Dynamic Regulation of Histone Chaperone Nucleoplasmin

Small-angle X-ray scattering (SAXS) analysis of nucleoplasmin (Npm) Core+A2 truncation (1-145) bound to five H2A/H2B dimers. (Top) SAXs envelope of the pentameric complex (pink) with the best nuclear magnetic resonance (NMR)–restrained SAXS hybrid model inside. (Bottom) SAXS curve of the complex (purple dots). Simulated SAXS curve (black line) from the best-scoring structural model. [From Warren, C., et al. “Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release.” Nat. Commun. 8, 2215 (2017). DOI:10.1038/s41467-017-02308-3. Reused under a Creative Commons license (CC BY 4.0, https://creativecommons.org/licenses/by/4.0/​).]
Histones are eukaryotic cell nuclei proteins that package and order DNA into structural units called nucleosomes. Chromatin is the complex of DNA and proteins comprising the genome’s physiological form. As chromatin’s chief protein components, histones act as spools around which DNA winds, playing a role in gene regulation. A chaperone protein assists in the folding and unfolding of macromolecules, such as in the assembly of nucleosomes from folded histones and DNA. Nucleoplasmin (Npm) is a highly conserved embryonic histone chaperone, responsible for the maternal storage and zygotic release of histones H2A and H2B. Npm contains a pentameric N-terminal Core domain and an intrinsically disordered C-terminal Tail domain. Although intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning have remained unclear.

This study, using the Xenopus laevis Npm Tail domain, unveils the architecture of the Npm histone complex and a mechanism of histone chaperone regulation. It demonstrates that intramolecular regulation of the histone chaperone Npm controls histone binding and release—a key process in the earliest stages of embryonic development. Structural analyses enabled model constructions of both the Npm Tail domain and the pentameric complex, revealing that the Tail domain controls the binding of histones through specific, electrostatic interactions. Functional analyses demonstrated that these competitive interactions negatively regulate Npm histone chaperone activity in vitro. Data from these studies establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites.

Warren, C., et al. “Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release.” Nat. Commun. 8, 2215 (2017). DOI:10.1038/s41467-017-02308-3.

Instruments and Facilities Used: Bruker 600 nuclear magnetic resonance (NMR) and Inova 600 NMR instruments in the Albert Einstein College of Medicine (AECM) Einstein Structural NMR Resource; and bio–small-angle X-ray scattering (bio-SAXS) beamline 4-2, SLAC National Accelerator Laboratory’s Stanford Synchrotron Radiation Lightsource (SSRL).

“Zip-code” Mechanism for Hormone Signaling Revealed

Crystal structure of multikinase inhibitor sKLB bound to fibroblast growth factor (FGF) FGF21CT reveals two distinct binding sites. Structure of extracellular domain of β-Klotho “longevity” protein (green ribbons) bound to FGF21 hormone (salmon ball-and-stick) unveils critical molecular interactions required for hormone binding and cell activation. Yellow sticks denote nitrogen (N)-linked glycans. Grey dashed lines denote regions that do not exhibit significant electron density. About 30 angsroms (Å) apart, the FGF binding sites on the β-Klotho D1 and D2 domains are located on the opposite side of the molecule from the flexible linker that connects the two glycosidase domains and may contribute to the interdomain dynamic properties that enable complex formation with ligands and FGF receptors (FGFRs). [Reprinted by permission from Springer Nature: Lee, S., et al. “Structures of β-klotho reveal a ‘zip code’-like mechanism for endocrine FGF signaling.” Nature 553, 501–505 (2018). DOI:10.1038/nature25010. Copyright 2018]
Named after the Greek goddess who spun the thread of life, Klotho proteins play an important role in the regulation of longevity and metabolism. Alpha-Klotho is a membrane-spanning protein expressed predominantly in the kidney, as well as in the brain. Mice lacking α-klotho exhibit a range of signs associated with aging and have elevated blood phosphate levels. Like α-klotho, β-klotho functions as a co-receptor for endocrine fibroblast growth factors (FGFs). FGF21 is secreted from the liver following fasting, acting in fat cells and the brain to induce metabolic adaptation to fasting and responses to stress. Although FGF receptors (FGFRs) are expressed in a wide range of tissues, expression of the β-klotho “longevity” protein in the liver, fat, and brain restricts the target organs of these endocrine FGFs.

In a recent Yale Medical School–led study, researchers revealed the three-dimensional (3D), high-resolution structure of β-Klotho, illuminating its intricate mechanism and potential for antiaging therapeutics, as well as for treating a wide range of medical conditions. X-ray crystallography data revealed critical molecular interactions required for hormone binding and cell activation. The specific “zip-code”–like interactions of β-klotho receptors appear to regulate critical metabolic processes in the liver, kidneys, and brain, among other organs. Analysis yielded several insights, including that β-Klotho is the primary receptor that binds to FGF21, a key hormone that stimulates insulin sensitivity and glucose metabolism, causing weight loss. The researchers believe this new understanding can guide the development of therapies by improving the biological activity of FGF21. Also found were a new variant of FGF21 that has 10 times higher potency and cellular activity and the evidence of how a structurally related enzyme (glycosidase) that breaks down sugars evolved into a receptor for a hormone that lowers blood sugar. Researchers believe the untangled β-Klotho structure presents a platform for exploring and developing agents that either enhance or block the pathway, enabling therapies for conditions such as liver cancer and bone diseases.

Lee, S., et al. “Structures of β-klotho reveal a ‘zip code’-like mechanism for endocrine FGF signaling.” Nature 553, 501–505 (2018). DOI:10.1038/nature25010.

Instruments and Facilities Used: X-ray data were collected from β-klotho–receptor crystals at Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, beamline 14-1 as part of the program to aid National Synchrotron Light Source (NSLS) users prior to NSLS-II operations.

Structure of a Flavoenzyme Assembly Intermediate

(a) The structure of the FrdA-SdhE flavoenzyme assembly intermediate: flavoprotein subunit FrdA (cyan), assembly factor SdhE (green), flavin adenine dinucleotide FAD (orange sticks), and malonate (yellow sticks). The boxed region highlights the covalent interaction between the FAD and the enzyme. (b) Overlay of the flavin-binding domains of the FrdA subunit from the FrdA-SdhE intermediate (cyan) and the FrdA subunit from the mature assembled FrdABCD complex (gray). A rotation of 10.8° is observed in the capping domain of the assembly intermediate when compared to assembled FrdABCD. [From Sharma, P., et al. “Crystal structure of an assembly intermediate of respiratory Complex II,” Nat. Commun. 9, 274 (2018). DOI:10.1038/s41467-017-02713-8. Reused under a Creative Commons license (CC BY 4.0, https://creativecommons.org/licenses/by/4.0/​.)]
Enzymes frequently depend on an electron transport cofactor for executing catalytic functions such as reduction-oxidation (redox) reactions. For flavoenzymes, the cofactor is flavin adenine dinucleotide (FAD), whose binding type with the enzyme impacts the redox potential and thus reaction chemistry, such as for metabolism and detoxification. Researchers in this study discovered that the structure of an assembled flavoenzyme intermediate reveals the mechanism of covalent flavin binding in respiration. Assembly factors include SdhAF2 in humans, SdhE in Escherichia coli, and Sdh5 in yeast. Other revelations include that mitochondrial flavoenzymes drive both noncovalent and covalent redox reactions and that the assembly factor (SdhE, a small protein of ~90 to 140 amino acids, conserved in all kingdoms) in the structure of the SdhE:FrdA complex with covalent FAD stabilizes a conformation of the flavoprotein subunit FrdA that favors succinate oxidation.

Researchers fixed the E. coli FrdA-SdhE intermediate via site-specific crosslinking, resolving the structure to 2.6 angstroms (Å). This study identified that SdhE stabilizes an FrdA conformation that likely enables the mechanism of autocatalytic covalent flavinylation. FrdA’s FAD-binding domain and capping domain both interact with SdhE, but structural data revealed a 10.8° difference in their angles. The investigators believe that domain rotation affects flavinylation, showing that enzymes are tuned to catalyze reactions in different ways and that conformational diversity can directly relate to catalytic mechanism diversity.

Sharma, P., et al. “Crystal structure of an assembly intermediate of respiratory Complex II.” Nat. Commun. 9, 274 (2018). DOI:10.1038/s41467-017-02713-8.

Instruments and Facilities Used: Small angle X-ray scattering (SAXS) and diffraction and mass spectrometry analysis using beamline 9-2 at Stanford Synchrotron Radiation Lightsource (SSRL) at SLAC National Accelerator Laboratory (SLAC).

A Regional Model for Uranium Redox and Mobility

Anoxic organic-enriched sediments strongly accumulate uranium as U(IV), sulfide, and other reduced species. Seasonal reduction-oxidation (redox) cycling triggered by changing water tables can intermittently mobilize these species. [Courtesy Vincent Noël and John Bargar, Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory]
Uranium (U) contamination stubbornly persists as a challenging and costly water quality concern at former uranium ore processing sites across the Upper Colorado River Basin (UCRB). Plumes at these sites are not self-attenuating via natural flushing by groundwater as originally expected. Recent studies at the Rifle, Colo., legacy site suggest that organic-enriched anoxic sediments create conditions that promote reduction of U(VI) to relatively immobile U(IV), causing it to accumulate locally under persistently saturated and anoxic conditions. However, incursion of oxidants into reduced sediments could transform contaminants, allowing these sediments to act as secondary sources of uranium. Oxidant incursions take place during periods of changing water tables, which occur in UCRB throughout the year. If these sediments were regionally common in the UCRB and exposed to varying reduction-oxidation (redox) conditions, then they could contribute to maintaining the longevity of regional uranium plumes.

To investigate these issues, researchers examined the occurrence and distribution of reduced and oxidized iron (Fe), sulfur (S), and U species in sediment cores spanning dry and oxic to wet and reduced conditions at three different UCRB sites. Detailed molecular characterization involved chemical extractions, X-ray absorption spectroscopy (XAS), Mössbauer spectroscopy, and X-ray microspectroscopy. This work demonstrates that anoxic organic-enriched sediments occur at all sites, strongly accumulate sulfides and U, and are exposed to strong seasonal redox cycles. Uranium was found to be present as U(IV) complexed to sediment-associated organic carbon and possibly to mineral surfaces. This finding is significant because complexed U(IV) is relatively susceptible to oxidative mobilization. Sediment particle size, organic carbon content, and pore saturation control redox conditions in sediments and thus strongly influence Fe, S, and U biogeochemistry. These findings help to illuminate the mechanistic linkages between hydrology, sediment texture, and biogeochemistry. They further provide enhanced contextual and conceptual underpinnings to support reactive transport modeling of uranium, other contaminants, and nutrients in redox-variable floodplains—a subject of importance to BER research missions. Cyclic redox variability has major implications for mobility of carbon (C), nitrogen (N), and metal contaminants in groundwater and surface waters. Redox-variable, organic-enriched sediments mediate the mobility of C, N, Fe, S, U, and metal contaminants regionally in the UCRB. Organic-enriched sediments were established to regionally mediate groundwater quality within the UCRB.

Noël, V., et al. “Understanding controls on redox processes in floodplain sediments of the Upper Colorado River Basin.” Sci. Total Environ. 603–604, 663–675 (2017). DOI:10.1016/j.scitotenv.2017.01.109.

Noël, V., et al. “Redox constraints over U(IV) mobility in the floodplains of Upper Colorado River Basin.” Environ. Sci. Technol. 51(19), 10954–10964 (2017). DOI:10.1021/acs.est.7b02203.

Instruments and Facilities Used: X-ray absorption spectroscopy and X-ray microprobe mapping at Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory (SLAC), and Mössbauer spectroscopy at Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL).

New Chemical Strategy for Conversion of CO2 to Biomass

acetone carboxylase
Upon nucleotide binding, acetone carboxylase undergoes conformational shifts, opening an internal solvent channel. (a) Ligand-free structure showing a substrate channel (grey) linking the nucleotide binding site to the outside solvent and allowing adenosine triphosphate (ATP) cofactor and carbon dioxide (CO2) substrate to enter. Access to the manganese (Mn) active site is closed by an α-helix in the path. (b) The adenosine monophosphate (AMP)–bound structure shows an opening of an internal channel (grey) linking the nucleotide binding site to the Mn active site. The blocking helix becomes a disordered loop when AMP is bound. [From Mus, F., et al. “Structural Basis for the Mechanism of ATP-Dependent Acetone Carboxylation.” Nat. Sci. Rep. 7, Article 7234 (2017). DOI:10.1038/s41598-017-06973-8. Reused under a Creative Commons license (CC by 4.0, https://creativecommons.org/licenses/by/4.0/).]
A research team has uncovered a new mechanism for enzyme-mediated carbon dioxide (CO2) capture and conversion. They have revealed new and unique elements of carboxylation chemistry (CC) that could be used in catalytic strategies for converting CO2 into chemical feedstock or biomass. The crystal structure of acetone carboxylase (AC; enzyme involved in biodegradation by bacteria) was solved using X-ray crystallographic data. The inactive, unbound (apo) structure shows a substrate channel blocked off from the manganese (Mn) active site where CO2 conversion takes place. An adenosine monophosphate (AMP)–bound structure contains large conformational changes that open the channel to the active site. Highly reactive intermediates in the channel are protected from outside solvent as they are transported to the Mn active site for CO2 conversion. Stepwise mechanisms of carboxylation reactions differ in essential ways with respect to co-substrate, co-factor, and metal requirements. Knowledge of these mechanisms provides the basis for an increased fundamental understanding of CC, contributing to future strategies for CO2 capture and conversion to biomass. These in turn may mitigate the effects of increasing concentrations of CO2 on the global climate.

Mus, F., et al. “Structural Basis for the Mechanism of ATP-Dependent Acetone Carboxylation.” Nat. Sci. Rep. 7, Article 7234 (2017). [DOI:10.1038/s41598-017-06973-8].

Instruments and Facilities Used: X-ray crystallographic data from the Stanford Synchrotron Lightsource (SSLS) at SLAC National Accelerator Laboratory (SLAC). Structural data from macromolecular crystallography were measured on SSLS beamline 12-2; Advanced Photon Source (APS) at Argonne National Laboratory (ANL); and the Diamond Light Source, Oxfordshire, United Kingdom.

Engineering Better Plant Cultivars for Iron Uptake by Modifying the Fe Deficiency Response in Arabidopsis thaliana

Root tip scans
(Left) Representative synchrotron X-ray fluorescent (SXRF) scans of root tips of plants grown for 7 days on B5 medium and then transferred to iron (Fe) medium for 3 days. (Middle) SXRF scan of leaves showing Fe (red). (Right) SXRF scan showing Fe (red), zinc (green), manganese (blue) localization in developed, green siliques. [Reproduced from Hindt, M. N., et al. “BRUTUS and Its Paralogs, BTS LIKE1 and BTS LIKE2, Encode Important Negative Regulators of the Iron Deficiency Response in Arabidopsis thaliana.” Metallomics 9(7), 876–890 (2017) with permission of The Royal Society of Chemistry. 10.1039/C7MT00152E]
Many populations in developing countries rely on plants for dietary iron (Fe)—essential for plant growth, crop yields, and human health, but, due to its low or limited solubility, Fe is sparingly available in neutral or basic soils and thus not readily accessible in the rhizosphere. Leading to a restricted Fe content in many plants, this low solubility is a major factor contributing to the widespread prevalence of Fe deficiency anemia in people with plant-based diets. Thus, increasing plant Fe acquisition and storage may have profound impacts on plant and human nutrition and can be achieved by manipulating genes and related mechanisms governing Fe homeostasis in plants. However, understanding the balance between positive and negative regulation of the Fe deficiency response is essential for efforts to engineer plants having a sufficient but not toxic level of Fe. Although plants often are challenged with Fe deficiency, no environment remains constant, making Fe availability in the rhizosphere dependent on many factors. When sufficient Fe is available, plants must effectively suppress Fe-deficiency response to avoid excessive uptake.

A research team has identified a novel Fe-binding domain allele called BTS in a mutagenesis screen for altered Fe accumulation (bts-3). Data showed that bts-3 is more tolerant than wild type to Fe-deficient conditions and that bts-3 is sensitive to Fe-sufficient conditions and accumulates excessive Fe. A triple mutant with loss of both BTS paralogs and a partial loss of BTS expression exhibits even greater tolerance to Fe-deficient conditions and increased Fe accumulation without any resulting Fe toxicity effects, with the mutations also changing their uptake of important minerals such as zinc (Zn) and manganese (Mn). Genetic knockdowns and modifications of the proteins have been implicated in regulating plant uptake of Fe. This work will lead to greater understanding of plant Fe homeostasis to inform efforts for improved crops. Identifying natural variants of these genes in crop species may lead to traditional breeding efforts to generate higher-Fe cultivars.

Hindt, M. N., et al. “BRUTUS and its Paralogs, BTS LIKE1 and BTS LIKE2, Encode Important Negative Regulators of the Iron Deficiency Response in Arabidopsis thaliana.” Metallomics 9(7), 876–890 (2017). [DOI:10.1039/C7MT00152E].

Instruments and Facilities Used: PerkinElmer LAS Ltd, Seer Green, United Kingdom, and Elemental Scientific Inc., Omaha, Neb.; synchrotron X-ray fluorescence (SXRF) at National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory; Stanford Synchrotron Lightsource (SSLS) at SLAC National Accelerator Laboratory (SLAC); real-time quantitative PCR (Step One Plus Real Time PCR System using Applied Biosystems Version 2.2.3; Advanced Photon Source (APS) at Argonne National Laboratory (ANL); and Australian Synchrotron, Victoria. Two-dimensional SXRF analysis was performed at various X-ray microprobe beamlines: microarray analysis and X-ray fluorescence imaging (XFI) on SSLS beamline 2-3 at SLAC; X26A and X27A of NSLS; XFM beamline of the Australian Synchrotron; APS beamline 2-ID-D. Microarray analysis performed at Geisel School of Medicine in the Genomics Shared Resource at Dartmouth College. Elemental concentration analysis (inductively coupled plasma-mass spectrometry (ICP-MS, PerkinElmer NexION 300D equipped with Elemental Scientific Inc. autosampler and Apex HF sample introduction system at PerkinElmer LAS Ltd, Seer Green, U.K., and Elemental Scientific Inc., Omaha, Neb., respectively, in the standard mode.

Probing S-layer Protein Structural Dynamics by SAXS

Biophysical Journal Cover Image
Calcium mediates the structural state of the Caulobacter crescentus surface layer protein, RsaA. Image featured on the cover of Biophysical Journal.

All archaea, and many bacteria, possess a protein shell referred to as a surface layer (S-layer), which usually consist of a single protein that self-assembles into a two-dimensional (2D) crystal lattice.  Studies have revealed the structural dynamics of this S-layer protein from the model bacterium Caulobacter crescentus, called RsaA. Using small angle scattering and diffraction (SAXS/D) techniques, multiple structural states of RsaA were successfully characterized including monomeric, aggregated, and crystalline states (see figure), with only monomeric Rssa forming 2D crystals. Enabling differentiation of the discrete states, these results rationalize physiological data implicating RsaA as a player in environmental adaptation of C. crescentus. The findings also provide a biochemical and physiological basis for RsaA’s calcium (Ca)-binding behavior, which extends far beyond Ca’s usual role in S-layer biology of aiding biogenesis or oligomerization, and demonstrate a connection to cellular fitness. Further characterization using slow and fast time-resolved SAXS/D methods is ongoing.

SAXS/D data
Small angle X-ray scattering and diffraction (SAXS/D) data of five solutions with different concentrations of the Caulobacter crescentus S-layer protein, RsaA, in the presence of calcium (Ca). Scattering profiles indicate concentration-dependent crystallization. Automatic indexing of the numbered peaks yielded a hexagonal crystal lattice consistent with predictions and denoted by Miller indices. (Top) The diffraction pattern obtained for the highest concentration used (8 mg/ml) shows powder rings. (Bottom) Transmission electron microscopy of the 8 mg/ml RsaA in the presence of 10 millimole per Liter (mm/L) of calcium chloride (CaCl2) (scale bar 200 nm). [Reprinted from Herrmann, J., et al. “Environmental Calcium Controls Alternate Physical States of the Caulobacter Surface Layer.” Biophys. J. 112(9), 1841–1851 (2017). DOI:10.1016/j.bpj.2017.04.003. Copyright 2017, with permission from Elsevier.]
Herrmann, J., et al. “Environmental Calcium Controls Alternate Physical States of the Caulobacter Surface Layer,” Biophys. J. 112(9), 1841–1851 (2017). [DOI:10.1016/j.bpj.2017.04.003].

Instruments and Facilities Used: Stanford ChEM-H Macromolecular Structure Knowledge Center,  Stanford Department of Structural Biology Electron Microscopy Center, Stanford Synchrotron Radiation Lightsource (SSRL) at SLAC National Accelerator Laboratory (SLAC). Beamlines or instruments used: transmission electron microscopy (TEM) and small angle X-ray scattering and diffraction (SAXS/D) at SSRL beamline 4-2 at SLAC.

Understanding Nitrogen Fixation in Bacteria

 

Nitrogen fixation is required for all forms of life, being essential for the biosynthesis of molecules that are used in creating plants and organisms.  Nitrogenase is the only known enzyme capable of performing this multi-electron reduction, and understanding how it does this conversion is of high importance also for the production of ammonia (as fertilizer), for energy efficiency (as industrial processes to produce ammonia consumes enormous amounts of energy), and for global warming (capturing N2).  The structure of the CO inhibitor bound to the FeMo-cofactor active site in nitrogenase at high resolution provides insight into a catalytic competent state, establishes the importance of a bridging S atom, and indicates how N2 might bind during turn-over. Stanford Synchrotron Radiation Lightsource.

Spatzal, K.A. Perez, O. Einsle, J.B. Howard, D.C. Rees, “Ligand binding to the FeMo-cofactor: Structures of CO-bound and reactivated nitrogenase” Science 345, 1620-1623 (2014), doi: 10.1126/science.1256679

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