FACT caught in the act of manipulating the nucleosome.

The organization of genomic DNA into nucleosomes profoundly affects all DNA-related processes in eukaryotes. The histone chaperone known as 'facilitates chromatin transcription' (FACT1) (consisting of subunits SPT16 and SSRP1) promotes both disassembly and reassembly of nucleosomes during gene transcription, DNA replication and DNA repair2. However, the mechanism by which FACT causes these opposing outcomes is unknown. Here we report two cryo-electron-microscopic structures of human FACT in complex with partially assembled subnucleosomes, with supporting biochemical and hydrogen-deuterium exchange data. We find that FACT is engaged in extensive interactions with nucleosomal DNA and all histone variants. The large DNA-binding surface on FACT appears to be protected by the carboxy-terminal domains of both of its subunits, and this inhibition is released by interaction with H2A-H2B, allowing FACT-H2A-H2B to dock onto a complex containing DNA and histones H3 and H4 (ref. 3). SPT16 binds nucleosomal DNA and tethers H2A-H2B through its carboxy-terminal domain by acting as a placeholder for DNA. SSRP1 also contributes to DNA binding, and can assume two conformations, depending on whether a second H2A-H2B dimer is present. Our data suggest a compelling mechanism for how FACT maintains chromatin integrity during polymerase passage, by facilitating removal of the H2A-H2B dimer, stabilizing intermediate subnucleosomal states and promoting nucleosome reassembly. Our findings reconcile discrepancies regarding the many roles of FACT and underscore the dynamic interactions between histone chaperones and nucleosomes.

Mechanism of RanGTP priming H2A-H2B release from Kap114 in an atypical RanGTP•Kap114•H2A-H2B complex.

Previously, we showed that the nuclear import receptor Importin-9 wraps around the H2A-H2B core to chaperone and transport it from the cytoplasm to the nucleus. However, unlike most nuclear import systems where RanGTP dissociates cargoes from their importins, RanGTP binds stably to the Importin-9•H2A-H2B complex, and formation of the ternary RanGTP•Importin-9•H2A-H2B complex facilitates H2A-H2B release to the assembling nucleosome. It was unclear how RanGTP and the cargo H2A-H2B can bind simultaneously to an importin, and how interactions of the three components position H2A-H2B for release. Here, we show cryo-EM structures of Importin-9•RanGTP and of its yeast homolog Kap114, including Kap114•RanGTP, Kap114•H2A-H2B, and RanGTP•Kap114•H2A-H2B, to explain how the conserved Kap114 binds H2A-H2B and RanGTP simultaneously and how the GTPase primes histone transfer to the nucleosome. In the ternary complex, RanGTP binds to the N-terminal repeats of Kap114 in the same manner as in the Kap114/Importin-9•RanGTP complex, and H2A-H2B binds via its acidic patch to the Kap114 C-terminal repeats much like in the Kap114/Importin-9•H2A-H2B complex. Ran binds to a different conformation of Kap114 in the ternary RanGTP•Kap114•H2A-H2B complex. Here, Kap114 no longer contacts the H2A-H2B surface proximal to the H2A docking domain that drives nucleosome assembly, positioning it for transfer to the assembling nucleosome or to dedicated H2A-H2B chaperones in the nucleus.

Leveraging intrinsic flexibility to engineer enhanced enzyme catalytic activity.

Dynamic motions of enzymes occurring on a broad range of timescales play a pivotal role in all steps of the reaction pathway, including substrate binding, catalysis, and product release. However, it is unknown whether structural information related to conformational flexibility can be exploited for the directed evolution of enzymes with higher catalytic activity. Here, we show that mutagenesis of residues exclusively located at flexible regions distal to the active site of Homo sapiens kynureninase (HsKYNase) resulted in the isolation of a variant (BF-HsKYNase) in which the rate of the chemical step toward kynurenine was increased by 45-fold. Mechanistic pre­steady-state kinetic analysis of the wild type and the evolved enzyme shed light on the underlying effects of distal mutations (>10 Å from the active site) on the rate-limiting step of the catalytic cycle. Hydrogen-deuterium exchange coupled to mass spectrometry and molecular dynamics simulations revealed that the amino acid substitutions in BF-HsKYNase allosterically affect the flexibility of the pyridoxal-5'-phosphate (PLP) binding pocket, thereby impacting the rate of chemistry, presumably by altering the conformational ensemble and sampling states more favorable to the catalyzed reaction.

A positive spin on the centromere.

The properties of centromeric nucleosomes have been the subject of considerable debate and controversy. Furuyama and Henikoff (2009) now provide surprising evidence that centromeric nucleosomes wrap DNA in an orientation that is opposite to that of canonical nucleosomes.

Hydrogen-deuterium exchange mass spectrometry of Mtr4 with diverse RNAs reveals substrate-dependent dynamics and interfaces in the arch.

Mtr4 is a eukaryotic RNA helicase required for RNA decay by the nuclear exosome. Previous studies have shown how RNA en route to the exosome threads through the highly conserved helicase core of Mtr4. Mtr4 also contains an arch domain, although details of potential interactions between the arch and RNA have been elusive. To understand the interaction of Saccharomyces cerevisiae Mtr4 with various RNAs, we have characterized RNA binding in solution using hydrogen-deuterium exchange mass spectrometry, and affinity and unwinding assays. We have identified RNA interactions within the helicase core that are consistent with existing structures and do not vary between tRNA, single-stranded RNA and double-stranded RNA constructs. We have also identified novel RNA interactions with a region of the arch known as the fist or KOW. These interactions are important for RNA unwinding and vary in strength depending on RNA structure and length. They account for Mtr4 discrimination between different RNAs. These interactions further drive Mtr4 to adopt a closed conformation characterized by reduced dynamics of the arch arm and intra-domain contacts between the fist and helicase core.

HD-eXplosion: visualization of hydrogen-deuterium exchange data as chiclet and volcano plots with statistical filtering.

SUMMARY: Hydrogen-Deuterium eXchange coupled to mass spectrometry is a powerful tool for the analysis of protein dynamics and interactions. Bottom-up experiments looking at deuterium uptake differences between various conditions are the most common. These produce multi-dimensional data that can be challenging to depict in a single visual format. Each user must also set significance thresholds to define meaningful differences and make these apparent in data presentation. To assist in this process, we have created HD-eXplosion, an open-source, web-based application for the generation of chiclet and volcano plots with statistical filters. HD-eXplosion fills a void in available software packages and produces customizable plots that are publication quality. AVAILABILITY AND IMPLEMENTATION: The HD-eXplosion application is available at http://hd-explosion.utdallas.edu. The source code can be found at https://github.com/HD-Explosion.

Comprehensive analysis of histone-binding proteins with multi-angle light scattering.

Interactions between histones and their binding partners are an important aspect of chromatin biology. Determining the stoichiometry of histone-containing complexes is an important pre-requisite for performing in vitro biochemical, biophysical and structural analyses. In this article, we detail how Size Exclusion Chromatography (SEC) coupled to Multi-Angle Light Scattering (MALS) can be used to study histone chaperones and their complexes. Our protocol details system setup, sample preparation, data collection, and data interpretation. We provide tips on designing an informative SEC-MALS experiment, using histone chaperones Nap1 and Vps75 as demonstrative examples. We outline recommendations to overcome specific challenges such as protein oligomerization, heterogeneity, and non-specific binding. We find SEC-MALS to be a robust and user-friendly approach for characterizing histone-binding proteins and their complexes.

Chaperone Nap1 shields histone surfaces used in a nucleosome and can put H2A-H2B in an unconventional tetrameric form.

The histone H2A-H2B heterodimer is an integral component of the nucleosome. The cellular localization and deposition of H2A-H2B into chromatin is regulated by numerous factors, including histone chaperones such as nucleosome assembly protein 1 (Nap1). We use hydrogen-deuterium exchange coupled to mass spectrometry to characterize H2A-H2B and Nap1. Unexpectedly, we find that at low ionic strength, the α helices in H2A-H2B are frequently sampling partially disordered conformations and that binding to Nap1 reduces this conformational sampling. We identify the interaction surface between H2A-H2B and Nap1 and confirm its relevance both in vitro and in vivo. We show that two copies of H2A-H2B bound to a Nap1 homodimer form a tetramer with contacts between H2B chains similar to those in the four-helix bundle structural motif. The organization of the complex reveals that Nap1 competes with histone-DNA and interhistone interactions observed in the nucleosome, thereby regulating the availability of histones for chromatin assembly.

Characterization of Caenorhabditis elegans Nucleosome Assembly Protein 1 Uncovers the Role of Acidic Tails in Histone Binding.

Nucleosome assembly proteins (Naps) influence chromatin dynamics by directly binding to histones. Here we provide a comprehensive structural and biochemical analysis of a Nap protein from Caenorhabditis elegans (CeNap1). CeNap1 naturally lacks the acidic N-terminal tail and has a short C-terminal tail compared to many other Nap proteins. Comparison of CeNap1 with full length and tail-less constructs of Saccharomyces cerevisiae Nap1 uncovers the role of these tails in self-association, histone binding, and Nap competition with DNA for H2A-H2B. We find that the presence of tails influences the stoichiometry of H2A-H2B binding and is required to complete the interactions between H2A-H2B and DNA. The absolute stoichiometry of the Nap protein and H2A-H2B complex is 2:1 or 2:2, with only a very small population of higher-order oligomers occurring at 150 mM NaCl. We also show that H3-H4 binds differently than H2A-H2B and that an (H3-H4)2 tetramer can simultaneously bind two Nap2 protein homodimers.

IroT/MavN Is a Legionella Transmembrane Fe(II) Transporter: Metal Selectivity and Translocation Kinetics Revealed by in Vitro Real-Time Transport.

In intravacuolar pathogens, iron is essential for growth and virulence. In Legionella pneumophila, a putative transmembrane protein inserted on the surface of the host pathogen-containing vacuole, IroT/MavN, facilitates intravacuolar iron acquisition from the host by an unknown mechanism, bypassing the problem of Fe(III) insolubility and mobilization. We developed a platform for purification and reconstitution of IroT in artificial lipid bilayer vesicles (proteoliposomes). By encapsulating the fluorescent reporter probe Fluozin-3, we reveal, by real-time metal transport assays, that IroT is a high-affinity iron transporter selective for Fe(II) over other essential transition metals. Mutational analysis reveals important residues in the transmembrane helices, soluble domains, and loops important for substrate recognition and translocation. The work establishes the substrate transport properties in a novel transporter family important for iron acquisition at the host-pathogen intravacuolar interface and provides chemical tools for a comparative investigation of the translocation properties in other iron transporter families.

Regulating the Uptake of Viral Nanoparticles in Macrophage and Cancer Cells via a pH Switch.

Controlling the uptake of nanomaterials into phagocytes is a challenging problem. We describe an approach to inhibit the cellular uptake by macrophages and HeLa cells of nanoparticles derived from bacteriophage Qß by conjugating negatively charged terminal hexanoic acid moieties onto its surface. Additionally, we show hydrazone linkers can be installed between the surface of Qß and the terminal hexanoic acid moieties, resulting in a pH-responsive conjugate that, in acidic conditions, can release the terminal hexanoic acid moiety and allow for the uptake of the Qß nanoparticle. The installation of the "pH switch" did not change the structure-function properties of the hexanoic acid moiety and the uptake of the Qß conjugates by macrophages.

Molecular Hyperthermia: Spatiotemporal Protein Unfolding and Inactivation by Nanosecond Plasmonic Heating.

Spatiotemporal control of protein structure and activity in biological systems has important and broad implications in biomedical sciences as evidenced by recent advances in optogenetic approaches. Here, this study demonstrates that nanosecond pulsed laser heating of gold nanoparticles (GNP) leads to an ultrahigh and ultrashort temperature increase, coined as "molecular hyperthermia", which causes selective unfolding and inactivation of proteins adjacent to the GNP. Protein inactivation is highly dependent on both laser pulse energy and GNP size, and has a well-defined impact zone in the nanometer scale. It is anticipated that the fine control over protein structure and function enabled by this discovery will be highly enabling within a number of arenas, from probing the biophysics of protein folding/unfolding to the nanoscopic manipulation of biological systems via an optical trigger, to developing novel therapeutics for disease treatment without genetic modification.

Fluorescent Functionalization across Quaternary Structure in a Virus-like Particle.

Proteinaceous nanomaterials and, in particular, virus-like particles (VLPs) have emerged as robust and uniform platforms that are seeing wider use in biomedical research. However, there are a limited number of bioconjugation reactions for functionalizing the capsids, and very few of those involve functionalization across the supramolecular quaternary structure of protein assemblies. In this work, we exploit the recently described dibromomaleimide moiety as part of a bioconjugation strategy on VLP Qß to break and rebridge the exposed and structurally important disulfides in good yields. Not only was the stability of the quaternary structure retained after the reaction, but the newly functionalized particles also became brightly fluorescent and could be tracked in vitro using a commercially available filter set. Consequently, we show that this highly efficient bioconjugation reaction not only introduces a new functional handle "between" the disulfides of VLPs without compromising their thermal stability but also can be used to create a fluorescent probe.

The right place at the right time: chaperoning core histone variants.

Histone proteins dynamically regulate chromatin structure and epigenetic signaling to maintain cell homeostasis. These processes require controlled spatial and temporal deposition and eviction of histones by their dedicated chaperones. With the evolution of histone variants, a network of functionally specific histone chaperones has emerged. Molecular details of the determinants of chaperone specificity for different histone variants are only slowly being resolved. A complete understanding of these processes is essential to shed light on the genuine biological roles of histone variants, their chaperones, and their impact on chromatin dynamics.

Towards a mechanism for histone chaperones.

Histone chaperones can be broadly defined as histone-binding proteins that influence chromatin dynamics in an ATP-independent manner. Their existence reflects the importance of chromatin homeostasis and the unique and unusual biochemistry of the histone proteins. Histone supply and demand at chromatin is regulated by a network of structurally and functionally diverse histone chaperones. At the core of this network is a mechanistic variability that is only beginning to be appreciated. In this review, we highlight the challenges in determining histone chaperone mechanism and discuss possible mechanisms in the context of nucleosome thermodynamics. We discuss how histone chaperones prevent promiscuous histone interactions, and consider if this activity represents the full extent of histone chaperone function in governing chromatin dynamics. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.

Fluorescence strategies for high-throughput quantification of protein interactions.

Advances in high-throughput characterization of protein networks in vivo have resulted in large databases of unexplored protein interactions that occur during normal cell function. Their further characterization requires quantitative experimental strategies that are easy to implement in laboratories without specialized equipment. We have overcome many of the previous limitations to thermodynamic quantification of protein interactions, by developing a series of in-solution fluorescence-based strategies. These methods have high sensitivity, a broad dynamic range, and can be performed in a high-throughput manner. In three case studies we demonstrate how fluorescence (de)quenching and fluorescence resonance energy transfer can be used to quantitatively probe various high-affinity protein-DNA and protein-protein interactions. We applied these methods to describe the preference of linker histone H1 for nucleosomes over DNA, the ionic dependence of the DNA repair enzyme PARP1 in DNA binding, and the interaction between the histone chaperone Nap1 and the histone H2A-H2B heterodimer.

Molecular basis of RanGTP-activated nucleosome assembly with Histones H2A-H2B bound to Importin-9.

Padavannil et al. 2019 show that Importin-9 (Imp9) transports Histones H2A-H2B from the cytoplasm to the nucleus using a non-canonical mechanism whereby binding of a GTP-bound Ran GTPase (RanGTP) fails to evict the H2A-H2B cargo. Instead, a stable complex forms, comprised of equimolar RanGTP, Imp9, and H2A-H2B. Unlike the binary Imp9•H2A-H2B complex, this RanGTP•Imp9•H2A-H2B ternary complex can release H2A-H2B to an assembling nucleosome. Here, we define the molecular basis for this RanGTP-activated nucleosome assembly by Imp9. We use hydrogen-deuterium exchange coupled with mass spectrometry and compare the dynamics and interfaces of the RanGTP•Imp9•H2A-H2B ternary complex to those in the Imp9•H2A-H2B or Imp9•RanGTP binary complexes. Our data are consistent with the Imp9•H2A-H2B structure by Padavannil et al. 2019 showing that Imp9 HEAT repeats 4-5 and 18-19 contact H2A-H2B, as well as many homologous importin•RanGTP structures showing that importin HEAT repeats 1 and 3, and the h8 loop, contact RanGTP. We show that Imp9 stabilizes H2A-H2B beyond the direct binding site, similar to other histone chaperones. Importantly, we reveal that binding of RanGTP releases H2A-H2B interaction at Imp9 HEAT repeats 4-5, but not 18-19. This exposes DNA- and histone-binding surfaces of H2A-H2B, thereby facilitating nucleosome assembly. We also reveal that RanGTP has a weaker affinity for Imp9 when H2A-H2B is bound. This may ensure that H2A-H2B is only released in high RanGTP concentrations near chromatin. We delineate the molecular link between the nuclear import of H2A-H2B and its deposition into chromatin by Imp9. Significance: Imp9 is the primary importin for shuttling H2A-H2B from the cytoplasm to the nucleus. It employs an unusual mechanism where the binding of RanGTP alone is insufficient to release H2A-H2B. The resulting stable RanGTP•Imp9•H2A-H2B complex gains nucleosome assembly activity as H2A-H2B can be deposited onto an assembling nucleosome. We show that H2A-H2B is allosterically stabilized via interactions with both N- and C-terminal portions of Imp9, reinforcing its chaperone-like behavior. RanGTP binding causes H2A-H2B release from the N-terminal portion of Imp9 only. The newly-exposed H2A-H2B surfaces can interact with DNA or H3-H4 in nucleosome assembly. Imp9 thus plays a multi-faceted role in histone import, storage, and deposition regulated by RanGTP, controlling histone supply in the nucleus and to chromatin.

Molecular basis of RanGTP-activated release of Histones H2A-H2B from Importin-9.

Imp9 is the primary importin for shuttling H2A-H2B from the cytoplasm to the nucleus. It employs an unusual mechanism where the binding of RanGTP is insufficient to release H2A-H2B. The resulting stable RanGTP·Imp9·H2A-H2B complex gains nucleosome assembly activity with H2A-H2B able to be deposited into an assembling nucleosome in vitro. Using hydrogen-deuterium exchange coupled with mass spectrometry (HDX), we show that Imp9 stabilizes H2A-H2B beyond the direct-binding site, like other histone chaperones. HDX also shows that binding of RanGTP releases H2A-H2B contacts at Imp9 HEAT repeats 4-5, but not 18-19. DNA- and histone-binding surfaces of H2A-H2B are exposed in the ternary complex, facilitating nucleosome assembly. We also reveal that RanGTP has a weaker affinity for Imp9 when H2A-H2B is bound. Imp9 thus provides a connection between the nuclear import of H2A-H2B and its deposition into chromatin.

Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses.

To address the ongoing SARS-CoV-2 pandemic and prepare for future coronavirus outbreaks, understanding the protective potential of epitopes conserved across SARS-CoV-2 variants and coronavirus lineages is essential. We describe a highly conserved, conformational S2 domain epitope present only in the prefusion core of ß-coronaviruses: SARS-CoV-2 S2 apex residues 980-1006 in the flexible hinge. Antibody RAY53 binds the native hinge in MERS-CoV and SARS-CoV-2 spikes on the surface of mammalian cells and mediates antibody-dependent cellular phagocytosis and cytotoxicity against SARS-CoV-2 spike in vitro. Hinge epitope mutations that ablate antibody binding compromise pseudovirus infectivity, but changes elsewhere that affect spike opening dynamics, including those found in Omicron BA.1, occlude the epitope and may evade pre-existing serum antibodies targeting the S2 core. This work defines a third class of S2 antibody while providing insights into the potency and limitations of S2 core epitope targeting.

Using hydrogen-deuterium exchange mass spectrometry to characterize Mtr4 interactions with RNA.

Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) is a valuable technique to investigate the dynamics of protein systems. The approach compares the deuterium uptake of protein backbone amides under multiple conditions to characterize protein conformation and interaction. HDX-MS is versatile and can be applied to diverse ligands, however, challenges remain when it comes to exploring complexes containing nucleic acids. In this chapter, we present procedures for the optimization and application of HDX-MS to studying RNA-binding proteins and use the RNA helicase Mtr4 as a demonstrative example. We highlight considerations in designing on-exchange, bottom-up, comparative studies on proteins with RNA. Our protocol details preliminary testing and optimization of experimental parameters. Difficulties arising from the inclusion of RNA, such as signal repression and sample carryover, are addressed. We discuss how chromatography parameters can be adjusted depending on the issues presented by the RNA, emphasizing reproducible peptide recovery in the absence and presence of RNA. Methods for visualization of HDX data integrated with statistical analysis are also reviewed with examples. These protocols can be applied to future studies of various RNA-protein complexes.