and improve predicted AlphaFold 3 RNA:RNA complex structures to reflect its solution structure. Our work demonstrates that in silico modeling, SAXS, and CM-SANS can be used in concert to directly analyze conformational changes within RNAs when in complex, enhancing our understanding of RNA structure in functional assemblies.

Host Tropism and Structural Biology of ABC Toxin Complexes.

ABC toxin complexes are a class of protein toxin translocases comprised of a multimeric assembly of protein subunits. Each subunit displays a unique composition, contributing to the formation of a syringe-like nano-machine with natural cargo carrying, targeting, and translocation capabilities. Many of these toxins are insecticidal, drawing increasing interest in agriculture for use as biological pesticides. The A subunit (TcA) is the largest subunit of the complex and contains domains associated with membrane permeation and targeting. The B and C subunits, TcB and TcC, respectively, package into a cocoon-like structure that contains a toxic peptide and are coupled to TcA to form a continuous channel upon final assembly. In this review, we outline the current understanding and gaps in the knowledge pertaining to ABC toxins, highlighting seven published structures of TcAs and how these structures have led to a better understanding of the mechanism of host tropism and toxin translocation. We also highlight similarities and differences between homologues that contribute to variations in host specificity and conformational change. Lastly, we review the biotechnological potential of ABC toxins as both pesticides and cargo-carrying shuttles that enable the transport of peptides into cells.

Revealing a hidden conducting state by manipulating the intracellular domains in KV10.1 exposes the coupling between two gating mechanisms.

The KCNH family of potassium channels serves relevant physiological functions in both excitable and non-excitable cells, reflected in the massive consequences of mutations or pharmacological manipulation of their function. This group of channels shares structural homology with other voltage-gated K+ channels, but the mechanisms of gating in this family show significant differences with respect to the canonical electromechanical coupling in these molecules. In particular, the large intracellular domains of KCNH channels play a crucial role in gating that is still only partly understood. Using KCNH1(KV10.1) as a model, we have characterized the behavior of a series of modified channels that could not be explained by the current models. With electrophysiological and biochemical methods combined with mathematical modeling, we show that the uncovering of an open state can explain the behavior of the mutants. This open state, which is not detectable in wild-type channels, appears to lack the rapid flicker block of the conventional open state. Because it is accessed from deep closed states, it elucidates intermediate gating events well ahead of channel opening in the wild type. This allowed us to study gating steps prior to opening, which, for example, explain the mechanism of gating inhibition by Ca2+-Calmodulin and generate a model that describes the characteristic features of KCNH channels gating.

Metal ions in biomedically relevant macromolecular structures.

Understanding the functions of metal ions in biological systems is crucial for many aspects of research, including deciphering their roles in diseases and potential therapeutic use. Structural information about the molecular or atomic details of these interactions, generated by methods like X-ray crystallography, cryo-electron microscopy, or nucleic magnetic resonance, frequently provides details that no other method can. As with any experimental method, they have inherent limitations that sometimes lead to an erroneous interpretation. This manuscript highlights different aspects of structural data available for metal-protein complexes. We examine the quality of modeling metal ion binding sites across different structure determination methods, where different kinds of errors stem from, and how they can impact correct interpretations and conclusions.

Zig, Zag, and 'Zyme: leveraging structural biology to engineer disease resistance.

Dynamic host-pathogen interactions determine whether disease will occur. Pathogen effector proteins are central players in such disease development. On one hand, they improve susceptibility by manipulating host targets; on the other hand, they can trigger immunity after recognition by host immune receptors. A major research direction in the study of molecular plant pathology is to understand effector-host interactions, which has informed the development and breeding of crops with enhanced disease resistance. Recent breakthroughs on experiment- and artificial intelligence-based structure analyses significantly accelerate the development of this research area. Importantly, the detailed molecular insight of effector-host interactions enables precise engineering to mitigate disease. Here, we highlight a recent study by Xiao et al., who describe the structure of an effector-receptor complex that consists of a fungal effector, with polygalacturonase (PG) activity, and a plant-derived polygalacturonase-inhibiting protein (PGIP). PGs weaken the plant cell wall and produce immune-suppressive oligogalacturonides (OGs) as a virulence mechanism; however, PGIPs directly bind to PGs and alter their enzymatic activity. When in a complex with PGIPs, PGs produce OG polymers with longer chains that can trigger immunity. Xiao et al. demonstrate that a PGIP creates a new active site tunnel, together with a PG, which favors the production of long-chain OGs. In this way, the PGIP essentially acts as both a PG receptor and enzymatic manipulator, converting virulence to defense activation. Taking a step forward, the authors used the PG-PGIP complex structure as a guide to generate PGIP variants with enhanced long-chain OG production, likely enabling further improved disease resistance. This study discovered a novel mechanism by which a plant receptor plays a dual role to activate immunity. It also demonstrates how fundamental knowledge, obtained through structural analyses, can be employed to guide the design of proteins with desired functions in agriculture.

Exploring protein structural ensembles: Integration of sparse experimental data from electron paramagnetic resonance spectroscopy with molecular modeling methods.

Under physiological conditions, proteins continuously undergo structural fluctuations on different timescales. Some conformations are only sparsely populated, but still play a key role in protein function. Thus, meaningful structure-function frameworks must include structural ensembles rather than only the most populated protein conformations. To detail protein plasticity, modern structural biology combines complementary experimental and computational approaches. In this review, we survey available computational approaches that integrate sparse experimental data from electron paramagnetic resonance spectroscopy with molecular modeling techniques to derive all-atom structural models of rare protein conformations. We also propose strategies to increase the reliability and improve efficiency using deep learning approaches, thus advancing the field of integrative structural biology.

PPI-hotspotID for detecting protein-protein interaction hot spots from the free protein structure.

Experimental detection of residues critical for protein-protein interactions (PPI) is a time-consuming, costly, and labor-intensive process. Hence, high-throughput PPI-hot spot prediction methods have been developed, but they have been validated using relatively small datasets, which may compromise their predictive reliability. Here, we introduce PPI-hotspotID, a novel method for identifying PPI-hot spots using the free protein structure, and validated it on the largest collection of experimentally confirmed PPI-hot spots to date. We explored the possibility of detecting PPI-hot spots using (i) FTMap in the PPI mode, which identifies hot spots on protein-protein interfaces from the free protein structure, and (ii) the interface residues predicted by AlphaFold-Multimer. PPI-hotspotID yielded better performance than FTMap and SPOTONE, a webserver for predicting PPI-hot spots given the protein sequence. When combined with the AlphaFold-Multimer-predicted interface residues, PPI-hotspotID yielded better performance than either method alone. Furthermore, we experimentally verified several PPI-hotspotID-predicted PPI-hot spots of eukaryotic elongation factor 2. Notably, PPI-hotspotID can reveal PPI-hot spots not obvious from complex structures, including those in indirect contact with binding partners. PPI-hotspotID serves as a valuable tool for understanding PPI mechanisms and aiding drug design. It is available as a web server (https://ppihotspotid.limlab.dnsalias.org/) and open-source code (https://github.com/wrigjz/ppihotspotid/).

HLA A*24:02-restricted T cell receptors cross-recognize bacterial and preproinsulin peptides in type 1 diabetes.

CD8+ T cells destroy insulin-producing pancreatic ß cells in type 1 diabetes through HLA class I-restricted presentation of self-antigens. Combinatorial peptide library screening was used to produce a preferred peptide recognition landscape for a patient-derived T cell receptor (TCR) that recognized the preproinsulin-derived (PPI-derived) peptide sequence LWMRLLPLL in the context of disease risk allele HLA A*24:02. Data were used to generate a strong superagonist peptide, enabling production of an autoimmune HLA A*24:02-peptide-TCR structure by crystal seeding. TCR binding to the PPI epitope was strongly focused on peptide residues Arg4 and Leu5, with more flexibility at other positions, allowing the TCR to strongly engage many peptides derived from pathogenic bacteria. We confirmed an epitope from Klebsiella that was recognized by PPI-reactive T cells from 3 of 3 HLA A*24:02+ patients. Remarkably, the same epitope selected T cells from 7 of 8 HLA A*24+ healthy donors that cross-reacted with PPI, leading to recognition and killing of HLA A*24:02+ cells expressing PPI. These data provide a mechanism by which molecular mimicry between pathogen and self-antigens could have resulted in the breaking of self-tolerance to initiate disease.

Cryo-EM structure of the CBC-ALYREF complex.

In eukaryotes, RNAs transcribed by RNA Pol II are modified at the 5' end with a 7-methylguanosine (m7G) cap, which is recognized by the nuclear cap binding complex (CBC). The CBC plays multiple important roles in mRNA metabolism, including transcription, splicing, polyadenylation, and export. It promotes mRNA export through direct interaction with a key mRNA export factor, ALYREF, which in turn links the TRanscription and EXport (TREX) complex to the 5' end of mRNA. However, the molecular mechanism for CBC-mediated recruitment of the mRNA export machinery is not well understood. Here, we present the first structure of the CBC in complex with an mRNA export factor, ALYREF. The cryo-EM structure of CBC-ALYREF reveals that the RRM domain of ALYREF makes direct contact with both the NCBP1 and NCBP2 subunits of the CBC. Comparing CBC-ALYREF with other cellular complexes containing CBC and/or ALYREF components provides insights into the coordinated events during mRNA transcription, splicing, and export.