Structural basis of Irgb6 inactivation by Toxoplasma gondii through the phosphorylation of switch I.

Irgb6 is a priming immune-related GTPase (IRG) that counteracts Toxoplasma gondii. It is known to be recruited to the low virulent type II T. gondii parasitophorous vacuole (PV), initiating cell-autonomous immunity. However, the molecular mechanism by which immunity-related GTPases become inactivated after the parasite infection remains obscure. Here, we found that Thr95 of Irgb6 is prominently phosphorylated in response to low virulent type II T. gondii infection. We observed that a phosphomimetic T95D mutation in Irgb6 impaired its localization to the PV and exhibited reduced GTPase activity in vitro. Structural analysis unveiled an atypical conformation of nucleotide-free Irgb6-T95D, resulting from a conformational change in the G-domain that allosterically modified the PV membrane-binding interface. In silico docking corroborated the disruption of the physiological membrane binding site. These findings provide novel insights into a T. gondii-induced allosteric inactivation mechanism of Irgb6.

TEAD1 trapping by the Q353R-Lamin A/C causes dilated cardiomyopathy.

Mutations in the LMNA gene encoding Lamin A and C (Lamin A/C), major components of the nuclear lamina, cause laminopathies including dilated cardiomyopathy (DCM), but the underlying molecular mechanisms have not been fully elucidated. Here, by leveraging single-cell RNA sequencing (RNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), protein array, and electron microscopy analysis, we show that insufficient structural maturation of cardiomyocytes owing to trapping of transcription factor TEA domain transcription factor 1 (TEAD1) by mutant Lamin A/C at the nuclear membrane underlies the pathogenesis of Q353R-LMNA-related DCM. Inhibition of the Hippo pathway rescued the dysregulation of cardiac developmental genes by TEAD1 in LMNA mutant cardiomyocytes. Single-cell RNA-seq of cardiac tissues from patients with DCM with the LMNA mutation confirmed the dysregulated expression of TEAD1 target genes. Our results propose an intervention for transcriptional dysregulation as a potential treatment of LMNA-related DCM.

Structural model of microtubule dynamics inhibition by kinesin-4 from the crystal structure of KLP-12 -tubulin complex.

Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of the inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.

From meter-long structures that allow nerve cells to stretch across a body to miniscule 'hairs' required for lung cells to clear mucus, many life processes rely on cells sporting projections which have the right size for their role. Networks of hollow filaments known as microtubules shape these structures and ensure that they have the appropriate dimensions. Controlling the length of microtubules is therefore essential for organisms, yet how this process takes place is still not fully elucidated. Previous research has shown that microtubules continue to grow when their end is straight but stop when it is curved. A family of molecular motors known as kinesin-4 participate in this process, but the exact mechanisms at play remain unclear. To investigate, Tuguchi, Nakano, Imasaki et al. focused on the KLP-12 protein, a kinesin-4 equivalent which helps to controls the length of microtubules in the tiny worm Caenorhabditis elegans. They performed genetic manipulations and imaged the interactions between KLP-12 and the growing end of a microtubule using X-ray crystallography. This revealed that KLP-12 controls the length of neurons by inhibiting microtubule growth. It does so by modulating the curvature of the growing end of the filament to suppress its extension. A 'snapshot' of KLP-12 binding to a microtubule at the resolution of the atom revealed exactly how the protein helps to bend the end of the filament to prevent it from growing further. These results will help to understand how nerve cells are shaped. This may also provide insights into the molecular mechanisms for various neurodegenerative disorders caused by problems with the human equivalents of KLP-12, potentially leading to new therapies.

CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre through phase separation.

Microtubules are dynamic polymers consisting of αß-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αß-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αß-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.

Cells are able to hold their shape thanks to tube-like structures called microtubules that are made of hundreds of tubulin proteins. Microtubules are responsible for maintaining the uneven distribution of molecules throughout the cell, a phenomenon known as polarity that allows cells to differentiate into different types with various roles. A protein complex called the γ-tubulin ring complex (γ-TuRC) is necessary for microtubules to form. This protein helps bind the tubulin proteins together and stabilises microtubules. However, recent research has found that in highly polarized cells such as neurons, which have highly specialised regions, microtubules can form without γ-TuRC. Searching for the proteins that could be filling in for γ-TuRC in these cells some evidence has suggested that a group known as CAMSAPs may be involved, but it is not known how. To characterize the role of CAMSAPs, Imasaki, Kikkawa et al. studied how one of these proteins, CAMSAP2, interacts with tubulins. To do this, they reconstituted both CAMSAP2 and tubulins using recombinant biotechnology and mixed them in solution. These experiments showed that CAMSAP2 can help form microtubules by bringing together their constituent proteins so that they can bind to each other more easily. Once microtubules start to form, CAMSAP2 continues to bind to them, stabilizing them and enabling them to grow to full size. These results shed light on how polarity is established in cells such as neurons, muscle cells, and epithelial cells. Additionally, the ability to observe intermediate structures during microtubule formation can provide insights into the processes that these structures are involved in.

Erratum: Author Correction: High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity.

[This corrects the article DOI: 10.1038/s42003-019-0587-z.].

High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity.

Expression of human asparagine synthetase (ASNS) promotes metastatic progression and tumor cell invasiveness in colorectal and breast cancer, presumably by altering cellular levels of L-asparagine. Human ASNS is therefore emerging as a bona fide drug target for cancer therapy. Here we show that a slow-onset, tight binding inhibitor, which exhibits nanomolar affinity for human ASNS in vitro, exhibits excellent selectivity at 10 µM concentration in HCT-116 cell lysates with almost no off-target binding. The high-resolution (1.85 Å) crystal structure of human ASNS has enabled us to identify a cluster of negatively charged side chains in the synthetase domain that plays a key role in inhibitor binding. Comparing this structure with those of evolutionarily related AMP-forming enzymes provides insights into intermolecular interactions that give rise to the observed binding selectivity. Our findings demonstrate the feasibility of developing second generation human ASNS inhibitors as lead compounds for the discovery of drugs against metastasis.

A practical method for efficient and optimal production of Seleno-methionine-labeled recombinant protein complexes in the insect cells.

The use of Seleno-methionine (SeMet) incorporated protein crystals for single or multi-wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X-ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet-incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60-90% compared with native protein expression.

Structural insight into microtubule stabilization and kinesin inhibition by Tau family MAPs.

The Tau family microtubule-associated proteins (MAPs) promote microtubule stabilization and regulate microtubule-based motility. They share the C-terminal microtubule-binding domain, which includes three to five tubulin-binding repeats. Different numbers of repeats formed by alternative splicing have distinct effects on the activities of these proteins, and the distribution of these variants regulates fundamental physiological phenomena in cells. In this study, using cryo-EM, we visualized the MAP4 microtubule complex with the molecular motor kinesin-1. MAP4 bound to the C-terminal domains of tubulins along the protofilaments stabilizes the longitudinal contacts of the microtubule. The strongest bond of MAP4 was found around the intertubulin-dimer interface such that MAP4 coexists on the microtubule with kinesin-1 bound to the intratubulin-dimer interface as well. MAP4, consisting of five repeats, further folds and accumulates above the intertubulin-dimer interface, interfering with kinesin-1 movement. Therefore, these cryo-EM studies reveal new insight into the structural basis of microtubule stabilization and inhibition of kinesin motility by the Tau family MAPs.

Recent progress in structural biology: lessons from our research history.

The recent 'resolution revolution' in structural analyses of cryo-electron microscopy (cryo-EM) has drastically changed the research strategy for structural biology. In addition to X-ray crystallography and nuclear magnetic resonance spectroscopy, cryo-EM has achieved the structural analysis of biological molecules at near-atomic resolution, resulting in the Nobel Prize in Chemistry 2017. The effect of this revolution has spread within the biology and medical science fields affecting everything from basic research to pharmaceutical development by visualizing atomic structure. As we have used cryo-EM as well as X-ray crystallography since 2000 to elucidate the molecular mechanisms of the fundamental phenomena in the cell, here we review our research history and summarize our findings. In the first half of the review, we describe the structural mechanisms of microtubule-based motility of molecular motor kinesin by using a joint cryo-EM and X-ray crystallography method. In the latter half, we summarize our structural studies on transcriptional regulation by X-ray crystallography of in vitro reconstitution of a multi-protein complex.

Titer estimation for quality control (TEQC) method: A practical approach for optimal production of protein complexes using the baculovirus expression vector system.

The baculovirus expression vector system (BEVS) is becoming the method of choice for expression of many eukaryotic proteins and protein complexes for biochemical, structural and pharmaceutical studies. Significant technological advancement has made generation of recombinant baculoviruses easy, efficient and user-friendly. However, there is a tremendous variability in the amount of proteins made using the BEVS, including different batches of virus made to express the same proteins. Yet, what influences the overall production of proteins or protein complexes remains largely unclear. Many downstream applications, particularly protein structure determination, require purification of large quantities of proteins in a repetitive manner, calling for a reliable experimental set-up to obtain proteins or protein complexes of interest consistently. During our investigation of optimizing the expression of the Mediator Head module, we discovered that the 'initial infectivity' was an excellent indicator of overall production of protein complexes. Further, we show that this initial infectivity can be mathematically described as a function of multiplicity of infection (MOI), correlating recombinant protein yield and virus titer. All these findings led us to develop the Titer Estimation for Quality Control (TEQC) method, which enables researchers to estimate initial infectivity, titer/MOI values in a simple and affordable way, and to use these values to quantitatively optimize protein expressions utilizing BEVS in a highly reproducible fashion.

Structural Insights into the Altering Function of CRMP2 by Phosphorylation.

Collapsin response mediator protein 2 (CRMP2) regulates neuronal polarity by controlling microtubule dynamics. CRMP2 activity is regulated by semaphorin-induced phosphorylation at the C-terminal tail domain. Unphosphorylated CRMP2 induces effective axonal microtubule formation to give the axonal characteristics to a neurite, whereas phosphorylated CRMP2 leads to the apparently opposite effect, growth cone collapse. We have recently characterized the structural detail of CRMP2-induced axonal microtubule formation (Niwa et al. (2017) Sci. Rep., 7: 10681). CRMP2 forms the hetero-trimer with GTP-tubulin to induce effective axonal microtubule formation in the future axon. Phosphorylation of CRMP2 has been reported to decrease the affinity between CRMP2 and the microtubule, albeit the molecular mechanisms of how the phosphorylation of CRMP2 changes the structure to achieve distinct effects from unphosphorylated CRMP2 is not well understood. Here we performed a series of biochemical and structural analyses of phospho-mimic CRMP2. Phosphorylation of CRMP2 undergoes small conformational changes at the C-terminal tail with shifting the surface charge, which not only alters the interactions within the CRMP2 tetramer but also alters the interactions with GTP-tubulin. Consequently, phospho-mimic CRMP2 fails to form a hetero-trimer with GTP-tubulin, thus losing the ability to establish and maintain the axonal microtubules.Key words: CRMP2, phosphorylation, microtubule, axon, crystal structure.

Yeast Hrq1 shares structural and functional homology with the disease-linked human RecQ4 helicase.

The five human RecQ helicases participate in multiple processes required to maintain genome integrity. Of these, the disease-linked RecQ4 is the least studied because it poses many technical challenges. We previously demonstrated that the yeast Hrq1 helicase displays similar functions to RecQ4 in vivo, and here, we report the biochemical and structural characterization of these enzymes. In vitro, Hrq1 and RecQ4 are DNA-stimulated ATPases and robust helicases. Further, these activities were sensitive to DNA sequence and structure, with the helicases preferentially unwinding D-loops. Consistent with their roles at telomeres, telomeric repeat sequence DNA also stimulated binding and unwinding by these enzymes. Finally, electron microscopy revealed that Hrq1 and RecQ4 share similar structural features. These results solidify Hrq1 as a true RecQ4 homolog and position it as the premier model to determine how RecQ4 mutations lead to genomic instability and disease.

Expression and purification of functional human glycogen synthase-1 (hGYS1) in insect cells.

We have successfully expressed and purified active human glycogen synthase-1 (hGYS1). Successful production of the recombinant hGYS1 protein was achieved by co-expression of hGYS1 and rabbit glycogenin (rGYG1) using the MultiBac baculovirus expression system (BEVS). Functional measurements of activity ratios of hGYS1 in the absence and presence of glucose-6-phosphate and treatment with phosphatase indicate that the expressed protein is heavily phosphorylated. We used mass spectrometry to further characterize the sites of phosphorylation, which include most of the known regulatory phosphorylation sites, as well as several sites unique to the insect cell over-expression. Obtaining large quantities of functional hGYS1 will be invaluable for future structural studies as well as detailed studies on the effects on specific sites of phosphorylation.

Interaction of the mediator head module with RNA polymerase II.

Mediator, a large (21 polypeptides, MW ∼1 MDa) complex conserved throughout eukaryotes, plays an essential role in control of gene expression by conveying regulatory signals that influence the activity of the preinitiation complex. However, the precise mode of interaction between Mediator and RNA polymerase II (RNAPII), and the mechanism of regulation by Mediator remain elusive. We used cryo-electron microscopy and reconstituted in vitro transcription assays to characterize a transcriptionally-active complex including the Mediator Head module and components of a minimum preinitiation complex (RNAPII, TFIIF, TFIIB, TBP, and promoter DNA). Our results reveal how the Head interacts with RNAPII, affecting its conformation and function.

MultiBac: expanding the research toolbox for multiprotein complexes.

Protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. Among other applications, MultiBac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms.

Substrate specificity of lymphoid-specific tyrosine phosphatase (Lyp) and identification of Src kinase-associated protein of 55 kDa homolog (SKAP-HOM) as a Lyp substrate.

A missense single-nucleotide polymorphism in the gene encoding the lymphoid-specific tyrosine phosphatase (Lyp) has been identified as a causal factor in a wide spectrum of autoimmune diseases. Interestingly, the autoimmune-predisposing variant of Lyp appears to represent a gain-of-function mutation, implicating Lyp as an attractive target for the development of effective strategies for the treatment of many autoimmune disorders. Unfortunately, the precise biological functions of Lyp in signaling cascades and cellular physiology are poorly understood. Identification and characterization of Lyp substrates will help define the chain of molecular events coupling Lyp dysfunction to diseases. In the current study, we identified consensus sequence motifs for Lyp substrate recognition using an "inverse alanine scanning" combinatorial library approach. The intrinsic sequence specificity data led to the discovery and characterization of SKAP-HOM, a cytosolic adaptor protein required for proper activation of the immune system, as a bona fide Lyp substrate. To determine the molecular basis for Lyp substrate recognition, we solved crystal structures of Lyp in complex with the consensus peptide as well as the phosphopeptide derived from SKAP-HOM. Together with the biochemical data, the structures define the molecular determinants for Lyp substrate specificity and provide a solid foundation upon which novel therapeutics targeting Lyp can be developed for multiple autoimmune diseases.

Architecture of the Mediator head module.

Mediator is a key regulator of eukaryotic transcription, connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II). In the yeast Saccharomyces cerevisiae, Mediator comprises 25 subunits with a total mass of more than one megadalton (refs 5, 6) and is organized into three modules, called head, middle/arm and tail. Our understanding of Mediator assembly and its role in regulating transcription has been impeded so far by limited structural information. Here we report the crystal structure of the essential Mediator head module (seven subunits, with a mass of 223 kilodaltons) at a resolution of 4.3 ångströms. Our structure reveals three distinct domains, with the integrity of the complex centred on a bundle of ten helices from five different head subunits. An intricate pattern of interactions within this helical bundle ensures the stable assembly of the head subunits and provides the binding sites for general transcription factors and Pol II. Our structural and functional data suggest that the head module juxtaposes transcription factor IIH and the carboxy-terminal domain of the largest subunit of Pol II, thereby facilitating phosphorylation of the carboxy-terminal domain of Pol II. Our results reveal architectural principles underlying the role of Mediator in the regulation of gene expression.

Crystallization and X-ray diffraction studies of DNA-free and DNA-bound forms of EcoO109I DNA methyltransferase.

EcoO109I DNA methyltransferase (M.EcoO109I) is a type II modification enzyme from the EcoO109I restriction-modification system identified in Escherichia coli strain H709c. M.EcoO109I recognizes double-stranded RGGNCCY (where R = A or G, Y = T or C and N is any base) and transfers a methyl group to the C5 of the inner cytosines from S-adenosylmethionine. To reveal the mechanism of substrate recognition by M.EcoO109I, DNA-free and DNA-bound forms of M.EcoO109I were successfully crystallized. Crystals of the DNA-free and DNA-bound forms belonged to space groups P4(2)2(1)2, with unit-cell parameters a = b = 120.5, c = 79.8 Å, and P2(1), with unit-cell parameters a = 55.8, b = 77.4, c = 117.4 Å, ß = 93.5°, respectively.

Crystal structure of the human Hsmar1-derived transposase domain in the DNA repair enzyme Metnase.

Although the human genome is littered with sequences derived from the Hsmar1 transposon, the only intact Hsmar1 transposase gene exists within a chimeric SET-transposase fusion protein referred to as Metnase or SETMAR. Metnase retains many of the transposase activities including terminal inverted repeat (TIR) specific DNA-binding activity, DNA cleavage activity, albeit uncoupled from TIR-specific binding, and the ability to form a synaptic complex. However, Metnase has evolved as a DNA repair protein that is specifically involved in nonhomologous end joining. Here, we present two crystal structures of the transposase catalytic domain of Metnase revealing a dimeric enzyme with unusual active site plasticity that may be involved in modulating metal binding. We show through characterization of a dimerization mutant, F460K, that the dimeric form of the enzyme is required for its DNA cleavage, DNA-binding, and nonhomologous end joining activities. Of significance is the conservation of F460 along with residues that we propose may be involved in the modulation of metal binding in both the predicted ancestral Hsmar1 transposase sequence as well as in the modern enzyme. The Metnase transposase has been remarkably conserved through evolution; however, there is a clustering of substitutions located in alpha helices 4 and 5 within the putative DNA-binding site, consistent with loss of transposition specific DNA cleavage activity and acquisition of DNA repair specific cleavage activity.

Mediator head module structure and functional interactions.

We used single-particle electron microscopy to characterize the structure and subunit organization of the Mediator Head module that controls Mediator-RNA polymerase II (RNAPII) and Mediator-promoter interactions. The Head module adopts several conformations differing in the position of a movable jaw formed by the Med18-Med20 subcomplex. We also characterized, by structural, biochemical and genetic means, the interactions of the Head module with TATA-binding protein (TBP) and RNAPII subunits Rpb4 and Rpb7. TBP binds near the Med18-Med20 attachment point and stabilizes an open conformation of the Head module. Rpb4 and Rpb7 bind between the Head jaws, establishing contacts essential for yeast-cell viability. These results, and consideration of the structure of the Mediator-RNAPII holoenzyme, shed light on the stabilization of the pre-initiation complex by Mediator and suggest how Mediator might influence initiation by modulating polymerase conformation and interaction with promoter DNA.