High-resolution Structural Analysis Research Articles

Efficient Segmental Isotope Labeling of Integral Membrane Proteins for High-Resolution NMR Studies.

High-resolution structural NMR analyses of membrane proteins are challenging due to their large size, resulting in broad resonances and strong signal overlap. Among the isotope labeling methods that can remedy this situation, segmental isotope labeling is a suitable strategy to simplify NMR spectra and retain high-resolution structural information. However, protein ligation within integral membrane proteins is complicated since the hydrophobic protein fragments are insoluble, and the removal of ligation side-products is elaborate. Here, we show that a stabilized split-intein system can be used for rapid and high-yield protein trans-splicing of integral membrane proteins under denaturing conditions. This setup enables segmental isotope labeling experiments within folded protein domains for NMR studies. We show that high-quality NMR spectra of markedly reduced complexity can be obtained in detergent micelles and lipid nanodiscs. Of note, the nanodisc insertion step specifically selects for the ligated and correctly folded membrane protein and simultaneously removes ligation byproducts. Using this tailored workflow, we show that high-resolution NMR structure determination is strongly facilitated with just two segmentally isotope-labeled membrane protein samples. The presented method will be broadly applicable to structural and dynamical investigations of (membrane-) proteins and their complexes by solution and solid-state NMR but also other structural methods where segmental labeling is beneficial.

Honeycomb gold specimen supports enabling orthogonal focussed ion beam-milling of elongated cells for cryo-ET

Cryo-focussed ion beam (FIB)-milling is a powerful technique that opens up thick, cellular specimens to high-resolution structural analysis by electron cryotomography (cryo-ET). FIB-milled lamellae can be produced from cells on grids, or cut from thicker, high-pressure frozen specimens. However, these approaches can put geometrical constraints on the specimen that may be unhelpful, particularly when imaging structures within the cell that have a very defined orientation. For example, plunge frozen rod-shaped bacteria orient parallel to the plane of the grid, yet the Z-ring, a filamentous structure of the tubulin-like protein FtsZ and the key organiser of bacterial division, runs around the circumference of the cell such that it is perpendicular to the imaging plane. It is therefore difficult or impractical to image many complete rings with current technologies. To circumvent this problem, we have fabricated monolithic gold specimen supports with a regular array of cylindrical wells in a honeycomb geometry, which trap bacteria in a vertical orientation. These supports, which we call “honeycomb gold discs”, replace standard EM grids and when combined with FIB-milling enable the production of lamellae containing cross-sections through cells. The resulting lamellae are more stable and resistant to breakage and charging than conventional lamellae. The design of the honeycomb discs can be modified according to need and so will also enable cryo-ET and cryo-EM imaging of other specimens in otherwise difficult to obtain orientations.

High-resolution structure and biochemical properties of the LH1–RC photocomplex from the model purple sulfur bacterium, Allochromatium vinosum

The mesophilic purple sulfur phototrophic bacterium Allochromatium (Alc.) vinosum (bacterial family Chromatiaceae) has been a favored model for studies of bacterial photosynthesis and sulfur metabolism, and its core light-harvesting (LH1) complex has been a focus of numerous studies of photosynthetic light reactions. However, despite intense efforts, no high-resolution structure and thorough biochemical analysis of the Alc. vinosum LH1 complex have been reported. Here we present cryo-EM structures of the Alc. vinosum LH1 complex associated with reaction center (RC) at 2.24 Å resolution. The overall structure of the Alc. vinosum LH1 resembles that of its moderately thermophilic relative Alc. tepidum in that it contains multiple pigment-binding α- and β-polypeptides. Unexpectedly, however, six Ca ions were identified in the Alc. vinosum LH1 bound to certain α1/β1- or α1/β3-polypeptides through a different Ca2+-binding motif from that seen in Alc. tepidum and other Chromatiaceae that contain Ca2+-bound LH1 complexes. Two water molecules were identified as additional Ca2+-coordinating ligands. Based on these results, we reexamined biochemical and spectroscopic properties of the Alc. vinosum LH1–RC. While modest but distinct effects of Ca2+ were detected in the absorption spectrum of the Alc. vinosum LH1 complex, a marked decrease in thermostability of its LH1–RC complex was observed upon removal of Ca2+. The presence of Ca2+ in the photocomplex of Alc. vinosum suggests that Ca2+-binding to LH1 complexes may be a common adaptation in species of Chromatiaceae for conferring spectral and thermal flexibility on this key component of their photosynthetic machinery.

Lateral prefrontal cortex thickness is associated with stress but not cognitive fatigue in exhaustion disorder.

Impaired executive functioning, including cognitive fatigue, is a core feature of the long-term stress-related condition exhaustion disorder (ED). Recent research suggests that a key area for executive control, the lateral prefrontal cortex (LPFC), may be mechanistically linked to cognitive fatigue due to stress. Here, we therefore asked if and how stress, the LPFC and cognitive fatigue may be related in ED. We used a multimodal cross-sectional study design with high-resolution structural magnetic resonance imaging (MRI), self-reported measures, and path analysis modeling in 300 participants with ED. We found positive associations between stress and cognitive fatigue, and stress and LPFC thickness,but no association between LPFC thickness and cognitive fatigue. Furthermore, LPFC thickness did not mediate or moderate the association between stress and cognitive fatigue. These findings suggest that LPFC brain morphology is related to perceived stress levels but not cognitive fatigue, expanding previous research on the role of the LPFC in executive functioning. Moreover, the results support the notion that the LPFC may be mechanistically involved in stress-related executive function impairment but prompt further research into if and how this may be related to cognitive symptoms in ED.

Focused ion beam milling and MicroED structure determination of metal-organic framework crystals

We report new advancements in the determination and high-resolution structural analysis of beam-sensitive metal organic frameworks (MOFs) using microcrystal electron diffraction (MicroED) coupled with focused ion beam milling at cryogenic temperatures (cryo-FIB). A microcrystal of the beam-sensitive MOF, ZIF-8, was ion-beam milled in a thin lamella approximately 150 nm thick. MicroED data were collected from this thin lamella using an energy filter and a direct electron detector operating in counting mode. Using this approach, we achieved a greatly improved resolution of 0.59 Å with a minimal total exposure of only 0.64 e−/A2. These innovations not only improve model statistics but also further demonstrate that ion-beam milling is compatible with beam-sensitive materials, augmenting the capabilities of electron diffraction in MOF research.

Anti-InlA single-domain antibodies that inhibit the cell invasion of Listeria monocytogenes

Listeriosis, caused by infection with Listeria monocytogenes, is a severe disease with a high mortality rate. The L. monocytogenes virulence factor, internalin family protein InlA, which binds to the host receptor E-cadherin, is necessary to invade host cells. Here, we isolated two single-domain antibodies (VHHs) that bind to InlA with picomolar affinities from an alpaca immune library using the phage display method. These InlA-specific VHHs inhibited the binding of InlA to the extracellular domains of E-cadherin invitro as shown by biophysical interaction analysis. Furthermore, we determined that the VHHs inhibited the invasion of L.monocytogenes into host cells in culture. High-resolution X-ray structure analyses of the complexes of VHHs with InlA revealed that the VHHs bind to the same binding site as E-cadherin against InlA. We conclude that these VHHs have the potential for use as drugs to treat listeriosis.

Neurodegeneration or dysfunction in Phelan-McDermid syndrome? A multimodal approach with CSF and computational MRI

BackgroundPhelan-McDermid syndrome (PMS) is a rare multisystem disease with global developmental delay and autistic features. Genetically, the disease is based on a heterozygous deletion of chromosome 22q13.3 with involvement of at least part of the SHANK3 gene or heterozygous pathogenic variants in SHANK3. Pathophysiologically, this syndrome has been regarded as a synaptopathy, but current data suggest an additional concept, since axonal functions of neurons are also impaired, thus, the specific pathophysiological processes in this disease are not yet fully understood. Since symptoms of the autism spectrum, regression, and stagnation in development occur, we investigated whether neuroinflammatory and neurodegenerative processes may also play a role. To this end, we analysed biomarkers in cerebrospinal fluid (CSF) and parameters from magnetic resonance imaging with high-resolution structural T1w volumetry and diffusion tensor imaging analysis in 19 Phelan-McDermid syndrome patients.ResultsCSF showed no inflammation but abnormalities in tau protein and amyloid-ß concentrations, however, with no typical biomarker pattern as in Alzheimer’s disease. It could be demonstrated that these CSF changes were correlated with integrity losses of the fibres in the corticospinal tract as well as in the splenium and dorsal part of the cingulum. High CSF levels of tau protein were associated with loss of integrity of fibres in the corticospinal tract; lower levels of amyloid-ß were associated with decreasing integrity of fibre tracts of the splenium and posterior cingulate gyrus. Volumetric investigations showed global atrophy of the white matter, but not the grey matter, and particularly not in temporal or mesiotemporal regions, as is typical in later stages of Alzheimer’s disease.ConclusionsIn summary, alterations of neurodegenerative CSF markers in PMS individuals could be demonstrated which were correlated with structural connectivity losses of the corticospinal tract, the splenium, and the dorsal part of the cingulum, which can also be associated with typical clinical symptoms in these patients. These findings might represent a state of dysfunctional processes with ongoing degenerative and regenerative processes or a kind of accelerated aging. This study should foster further clinical diagnostics like tau- and amyloid-PET imaging as well as novel scientific approaches especially in basic research for further mechanistic proof.

Cross-Linked α-Synuclein as Inhibitor of Amyloid Formation.

The aggregation and amyloid formation of α-synuclein is associated with Parkinson's disease and other synucleinopathies. In its native, monomeric form α-synuclein is an intrinsically disordered protein represented by highly dynamic conformational ensembles. Inhibition of α-synuclein aggregation using small molecules, peptides, or proteins has been at the center of interest in recent years. Our aim was to explore the effects of cross-linking on the structure and aggregation/amyloid formation properties of α-synuclein. Comparative analysis of available high-resolution amyloid structures and representative structural models and MD trajectory of monomeric α-synuclein revealed that potential cross-links in the monomeric protein are mostly incompatible with the amyloid forms and thus might inhibit fibrillation. Monomeric α-synuclein has been intramolecularly chemically cross-linked under various conditions using different cross-linkers. We determined the location of cross-links and their frequency using mass spectrometry and found that most of them cannot be realized in the amyloid structures. The inhibitory potential of cross-linked proteins has been experimentally investigated using various methods, including thioflavin-T fluorescence and transmission electron microscopy. We found that conformational constraints applied by cross-linking fully blocked α-synuclein amyloid formation. Moreover, DTSSP-cross-linked molecules exhibited an inhibitory effect on the aggregation of unmodified α-synuclein as well.

High-throughput and high-resolution 3D-ED structure analysis through AI-based data collection and hybrid approach

High-throughput and high-resolution 3D-ED structure analysis through AI-based data collection and hybrid approach

Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-α-amino acids in vitro and in vivo

The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-l-α-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Here we report that pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants accept α-hydroxy, α-thio and N-formyl-l-α-amino acids, as well as α-carboxy acid monomers that are precursors to polyketide natural products. These monomers are accommodated and accepted by the translation apparatus in vitro; those with reactive nucleophiles are incorporated into proteins in vivo. High-resolution structural analysis of the complex formed between one PylRS enzyme and a m-substituted 2-benzylmalonic acid derivative revealed an active site that discriminates prochiral carboxylates and accommodates the large size and distinct electrostatics of an α-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges substantially from the α-amine of proteinogenic amino acids. These enzymes or derivatives thereof could synergize with natural or evolved ribosomes and/or translation factors to generate diverse sequence-defined non-protein heteropolymers.

Structural classification of peptide/HLA complexes enables accurate high-throughput modeling

Abstract The class I major histocompatibility complex (MHC-I) proteins display 8 to 15 residue peptides derived from endogenous proteins on the cell surface for immune surveillance. Accurate structural modeling of peptides bound to HLA (the human MHC) proteins has been mired by the inability to capture the central residues of the peptide. Here, an analysis of high-resolution X-ray crystal structures revealed that diverse peptide conformations arise as a result of local changes in the backbone configuration. As a result, we established an internal-coordinate based system to assess structural similarity between peptide backbones and found that peptide/HLA complexes, encompassing a wide range of allotypes and peptide sequences, present a discrete set of peptide backbone conformations. Leveraging these representative backbones and a regression model trained on a physically realistic scoring function, we developed RepPred, a structural modeling approach which predicts near native nonamer/HLA-A*02:01 complexes with high accuracy and throughput. Our method significantly outperformed five state-of-the-art peptide/HLA modeling approaches on the basis of structural accuracy. Thus, the structural information obtained from RepPred can be used to address long-standing immunology challenges such as predicting peptide immunogenicity. Supported by grants from NIH (R01AI143997, R35GM125034, U01DK112217).

Structural analysis of cancer-relevant TCR-CD3 and peptide-MHC complexes by cryoEM

The recognition of antigenic peptide-MHC (pMHC) molecules by T-cell receptors (TCR) initiates the T-cell mediated immune response. Structural characterization is key for understanding the specificity of TCR-pMHC interactions and informing the development of therapeutics. Despite the rapid rise of single particle cryoelectron microscopy (cryoEM), x-ray crystallography has remained the preferred method for structure determination of TCR-pMHC complexes. Here, we report cryoEM structures of two distinct full-length α/β TCR-CD3 complexes bound to their pMHC ligand, the cancer-testis antigen HLA-A2/MAGEA4 (230–239). We also determined cryoEM structures of pMHCs containing MAGEA4 (230–239) peptide and the closely related MAGEA8 (232–241) peptide in the absence of TCR, which provided a structural explanation for the MAGEA4 preference displayed by the TCRs. These findings provide insights into the TCR recognition of a clinically relevant cancer antigen and demonstrate the utility of cryoEM for high-resolution structural analysis of TCR-pMHC interactions.

Human CEACAM1 is targeted by a Streptococcus pyogenes adhesin implicated in puerperal sepsis pathogenesis

Life-threatening bacterial infections in women after childbirth, known as puerperal sepsis, resulted in classical epidemics and remain a global health problem. While outbreaks of puerperal sepsis have been ascribed to Streptococcus pyogenes, little is known about disease mechanisms. Here, we show that the bacterial R28 protein, which is epidemiologically associated with outbreaks of puerperal sepsis, specifically targets the human receptor CEACAM1. This interaction triggers events that would favor the development of puerperal sepsis, including adhesion to cervical cells, suppression of epithelial wound repair and subversion of innate immune responses. High-resolution structural analysis showed that an R28 domain with IgI3-like fold binds to the N-terminal domain of CEACAM1. Together, these findings demonstrate that a single adhesin-receptor interaction can drive the pathogenesis of bacterial sepsis and provide molecular insights into the pathogenesis of one of the most important infectious diseases in medical history.

Role of surfactants in electron cryo-microscopy film preparation

Single-particle electron cryo-microscopy (cryo-EM) has become an effective and straightforward approach to determine the structure of membrane proteins. However, obtaining cryo-EM grids of sufficient quality for high-resolution structural analysis remains a major bottleneck. One of the difficulties arises from the presence of detergents, which often leads to a lack of control of the ice thickness. Amphipathic polymers such as amphipols (APols) are detergent substitutes, which have proven to be valuable tools for cryo-EM studies. In this work, we investigate the physico-chemical behavior of APol- and detergent-containing solutions and show a correlation with the properties of vitreous thin films in cryo-EM grids. This study provides new insight on the potential of APols, allowing a better control of ice thickness while limiting protein adsorption at the air-water interface, as shown with the full-length mouse serotonin 5-HT3A receptor whose structure has been solved in APol. These findings may speed up the process of grid optimization to obtain high-resolution structures of membrane proteins.

Shifting the selectivity of pyrido[2,3-d]pyrimidin-7(8H)-one inhibitors towards the salt-inducible kinase (SIK) subfamily

Salt-inducible kinases 1–3 (SIK1-3) are key regulators of the LKB1-AMPK pathway and play an important role in cellular homeostasis. Dysregulation of any of the three isoforms has been associated with tumorigenesis in liver, breast, and ovarian cancers. We have recently developed the dual pan-SIK/group I p21-activated kinase (PAK) chemical probe MRIA9. However, inhibition of p21-activated kinases has been associated with cardiotoxicity in vivo, which complicates the use of MRIA9 as a tool compound. Here, we present a structure-based approach involving the back-pocket and gatekeeper residues, for narrowing the selectivity of pyrido[2,3-d]pyrimidin-7(8H)-one-based inhibitors towards SIK kinases, eliminating PAK activity. Optimization was guided by high-resolution crystal structure analysis and computational methods, resulting in a pan-SIK inhibitor, MR22, which no longer exhibited activity on STE group kinases and displayed excellent selectivity in a representative kinase panel. MR22-dependent SIK inhibition led to centrosome dissociation and subsequent cell-cycle arrest in ovarian cancer cells, as observed with MRIA9, conclusively linking these phenotypic effects to SIK inhibition. Taken together, MR22 represents a valuable tool compound for studying SIK kinase function in cells.

Interaction Patterns of Pyrazolopyrimidines with Receptor Proteins.

Heterocyclic compounds have a prominent role in medicinal chemistry and drug design. They are not only useful as medicinally active compounds but also as a modular structural scaffold for drug design. Therefore, heterocycles are present in many ligands that exhibit a broad spectrum of biological activities. Pyazolopyrimidines are nitrogen heterocycles and are part of many biologically active compounds and marketed drugs. This study examines the non-covalent interactions between the pyrazolopyrimidine rings and receptor proteins through data mining and analysis of high-resolution crystal structures deposited in the Protein Data Bank. The Protein Data Bank contains 471 crystal structures with pyrazolopyrimidine derivatives as ligands, among which 50% contains 1H-pyrazolo[3,4-d]pyrimidines (Pyp1), while 38% contains pyrazolo[1,5-a] pyrimidines (Pyp2). 1H-Pyrazolo[4,3-d]pyrimidines (Pyp3) are found in 11% of the structures, and no structural data is available for pyrazolo[1,5-c]pyrimidine isomers (Pyp4). Among receptor proteins, transferases are found in most examples (67.5%), followed by hydrolases (13.4%) and oxidoreductases (8.9%). Detailed analysis of structures to identify the most prevalent interactions of pyrazolopyrimidines with proteins shows that aromatic π···π interactions are present in ∼91% of the structures and hydrogen bonds/other polar contacts are present in ∼73% of the structures. The centroid-centroid distances (dcent) between the pyrazolopyrimidine rings and aromatic side chains of the proteins have been retrieved from crystal structures recorded at a high resolution (data resolution <2.0 Å). The average value of dcent in pyrazolopyrimidine-protein complexes is 5.32 Å. The information on the geometric parameters of aromatic interactions between the core pyrazolopyrimidine ring and the protein would be helpful in future in silico modeling studies on pyrazolopyrimidine-receptor complexes.

New structural analysis in North Dobrogea - a key region to unravel the tectonics of the Black Sea back-arc basin during the Mesozoic

High-resolution structural analysis of stratigraphically-controlled units within North Dobrogea (ND), based on fieldwork and the production of new cross-sections as well as a reconstruction of the Mesozoic paleo-stress regimes, has resulted in a revision of the tectonic events across the region as well as demonstrating the significance of tectonic inheritance. The observed structures are closely related to the major strike-slip faults of the Teisseyre-Tornquist Zone (TTZ) a lithospheric structure active during the early and middle Mesozoic. The significance of this zone has been underestimated in previous kinematic reconstructions examining the opening of the continental back-arc basin of the Black Sea. Integrating the present results with existing knowledge on the tectonic evolution of the Black Sea, suggests a new conceptual kinematic model for further testing, one that involves movement of the continental fragment of Moesia NW along the TTZ during the early and middle Mesozoic. Such a displacement would represent the westernmost occurrence of the Cimmerian orogeny in the region of the western Black Sea. The escape of Moesia to the NW could possibly explain the polyphase extension of the western Black Sea crust, which developed on the continental Eurasian Plate as a back-arc basin due to the N-directed subduction of Tethys.

Epoxidized graphene grid for highly efficient high-resolution cryoEM structural analysis

Functionalization of graphene is one of the most important fundamental technologies in a wide variety of fields including industry and biochemistry. We have successfully achieved a novel oxidative modification of graphene using photoactivated ClO2· as a mild oxidant and confirmed the oxidized graphene grid is storable with its functionality for at least three months under N2 atmosphere. Subsequent chemical functionalization enabled us to develop an epoxidized graphene grid (EG-grid™), which effectively adsorbs protein particles for electron cryomicroscopy (cryoEM) image analysis. The EG-grid dramatically improved the particle density and orientation distribution. The density maps of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were reconstructed at 1.99 and 2.16 Å resolution from only 504 and 241 micrographs, respectively. A sample solution of 0.1 mg ml−1 was sufficient to reconstruct a 3.10 Å resolution map of SARS-CoV-2 spike protein from 1163 micrographs. The map resolutions of β-galactosidase and apoferritin easily reached 1.81 Å and 1.29 Å resolution, respectively, indicating its atomic-resolution imaging capability. Thus, the EG-grid will be an extremely powerful tool for highly efficient high-resolution cryoEM structural analysis of biological macromolecules.

Structure and Mechanism of Human ABC Transporters.

ABC transporters are essential for cellular physiology. Humans have 48 ABC genes organized into seven distinct families. Of these genes, 44 (in five distinct families) encode for membrane transporters, of which several are involved in drug resistance and disease pathways resulting from transporter dysfunction. Over the last decade, advances in structural biology have vastly expanded our mechanistic understanding of human ABC transporter function, revealing details of their molecular arrangement, regulation, and interactions, facilitated in large part by advances in cryo-EM that have rendered hitherto inaccessible targets amenable to high-resolution structural analysis. As a result, experimentally determined structures of multiple members of each of the five families of ABC transporters in humans are now available. Here we review this recent progress, highlighting the physiological relevance of human ABC transporters and mechanistic insights gleaned from their direct structure determination. We also discuss the impact and limitations of model systems and structure prediction methods in understanding human ABC transporters and discuss current challenges and future research directions.

The Switch and Reciprocating Models for the Function of ABC Multidrug Exporters: Perspectives on Recent Research.

ATP-binding cassette (ABC) transporters comprise a large superfamily of primary active transporters, which are integral membrane proteins that couple energy to the uphill vectorial transport of substrates across cellular membranes, with concomitant hydrolysis of ATP. ABC transporters are found in all living organisms, coordinating mostly import in prokaryotes and export in eukaryotes. Unlike the highly conserved nucleotide binding domains (NBDs), sequence conservation in the transmembrane domains (TMDs) is low, with their divergent nature likely reflecting a need to accommodate a wide range of substrate types in terms of mass and polarity. An explosion in high resolution structural analysis over the past decade and a half has produced a wealth of structural information for ABCs. Based on the structures, a general mechanism for ABC transporters has been proposed, known as the Switch or Alternating Access Model, which holds that the NBDs are widely separated, with the TMDs and NBDs together forming an intracellular-facing inverted "V" shape. Binding of two ATPs and the substrate to the inward-facing conformation induces a transition to an outward conformation. Despite this apparent progress, certainty around the transport mechanism for any given ABC remains elusive. How substrate binding and transport is coupled to ATP binding and hydrolysis is not known, and there is a large body of biochemical and biophysical data that is at odds with the widely separated NBDs being a functional physiological state. An alternative Constant Contact model has been proposed in which the two NBSs operate 180 degrees out of phase with respect to ATP hydrolysis, with the NBDs remaining in close proximity throughout the transport cycle and operating in an asymmetric allosteric manner. The two models are discussed in the light of recent nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses of three ABC exporters.