Displaying a Protein Cage on a Protein Crystal by In-Cell Crystal Engineering.

The development of solid biomaterials has rapidly progressed in recent years in applications in bionanotechnology. The immobilization of proteins, such as enzymes, within protein crystals is being used to develop solid catalysts and functionalized materials. However, an efficient method for encapsulating protein assemblies has not yet been established. This work presents a novel approach to displaying protein cages onto a crystalline protein scaffold using in-cell protein crystal engineering. The polyhedra crystal (PhC) scaffold, which displays a ferritin cage, was produced by coexpression of polyhedrin monomer (PhM) and H1-ferritin (H1-Fr) monomer in Escherichia coli. The H1-tag is derived from the H1-helix of PhM. Our technique represents a unique strategy for immobilizing protein assemblies onto in-cell protein crystals and is expected to contribute to various applications in bionanotechnology.

Crystal structure of the in-cell Cry1Aa purified from Bacillus thuringiensis.

In-cell protein crystals which spontaneously crystallize in living cells, have recently been analyzed in investigations of their structures and biological functions. The crystals have been challenging to analyze structurally because of their small size. Therefore, the number of in-cell protein crystals in which the native structure has been determined is limited because most of the structures of in-cell crystals have been determined by recrystallization after dissolution. Some proteins have been reported to form intermolecular disulfide bonds in natural protein crystals that stabilize the crystals. Here, we focus on Cry1Aa, a cysteine-rich protein that crystallizes in Bacillus thuringiensis (Bt) and forms disulfide bonds. Previously, the full-length structure of 135 kDa Cry1Ac, which is the same size as Cry1Aa, was determined by recrystallization of dissolved protein from crystals purified from Bt cells. However, the formation of disulfide bonds has not been investigated because it was necessary to replace cysteine residues to prevent aggregation of the soluble protein. In this work, we succeeded in direct X-ray crystallographic analysis using crystals purified from Bt cells and characterized the cross-linked network of disulfide bonds within Cry1Aa crystals.

Elucidating polymorphs of crystal structures by intensity-based hierarchical clustering analysis of multiple diffraction data sets.

In macromolecular structure determination using X-ray diffraction from multiple crystals, the presence of different structures (structural polymorphs) necessitates the classification of the diffraction data for appropriate structural analysis. Hierarchical clustering analysis (HCA) is a promising technique that has so far been used to extract isomorphous data, mainly for single-structure determination. Although in principle the use of HCA can be extended to detect polymorphs, the absence of a reference to define the threshold used to group the isomorphous data sets (the `isomorphic threshold') poses a challenge. Here, unit-cell-based and intensity-based HCAs have been applied to data sets for apo trypsin and inhibitor-bound trypsin that were mixed post data acquisition to investigate the efficacy of HCA in classifying polymorphous data sets. Single-step intensity-based HCA successfully classified polymorphs with a certain `isomorphic threshold'. In data sets for several samples containing an unknown degree of structural heterogeneity, polymorphs could be identified by intensity-based HCA using the suggested `isomorphic threshold'. Polymorphs were also detected in single crystals using data collected using the continuous helical scheme. These findings are expected to facilitate the determination of multiple structural snapshots by exploiting automated data collection and analysis.

Arginine cluster introduction on framework region in anti-lysozyme antibody improved association rate constant by changing conformational diversity of CDR loops.

Antibodies are used for many therapeutic and biotechnological purposes. Because the affinity of an antibody to the antigen is critical for clinical efficacy of pharmaceuticals, many affinity maturation strategies have been developed. Although we previously reported an affinity maturation strategy in which the association rate of the antibody toward its antigen is improved by introducing a cluster of arginine residues into the framework region of the antibody, the detailed molecular mechanism responsible for this improvement has been unknown. In this study, we introduced five arginine residues into an anti-hen egg white lysozyme antibody (HyHEL10) Fab fragment to create the R5-mutant and comprehensively characterized the interaction between antibody and antigen using thermodynamic analysis, X-ray crystallography, and molecular dynamics (MD) simulations. Our results indicate that introduction of charged residues strongly enhanced the association rate, as previously reported, and the antibody-antigen complex structure was almost the same for the R5-mutant and wild-type Fabs. The MD simulations indicate that the mutation increased conformational diversity in complementarity-determining region loops and thereby enhanced the association rate. These observations provide the molecular basis of affinity maturation by R5 mutation.

Constrained catecholamines gain ß2AR selectivity through allosteric effects on pocket dynamics.

G protein-coupled receptors (GPCRs) within the same subfamily often share high homology in their orthosteric pocket and therefore pose challenges to drug development. The amino acids that form the orthosteric binding pocket for epinephrine and norepinephrine in the ß1 and ß2 adrenergic receptors (ß1AR and ß2AR) are identical. Here, to examine the effect of conformational restriction on ligand binding kinetics, we synthesized a constrained form of epinephrine. Surprisingly, the constrained epinephrine exhibits over 100-fold selectivity for the ß2AR over the ß1AR. We provide evidence that the selectivity may be due to reduced ligand flexibility that enhances the association rate for the ß2AR, as well as a less stable binding pocket for constrained epinephrine in the ß1AR. The differences in the amino acid sequence of the extracellular vestibule of the ß1AR allosterically alter the shape and stability of the binding pocket, resulting in a marked difference in affinity compared to the ß2AR. These studies suggest that for receptors containing identical binding pocket residues, the binding selectivity may be influenced in an allosteric manner by surrounding residues, like those of the extracellular loops (ECLs) that form the vestibule. Exploiting these allosteric influences may facilitate the development of more subtype-selective ligands for GPCRs.

Engineering of an in-cell protein crystal for fastening a metastable conformation of a target miniprotein.

Protein crystals can be utilized as porous scaffolds to capture exogenous molecules. Immobilization of target proteins using protein crystals is expected to facilitate X-ray structure analysis of proteins that are difficult to be crystallized. One of the advantages of scaffold-assisted structure determination is the analysis of metastable structures that are not observed in solution. However, efforts to fix target proteins within the pores of scaffold protein crystals have been limited due to the lack of strategies to control protein-protein interactions formed in the crystals. In this study, we analyze the metastable structure of the miniprotein, CLN025, which forms a ß-hairpin structure in solution, using a polyhedra crystal (PhC), an in-cell protein crystal. CLN025 is successfully fixed within the PhC scaffold by replacing the original loop region. X-ray crystal structure analysis and molecular dynamics (MD) simulation reveal that CLN025 is fixed as a helical structure in a metastable state by non-covalent interactions in the scaffold crystal. These results indicate that modulation of intermolecular interactions can trap various protein conformations in the engineered PhC and provides a new strategy for scaffold-assisted structure determination.

Cell-free protein crystallization for nanocrystal structure determination.

In-cell protein crystallization (ICPC) has been investigated as a technique to support the advancement of structural biology because it does not require protein purification and a complicated crystallization process. However, only a few protein structures have been reported because these crystals formed incidentally in living cells and are insufficient in size and quality for structure analysis. Here, we have developed a cell-free protein crystallization (CFPC) method, which involves direct protein crystallization using cell-free protein synthesis. We have succeeded in crystallization and structure determination of nano-sized polyhedra crystal (PhC) at a high resolution of 1.80 Å. Furthermore, nanocrystals were synthesized at a reaction scale of only 20 µL using the dialysis method, enabling structural analysis at a resolution of 1.95 Å. To further demonstrate the potential of CFPC, we attempted to determine the structure of crystalline inclusion protein A (CipA), whose structure had not yet been determined. We added chemical reagents as a twinning inhibitor to the CFPC solution, which enabled us to determine the structure of CipA at 2.11 Å resolution. This technology greatly expands the high-throughput structure determination method of unstable, low-yield, fusion, and substrate-biding proteins that have been difficult to analyze with conventional methods.

De novo Fc-based receptor dimerizers differentially modulate PlexinB1 function.

Signaling by single-pass transmembrane receptors often involves a formation of ligand-induced receptor dimers with particular conformation, and bivalent receptor binders can modulate receptor functions by inducing different receptor dimer conformations, although such agents are difficult to design. Here, we describe the generation of both antagonistic and agonistic receptor dimerizers toward PlexinB1 (PlxnB1), a receptor for semaphorin 4D (Sema4D), by grafting two different PlxnB1-binding peptides onto the human immunoglobulin G1 (IgG1) Fc protein. The function-modulating activity of a peptide Fc was strongly dependent on the type of the peptide as well as the grafting site, with the best variants showing activity at an nM concentration range. Structural analysis of each peptide-PlxnB1 complex revealed that the agonistic Fc dimerizes PlxnB1 in a face-to-face fashion similar to that induced by Sema4D, whereas antagonistic Fc would induce signaling-incompetent PlxnB1 dimer conformation, enforcing the idea that plexin activation is primarily controlled by the receptor orientation within the dimer.

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP.

Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including Escherichia coli RseP. Structure-based chemical modification and cross-linking experiments indicated that the RseP domains surrounding the active center undergo conformational changes to expose the substrate-binding site, suggesting that RseP has a gating mechanism to regulate substrate entry. Furthermore, mutational analysis suggests that a conserved electrostatic linkage between the transmembrane and peripheral membrane-associated domains mediates the conformational changes. In vivo cleavage assays also support that the substrate transmembrane helix is unwound by strand addition to the intramembrane ß sheet of RseP and is clamped by a conserved asparagine residue at the active center for efficient cleavage. This mechanism underlying the substrate binding, i.e., unwinding and clamping, appears common across distinct families of intramembrane proteases that cleave transmembrane segments.

Guidelines for de novo phasing using multiple small-wedge data collection. Corrigendum.

A figure in the article by Baba et al. [(2021), J. Synchrotron Rad. 28, 1284-1295] is corrected.

The structure of NLRP9 reveals a unique C-terminal region with putative regulatory function.

Nucleotide-binding and oligomerisation domain-like receptors (NLRs) can form inflammasomes that activate caspase-1 and pro-interleukin-1ß and induce pyroptosis. NLR family pyrin domain-containing 9 (NLRP9) forms an inflammasome and activates innate immune responses during virus infection, but little is known about this process. Here, we report the crystal and cryo-electron microscopy structures of NLRP9 in an ADP-bound state, revealing inactive and closed conformations of NLRP9 and its similarities to other structurally characterised NLRs. Moreover, we found a C-terminal region interacting with the concave surface of the leucine-rich repeat domain of NLRP9. This region is unique among NLRs and might be involved in the specific function of NLRP9. These data provide the structural basis for understanding the mechanism of NLRP9 regulation and activation.

Molecular mechanism of agonism and inverse agonism in ghrelin receptor.

Much effort has been invested in the investigation of the structural basis of G protein-coupled receptors (GPCRs) activation. Inverse agonists, which can inhibit GPCRs with constitutive activity, are considered useful therapeutic agents, but the molecular mechanism of such ligands remains insufficiently understood. Here, we report a crystal structure of the ghrelin receptor bound to the inverse agonist PF-05190457 and a cryo-electron microscopy structure of the active ghrelin receptor-Go complex bound to the endogenous agonist ghrelin. Our structures reveal a distinct binding mode of the inverse agonist PF-05190457 in the ghrelin receptor, different from the binding mode of agonists and neutral antagonists. Combining the structural comparisons and cellular function assays, we find that a polar network and a notable hydrophobic cluster are required for receptor activation and constitutive activity. Together, our study provides insights into the detailed mechanism of ghrelin receptor binding to agonists and inverse agonists, and paves the way to design specific ligands targeting ghrelin receptors.

Crystal structure of Tam41 cytidine diphosphate diacylglycerol synthase from a Firmicutes bacterium.

Translocator assembly and maintenance 41 (Tam41) catalyses the synthesis of cytidine diphosphate diacylglycerol (CDP-DAG), which is a high-energy intermediate phospholipid critical for generating cardiolipin in mitochondria. Although Tam41 is present almost exclusively in eukaryotic cells, a Firmicutes bacterium contains the gene encoding Tam41-type CDP-DAG synthase (FbTam41). FbTam41 converted phosphatidic acid (PA) to CDP-DAG using a ternary complex mechanism in vitro. Additionally, FbTam41 functionally substituted yeast Tam41 in vivo. These results demonstrate that Tam41-type CDP-DAG synthase functions in some prokaryotic cells. We determined the crystal structure of FbTam41 lacking the C-terminal 18 residues in the cytidine triphosphate (CTP)-Mg2+ bound form at a resolution of 2.6 Å. The crystal structure showed that FbTam41 contained a positively charged pocket that specifically accommodated CTP-Mg2+ and PA in close proximity. By using this structure, we constructed a model for the full-length structure of FbTam41 containing the last a-helix, which was missing in the crystal structure. Based on this model, we propose a molecular mechanism for CDP-DAG synthesis in bacterial cells and mitochondria.

A Genetically Encoded F-19 NMR Probe Reveals the Allosteric Modulation Mechanism of Cannabinoid Receptor 1.

Due to the lack of genetically encoded probes for fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR), its utility for probing eukaryotic membrane protein dynamics is limited. Here we report an efficient method for the genetic incorporation of an unnatural amino acid (UAA), 3'-trifluoromenthyl-phenylalanine (mtfF), into cannabinoid receptor 1 (CB1) in the Baculovirus Expression System. The probe can be inserted at any environmentally sensitive site, while causing minimal structural perturbation to the target protein. Using 19F NMR and X-ray crystallography methods, we discovered that the allosteric modulator Org27569 and agonists synergistically stabilize a previously unrecognized pre-active state. An allosteric modulation model is proposed to explain Org27569's distinct behavior. We demonstrate that our site-specific 19F NMR labeling method is a powerful tool in decoding the mechanism of GPCR allosteric modulation. This new method should be broadly applicable for uncovering conformational states for many important eukaryotic membrane proteins.

Guidelines for de novo phasing using multiple small-wedge data collection.

Intense micro-focus X-ray beamlines available at synchrotron facilities have achieved high-quality data collection even from the microcrystals of membrane proteins. The automatic data collection system developed at SPring-8, named ZOO, has contributed to many structure determinations of membrane proteins using small-wedge synchrotron crystallography (SWSX) datasets. The `small-wedge' (5-20°) datasets are collected from multiple crystals and then merged to obtain the final structure factors. To our knowledge, no systematic investigation on the dose dependence of data accuracy has so far been reported for SWSX, which is between `serial crystallography' and `rotation crystallography'. Thus, herein, we investigated the optimal dose conditions for experimental phasing with SWSX. Phase determination using anomalous scattering signals was found to be more difficult at higher doses. Furthermore, merging more homogeneous datasets grouped by hierarchical clustering with controlled doses mildly reduced the negative factors in data collection, such as `lack of signal' and `radiation damage'. In turn, as more datasets were merged, more probable phases could be obtained across a wider range of doses. Therefore, our findings show that it is essential to choose a lower dose than 10 MGy for de novo structure determination by SWSX. In particular, data collection using a dose of 5 MGy proved to be optimal in balancing the amount of signal available while reducing the amount of damage as much as possible.

The structure of an archaeal oligosaccharyltransferase provides insight into the strict exclusion of proline from the N-glycosylation sequon.

Oligosaccharyltransferase (OST) catalyzes oligosaccharide transfer to the Asn residue in the N-glycosylation sequon, Asn-X-Ser/Thr, where Pro is strictly excluded at position X. Considering the unique structural properties of proline, this exclusion may not be surprising, but the structural basis for the rejection of Pro residues should be explained explicitly. Here we determined the crystal structure of an archaeal OST in a complex with a sequon-containing peptide and dolichol-phosphate to a 2.7 Å resolution. The sequon part in the peptide forms two inter-chain hydrogen bonds with a conserved amino acid motif, TIXE. We confirmed the essential role of the TIXE motif and the adjacent regions by extensive alanine-scanning of the external loop 5. A Ramachandran plot revealed that the ring structure of the Pro side chain is incompatible with the ϕ backbone dihedral angle around -150° in the rigid sequon-TIXE structure. The present structure clearly provides the structural basis for the exclusion of Pro residues from the N-glycosylation sequon.

Endogenous agonist-bound S1PR3 structure reveals determinants of G protein-subtype bias.

Sphingosine-1-phosphate (S1P) regulates numerous important physiological functions, including immune response and vascular integrity, via its cognate receptors (S1PR1 to S1PR5); however, it remains unclear how S1P activates S1PRs upon binding. Here, we determined the crystal structure of the active human S1PR3 in complex with its natural agonist S1P at 3.2-Å resolution. S1P exhibits an unbent conformation in the long tunnel, which penetrates through the receptor obliquely. Compared with the inactive S1PR1 structure, four residues surrounding the alkyl tail of S1P (the "quartet core") exhibit orchestrating rotamer changes that accommodate the moiety, thereby inducing an active conformation. In addition, we reveal that the quartet core determines G protein selectivity of S1PR3. These results offer insight into the structural basis of activation and biased signaling in G protein-coupled receptors and will help the design of biased ligands for optimized therapeutics.

Short-lived intermediate in N2O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography.

Nitric oxide (NO) reductase from the fungus Fusarium oxysporum is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N2O) in the global nitrogen cycle. In this enzymatic reaction, the heme-bound NO is activated by the direct hydride transfer from NADH to generate a short-lived intermediate ( I ), a key state to promote N-N bond formation and N-O bond cleavage. This study applied time-resolved (TR) techniques in conjunction with photolabile-caged NO to gain direct experimental results for the characterization of the coordination and electronic structures of I TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe-NO coordination in I , with an elongated Fe-NO bond length (Fe-NO = 1.91 Å, Fe-N-O = 138°) in the absence of NAD+ TR-infrared (IR) spectroscopy detects the formation of I with an N-O stretching frequency of 1,290 cm-1 upon hydride transfer from NADH to the Fe3+-NO enzyme via the dissociation of NAD+ from a transient state, with an N-O stretching of 1,330 cm-1 and a lifetime of ca. 16 ms. Quantum mechanics/molecular mechanics calculations, based on these crystallographic and IR spectroscopic results, demonstrate that the electronic structure of I is characterized by a singly protonated Fe3+-NHO•- radical. The current findings provide conclusive evidence for the N2O generation mechanism via a radical-radical coupling of the heme nitroxyl complex with the second NO molecule.

XFEL Crystal Structures of Peroxidase Compound II.

Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The nature of the ferryl heme-and whether it is an FeIV =O or FeIV -OH species-is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated FeIV =O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Šand 1.50 Šcrystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), collected using the X-ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron-oxygen bond length in CcP (1.76 Å) is notably shorter than in APX (1.87 Å). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine-tuning is linked to the functional need for proton delivery to the heme.