Unique Structural Insights Research Articles

Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells.

Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in a heterogeneous mixture of undesirable editing outcomes. We recently leveraged a type I-F CRISPR-associated transposase (CAST) from the Pseudoalteromonas Tn7016 transposon (PseCAST) for DSB-free, RNA-guided DNA integration in human cells, taking advantage of its programmability and large payload capacity. PseCAST is the only characterized CAST system that has achieved human genomic DNA insertions, but multiple lines of evidence suggest that DNA binding may be a critical bottleneck that limits high-efficiency activity. Here we report structural determinants of target DNA recognition by the PseCAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), which revealed novel subtype-specific interactions and RNA-DNA heteroduplex features. By combining our structural data with target DNA library screens and rationally engineered protein mutations, we uncovered CAST variants that exhibit increased integration efficiency and modified PAM stringency. Structure predictions of key interfaces in the transpososome holoenzyme also revealed opportunities for the design of hybrid CASTs, which we leveraged to build chimeric systems that combine high-activity DNA binding and DNA integration modules. Collectively, our work provides unique structural insights into type I-F CAST systems while showcasing multiple diverse strategies to investigate and engineer new RNA-guided transposase architectures for human genome editing applications.

Selective modifications of lactose and N-acetyllactosamine with sulfate and aromatic bulky groups unveil unique structural insights in galectin-1-ligand recognition

Galectins, a family of endogenous glycan-binding proteins, play crucial roles in a broad range of physiological and pathological processes. Galectin-1 (Gal-1), a proto-type member of this family, is overexpressed in several cancers and plays critical roles in tumor-immune escape, angiogenesis and metastasis. Thus, generation of high-affinity Gal-1 inhibitors emerges as an attractive therapeutic approach for a wide range of neoplastic conditions. Small-molecule carbohydrate inhibitors based on lactose (Lac) and N-acetyllactosamine (LacNAc) structures have been tested showing different results. In this study, we evaluated Lac- and LacNAc-based compounds with specific chemical modifications at key positions as Gal-1 ligands by competitive solid-phase assays (SPA) and isothermal titration calorimetry (ITC). Both assays showed excellent correlation, highlighting that lactosides bearing bulky aromatic groups at the anomeric carbon and sulfate groups at the O3′ position exhibited the highest binding affinities. To dissect the atomistic determinants for preferential affinity of the different tested Gal-1 ligands, molecular docking simulations were conducted and PRODIGY-LIG structure-based method was employed to predict binding affinity in protein–ligand complexes. Notably, calculated binding free energies derived from the molecular docking were in accordance with experimental values determined by SPA and ITC, showing excellent correlation between theoretical and experimental approaches. Moreover, this analysis showed that 3′-O-sulfate groups interact with residues of the Gal-1 subsite B, mainly with Asn33, while the ester groups of the aromatic anomeric group interact with Gly69 and Thr70 at Gal-1 subsite E, extending deeper into the pocket, which could account for the enhanced binding affinity. This study contributes to the rational design of highly optimized Gal-1 inhibitors to be further studied in cancer models and other pathologic conditions.

Combining Surface-Induced Dissociation and Charge Detection Mass Spectrometry to Reveal the Native Topology of Heterogeneous Protein Complexes.

Charge detection mass spectrometry (CDMS) enables the direct mass measurement of heterogeneous samples on the megadalton scale, as the charge state for a single ion is determined simultaneously with the mass-to-charge ratio (m/z). Surface-induced dissociation (SID) is an effective activation method to dissociate non-intertwined, non-covalent protein complexes without extensive gas-phase restructuring, producing various subcomplexes reflective of the native protein topology. Here, we demonstrate that using CDMS after SID on an Orbitrap platform offers subunit connectivity, topology, proteoform information, and relative interfacial strengths of the intact macromolecular assemblies. SID dissects the capsids (∼3.7 MDa) of adeno-associated viruses (AAVs) into trimer-containing fragments (3mer, 6mer, 9mer, 15mer, etc.) that can be detected by the individual ion mass spectrometry (I2MS) approach on Orbitrap instruments. SID coupled to CDMS provides unique structural insights into heterogeneous assemblies that are not readily obtained by traditional MS measurements.

Investigation into aquaporin 1's conserved structural features and the C-terminal tail.

The integral membrane protein human aquaporin-1 (AQP1), for the most part, has been well characterized structurally. A few X-ray and cryo-EM structures exist, however information regarding several functionally important residues and motifs that are conserved throughout the aquaporin family have not been fully explored. As well, the long, flexible C-terminal tail is not well-characterized, even though evidence suggests it is important in several protein-protein interaction events that govern AQP1's translocation to the cellular membrane, and in turn its activity. These structural features are important in further understanding AQP1's function, as it becomes increasingly more medically relevant and a possible drug target, with its involvement in tumour cell migration and angiogenesis. In this work, we explored the feasibility of probing AQP1's C-terminal tail in solution using NMR spectroscopy and a novel polymer nanodisc platform. The nanodiscs encapsulating AQP1 have taken advantage of the styrene-maleic acid co-polymer to directly solubilize Pichia pastoris membranes. The resulting styrene-maleic acid lipid nanoparticles (SMALPs), suspended freely in solution, then act as a scaffold to study protein-protein interactions on the C-terminal tail of AQP1. Preliminary studies of the AQP1-SMALPs have resulted in well resolved 1H-15N HSQC spectra, and further microscopy and spectroscopy studies have determined the size and relative stability of the discs. Furthermore, we have also investigated several key conserved residues in AQP1, that through prior work, are hypothesized to be heavily involved in overall protein fold stability. Through mutagenesis experiments coupled with H/D exchange and Fourier transform infrared spectroscopy, we were able to monitor the differences in stability given the extent of H/D exchange at multiple temperature points. These experiments will yield unique structural insights into AQP1, and present a foundation towards discovering new protein partner interactions.

Prostate cancer histopathology using label-free multispectral deep-UV microscopy quantifies phenotypes of tumor aggressiveness and enables multiple diagnostic virtual stains

Identifying prostate cancer patients that are harboring aggressive forms of prostate cancer remains a significant clinical challenge. Here we develop an approach based on multispectral deep-ultraviolet (UV) microscopy that provides novel quantitative insight into the aggressiveness and grade of this disease, thus providing a new tool to help address this important challenge. We find that UV spectral signatures from endogenous molecules give rise to a phenotypical continuum that provides unique structural insight (i.e., molecular maps or “optical stains") of thin tissue sections with subcellular (nanoscale) resolution. We show that this phenotypical continuum can also be applied as a surrogate biomarker of prostate cancer malignancy, where patients with the most aggressive tumors show a ubiquitous glandular phenotypical shift. In addition to providing several novel “optical stains” with contrast for disease, we also adapt a two-part Cycle-consistent Generative Adversarial Network to translate the label-free deep-UV images into virtual hematoxylin and eosin (H&E) stained images, thus providing multiple stains (including the gold-standard H&E) from the same unlabeled specimen. Agreement between the virtual H&E images and the H&E-stained tissue sections is evaluated by a panel of pathologists who find that the two modalities are in excellent agreement. This work has significant implications towards improving our ability to objectively quantify prostate cancer grade and aggressiveness, thus improving the management and clinical outcomes of prostate cancer patients. This same approach can also be applied broadly in other tumor types to achieve low-cost, stain-free, quantitative histopathological analysis.

Metallation of sensitive fluoroarenes using a potassium TMP-zincate supported by a silyl(bis)amido ligand.

Combining a bulky bis(amide) and a reactive one-coordinate TMP (2,2,6,6-tetramethylpiperidide) ligand, a new mixed K/Zn heteroleptic base has been developed for regioselective zincation of fluoroarenes. This special ligand set allows for trapping and structural authentication of the first intermediates of direct Zn–H exchange of fluoroarenes obtained via deprotonative metallation, providing mechanistic insights of the processes involved.

An EDS1 heterodimer signalling surface enforces timely reprogramming of immunity genes in Arabidopsis

Plant intracellular NLR receptors recognise pathogen interference to trigger immunity but how NLRs signal is not known. Enhanced disease susceptibility1 (EDS1) heterodimers are recruited by Toll-interleukin1-receptor domain NLRs (TNLs) to transcriptionally mobilise resistance pathways. By interrogating the Arabidopsis EDS1 ɑ-helical EP-domain we identify positively charged residues lining a cavity that are essential for TNL immunity signalling, beyond heterodimer formation. Mutating a single, conserved surface arginine (R493) disables TNL immunity to an oomycete pathogen and to bacteria producing the virulence factor, coronatine. Plants expressing a weakly active EDS1R493A variant have delayed transcriptional reprogramming, with severe consequences for resistance and countering bacterial coronatine repression of early immunity genes. The same EP-domain surface is utilised by a non-TNL receptor RPS2 for bacterial immunity, indicating that the EDS1 EP-domain signals in resistance conferred by different NLR receptor types. These data provide a unique structural insight to early downstream signalling in NLR receptor immunity.

Reliable Biomolecular Structural Modelling with Small-Angle Scattering

The small-angle scattering (SAS) of X-rays or neutrons from biomolecules in solution can provide (1) unique structural insights, (2) information on the range of potential conformations present, (3) and powerful restraints for integrative/hybrid structural modelling (IHM). The rotational and ensemble averaging of the signal presents multiple challenges to the reliable utilisation of SAS data: (1) demonstrating SAS profile represents the species of interest, (2) reliably evaluating model fits, and (3) over-interpretation and over-fitting of data. A community driven project has been underway for more than a decade now to define the information that should be made available regarding sample quality, data acquisition and reduction, presentation of scattering data and validation, and modelling for biomolecular SAS experiments. These guidelines (Trewhella et al. (2017) Acta D73, 710) provide a reporting framework so that “readers can independently assess the quality of the data and the basis for any interpretations presented” concerning the results of bimolecular SAS experiments, including the generation or testing of 3D models. The validation task forces for SAS (Trewhella et al. (2013) Structure 21, 875) and for IHM (Sali et al. (2015) Structure 23, 1156) of the world-wide Protein Data Bank are additionally addressing the complex issues concerning the archiving and validation of IHM models that depend upon computational methods and disparate data from multiple techniques (Burley et al. (2017) Structure 25, 1317). Finally, the question of how many parameters are justified by the model evidence inherent in the SAS profile is an important question to answer when modelling conformational ensembles (Potrzebowski, Trewhella and Andr⌈ (2018) in review PLOS Computational Biology). This presentation will address the recommended reporting framework and its rationale, the reliable incorporation of SAS data in IHM, and the strengths and limits of SAS in ensemble modelling.

The Functional Significance of Intrinsically Disordered Protein Regions Encoded by the Diabetes Gene Clec16A

Recent advances in structural biology implicate intrinsically disordered proteins (IDPs) and/or intrinsically disordered protein regions (IDPRs), which lack a fixed 3-dimensional structure, in several human diseases including diabetes. However, comprehensive structure-to-function characterizations of IDPs or IDPRs in the context of diabetes is lacking. Using in silico predictive algorithms, we observe that the diabetes-associated gene encoding the E3 ligase and mitophagy regulator Clec16a, contains two comparatively large putative IDPRs. Mitochondrial function and mass, governed by a balance of biogenesis and selective mitochondrial turnover (mitophagy), is crucial to maintain insulin secretion and glucose homeostasis. We previously identified Clec16a to regulate pancreatic beta-cell function, insulin release, and glucose homeostasis through mitophagy. Little is known regarding the structure-to-function implications of the putative IDPRs within Clec16a. We hypothesized that diabetes-associated Clec16a IDPRs control structure-function relationships of consequence for beta-cell competence. Utilizing a mouse model lacking a putative C-terminal Clec16a IDPR, we observed that IDPR-deficientmice had impaired glucose homeostasis, deficient insulin secretion, abnormal β-cell mitochondrial morphology/respiration, and impaired mitophagic flux. Disorder-to-order (D-O) mutagenesis revealed that Clec16a IDPRs are vital for binding, stabilization, and ubiquitination of its principal mitophagy effector Nrdp1. Taken together, we identify novel roles for IDPRs in the function of a key diabetes-associated protein. Extension of these structure-function relationships within other diabetes-associated proteins may provide a unique structural insight into diabetes pathogenesis and elucidate novel approaches to protect beta-cells.

Selective Utilization of Benzimidazolyl-Norcobamides as Cofactors by the Tetrachloroethene Reductive Dehalogenase of Sulfurospirillum multivorans.

The organohalide-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B12, which serves as cofactor of the tetrachloroethene (PCE) reductive dehalogenase (PceA). As previously reported, a replacement of the adeninyl moiety, the lower base of the cofactor, by exogenously applied 5,6-dimethylbenzimidazole led to inactive PceA. To explore the general effect of benzimidazoles on the PCE metabolism, the susceptibility of the organism for guided biosynthesis of various singly substituted benzimidazolyl-norcobamides was investigated, and their use as cofactor by PceA was analyzed. Exogenously applied 5-methylbenzimidazole (5-MeBza), 5-hydroxybenzimidazole (5-OHBza), and 5-methoxybenzimidazole (5-OMeBza) were found to be efficiently incorporated as lower bases into norcobamides (NCbas). Structural analysis of the NCbas by nuclear magnetic resonance spectroscopy uncovered a regioselectivity in the utilization of these precursors for NCba biosynthesis. When 5-MeBza was added, a mixture of 5-MeBza-norcobamide and 6-MeBza-norcobamide was formed, and the PceA enzyme activity was affected. In the presence of 5-OHBza, almost exclusively 6-OHBza-norcobamide was produced, while in the presence of 5-OMeBza, predominantly 5-OMeBza-norcobamide was detected. Both NCbas were incorporated into PceA, and no negative effect on the PceA activity was observed. In crystal structures of PceA, both NCbas were bound in the base-off mode with the 6-OHBza and 5-OMeBza lower bases accommodated by the same solvent-exposed hydrophilic pocket that harbors the adenine as the lower base of authentic norpseudo-B12 In this study, a selective production of different norcobamide isomers containing singly substituted benzimidazoles as lower bases is shown, and unique structural insights into their utilization as cofactors by a cobamide-containing enzyme are provided.IMPORTANCE Guided biosynthesis of norcobamides containing singly substituted benzimidazoles as lower bases by the organohalide-respiring epsilonproteobacterium Sulfurospirillum multivorans is reported. An unprecedented specificity in the formation of norcobamide isomers containing hydroxylated or methoxylated benzimidazoles was observed that implicated a strict regioselectivity of the norcobamide biosynthesis in the organism. In contrast to 5,6-dimethylbenzimidazolyl-norcobamide, the incorporation of singly substituted benzimidazolyl-norcobamides as a cofactor into the tetrachloroethene reductive dehalogenase was not impaired. The enzyme was found to be functional with different isomers and not limited to the use of adeninyl-norcobamide. Structural analysis of the enzyme equipped with either adeninyl- or benzimidazolyl-norcobamide cofactors visualized for the first time structurally different cobamides bound in base-off conformation to the cofactor-binding site of a cobamide-containing enzyme.

Dispersing perylene diimide/SWCNT hybrids: structural insights at the molecular level and fabricating advanced materials.

The unique properties of carbon nanotubes (CNT) are advantageous for emerging applications. Yet, the CNT insolubility hampers their potential. Approaches based on covalent and noncovalent methodologies have been tested to realize stable dispersions of CNTs. Noncovalent approaches are of particular interest as they preserve the CNT's structures and properties. We report on hybrids, in which perylene diimide (PDI) amphiphiles are noncovalently immobilized onto single wall carbon nanotubes (SWCNT). The resulting hybrids were dispersed and exfoliated both in water and organic solvents in the presence of two different PDI derivatives, PP2b and PP3a. The dispersions were investigated using cryogenic transmission electron microscopy (cryo-TEM), providing unique structural insights into the exfoliation. A helical arrangement of PP2b assemblies on SWCNTs dominates in aqueous dispersions, while a single layer of PP2b and PP3a was found on SWCNTs in organic dispersions. The dispersions were probed by steady-state and time-resolved spectroscopies, revealing appreciable charge redistribution in the ground state, and an efficient electron transfer from SWCNTs to PDIs in the excited state. We also fabricated hybrid materials from the PP2b/SWCNT dispersions. A supramolecular membrane was prepared from aqueous dispersions and used for size-selective separation of gold nanoparticles. Hybrid buckypaper films were prepared from the organic dispersions. In the latter, high conductivity results from enhanced electronic communication and favorable morphology within the hybrid material. Our findings shed light onto SWCNT/dispersant molecular interactions, and introduce a versatile approach toward universal solution processing of SWCNT-based materials.

Multinuclear solid-state NMR characterization of the Brønsted/Lewis acid properties in the BP HAMS-1B (H-[B]-ZSM-5) borosilicate molecular sieve using adsorbed TMPO and TBPO probe molecules

The acid properties of a dehydrated borosilicate, HAMS-1B (H-[B]-ZSM-5), including the acid types, strengths, location, and quantities are investigated by means of trialkylphosphine oxides through multinuclear 1D/2D MAS NMR experiments. 11B DQF-STMAS combined with 1H MAS NMR studies revealed B–OH and distinct Si–OH protons associated with trigonal boron. 31P NMR spectra of TMPO-treated HAMS-1B reveal three Brønsted and three Lewis acid sites. We have found a number of limitations applying the TMPO/TBPO method to identify internal/external acidity. Therefore, we propose a new approach to unambiguously discriminate external/internal acid sites by treating a pore-free and pore-blocked HAMS-1B zeolite. This method provided unique structural insight regarding the identification of boron species/coordinations associated with Brønsted/Lewis acid sites. Additionally, ICP analysis in tandem with solid-state NMR enabled full assignment of the detected internal/external acid species and the study of their acid strength. Moreover, we identify the nature of TMPO complexes arising from Brønsted/Lewis interactions.

Structural and molecular insights into novel surface‐exposed mucus adhesins from Lactobacillus reuteri human strains

The mucus layer covering the gastrointestinal tract is the first point of contact of the intestinal microbiota with the host. Cell surface macromolecules are critical for adherence of commensal bacteria to mucus but structural information is scarce. Here we report the first molecular and structural characterization of a novel cell-surface protein, Lar_0958 from Lactobacillus reuteri JCM 1112(T) , mediating adhesion of L. reuteri human strains to mucus. Lar_0958 is a modular protein of 133 kDa containing six repeat domains, an N-terminal signal sequence and a C-terminal anchoring motif (LPXTG). Lar_0958 homologues are expressed on the cell-surface of L. reuteri human strains, as shown by flow-cytometry and immunogold microscopy. Adhesion of human L. reuteri strains to mucus in vitro was significantly reduced in the presence of an anti-Lar_0958 antibody and Lar_0958 contribution to adhesion was further confirmed using a L. reuteri ATCC PTA 6475 lar_0958 KO mutant (6475-KO). The X-ray crystal structure of a single Lar_0958 repeat, determined at 1.5 Å resolution, revealed a divergent immunoglobulin (Ig)-like β-sandwich fold, sharing structural homology with the Ig-like inter-repeat domain of internalins of the food borne pathogen Listeria monocytogenes. These findings provide unique structural insights into cell-surface protein repeats involved in adhesion of Gram-positive bacteria to the intestine.

Development of a High Throughput X-Ray Footprinting Facility at the Advanced Light Source to Study the Structure and Dynamics of Complex Biological Macromolecules

Radiolytic footprinting is an increasingly popular method for structural elucidation of macromolecules in the solution state. X-ray footprinting in particular has developed in the past decade into a nearly routine technique and had been applied to a diverse range of biological systems, yielding unique structural insights impossible to obtain using the more standard structural methods of crystallography, NMR, electron microscopy and small angle scattering. The technique was pioneered at the National Synchrotron Light Source (NSLS) over the past decade; now with the user community continuing to grow and the anticipated closure of this facility, it is imperative that x-ray footprinting continue to be developed. Towards this end we are building a new footprinting beamline at the Advanced Light Source (ALS) synchrotron which will not only support the NSLS users during the commissioning of the NSLS-II, but will provide unique capabilities for the continued development of the method. In particular, our preliminary data show that flux densities produced by an ALS focused white-light bend magnet beamline are high enough to allow microsecond exposures yielding sufficient modification to conduct footprinting experiments. In addition, in-house mass spectrometry availability and expertise will allow the installation of a mass spectrometer instrumentation directly at the beamline. Combined with commercially available tools for automated sample handling, rapid sample digestion and data processing methods, this will allow rapid screening of a large number of samples and will be especially important for samples sensitive to degradation. The high-throughput methodology and microsecond regime made available at the ALS beamline will be highly complementary to the planned NSLS-II footprinting beamline, and both resources will be necessary to accommodate the growing national user community.

ChemInform Abstract: Applications of High‐Resolution 1H Solid‐State NMR

AbstractReview: 310 refs.

How Random are Intrinsically Disordered Proteins? A Small Angle Scattering Perspective

While the crucial role of intrinsically disordered proteins (IDPs) in the cell cycle is now recognized, deciphering their molecular mode of action at the structural level still remains highly challenging and requires a combination of many biophysical approaches. Among them, small angle X-ray scattering (SAXS) has been extremely successful in the last decade and has become an indispensable technique for addressing many of the fundamental questions regarding the activities of IDPs. After introducing some experimental issues specific to IDPs and in relation to the latest technical developments, this article presents the interest of the theory of polymer physics to evaluate the flexibility of fully disordered proteins. The different strategies to obtain 3-dimensional models of IDPs, free in solution and associated in a complex, are then reviewed. Indeed, recent computational advances have made it possible to readily extract maximum information from the scattering curve with a special emphasis on highly flexible systems, such as multidomain proteins and IDPs. Furthermore, integrated computational approaches now enable the generation of ensembles of conformers to translate the unique flexible characteristics of IDPs by taking into consideration the constraints of more and more various complementary experiment. In particular, a combination of SAXS with high-resolution techniques, such as x-ray crystallography and NMR, allows us to provide reliable models and to gain unique structural insights about the protein over multiple structural scales. The latest neutron scattering experiments also promise new advances in the study of the conformational changes of macromolecules involving more complex systems.

BiotecVisions 2011, September

BiotecVisions 2011, September

Insights into protein folding from NMR.

NMR has emerged as an important tool for studies of protein folding because of the unique structural insights it can provide into many aspects of the folding process. Applications include measurements of kinetic folding events and structural characterization of folding intermediates, partly folded states, and unfolded states. Kinetic information on a time scale of milliseconds or longer can be obtained by real-time NMR experiments and by quench-flow hydrogen-exchange pulse labeling. Although NMR cannot provide direct information on the very rapid processes occurring during the earliest stages of protein folding, studies of isolated peptide fragments provide insights into likely protein folding initiation events. Multidimensional NMR techniques are providing new information on the structure and dynamics of protein folding intermediates and both partly folded and unfolded states.