PTCHD1 Binds Cholesterol but Not Sonic Hedgehog, Suggesting a Distinct Cellular Function.

Deleterious mutations in the X-linked Patched domain-containing 1 (PTCHD1) gene may account for up to 1% of autism cases. Despite this, the PTCHD1 protein remains poorly understood. Structural similarities to Patched family proteins point to a role in sterol transport, but this hypothesis has not been verified experimentally. Additionally, PTCHD1 has been suggested to be involved in Hedgehog signalling, but thus far, the experimental results have been conflicting. To enable a variety of biochemical and structural experiments, we developed a method for expressing PTCHD1 in Spodoptera frugiperda cells, solubilising it in glycol-diosgenin, and purifying it to homogeneity. In vitro and in silico experiments show that PTCHD1 function is not interchangeable with Patched 1 (PTCH1) in canonical Hedgehog signalling, since it does not repress Smoothened in Ptch1-/- mouse embryonic fibroblasts and does not bind Sonic Hedgehog. However, we found that PTCHD1 binds cholesterol similarly to PTCH1. Furthermore, we identified 13 PTCHD1-specific protein interactors through co-immunoprecipitation and demonstrated a link to cell stress responses and RNA stress granule formation. Thus, our results support the notion that despite structural similarities to other Patched family proteins, PTCHD1 may have a distinct cellular function.

The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins.

An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT™ system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT™ system have been reported, thereby reducing final yields. Therefore, controlling the protein-splicing activity of inteins has become critical. Here we utilized conditional protein splicing by salt conditions. We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides. Moreover, we described a method for the amidation using the same IIST system and demonstrated 15N-labeling of the C-terminal amide group of a single domain antibody (VHH).

NMR Structure Determinations of Small Proteins Using only One Fractionally 20% 13C- and Uniformly 100% 15N-Labeled Sample.

Uniformly 13C- and 15N-labeled samples ensure fast and reliable nuclear magnetic resonance (NMR) assignments of proteins and are commonly used for structure elucidation by NMR. However, the preparation of uniformly labeled samples is a labor-intensive and expensive step. Reducing the portion of 13C-labeled glucose by a factor of five using a fractional 20% 13C- and 100% 15N-labeling scheme could lower the total chemical costs, yet retaining sufficient structural information of uniformly [13C, 15N]-labeled sample as a result of the improved sensitivity of NMR instruments. Moreover, fractional 13C-labeling can facilitate reliable resonance assignments of sidechains because of the biosynthetic pathways of each amino-acid. Preparation of only one [20% 13C, 100% 15N]-labeled sample for small proteins (<15 kDa) could also eliminate redundant sample preparations of 100% 15N-labeled and uniformly 100% [13C, 15N]-labeled samples of proteins. We determined the NMR structures of a small alpha-helical protein, the C domain of IgG-binding protein A from Staphylococcus aureus (SpaC), and a small beta-sheet protein, CBM64 module using [20% 13C, 100% 15N]-labeled sample and compared with the crystal structures and the NMR structures derived from the 100% [13C, 15N]-labeled sample. Our results suggest that one [20% 13C, 100% 15N]-labeled sample of small proteins could be routinely used as an alternative to conventional 100% [13C, 15N]-labeling for backbone resonance assignments, NMR structure determination, 15N-relaxation analysis, and ligand-protein interaction.

Mutations in the U11/U12-65K protein associated with isolated growth hormone deficiency lead to structural destabilization and impaired binding of U12 snRNA.

Mutations in the components of the minor spliceosome underlie several human diseases. A subset of patients with isolated growth hormone deficiency (IGHD) harbors mutations in the RNPC3 gene, which encodes the minor spliceosome-specific U11/U12-65K protein. Although a previous study showed that IGHD patient cells have defects in U12-type intron recognition, the biochemical effects of these mutations on the 65K protein have not been characterized. Here, we show that a proline-to-threonine missense mutation (P474T) and a nonsense mutation (R502X) in the C-terminal RNA recognition motif (C-RRM) of the 65K protein impair the binding of 65K to U12 and U6atac snRNAs. We further show that the nonsense allele is targeted to the nonsense-mediated decay (NMD) pathway, but in an isoform-specific manner, with the nuclear-retained 65K long-3'UTR isoform escaping the NMD pathway. In contrast, the missense P474T mutation leads, in addition to the RNA-binding defect, to a partial defect in the folding of the C-RRM and reduced stability of the full-length protein, thus reducing the formation of U11/U12 di-snRNP complexes. We propose that both the C-RRM folding defect and NMD-mediated decrease in the levels of the U11/U12-65K protein reduce formation of the U12-type intron recognition complex and missplicing of a subset of minor introns leading to pituitary hypoplasia and a subsequent defect in growth hormone secretion.

Heterogeneous dynamics in partially disordered proteins.

Importance of disordered protein regions is increasingly recognized in biology, but their characterization remains challenging due to the lack of suitable experimental and theoretical methods. NMR experiments can detect multiple timescale dynamics and structural details of disordered protein regions, but their detailed interpretation is often difficult. Here we combine protein backbone 15N spin relaxation data with molecular dynamics (MD) simulations to detect not only heterogeneous dynamics of large partially disordered proteins but also their conformational ensembles. We observed that the rotational dynamics of folded regions in partially disordered proteins is dominated by similar rigid body rotation as in globular proteins, thereby being largely independent of flexible disordered linkers. Disordered regions, on the other hand, exhibit complex rotational motions with multiple timescales below ∼30 ns which are difficult to detect from experimental data alone, but can be captured by MD simulations. Combining MD simulations and backbone 15N spin relaxation data, measured applying segmental isotopic labeling with salt-inducible split intein, we resolved the conformational ensemble and dynamics of partially disordered periplasmic domain of TonB protein from Helicobacter pylori containing 250 residues. To demonstrate the universality of our approach, it was applied also to the partially disordered region of chicken Engrailed 2. Our results pave the way in understanding how TonB transfers energy from inner membrane to the outer membrane receptors in Gram-negative bacteria, as well as the function of other proteins with disordered domains.

An off-the-Shelf Approach for the Production of Fc Fusion Proteins by Protein Trans-Splicing towards Generating a Lectibody In Vitro.

Monoclonal antibodies, engineered antibodies, and antibody fragments have become important biological therapeutic platforms. The IgG format with bivalent binding sites has a modular structure with different biological roles, i.e., effector and binding functions, in different domains. We demonstrated the reconstruction of an IgG-like domain structure in vitro by protein ligation using protein trans-splicing. We produced various binding domains to replace the binding domain of IgG from Escherichia coli and the Fc domain of human IgG from Brevibacillus choshinensis as split-intein fusions. We showed that in vitro protein ligation could produce various Fc-fusions at the N-terminus in vitro from the independently produced domains from different organisms. We thus propose an off-the-shelf approach for the combinatorial production of Fc fusions in vitro with several distinct binding domains, particularly from naturally occurring binding domains. Antiviral lectins from algae are known to inhibit virus entry of HIV and SARS coronavirus. We demonstrated that a lectin could be fused with the Fc-domain in vitro by protein ligation, producing an IgG-like molecule as a "lectibody". Such an Fc-fusion could be produced in vitro by this approach, which could be an attractive method for developing potential therapeutic agents against rapidly emerging infectious diseases like SARS coronavirus without any genetic fusion and expression optimization.

The Convergence of the Hedgehog/Intein Fold in Different Protein Splicing Mechanisms.

Protein splicing catalyzed by inteins utilizes many different combinations of amino-acid types at active sites. Inteins have been classified into three classes based on their characteristic sequences. We investigated the structural basis of the protein splicing mechanism of class 3 inteins by determining crystal structures of variants of a class 3 intein from Mycobacterium chimaera and molecular dynamics simulations, which suggested that the class 3 intein utilizes a different splicing mechanism from that of class 1 and 2 inteins. The class 3 intein uses a bond cleavage strategy reminiscent of proteases but share the same Hedgehog/INTein (HINT) fold of other intein classes. Engineering of class 3 inteins from a class 1 intein indicated that a class 3 intein would unlikely evolve directly from a class 1 or 2 intein. The HINT fold appears as structural and functional solution for trans-peptidyl and trans-esterification reactions commonly exploited by diverse mechanisms using different combinations of amino-acid types for the active-site residues.

Off-Pathway-Sensitive Protein-Splicing Screening Based on a Toxin/Antitoxin System.

Protein-splicing domains are frequently used engineering tools that find application in the in vivo and in vitro ligation of protein domains. Directed evolution is among the most promising technologies used to advance this technology. However, the available screening systems for protein-splicing activity are associated with bottlenecks such as the selection of pseudo-positive clones arising from off-pathway reaction products or fragment complementation. Herein, we report a stringent screening method for protein-splicing activity in cis and trans, that exclusively selects productively splicing domains. By fusing splicing domains to an intrinsically disordered region of the antidote from the Escherichia coli CcdA/CcdB type II toxin/antitoxin system, we linked protein splicing to cell survival. The screen allows selecting novel cis- and trans-splicing inteins catalyzing productive highly efficient protein splicing, for example, from directed-evolution approaches or the natural intein sequence space.

Crystal structures of CDC21-1 inteins from hyperthermophilic archaea reveal the selection mechanism for the highly conserved homing endonuclease insertion site.

Self-splicing inteins are mobile genetic elements invading host genes via nested homing endonuclease (HEN) domains. All HEN domains residing within inteins are inserted at a highly conserved insertion site. A purifying selection mechanism directing the location of the HEN insertion site has not yet been identified. In this work, we solved the three-dimensional crystal structures of two inteins inserted in the cell division control protein 21 of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii. A comparison between the structures provides the structural basis for the thermo-stabilization mechanism of inteins that have lost the HEN domain during evolution. The presence of an entire extein domain in the intein structure from Pyrococcus horikoshii suggests the selection mechanism for the highly conserved HEN insertion point.

Conformational dynamics are a key factor in signaling mediated by the receiver domain of a sensor histidine kinase from Arabidopsis thaliana.

Multistep phosphorelay (MSP) cascades mediate responses to a wide spectrum of stimuli, including plant hormonal signaling, but several aspects of MSP await elucidation. Here, we provide first insight into the key step of MSP-mediated phosphotransfer in a eukaryotic system, the phosphorylation of the receiver domain of the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1RD) from Arabidopsis thaliana We observed that the crystal structures of free, Mg2+-bound, and beryllofluoridated CKI1RD (a stable analogue of the labile phosphorylated form) were identical and similar to the active state of receiver domains of bacterial response regulators. However, the three CKI1RD variants exhibited different conformational dynamics in solution. NMR studies revealed that Mg2+ binding and beryllofluoridation alter the conformational equilibrium of the ß3-α3 loop close to the phosphorylation site. Mutations that perturbed the conformational behavior of the ß3-α3 loop while keeping the active-site aspartate intact resulted in suppression of CKI1 function. Mechanistically, homology modeling indicated that the ß3-α3 loop directly interacts with the ATP-binding site of the CKI1 histidine kinase domain. The functional relevance of the conformational dynamics observed in the ß3-α3 loop of CKI1RD was supported by a comparison with another A. thaliana histidine kinase, ETR1. In contrast to the highly dynamic ß3-α3 loop of CKI1RD, the corresponding loop of the ETR1 receiver domain (ETR1RD) exhibited little conformational exchange and adopted a different orientation in crystals. Biochemical data indicated that ETR1RD is involved in phosphorylation-independent signaling, implying a direct link between conformational behavior and the ability of eukaryotic receiver domains to participate in MSP.

Segmental isotopic labeling by asparaginyl endopeptidase-mediated protein ligation.

Segmental isotopic labeling can facilitate NMR studies of large proteins, multi-domain proteins, and proteins with repetitive sequences by alleviating NMR signal overlaps. Segmental isotopic labeling also allows us to investigate an individual domain in the context of a full-length protein by NMR. Several established methods are available for segmental isotopic labeling such as intein-mediated ligation, but each has specific requirements and limitations. Here, we report an enzymatic approach using bacterially produced asparagine endopeptidase from Oldenlandia affinis for segmental isotopic labeling of a protein with repetitive sequences, a designed armadillo repeat protein, by overcoming some of the shortcomings of enzymatic ligation for segmental isotopic labeling.

Evaluation of blood adsorption onto dialysis membranes by time-of-flight secondary ion mass spectrometry and near-field infrared microscopy.

Blood adsorption onto the inside surface of hollow fiber dialysis membranes was investigated by means of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and near-field infrared microscopy (NFIR) in order to evaluate the biocompatibility and permeability of dialysis membranes. TOF-SIMS is useful for the imaging of particular molecules with a high spatial resolution of approximately 100 nm. In contrast, infrared spectra provide quantitative information and NFIR enables analysis with a high spatial resolution of less than 1 µm, which is close to the resolution of TOF-SIMS. A comparison was made of one of the most widely used dialysis membranes made of polysulfone (PSf), that has an asymmetric and inhomogeneous pore structure, and a newly developed asymmetric cellulose triacetate (ATA) membrane that also has an asymmetric pore structure, even though the conventional cellulose triacetate membrane has a symmetric and homogeneous pore structure. As a result, it was demonstrated that blood adsorption on the inside surface of the ATA membrane is more reduced than that on the PSf membrane. Graphical abstract Analysis of blood adsorption on inside surface of hollow fiber membrane.

Improved molecular replacement by density- and energy-guided protein structure optimization.

Molecular replacement procedures, which search for placements of a starting model within the crystallographic unit cell that best account for the measured diffraction amplitudes, followed by automatic chain tracing methods, have allowed the rapid solution of large numbers of protein crystal structures. Despite extensive work, molecular replacement or the subsequent rebuilding usually fail with more divergent starting models based on remote homologues with less than 30% sequence identity. Here we show that this limitation can be substantially reduced by combining algorithms for protein structure modelling with those developed for crystallographic structure determination. An approach integrating Rosetta structure modelling with Autobuild chain tracing yielded high-resolution structures for 8 of 13 X-ray diffraction data sets that could not be solved in the laboratories of expert crystallographers and that remained unsolved after application of an extensive array of alternative approaches. We estimate that the new method should allow rapid structure determination without experimental phase information for over half the cases where current methods fail, given diffraction data sets of better than 3.2 Å resolution, four or fewer copies in the asymmetric unit, and the availability of structures of homologous proteins with >20% sequence identity.

Intermolecular domain swapping induces intein-mediated protein alternative splicing.

Protein sequences are diversified on the DNA level by recombination and mutation and can be further increased on the RNA level by alternative RNA splicing, involving introns that have important roles in many biological processes. The protein version of introns (inteins), which catalyze protein splicing, were first reported in the 1990s. The biological roles of protein splicing still remain elusive because inteins neither provide any clear benefits nor have an essential role in their host organisms. We now report protein alternative splicing, in which new protein sequences can be produced by protein recombination by intermolecular domain swapping of inteins, as elucidated by NMR spectroscopy and crystal structures. We demonstrate that intein-mediated protein alternative splicing could be a new strategy to increase protein diversity (that is, functions) without any modification in genetic backgrounds. We also exploited it as a post-translational protein conformation-driven switch of protein functions (for example, as highly specific protein interference).

Tandem SUMO fusion vectors for improving soluble protein expression and purification.

Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate efficient protein purification and to improve soluble overexpression of proteins. Various purification and fusion tags have been widely used for overexpression in Escherichia coli. However, these tags might interfere with biological functions and/or structural investigations of the protein of interest. Therefore, an additional purification step to remove fusion tags by proteolytic digestion might be required. Here, we describe a set of new vectors in which yeast SUMO (SMT3) was used as the highly specific recognition sequence of ubiquitin-like protease 1, together with other commonly used solubility enhancing proteins, such as glutathione S-transferase, maltose binding protein, thioredoxin and trigger factor for optimizing soluble expression of protein of interest. This tandem SUMO (T-SUMO) fusion system was tested for soluble expression of the C-terminal domain of TonB from different organisms and for the antiviral protein scytovirin.

ToF-SIMS observation for evaluating the interaction between amyloid ß and lipid membranes.

The adsorption behaviour of amyloid beta (Aß), thought to be a key peptide for understanding Alzheimer's disease, was investigated by means of time-of-flight secondary ion mass spectrometry (ToF-SIMS). Aß aggregates depending on the lipid membrane condition though it has not been fully understood yet. In this study, Aß samples on different lipid membranes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), were observed with ToF-SIMS and the complex ToF-SIMS data of the Aß samples was interpreted using data analysis techniques such as principal component analysis (PCA), gentle-SIMS (G-SIMS) and g-ogram. DOPC and DMPC are liquid crystal at room temperature, while DPPC is gel at room temperature. As primary ion beams, Bi3(+) and Ar cluster ion beams were used and the effect of an Ar cluster ion for evaluating biomolecules was also studied. The secondary ion images of the peptide fragment ions indicated by G-SIMS and g-ogram were consistent with the PCA results. It is suggested that Aß is adsorbed homogeneously on the liquid-crystalline-phase lipid membranes, while it aggregates along the lipid on the gel-phase lipid membrane. Moreover, in the results using the Ar cluster, the influence of contamination was reduced.

Hidden photoinduced reactivity of the blue fluorescent protein mKalama1.

Understanding the photoinduced dynamics of fluorescent proteins is essential for their applications in bioimaging. Despite numerous studies on the ultrafast dynamics, the delayed response of these proteins, which often results in population of kinetically trapped dark states of various origins, is largely unexplored. Here, by using transient absorption spectroscopy spanning the time scale from picoseconds to seconds, we reveal a hidden reactivity of the bright blue-light emitting protein mKalama1 previously thought to be inert. This protein shows no excited-state proton transfer during its nanosecond excited-state lifetime; however, its tyrosine-based chromophore undergoes deprotonation coupled to non-radiative electronic relaxation. Such deprotonation causes distinct optical absorption changes in the broad UV-to-NIR spectral range (ca. 300-800 nm); the disappearance of the transient absorption signal has a complex nature and spans the whole microsecond-to-second time scale. The mechanisms underlying the relaxation kinetics are disclosed based on the X-ray structural analysis of mKalama1 and the high-level electronic structure calculations of proposed intermediates in the photocycle. We conclude that the non-radiative excited-state decay includes two major branches: internal conversion coupled to intraprotein proton transfer, where a conserved residue E222 serves as the proton acceptor; and ionization induced by two consecutive resonant absorption events, followed by deprotonation of the chromophore radical cation to bulk solvent through a novel water-mediated proton-wire pathway. Our findings open up new perspectives on the dynamics of fluorescent proteins as tracked by its optical transient absorption in the time domain extending up to seconds.

Structural basis for complement evasion by Lyme disease pathogen Borrelia burgdorferi.

Borrelia burgdorferi spirochetes that cause Lyme borreliosis survive for a long time in human serum because they successfully evade the complement system, an important arm of innate immunity. The outer surface protein E (OspE) of B. burgdorferi is needed for this because it recruits complement regulator factor H (FH) onto the bacterial surface to evade complement-mediated cell lysis. To understand this process at the molecular level, we used a structural approach. First, we solved the solution structure of OspE by NMR, revealing a fold that has not been seen before in proteins involved in complement regulation. Next, we solved the x-ray structure of the complex between OspE and the FH C-terminal domains 19 and 20 (FH19-20) at 2.83 Å resolution. The structure shows that OspE binds FH19-20 in a way similar to, but not identical with, that used by endothelial cells to bind FH via glycosaminoglycans. The observed interaction of OspE with FH19-20 allows the full function of FH in down-regulation of complement activation on the bacteria. This reveals the molecular basis for how B. burgdorferi evades innate immunity and suggests how OspE could be used as a potential vaccine antigen.

Photothermal conversion of CO2 into CH4 with H2 over Group VIII nanocatalysts: an alternative approach for solar fuel production.

The photothermal conversion of CO2 provides a straightforward and effective method for the highly efficient production of solar fuels with high solar-light utilization efficiency. This is due to several crucial features of the Group VIII nanocatalysts, including effective energy utilization over the whole range of the solar spectrum, excellent photothermal performance, and unique activation abilities. Photothermal CO2 reaction rates (mol h(-1) g(-1)) that are several orders of magnitude larger than those obtained with photocatalytic methods (µmol h(-1) g(-1)) were thus achieved. It is proposed that the overall water-based CO2 conversion process can be achieved by combining light-driven H2 production from water and photothermal CO2 conversion with H2. More generally, this work suggests that traditional catalysts that are characterized by intense photoabsorption will find new applications in photo-induced green-chemistry processes.

The three-dimensional structure of the Vint domain from Tetrahymena thermophila suggests a ligand-regulated cleavage mechanism by the HINT fold.

Vint proteins have been identified in unicellular metazoans as a novel hedgehog-related gene family, merging the von Willebrand factor type A domain and the Hedgehog/INTein (HINT) domains. We present the first three-dimensional structure of the Vint domain from Tetrahymena thermophila corresponding to the auto-processing domain of hedgehog proteins, shedding light on the unique features, including an adduct recognition region (ARR). Our results suggest a potential binding between the ARR and sulfated glycosaminoglycans like heparin sulfate. Moreover, we uncover a possible regulatory role of the ARR in the auto-processing by Vint domains, expanding our understanding of the HINT domain evolution and their use in biotechnological applications. Vint domains might have played a crucial role in the transition from unicellular to multicellular organisms.