Enantiomer-specific activities of an LRH-1 and SF-1 dual agonist.

Chirality is an important consideration in drug development: it can influence recognition of the intended target, pharmacokinetics, and off-target effects. Here, we investigate how chirality affects the activity and mechanism of action of RJW100, a racemic agonist of the nuclear receptors liver receptor homolog-1 (LRH-1) and steroidogenic factor-1 (SF-1). LRH-1 and SF-1 modulators are highly sought as treatments for metabolic and neoplastic diseases, and RJW100 has one of the few scaffolds shown to activate them. However, enantiomer-specific effects on receptor activation are poorly understood. We show that the enantiomers have similar binding affinities, but RR-RJW100 stabilizes both receptors and is 46% more active than SS-RJW100 in LRH-1 luciferase reporter assays. We present an LRH-1 crystal structure that illuminates striking mechanistic differences: SS-RJW100 adopts multiple configurations in the pocket and fails to make an interaction critical for activation by RR-RJW100. In molecular dynamics simulations, SS-RJW100 attenuates intramolecular signalling important for coregulator recruitment, consistent with previous observations that it weakly recruits coregulators in vitro. These studies provide a rationale for pursuing enantiomerically pure RJW100 derivatives: they establish RR-RJW100 as the stronger LRH-1 agonist and identify a potential for optimizing the SS-RJW100 scaffold for antagonist design.

Gene- and Species-Specific Hox mRNA Translation by Ribosome Expansion Segments.

Ribosomes have been suggested to directly control gene regulation, but regulatory roles for ribosomal RNA (rRNA) remain largely unexplored. Expansion segments (ESs) consist of multitudes of tentacle-like rRNA structures extending from the core ribosome in eukaryotes. ESs are remarkably variable in sequence and size across eukaryotic evolution with largely unknown functions. In characterizing ribosome binding to a regulatory element within a Homeobox (Hox) 5' UTR, we identify a modular stem-loop within this element that binds to a single ES, ES9S. Engineering chimeric, "humanized" yeast ribosomes for ES9S reveals that an evolutionary change in the sequence of ES9S endows species-specific binding of Hoxa9 mRNA to the ribosome. Genome editing to site-specifically disrupt the Hoxa9-ES9S interaction demonstrates the functional importance for such selective mRNA-rRNA binding in translation control. Together, these studies unravel unexpected gene regulation directly mediated by rRNA and how ribosome evolution drives translation of critical developmental regulators.

Structural insights into TAZ2 domain-mediated CBP/p300 recruitment by transactivation domain 1 of the lymphopoietic transcription factor E2A.

The E-protein transcription factors guide immune cell differentiation, with E12 and E47 (hereafter called E2A) being essential for B-cell specification and maturation. E2A and the oncogenic chimera E2A-PBX1 contain three transactivation domains (ADs), with AD1 and AD2 having redundant, independent, and cooperative functions in a cell-dependent manner. AD1 and AD2 both mediate their functions by binding to the KIX domain of the histone acetyltransferase paralogues CREB-binding protein (CBP) and E1A-binding protein P300 (p300). This interaction is necessary for B-cell maturation and oncogenesis by E2A-PBX1 and occurs through conserved ΦXXΦΦ motifs (with Φ denoting a hydrophobic amino acid) in AD1 and AD2. However, disruption of this interaction via mutation of the KIX domain in CBP/p300 does not completely abrogate binding of E2A and E2A-PBX1. Here, we determined that E2A-AD1 and E2A-AD2 also interact with the TAZ2 domain of CBP/p300. Characterization of the TAZ2:E2A-AD1(1-37) complex indicated that E2A-AD1 adopts an α-helical structure and uses its ΦXXΦΦ motif to bind TAZ2. Whereas this region overlapped with the KIX recognition region, key KIX-interacting E2A-AD1 residues were exposed, suggesting that E2A-AD1 could simultaneously bind both the KIX and TAZ2 domains. However, we did not detect a ternary complex involving E2A-AD1, KIX, and TAZ2 and found that E2A containing both intact AD1 and AD2 is required to bind to CBP/p300. Our findings highlight the structural plasticity and promiscuity of E2A-AD1 and suggest that E2A binds both the TAZ2 and KIX domains of CBP/p300 through AD1 and AD2.

Structures of a RAG-like transposase during cut-and-paste transposition.

Transposons have had a pivotal role in genome evolution1 and are believed to be the evolutionary progenitors of the RAG1-RAG2 recombinase2, an essential component of the adaptive immune system in jawed vertebrates3. Here we report one crystal structure and five cryo-electron microscopy structures of Transib4,5, a RAG1-like transposase from Helicoverpa zea, that capture the entire transposition process from the apo enzyme to the terminal strand transfer complex with transposon ends covalently joined to target DNA, at resolutions of 3.0-4.6 Å. These structures reveal a butterfly-shaped complex that undergoes two cycles of marked conformational changes in which the 'wings' of the transposase unfurl to bind substrate DNA, close to execute cleavage, open to release the flanking DNA and close again to capture and attack target DNA. Transib possesses unique structural elements that compensate for the absence of a RAG2 partner, including a loop that interacts with the transposition target site and an accordion-like C-terminal tail that elongates and contracts to help to control the opening and closing of the enzyme and assembly of the active site. Our findings reveal the detailed reaction pathway of a eukaryotic cut-and-paste transposase and illuminate some of the earliest steps in the evolution of the RAG recombinase.

Transposon molecular domestication and the evolution of the RAG recombinase.

Domestication of a transposon (a DNA sequence that can change its position in a genome) to give rise to the RAG1-RAG2 recombinase (RAG) and V(D)J recombination, which produces the diverse repertoire of antibodies and T cell receptors, was a pivotal event in the evolution of the adaptive immune system of jawed vertebrates. The evolutionary adaptations that transformed the ancestral RAG transposase into a RAG recombinase with appropriately regulated DNA cleavage and transposition activities are not understood. Here, beginning with cryo-electron microscopy structures of the amphioxus ProtoRAG transposase (an evolutionary relative of RAG), we identify amino acid residues and domains the acquisition or loss of which underpins the propensity of RAG for coupled cleavage, its preference for asymmetric DNA substrates and its inability to perform transposition in cells. In particular, we identify two adaptations specific to jawed-vertebrates-arginine 848 in RAG1 and an acidic region in RAG2-that together suppress RAG-mediated transposition more than 1,000-fold. Our findings reveal a two-tiered mechanism for the suppression of RAG-mediated transposition, illuminate the evolution of V(D)J recombination and provide insight into the principles that govern the molecular domestication of transposons.

Generating Novel Materials Using the Intrinsically Disordered Protein Ubx.

The development of functionalized materials is needed to enable diverse applications. Protein-based materials are typically biocompatible and biodegradable and can exhibit a wide variety of useful mechanical properties. Most importantly, gene fusion enables facile incorporation of active proteins into the materials. However, many protocols rely on denaturing conditions to stimulate materials formation. These conditions would be expected to inactivate any appended functional proteins. This chapter describes methods to create protein fibers and films in a mild aqueous buffer near neutral pH. This facile, inexpensive single-pot approach to materials assembly does not require any special equipment. Also included in this chapter are methods to fuse fibers to form fiber bundles, and to use fibers for cell culture. Although these methods were developed to generate materials from the Drosophila Hox transcription factor Ultrabithorax, they may also work for other self-assembling proteins, many of which have sequence features in common with Ubx.

Cracking the DNA Code for V(D)J Recombination.

To initiate V(D)J recombination for generating the adaptive immune response of vertebrates, RAG1/2 recombinase cleaves DNA at a pair of recombination signal sequences, the 12- and 23-RSS. We have determined crystal and cryo-EM structures of RAG1/2 with DNA in the pre-reaction and hairpin-forming complexes up to 2.75 Å resolution. Both protein and DNA exhibit structural plasticity and undergo dramatic conformational changes. Coding-flank DNAs extensively rotate, shift, and deform for nicking and hairpin formation. Two intertwined RAG1 subunits crisscross four times between the asymmetric pair of severely bent 12/23-RSS DNAs. Location-sensitive bending of 60° and 150° in 12- and 23-RSS spacers, respectively, must occur for RAG1/2 to capture the nonamers and pair the heptamers for symmetric double-strand breakage. DNA pairing is thus sequence-context dependent and structure specific, which partly explains the "beyond 12/23" restriction. Finally, catalysis in crystallo reveals the process of DNA hairpin formation and its stabilization by interleaved base stacking.

Dense Bicoid hubs accentuate binding along the morphogen gradient.

Morphogen gradients direct the spatial patterning of developing embryos; however, the mechanisms by which these gradients are interpreted remain elusive. Here we used lattice light-sheet microscopy to perform in vivo single-molecule imaging in early Drosophila melanogaster embryos of the transcription factor Bicoid that forms a gradient and initiates patterning along the anteroposterior axis. In contrast to canonical models, we observed that Bicoid binds to DNA with a rapid off rate throughout the embryo such that its average occupancy at target loci is on-rate-dependent. We further observed Bicoid forming transient "hubs" of locally high density that facilitate binding as factor levels drop, including in the posterior, where we observed Bicoid binding despite vanishingly low protein levels. We propose that localized modulation of transcription factor on rates via clustering provides a general mechanism to facilitate binding to low-affinity targets and that this may be a prevalent feature of other developmental transcription factors.

Structural changes upon peroxynitrite-mediated nitration of peroxiredoxin 2; nitrated Prx2 resembles its disulfide-oxidized form.

Peroxiredoxins are cys-based peroxidases that function in peroxide detoxification and H2O2-induced signaling. Human Prx2 is a typical 2-Cys Prx arranged as pentamers of head-to-tail homodimers. During the catalytic mechanism, the active-site cysteine (CP) cycles between reduced, sulfenic and disulfide state involving conformational as well as oligomeric changes. Several post-translational modifications were shown to affect Prx activity, in particular CP overoxidation which leads to inactivation. We have recently reported that nitration of Prx2, a post-translational modification on non-catalytic tyrosines, unexpectedly increases its peroxidase activity and resistance to overoxidation. To elucidate the cross-talk between this post-translational modification and the enzyme catalysis, we investigated the structural changes of Prx2 after nitration. Analytical ultracentrifugation, UV absorption, circular dichroism, steady-state and time-resolved fluorescence were used to connect catalytically relevant redox changes with tyrosine nitration. Our results show that the reduced nitrated Prx2 structurally resembles the disulfide-oxidized native form of the enzyme favoring a locally unfolded conformation that facilitates disulfide formation. These results provide structural basis for the kinetic analysis previously reported, the observed increase in activity and the resistance to overoxidation of the peroxynitrite-treated enzyme.

Molecular Mechanism of V(D)J Recombination from Synaptic RAG1-RAG2 Complex Structures.

Diverse repertoires of antigen-receptor genes that result from combinatorial splicing of coding segments by V(D)J recombination are hallmarks of vertebrate immunity. The (RAG1-RAG2)2 recombinase (RAG) recognizes recombination signal sequences (RSSs) containing a heptamer, a spacer of 12 or 23 base pairs, and a nonamer (12-RSS or 23-RSS) and introduces precise breaks at RSS-coding segment junctions. RAG forms synaptic complexes only with one 12-RSS and one 23-RSS, a dogma known as the 12/23 rule that governs the recombination fidelity. We report cryo-electron microscopy structures of synaptic RAG complexes at up to 3.4 Å resolution, which reveal a closed conformation with base flipping and base-specific recognition of RSSs. Distortion at RSS-coding segment junctions and base flipping in coding segments uncover the two-metal-ion catalytic mechanism. Induced asymmetry involving tilting of the nonamer-binding domain dimer of RAG1 upon binding of HMGB1-bent 12-RSS or 23-RSS underlies the molecular mechanism for the 12/23 rule.

Using Molecular Dynamics Simulations as an Aid in the Prediction of Domain Swapping of Computationally Designed Protein Variants.

In standard implementations of computational protein design, a positive-design approach is used to predict sequences that will be stable on a given backbone structure. Possible competing states are typically not considered, primarily because appropriate structural models are not available. One potential competing state, the domain-swapped dimer, is especially compelling because it is often nearly identical with its monomeric counterpart, differing by just a few mutations in a hinge region. Molecular dynamics (MD) simulations provide a computational method to sample different conformational states of a structure. Here, we tested whether MD simulations could be used as a post-design screening tool to identify sequence mutations leading to domain-swapped dimers. We hypothesized that a successful computationally designed sequence would have backbone structure and dynamics characteristics similar to that of the input structure and that, in contrast, domain-swapped dimers would exhibit increased backbone flexibility and/or altered structure in the hinge-loop region to accommodate the large conformational change required for domain swapping. While attempting to engineer a homodimer from a 51-amino-acid fragment of the monomeric protein engrailed homeodomain (ENH), we had instead generated a domain-swapped dimer (ENH_DsD). MD simulations on these proteins showed increased B-factors derived from MD simulation in the hinge loop of the ENH_DsD domain-swapped dimer relative to monomeric ENH. Two point mutants of ENH_DsD designed to recover the monomeric fold were then tested with an MD simulation protocol. The MD simulations suggested that one of these mutants would adopt the target monomeric structure, which was subsequently confirmed by X-ray crystallography.

CTCF controls HOXA cluster silencing and mediates PRC2-repressive higher-order chromatin structure in NT2/D1 cells.

HOX cluster genes are activated sequentially in their positional order along the chromosome during vertebrate development. This phenomenon, known as temporal colinearity, depends on transcriptional silencing of 5' HOX genes. Chromatin looping was recently identified as a conserved feature of silent HOX clusters, with CCCTC-binding factor (CTCF) binding sites located at the loop bases. However, the potential contribution of CTCF to HOX cluster silencing and the underlying mechanism have not been established. Here, we demonstrate that the HOXA locus is organized by CTCF into chromatin loops and that CTCF depletion causes significantly enhanced activation of HOXA3 to -A7, -A9 to -A11, and -A13 in response to retinoic acid, with the highest effect observed for HOXA9. Our subsequent analyses revealed that CTCF facilitates the stabilization of Polycomb repressive complex 2 (PRC2) and trimethylated lysine 27 of histone H3 (H3K27me3) at the human HOXA locus. Our results reveal that CTCF functions as a controller of HOXA cluster silencing and mediates PRC2-repressive higher-order chromatin structure.

Single molecule visualization and characterization of Sox2-Pax6 complex formation on a regulatory DNA element using a DNA origami frame.

We report the use of atomic force microscopy (AFM) to study Sox2-Pax6 complex formation on the regulatory DNA element at a single molecule level. Using an origami DNA scaffold containing two DNA strands with different levels of tensile force, we confirmed that DNA bending is necessary for Sox2 binding. We also demonstrated that two transcription factors bind cooperatively by observing the increased occupancy of Sox2-Pax6 on the DNA element compared to that of Sox2 alone.

The basic helix-loop-helix region of the transcriptional repressor hairy and enhancer of split 1 is preorganized to bind DNA.

Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions.

Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans.

The nematode Caenorhabditis elegans protein CEH-37 belongs to the paired OTD/OTX family of homeobox-containing homeodomain proteins. CEH-37 shares sequence similarity with homeodomain proteins, although it specifically binds to double-stranded C. elegans telomeric DNA, which is unusual to homeodomain proteins. Here, we report the solution structure of CEH-37 homeodomain and molecular interaction with double-stranded C. elegans telomeric DNA using nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that CEH-37 homeodomain is composed of a flexible N-terminal region and three α-helices with a helix-turn-helix (HTH) DNA binding motif. Data from size-exclusion chromatography and fluorescence spectroscopy reveal that CEH-37 homeodomain interacts strongly with double-stranded C. elegans telomeric DNA. NMR titration experiments identified residues responsible for specific binding to nematode double-stranded telomeric DNA. These results suggest that C. elegans homeodomain protein, CEH-37 could play an important role in telomere function via DNA binding.

Suppression of sampling artefacts in high-resolution four-dimensional NMR spectra using signal separation algorithm.

The development of non-uniform sampling (NUS) strategies permits to obtain high-dimensional spectra with increased resolution in significantly reduced experimental time. We extended a previously proposed signal separation algorithm (SSA) to process sparse four-dimensional NMR data. It is employed for two experiments carried out for a partially unstructured 114-residue construct of chicken Engrailed 2 protein, namely 4D HCCH-TOCSY and 4D C,N-edited NOESY. The SSA allowed us to obtain high-quality spectra using only as little as 0.16% of the available samples, with low sampling artefacts approaching the thermal noise level in most spectral regions. It is demonstrated that NUS 4D HCCH-TOCSY is dominated by sampling noise and requires efficient artefact suppression. On the other hand, 4D C,N-edited NOESY is a particularly attractive experiment for NUS, as the absence of diagonal peaks renders the problem of artefacts less critical. We also present a transverse-relaxation optimized sequence for HMQC that is especially designed for longer evolution periods in the indirectly detected proton dimension in high-dimensional pulse sequences. In conjunction with novel sampling strategies and efficient processing methods, this improvement enabled us to obtain unique structural information about aliphatic-amide contacts.

Client-loading conformation of the Hsp90 molecular chaperone revealed in the cryo-EM structure of the human Hsp90:Hop complex.

Hsp90 is an essential molecular chaperone required for the folding and activation of many hundreds of cellular "client" proteins. The ATP-dependent chaperone cycle involves significant conformational rearrangements of the Hsp90 dimer and interaction with a network of cochaperone proteins. Little is known about the mechanism of client protein binding or how cochaperone interactions modulate Hsp90 conformational states. We have determined the cryo-EM structure of the human Hsp90:Hop complex that receives client proteins from the Hsp70 chaperone. Hop stabilizes an alternate Hsp90 open state, where hydrophobic client-binding surfaces have converged and the N-terminal domains have rotated and match the closed, ATP conformation. Hsp90 is thus simultaneously poised for client loading by Hsp70 and subsequent N-terminal dimerization and ATP hydrolysis. Upon binding of a single Hsp70, the Hsp90:Hop conformation remains essentially unchanged. These results identify distinct functions for the Hop cochaperone, revealing an asymmetric mechanism for Hsp90 regulation and client loading.

DNA compaction by the higher-order assembly of PRH/Hex homeodomain protein oligomers.

Protein self-organization is essential for the establishment and maintenance of nuclear architecture and for the regulation of gene expression. We have shown previously that the Proline-Rich Homeodomain protein (PRH/Hex) self-assembles to form oligomeric complexes that bind to arrays of PRH binding sites with high affinity and specificity. We have also shown that many PRH target genes contain suitably spaced arrays of PRH sites that allow this protein to bind and regulate transcription. Here, we use analytical ultracentrifugation and electron microscopy to further characterize PRH oligomers. We use the same techniques to show that PRH oligomers bound to long DNA fragments self-associate to form highly ordered assemblies. Electron microscopy and linear dichroism reveal that PRH oligomers can form protein-DNA fibres and that PRH is able to compact DNA in the absence of other proteins. Finally, we show that DNA compaction is not sufficient for the repression of PRH target genes in cells. We conclude that DNA compaction is a consequence of the binding of large PRH oligomers to arrays of binding sites and that PRH is functionally and structurally related to the Lrp/AsnC family of proteins from bacteria and archaea, a group of proteins formerly thought to be without eukaryotic equivalents.

Initial stages of V(D)J recombination: the organization of RAG1/2 and RSS DNA in the postcleavage complex.

To obtain structural information on the early stages of V(D)J recombination, we isolated a complex of the core RAG1 and RAG2 proteins with DNA containing a pair of cleaved recombination signal sequences (RSS). Stoichiometric and molecular mass analysis established that this signal-end complex (SEC) contains two protomers each of RAG1 and RAG2. Visualization of the SEC by negative-staining electron microscopy revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N termini of RAG1 and RAG2 are positioned at opposing ends of the complex, and the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near the RAG1 N termini. These first images of the V(D)J recombinase in its postcleavage state provide a framework for modeling RAG domains and their interactions with DNA.

Improvement on a simplified model for protein folding simulation.

Improvements were made on a simplified protein model--the Ramachandran model-to achieve better computer simulation of protein folding. To check the validity of such improvements, we chose the ultrafast folding protein Engrailed Homeodomain as an example and explored several aspects of its folding. The engrailed homeodomain is a mainly alpha-helical protein of 61 residues from Drosophila melanogaster. We found that the simplified model of Engrailed Homeodomain can fold into a global minimum state with a tertiary structure in good agreement with its native structure.