Understanding the effects of ethanol on the liposome bilayer structure using microfluidic-based time-resolved small-angle X-ray scattering and molecular dynamics simulations.

Lipid nanoparticles (LNPs) are essential carrier particles in drug delivery systems, particularly in ribonucleic acid delivery. In preparing lipid-based nanoparticles, microfluidic-based ethanol injection may produce precisely size-controlled nanoparticles. Ethanol is critical in LNP formation and post-treatment processes and affects liposome size, structure, lamellarity, and drug-loading efficiency. However, the effects of time-dependent changes in the ethanol concentration on the structural dynamics of liposomes are not clearly understood. Herein, we investigated ethanol-induced lipid bilayer changes in liposomes on a time scale from microseconds to tens of seconds using a microfluidic-based small-angle X-ray scattering (SAXS) measurement system coupled with molecular dynamics (MD) simulations. The time-resolved SAXS measurement system revealed that single unilamellar liposomes were converted to multilamellar liposomes within 0.8 s of contact with ethanol, and the d-spacing was decreased from 6.1 (w/o ethanol) to 4.4 nm (80% ethanol) with increasing ethanol concentration. We conducted 1 µs MD simulations to understand the molecular-level structural changes in the liposomes. The MD simulations revealed that the changes in the lamellar structure caused by ethanol at the molecular level could explain the structural changes in the liposomes observed via time-resolved SAXS. Therefore, the post-treatment process to remove residual ethanol is critical in liposome formation.

Structure of full-length ERGIC-53 in complex with MCFD2 for cargo transport.

ERGIC-53 transports certain subsets of newly synthesized secretory proteins and membrane proteins from the endoplasmic reticulum to the Golgi apparatus. Despite numerous structural and functional studies since its identification, the overall architecture and mechanism of action of ERGIC-53 remain unclear. Here we present cryo-EM structures of full-length ERGIC-53 in complex with its functional partner MCFD2. These structures reveal that ERGIC-53 exists as a homotetramer, not a homohexamer as previously suggested, and comprises a four-leaf clover-like head and a long stalk composed of three sets of four-helix coiled-coil followed by a transmembrane domain. 3D variability analysis visualizes the flexible motion of the long stalk and local plasticity of the head region. Notably, MCFD2 is shown to possess a Zn2+-binding site in its N-terminal lid, which appears to modulate cargo binding. Altogether, distinct mechanisms of cargo capture and release by ERGIC- 53 via the stalk bending and metal binding are proposed.

Lipid cubic phase with an organic-inorganic hybrid structure formed by organoalkoxysilane lipid.

A lipid cubic phase encompassing a cross-linked siloxane structure was formed by the self-assembly of a synthetic organoalkoxysilane lipid in water. The spontaneous sol-gel reaction of the alkoxysilane moiety on the lipid head group produced an organic-inorganic hybrid material with a double gyroid Ia3d cubic structure.

SAXS/WAXS data of conformationally flexible ribose binding protein.

Modern artificial intelligence-based protein structure prediction methods, such as Alphafold2, can predict structures of folded proteins with reasonable accuracy. However, Alphafold2 provides a static view of a protein, which does not show the conformational variability of the protein, domain movement in a multi-domain protein, or ligand-induced conformational changes it might undergo in solution. Small-angle X-ay scattering (SAXS) and wide-angle X-ray scattering (WAXS) are solution techniques that can aid in integrative modeling of conformationally flexible proteins, or in validating their predicted ensemble structures. While SAXS is sensitive to global structural features, WAXS can expand the scope of structural modeling by including information about local structural changes. We present SAXS and WAXS datasets obtained from conformationally flexible d-ribose binding protein (RBP) from Escherichia coli in the ribose bound and unbound forms. SAXS/WAXS datasets of RBP provided here may aid in method development efforts for more accurate prediction of structural ensembles of conformationally flexible proteins, and their conformational changes.

Conformation state-specific monobodies regulate the functions of flexible proteins through conformation trapping.

Synthetic binding proteins have emerged as modulators of protein functions through protein-protein interactions (PPIs). Because PPIs are influenced by the structural dynamics of targeted proteins, investigating whether the synthetic-binders-based strategy is applicable for proteins with large conformational changes is important. This study demonstrates the applicability of monobodies (fibronectin type-III domain-based synthetic binding proteins) in regulating the functions of proteins that undergo tens-of-angstroms-scale conformational changes, using an example of the A55C/C77S/V169C triple mutant (Adktm ; a phosphoryl transfer-catalyzing enzyme with a conformational change between OPEN/CLOSED forms). Phage display successfully developed monobodies that recognize the OPEN form (substrate-unbound form), but not the CLOSED form of Adktm . Two OPEN form-specific clones (OP-2 and OP-4) inhibited Adktm kinase activity. Epitope mapping with a yeast-surface display/flow cytometry indicated that OP-2 binds to the substrate-entry side of Adktm , whereas OP-4 binding occurs at another site. Small angle X-ray scattering  coupled with size-exclusion chromatography (SEC-SAXS) indicated that OP-4 binds to the hinge side opposite to the substrate-binding site of Adktm , retaining the whole OPEN-form structure of Adktm . Titration of the OP-4-Adktm complex with Ap5 A, a transition-state analog of Adktm , showed that the conformational shift to the CLOSED form was suppressed although Adktm retained the OPEN-form (i.e., substrate-binding ready form). These results show that OP-4 captures and stabilizes the OPEN-form state, thereby affecting the hinge motion. These experimental results indicate that monobody-based modulators can regulate the functions of proteins that show tens-of-angstroms-scale conformational changes, by trapping specific conformational states generated during large conformational change process that is essential for function exertion.

The two-step cargo recognition mechanism of heterotrimeric kinesin.

Kinesin-driven intracellular transport is essential for various cell biological events and thus plays a crucial role in many pathological processes. However, little is known about the molecular basis of the specific and dynamic cargo-binding mechanism of kinesins. Here, an integrated structural analysis of the KIF3/KAP3 and KIF3/KAP3-APC complexes unveils the mechanism by which KIF3/KAP3 can dynamically grasp APC in a two-step manner, which suggests kinesin-cargo recognition dynamics composed of cargo loading, locking, and release. Our finding is the first demonstration of the two-step cargo recognition and stabilization mechanism of kinesins, which provides novel insights into the intracellular trafficking machinery.

Targeting transglutaminase 2 mediated exostosin glycosyltransferase 1 signaling in liver cancer stem cells with acyclic retinoid.

Transglutaminase 2 (TG2) is a multifunctional protein that promotes or suppresses tumorigenesis, depending on intracellular location and conformational structure. Acyclic retinoid (ACR) is an orally administered vitamin A derivative that prevents hepatocellular carcinoma (HCC) recurrence by targeting liver cancer stem cells (CSCs). In this study, we examined the subcellular location-dependent effects of ACR on TG2 activity at a structural level and characterized the functional role of TG2 and its downstream molecular mechanism in the selective depletion of liver CSCs. A binding assay with high-performance magnetic nanobeads and structural dynamic analysis with native gel electrophoresis and size-exclusion chromatography-coupled multi-angle light scattering or small-angle X-ray scattering showed that ACR binds directly to TG2, induces oligomer formation of TG2, and inhibits the transamidase activity of cytoplasmic TG2 in HCC cells. The loss-of-function of TG2 suppressed the expression of stemness-related genes, spheroid proliferation and selectively induced cell death in an EpCAM+ liver CSC subpopulation in HCC cells. Proteome analysis revealed that TG2 inhibition suppressed the gene and protein expression of exostosin glycosyltransferase 1 (EXT1) and heparan sulfate biosynthesis in HCC cells. In contrast, high levels of ACR increased intracellular Ca2+ concentrations along with an increase in apoptotic cells, which probably contributed to the enhanced transamidase activity of nuclear TG2. This study demonstrates that ACR could act as a novel TG2 inhibitor; TG2-mediated EXT1 signaling is a promising therapeutic target in the prevention of HCC by disrupting liver CSCs.

MOLASS: Software for automatic processing of matrix data obtained from small-angle X-ray scattering and UV-visible spectroscopy combined with size-exclusion chromatography.

Recent small-angle X-ray scattering (SAXS) for biological macromolecules (BioSAXS) is generally combined with size-exclusion chromatography (SEC-SAXS) at synchrotron facilities worldwide. For SEC-SAXS analysis, the final scattering profile for the target molecule is calculated from a large volume of continuously collected data. It would be ideal to automate this process; however, several complex problems exist regarding data measurement and analysis that have prevented automation. Here, we developed the analytical software MOLASS (Matrix Optimization with Low-rank factorization for Automated analysis of SEC-SAXS) to automatically calculate the final scattering profiles for solution structure analysis of target molecules. In this paper, the strategies for automatic analysis of SEC-SAXS data are described, including correction of baseline-drift using a low percentile method, optimization of peak decompositions composed of multiple scattering components using modified Gaussian fitting against the chromatogram, and rank determination for extrapolation to infinite dilution. In order to easily calculate each scattering component, the Moore-Penrose pseudo-inverse matrix is adopted as a basic calculation. Furthermore, this analysis method, in combination with UV-visible spectroscopy, led to better results in terms of accuracy in peak decomposition. Therefore, MOLASS will be able to smoothly suggest to users an accurate scattering profile for the subsequent structural analysis.

Ca2+ -induced structural changes and intramolecular interactions in N-terminal region of diacylglycerol kinase alpha.

Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that modulate the levels of lipid messengers, diacylglycerol, and phosphatidic acid. Recently, increasing attention has been paid to its α isozyme (DGKα) as a potential target for cancer immunotherapy. However, little progress has been made on the structural biology of DGKs, and a detailed understanding of the Ca2+ -triggered activation of DGKα, for which the N-terminal domains likely play a critical role, remains unclear. We have recently shown that Ca2+ binding to DGKα-EF induces conformational changes from a protease-susceptible "open" conformation in the apo state to a well-folded one in its holo state. Here, we further studied the structural properties of DGKα N-terminal (RVH and EF) domains using a series of biophysical techniques. We first revealed that the N-terminal RVH domain is a novel Ca2+ -binding domain, but the Ca2+ -induced conformational changes mainly occur in the EF domain. This was corroborated by NMR experiments showing that the EF domain adopts a molten-globule like structure in the apo state. Further analyses using SEC-SAXS and NMR indicate that the partially unfolded EF domain interacts with RVH domain, likely via hydrophobic interactions in the absence of Ca2+ , and this interaction is modified in the presence of Ca2+ . Taken together, these results present novel insights into the structural rearrangement of DGKα N-terminal domains upon binding to Ca2+ , which is essential for the activation of the enzyme.

K-edge anomalous SAXS for protein solution structure modeling.

K-edge anomalous SAXS intensity was measured from a small, dimeric, partly unstructured protein segment of myosin X by using cupric ions bound to its C-terminal polyhistidine tags. Energy-dependent anomalous SAXS can provide key location-specific information about metal-labeled protein structures in solution that cannot be obtained from routine SAXS analysis. However, anomalous SAXS is seldom used for protein research due to practical difficulties, such as a lack of generic multivalent metal-binding tags and the challenges of measuring weak anomalous signal at the metal absorption edge. This pilot feasibility study suggests that weak K-edge anomalous SAXS signal can be obtained from transition metals bound to terminally located histidine tags of small proteins. The measured anomalous signal can provide information about the distribution of all metal-protein distances in the complex. Such an anomalous SAXS signal can assist in the modeling and validation of structured or unstructured proteins in solution and may potentially become a new addition to the repertoire of techniques in integrative structural biology.

Spectroscopic and structural characteristics of a dual-light sensor protein, PYP-phytochrome related protein.

PYP-phytochrome related (Ppr) protein contains the two light sensor domains, photoactive yellow protein (PYP) and bacteriophytochrome (Bph), which mainly absorb blue and red light by the chromophores of p-coumaric acid (pCA) and biliverdin (BV), respectively. As a result, Ppr has the ability to photoactivate both domains together or separately. We investigated the photoreaction of each photosensor domain under different light irradiation conditions and clarified the inter-dependency between these domains. Within the first 10 s of blue light illumination, Ppr (Holo-Holo-Ppr) accompanied by both pCA and BV demonstrated spectrum changes reflecting PYPL accumulation, which can also be observed in Ppr containing only pCA (Holo-Apo-Ppr), and a fragment of Ppr lacking the C-terminal Bph domain. Although Holo-Apo-Ppr showed PYPL as a major photoproduct under blue light, as seen in the Bph-truncated Ppr, the equilibrium in the Holo-Holo-Ppr was shifted from PYPL to PYPM as the reaction progresses under blue light. Concomitantly, the spectrum of Bph exhibited subtle but distinguishable alteration. Together with the fact, it can be proposed that Bph with BV influences the photoreaction of PYP in Ppr, and vice versa. SAXS measurements revealed substantial tertiary structure changes in Holo-Holo-Ppr under continuous blue light irradiation within the first 5 min time domain. Interestingly, the changes in tertiary structure were partially suppressed by photoactivation of the Bph domain. These observations indicate that the photoreactions of the PYP and Bph domains are coupled with each other, and that the interplay realizes the structural switch, which might be involved in downstream signal transduction.

Optimal Mutant Model of Human S100A3 Protein Citrullinated at Arg51 by Peptidylarginine Deiminase Type III and Its Solution Structural Properties.

S100A3 protein, a member of the EF-hand-type Ca2+-binding S100 protein family, undergoes a Ca2+-/Zn2+-induced structural change to a tetrameric state upon specific citrullination of R51 in human hair cuticular cells. To elucidate the underlying mechanism, we prepared recombinant mutant S100A3 proteins, including R51A, R51C, R51E, R51K, and R51Q, as potential models of post-translationally modified S100A3 and evaluated their biophysical and biochemical properties relative to wild-type (WT) S100A3 and WT citrullinated in vitro. Size exclusion chromatography (SEC) showed that R51Q formed a tetramer in the presence of Ca2+, while Ca2+ titration monitored by Trp fluorescence indicated that R51Q had Ca2+-binding properties similar to those of citrullinated S1003A. We therefore concluded that R51Q is the optimal mutant model of post-translationally modified S100A3. We compared the solution structure of WT S100A3 and the R51Q mutant in the absence and presence of Ca2+ and Zn2+ by SEC-small-angle X-ray scattering. The radius of gyration of R51Q in the metal-free state was almost the same as that of WT; however, it increased by ∼1.5-fold in the presence of Ca2+/Zn2+, indicating a large expansion in molecular size. By contrast, addition of Ca2+/Zn2+ to WT led to nonspecific aggregation in SEC analysis and dynamic light scattering, suggesting that citrullination of S100A3 is essential for stabilization of the Ca2+-/Zn2+-bound state. These findings will lead to the further development of structural analyses for the Ca2+-/Zn2+-bound S100A3.

Structure of tRNA methyltransferase complex of Trm7 and Trm734 reveals a novel binding interface for tRNA recognition.

The complex between Trm7 and Trm734 (Trm7-Trm734) from Saccharomyces cerevisiae catalyzes 2'-O-methylation at position 34 in tRNA. We report biochemical and structural studies of the Trm7-Trm734 complex. Purified recombinant Trm7-Trm734 preferentially methylates tRNAPhe transcript variants possessing two of three factors (Cm32, m1G37 and pyrimidine34). Therefore, tRNAPhe, tRNATrp and tRNALeu are specifically methylated by Trm7-Trm734. We have solved the crystal structures of the apo and S-adenosyl-L-methionine bound forms of Trm7-Trm734. Small angle X-ray scattering reveals that Trm7-Trm734 exists as a hetero-dimer in solution. Trm7 possesses a Rossmann-fold catalytic domain, while Trm734 consists of three WD40 ß-propeller domains (termed BPA, BPB and BPC). BPA and BPC form a unique V-shaped cleft, which docks to Trm7. The C-terminal region of Trm7 is required for binding to Trm734. The D-arm of substrate tRNA is required for methylation by Trm7-Trm734. If the D-arm in tRNAPhe is docked onto the positively charged area of BPB in Trm734, the anticodon-loop is located near the catalytic pocket of Trm7. This model suggests that Trm734 is required for correct positioning of tRNA for methylation. Additionally, a point-mutation in Trm7, which is observed in FTSJ1 (human Trm7 ortholog) of nosyndromic X-linked intellectual disability patients, decreases the methylation activity.

Construction of a Quadrangular Tetramer and a Cage-Like Hexamer from Three-Helix Bundle-Linked Fusion Proteins.

Self-assembled protein nanostructures have gained interest, owing to their potential applications in biomaterials; however, successful design and construction of protein nanostructures are limited. Herein, we constructed fusion protein 1 by linking the C-terminus of a dimerization domain and the N-terminus of another dimerization domain with a three-helix bundle protein, where it self-assembled mainly into tetramers. By replacing the C-terminal dimerization domain of 1 with a trimerization domain (fusion protein 2), hexamers were mainly obtained. According to ab initio structural models reconstructed from the small-angle X-ray scattering data, the tetramer of 1 and hexamer of 2 adopted quadrangle and cage-like structures, respectively, although they were combinations of different conformations. High-speed atomic force microscopy observations indicated that the tetramer and hexamer exhibit conformational dynamics. These results show that the present method utilizing three-helix bundle-linked fusion proteins is useful in the construction of protein nanostructures.

Structure of MHC class I-like MILL2 reveals heparan-sulfate binding and interdomain flexibility.

The MILL family, composed of MILL1 and MILL2, is a group of nonclassical MHC class I molecules that occur in some orders of mammals. It has been reported that mouse MILL2 is involved in wound healing; however, the molecular mechanisms remain unknown. Here, we determine the crystal structure of MILL2 at 2.15 Å resolution, revealing an organization similar to classical MHC class I. However, the α1-α2 domains are not tightly fixed on the α3-ß2m domains, indicating unusual interdomain flexibility. The groove between the two helices in the α1-α2 domains is too narrow to permit ligand binding. Notably, an unusual basic patch on the α3 domain is involved in the binding to heparan sulfate which is essential for MILL2 interactions with fibroblasts. These findings suggest that MILL2 has a unique structural architecture and physiological role, with binding to heparan sulfate proteoglycans on fibroblasts possibly regulating cellular recruitment in biological events.

Crystal structure of the flexible tandem repeat domain of bacterial cellulose synthesis subunit C.

Bacterial cellulose (BC) is synthesized and exported through the cell membrane via a large protein complex (terminal complex) that consists of three or four subunits. BcsC is a little-studied subunit considered to export BC to the extracellular matrix. It is predicted to have two domains: a tetratrico peptide repeat (TPR) domain and a ß-barrelled outer membrane domain. Here we report the crystal structure of the N-terminal part of BcsC-TPR domain (Asp24-Arg272) derived from Enterobacter CJF-002. Unlike most TPR-containing proteins which have continuous TPR motifs, this structure has an extra α-helix between two clusters of TPR motifs. Five independent molecules in the crystal had three different conformations that varied at the hinge of the inserted α-helix. Such structural feature indicates that the inserted α-helix confers flexibility to the chain and changes the direction of the TPR super-helix, which was also suggested by structural analysis of BcsC-TPR (Asp24-Leu664) in solution by size exclusion chromatography-small-angle X-ray scattering. The flexibility at the α-helical hinge may play important role for exporting glucan chains.

Neutron crystallography of photoactive yellow protein reveals unusual protonation state of Arg52 in the crystal.

Because of its high pKa, arginine (Arg) is believed to be protonated even in the hydrophobic environment of the protein interior. However, our neutron crystallographic structure of photoactive yellow protein, a light sensor, demonstrated that Arg52 adopts an electrically neutral form. We also showed that the hydrogen bond between the chromophore and Glu46 is a so-called low barrier hydrogen bond (LBHB). Because both the neutral Arg and LBHB are unusual in proteins, these observations remain controversial. To validate our findings, we carried out neutron crystallographic analysis of the E46Q mutant of PYP. The resultant structure revealed that the proportion of the cationic form is higher in E46Q than in WT, although the cationic and neutral forms of Arg52 coexist in E46Q. These observations were confirmed by the occupancy of the deuterium atom bound to the N η1 atom combined with an alternative conformation of the N(η2)D2 group comprising sp2 hybridisation. Based on these results, we propose that the formation of the LBHB decreases the proton affinity of Arg52, stabilizing the neutral form in the crystal.

Probing the early stages of photoreception in photoactive yellow protein with ultrafast time-domain Raman spectroscopy.

Unveiling the nuclear motions of photoreceptor proteins in action is a crucial goal in protein science in order to understand their elaborate mechanisms and how they achieve optimal selectivity and efficiency. Previous studies have provided detailed information on the structures of intermediates that appear during the later stages (>ns) of such photoreception cycles, yet the initial events immediately after photoabsorption remain unclear because of experimental challenges in monitoring nuclear rearrangements on ultrafast timescales, including protein-specific low-frequency motions. Using time-domain Raman probing with sub-7-fs pulses, we obtain snapshot vibrational spectra of photoactive yellow protein and a mutant with high sensitivity, providing insights into the key responses that drive photoreception. Our data show a drastic intensity drop of the excited-state marker band at 135 cm-1 within a few hundred femtoseconds, suggesting a rapid weakening of the hydrogen bond that anchors the chromophore. We also track formation of the first ground-state intermediate over the first few picoseconds and fully characterize its vibrational structure, revealing a substantially-twisted cis conformation.

Unambiguous Determination of Protein Arginine Ionization States in Solution by NMR Spectroscopy.

Because arginine residues in proteins are expected to be in their protonated form almost without exception, reports demonstrating that a protein arginine residue is charge-neutral are rare and potentially controversial. Herein, we present a 13 C-detected NMR experiment for probing individual arginine residues in proteins notwithstanding the presence of chemical and conformational exchange effects. In the experiment, the 15 Nη and 15 Nϵ chemical shifts of an arginine head group are correlated with that of the directly attached 13 Cζ . In the resulting spectrum, the number of protons in the arginine head group can be obtained directly from the 15 N-1 H scalar coupling splitting pattern. We applied this method to unambiguously determine the ionization state of the R52 side chain in the photoactive yellow protein from Halorhodospira halophila. Although only three Hη atoms were previously identified by neutron crystallography, we show that R52 is predominantly protonated in solution.