Scyllatoxin-based peptide design for E. coli expression and HIV gp120 binding.

Targeting the hydrophobic Phe43 pocket of HIV's envelope glycoprotein gp120 is a critical strategy for antiviral interventions due to its role in interacting with the host cell's CD4. Previous inhibitors, including small molecules and CD4 mimetic peptides based on scyllatoxin, have demonstrated significant binding and neutralization capabilities but were often chemically synthesized or contained non-canonical amino acids. Microbial expression using natural amino acids offers advantages such as cost-effectiveness, scalability, and efficient production of fusion proteins. In this study, we enhanced the previous scyllatoxin-based synthetic peptide by substituting natural amino acids and successfully expressed it in E. coli. The peptide was optimized by mutating the C-terminal amidated valine to valine and glutamine, and by reducing the disulfide bonds from three to two. Circular dichroism confirmed proper secondary structure formation, and fluorescence polarization analysis revealed specific, concentration-dependent binding to HIV gp120, supported by molecular dynamics simulations. These findings indicate the potential for scalable microbial production of effective antiviral peptides, with significant applications in pharmaceutical development for HIV treatment.

ANCA: artificial nucleic acid circuit with argonaute protein for one-step isothermal detection of antibiotic-resistant bacteria.

Endonucleases have recently widely used in molecular diagnostics. Here, we report a strategy to exploit the properties of Argonaute (Ago) proteins for molecular diagnostics by introducing an artificial nucleic acid circuit with Ago protein (ANCA) method. The ANCA is designed to perform a continuous autocatalytic reaction through cross-catalytic cleavage of the Ago protein, enabling one-step, amplification-free, and isothermal DNA detection. Using the ANCA method, carbapenemase-producing Klebsiella pneumoniae (CPKP) are successfully detected without DNA extraction and amplification steps. In addition, we demonstrate the detection of carbapenem-resistant bacteria in human urine and blood samples using the method. We also demonstrate the direct identification of CPKP swabbed from surfaces using the ANCA method in conjunction with a three-dimensional nanopillar structure. Finally, the ANCA method is applied to detect CPKP in rectal swab specimens from infected patients, achieving sensitivity and specificity of 100% and 100%, respectively. The developed method can contribute to simple, rapid and accurate diagnosis of CPKP, which can help prevent nosocomial infections.

Enhancing the thermostability and activity of glycosyltransferase UGT76G1 via computational design.

The diterpene glycosyltransferase UGT76G1, derived from Stevia rebaudiana, plays a pivotal role in the biosynthesis of rebaudioside A, a natural sugar substitute. Nevertheless, its potential for industrial application is limited by certain enzymatic characteristics, notably thermostability. To enhance the thermostability and enzymatic activity, we employed a computational design strategy, merging stabilizing mutation scanning with a Rosetta-based protein design protocol. Compared to UGT76G1, the designed variant 76_4 exhibited a 9 °C increase in apparent Tm, a 2.55-fold increase rebaudioside A production capacity, and a substantial 11% reduction in the undesirable byproduct rebaudioside I. Variant 76_7 also showed a 1.91-fold enhancement rebaudioside A production capacity, which was maintained up to 55 °C, while the wild-type lost most of its activity. These results underscore the efficacy of structure-based design in introducing multiple mutations simultaneously, which significantly improves the enzymatic properties of UGT76G1. This strategy provides a method for the development of efficient, thermostable enzymes for industrial applications.

Design of hypoxia responsive CRISPR-Cas9 for target gene regulation.

The CRISPR-Cas9 system is a widely used gene-editing tool, offering unprecedented opportunities for treating various diseases. Controlling Cas9/dCas9 activity at specific location and time to avoid undesirable effects is very important. Here, we report a conditionally active CRISPR-Cas9 system that regulates target gene expression upon sensing cellular environmental change. We conjugated the oxygen-sensing transcription activation domain (TAD) of hypoxia-inducing factor (HIF-1α) with the Cas9/dCas9 protein. The Cas9-TAD conjugate significantly increased endogenous target gene cleavage under hypoxic conditions compared with that under normoxic conditions, whereas the dCas9-TAD conjugate upregulated endogenous gene transcription. Furthermore, the conjugate system effectively downregulated the expression of SNAIL, an essential gene in cancer metastasis, and upregulated the expression of the tumour-related genes HNF4 and NEUROD1 under hypoxic conditions. Since hypoxia is closely associated with cancer, the hypoxia-dependent Cas9/dCas9 system is a novel addition to the molecular tool kit that functions in response to cellular signals and has potential application for gene therapeutics.

Mitochondrial matrix protein LETMD1 maintains thermogenic capacity of brown adipose tissue in male mice.

Brown adipose tissue (BAT) has abundant mitochondria with the unique capability of generating heat via uncoupled respiration. Mitochondrial uncoupling protein 1 (UCP1) is activated in BAT during cold stress and dissipates mitochondrial proton motive force generated by the electron transport chain to generate heat. However, other mitochondrial factors required for brown adipocyte respiration and thermogenesis under cold stress are largely unknown. Here, we show LETM1 domain-containing protein 1 (LETMD1) is a BAT-enriched and cold-induced protein required for cold-stimulated respiration and thermogenesis of BAT. Proximity labeling studies reveal that LETMD1 is a mitochondrial matrix protein. Letmd1 knockout male mice display aberrant BAT mitochondria and fail to carry out adaptive thermogenesis under cold stress. Letmd1 knockout BAT is deficient in oxidative phosphorylation (OXPHOS) complex proteins and has impaired mitochondrial respiration. In addition, BAT-specific Letmd1 deficient mice exhibit phenotypes identical to those observed in Letmd1 knockout mice. Collectively, we demonstrate that the BAT-enriched mitochondrial matrix protein LETMD1 plays a tissue-autonomous role that is essential for BAT mitochondrial function and thermogenesis.

Mutational and structural analyses of UdgX: insights into the active site pocket architecture and its evolution.

UdgX excises uracil from uracil-containing DNA to concurrently form a covalent bond with the resulting AP-DNA. Structurally, UdgX is highly similar to family-4 UDGs (F4-UDGs). However, UdgX is unique in possessing a flexible R-loop (105KRRIH109). Among the class-defining motifs, while its motif A (51GEQPG55) diverged to possess Q53 in place of A53/G53 in F4-UDGs, motif B [178HPS(S/A)(L/V)(L/V)R184] has remained unchanged. Previously, we proposed an SN1 mechanism resulting in a covalent bond between H109 and AP-DNA. In this study, we investigated several single/double mutants of UdgX. The H109A, H109S, H109G, H109Q, H109C and H109K mutants gain conventional UDG activity to varying levels. The crystal structures of UdgX mutants show topological changes in their active sites, rationalizing their UDG activities. The E52Q, E52N and E52A mutants reveal that E52 forms a catalytic dyad with H109 to enhance its nucleophilicity. The Q53A mutant supports that UdgX specific evolution of Q53 occurred essentially to stabilize the R-loop conformation. The R184A mutation (motif B) supports the role of R184 in substrate-binding. Taken together, the structural, bioinformatics, and mutational studies suggest that UdgX diverged from F4-UDGs, and the emergence of the characteristic R-loop in UdgX is functionally assisted by A53/G53 to Q53 changes in motif A.

The Distinctive Permutated Domain Structure of Periplasmic α-Amylase (MalS) from Glycoside Hydrolase Family 13 Subfamily 19.

Periplasmic α-amylase MalS (EC. 3.2.1.1), which belongs to glycoside hydrolase (GH) family 13 subfamily 19, is an integral component of the maltose utilization pathway in Escherichia coli K12 and used among Ecnterobacteriaceae for the effective utilization of maltodextrin. We present the crystal structure of MalS from E. coli and reveal that it has unique structural features of circularly permutated domains and a possible CBM69. The conventional C-domain of amylase consists of amino acids 120-180 (N-terminal) and 646-676 (C-terminal) in MalS, and the whole domain architecture shows the complete circular permutation of C-A-B-A-C in domain order. Regarding substrate interaction, the enzyme has a 6-glucosyl unit pocket binding it to the non-reducing end of the cleavage site. Our study found that residues D385 and F367 play important roles in the preference of MalS for maltohexaose as an initial product. At the active site of MalS, ß-CD binds more weakly than the linear substrate, possibly due to the positioning of A402. MalS has two Ca2+ binding sites that contribute significantly to the thermostability of the enzyme. Intriguingly, the study found that MalS exhibits a high binding affinity for polysaccharides such as glycogen and amylopectin. The N domain, of which the electron density map was not observed, was predicted to be CBM69 by AlphaFold2 and might have a binding site for the polysaccharides. Structural analysis of MalS provides new insight into the structure-evolution relationship in GH13 subfamily 19 enzymes and a molecular basis for understanding the details of catalytic function and substrate binding of MalS.

Acceptor dependent catalytic properties of GH57 4-α-glucanotransferase from Pyrococcus sp. ST04.

The 4-α-glucanotransferase (4-α-GTase or amylomaltase) is an essential enzyme in maltodextrin metabolism. Generally, most bacterial 4-α-GTase is classified into glycoside hydrolase (GH) family 77. However, hyperthermophiles have unique 4-α-GTases belonging to GH family 57. These enzymes are the main amylolytic protein in hyperthermophiles, but their mode of action in maltooligosaccharide utilization is poorly understood. In the present study, we investigated the catalytic properties of 4-α-GTase from the hyperthermophile Pyrococcus sp. ST04 (PSGT) in the presence of maltooligosaccharides of various lengths. Unlike 4-α-GTases in GH family 77, GH family 57 PSGT produced maltotriose in the early stage of reaction and preferred maltose and maltotriose over glucose as the acceptor. The kinetic analysis showed that maltotriose had the lowest KM value, which increased amylose degradation activity by 18.3-fold. Structural models of PSGT based on molecular dynamic simulation revealed two aromatic amino acids interacting with the substrate at the +2 and +3 binding sites, and the mutational study demonstrated they play a critical role in maltotriose binding. These results clarify the mode of action in carbohydrate utilization and explain acceptor binding mechanism of GH57 family 4-α-GTases in hyperthermophilic archaea.

Ligation-free isothermal nucleic acid amplification.

In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.

One-step synthesis of glycogen-type polysaccharides from maltooctaose and its structural characteristics.

The one-step synthesis of glycogen-type polysaccharides from maltooctaose (G8) was accomplished based on the new findings of the catalytic mechanism of glycogen branching enzymes (GBEs) from Vibrio vulnificus, Deinococcus geothermalis, and Escherichia coli. GBEs from D. geothermalis and E. coli used maltononaose and maltotridecaose as the minimum, respectively, while V. vulnificus GBE (VvGBE) catalyzed the surprisingly small substrate, G8, into polysaccharides. Intriguingly, all three GBEs catalyzed α-1,4-transglycosylation at the early reaction stage of transglycosylation. VvGBE successfully converted the smallest substrate (G8) into two highly branched polysaccharides (HBP), in which the big polysaccharide (1.49 × 105 Da) exhibited structural properties similar to glycogen. Both HBPs had similar side chain distribution with a very short average degree of polymerization compared with mussel glycogen. As a molecular biology reagent, these nucleotide-free HBPs significantly increased the mRNA extraction efficiency of mammalian cells. Our results provide a new approach to the synthesis of novel polysaccharides.

Binding mode of brazzein to the taste receptor based on crystal structure and docking simulation.

Several natural substances including protein produce sweet taste. Brazzein, derived from the plant Pentadipladra brazzeana, is one of the sweet proteins that bind to the taste receptor with stronger sweetness than sugar. Mutations of this protein affect its flavour, yielding higher sweetness in D29K and lower sweetness in R43A. To elucidate its sweet mechanism in the taste receptor, we determined the structures of two variants, D29K and R43A, to a resolution of 1.5 Å and 1.3 Å, respectively. Structures of the brazzein exhibit two α-helix and three ß-sheets connected by four disulfide bonds with a significantly altered electrostatic distribution on the surface. Using the high-resolution structure data and models of the taste receptors T1R2 and T1R3 in the AlphaFold Protein Structure Database, we performed a docking calculation on the receptors and report that brazzein is bound between the two cysteine rich domains (CRDs) of the heterodimer protein complex. Substitution to lysine in D29K resulted in an increased number of hydrogen bonds in the T1R2 receptor, while substitution to alanine in R43A ablated a polar interaction in the T1R3 receptor. The significantly altered interaction of the variants at the interface is consistent with a change of the sweetness. The high-resolution structure and the docking model in this study may provide a structural basis to understand the flavour mechanism induced by the sweet protein.

Dimeric architecture of maltodextrin glucosidase (MalZ) provides insights into the substrate recognition and hydrolysis mechanism.

Maltodextrin glucosidase (MalZ) is a key enzyme in the maltose utilization pathway in Escherichia coli that liberates glucose from the reducing end of the short malto-oligosaccharides. Unlike other enzymes in the GH13_21 subfamily, the hydrolytic activity of MalZ is limited to maltodextrin rather than long starch substrates, forming various transglycosylation products in α-1,3, α-1,4 or α-1,6 linkages. The mechanism for the substrate binding and hydrolysis of this enzyme is not well understood yet. Here, we present the dimeric crystal structure of MalZ, with the N-domain generating a unique substrate binding groove. The N-domain bears CBM34 architecture and forms a part of the active site in the catalytic domain of the adjacent molecule. The groove found between the N-domain and catalytic domain from the adjacent molecule, shapes active sites suitable for short malto-oligosaccharides, but hinders long stretches of oligosaccharides. The conserved residue of E44 protrudes at subsite +2, elucidating the hydrolysis pattern of the substrate by the glucose unit from the reducing end. The structural analysis provides a molecular basis for the substrate specificity and the enzymatic property, and has potential industrial application for protein engineering.

The emerging genetic diversity of hereditary spastic paraplegia in Korean patients.

Hereditary Spastic Paraplegias (HSP) are a group of rare inherited neurological disorders characterized by progressive loss of corticospinal motor-tract function. Numerous patients with HSP remain undiagnosed despite screening for known genetic causes of HSP. Therefore, identification of novel genetic variations related to HSP is needed. In this study, we identified 88 genetic variants in 54 genes from whole-exome data of 82 clinically well-defined Korean HSP families. Fifty-six percent were known HSP genes, and 44% were composed of putative candidate HSP genes involved in the HSPome and originally reported neuron-related genes, not previously diagnosed in HSP patients. Their inheritance modes were 39, de novo; 33, autosomal dominant; and 10, autosomal recessive. Notably, ALDH18A1 showed the second highest frequency. Fourteen known HSP genes were firstly reported in Koreans, with some of their variants being predictive of HSP-causing protein malfunction. SPAST and REEP1 mutants with unknown function induced neurite abnormality. Further, 54 HSP-related genes were closely linked to the HSP progression-related network. Additionally, the genetic spectrum and variation of known HSP genes differed across ethnic groups. These results expand the genetic spectrum for HSP and may contribute to the accurate diagnosis and treatment for rare HSP.

Detection of Infectious Viruses Using CRISPR-Cas12-Based Assay.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease-19 (COVID-19), has severely influenced public health and economics. For the detection of SARS-CoV-2, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)-based assays have been emerged because of their simplicity, sensitivity, specificity, and wide applicability. Herein, we have developed a CRISPR-Cas12-based assay for the detection of SARS-CoV-2. In the assay, the target amplicons are produced by isothermal reverse transcription recombinase polymerase amplification (RT-RPA) and recognized by a CRISPR-Cas12a/guide RNA (gRNA) complex that is coupled with the collateral cleavage activity of fluorophore-tagged probes, allowing either a fluorescent measurement or naked-eye detection on a lateral flow paper strip. This assay enables the sensitive detection of SARS-CoV-2 at a low concentration of 10 copies per sample. Moreover, the reliability of the method is verified by using nasal swabs and sputum of COVID-19 patients. We also proved that the current assay can be applied to other viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), with no major changes to the basic scheme of testing. It is anticipated that the CRISPR-Cas12-based assay has the potential to serve as a point-of-care testing (POCT) tool for a wide range of infectious viruses.

Aglycosylated antibody-producing mice for aglycosylated antibody-lectin coupled immunoassay for the quantification of tumor markers (ALIQUAT).

Targeting aberrant glycoforms has been validated for in vitro cancer diagnostic development, and several assays are currently in routine clinical use. Because N-glycans in Fc region of antibodies show cross-reactivity with various lectins, high-quality aglycosylated antibodies are exceptionally important for immunoassay platform-based quantitative measurements. Previously, aglycosylated antibody acquisition relied on incomplete, uneconomical and onerous enzymatic and chemical methods. Here, we edited four murine immunoglobulin G genes using adenine base-editing and homology-directed recombination (HDR)-mediated gene editing methods to generate aglycosylated antibody-producing mice. Resulting aglycosylated antibodies showed required analytical performances without compromised protein stability. Thus, this aglycosylated monoclonal antibody-lectin coupled immunoassay for the quantification of tumour markers (ALIQUAT) method can provide a robust, versatile and accessible immunoassay platform to quantify specific glycoforms in precision cancer diagnostics. Moreover, the engineered mice can be used as a host to produce various aglycosylated antibodies in a convenient and robust fashion, thereby expanding in vitro diagnostic development opportunities that utilize glycoforms as a disease-specific biomarkers.

Tetrameric architecture of an active phenol-bound form of the AAA+ transcriptional regulator DmpR.

The Pseudomonas putida phenol-responsive regulator DmpR is a bacterial enhancer binding protein (bEBP) from the AAA+ ATPase family. Even though it was discovered more than two decades ago and has been widely used for aromatic hydrocarbon sensing, the activation mechanism of DmpR has remained elusive. Here, we show that phenol-bound DmpR forms a tetramer composed of two head-to-head dimers in a head-to-tail arrangement. The DmpR-phenol complex exhibits altered conformations within the C-termini of the sensory domains and shows an asymmetric orientation and angle in its coiled-coil linkers. The structural changes within the phenol binding sites and the downstream ATPase domains suggest that the effector binding signal is propagated through the coiled-coil helixes. The tetrameric DmpR-phenol complex interacts with the σ54 subunit of RNA polymerase in presence of an ATP analogue, indicating that DmpR-like bEBPs tetramers utilize a mechanistic mode distinct from that of hexameric AAA+ ATPases to activate σ54-dependent transcription.

Crystal structure of the mouse endonuclease G.

Endonuclease G (EndoG) is a mitochondrial enzyme that responds to apoptotic stimuli by translocating to the nucleus and cleaving the chromatin DNA. The molecular mechanism of EndoG still remains unknown in higher organisms. Here, we determined the crystal structure of mouse EndoG at ∼1.96 Å resolution. The EndoG shows an altered dimeric configuration in which N-terminal region of one subunit interact to the other subunit in dimer. The deletion of this region that is highly conserved in mammalian EndoGs resulted in a monomer with significantly reduced activity suggesting the association of the dimeric arrangement into the nuclease activity. Furthermore, we observed a large conformational change in the loop of the active site groove in EndoG, which corresponds to the DNA binding region. Intriguingly, EndoG dimers are linked by oxidation of the reactive cysteine 110 in this flexible loop to form a long oligomeric chain in the crystal lattice. The structural analysis and ensuing biochemical data suggest that this flexible loop region in the active site is important to the regulation of EndoG nuclease function in mouse.

Crystal structure of the Csm5 subunit of the type III-A CRISPR-Cas system.

The Csm complex eliminates foreign RNA and DNA in the microbial defense CRISPR-Cas system. Csm5, one of the five subunits in the complex, facilitates crRNA maturation and target RNA binding in the type III system. However, the exact functional mechanism of Csm5 has remained elusive. Here, we report the crystal structure of the apo form of the Csm5 subunit at a resolution of 2.6 Å. Structural comparison of amino acids in the complex bound to RNA exhibits notable conformational changes in the crRNA and the target RNA binding sites. Shifts in the ß-hairpin motif (ß5-ß6), α13 helix (resides 352-383), and G-rich loop (residues 335-337) in the C-terminal domain indicate an induced movement by crRNA binding. The positively charged residues (Lys 92, Arg 95 and Lys 96) located in the ß-α4 loop of the target RNA interface show high conformational flexibility, while three-helix bundles (α1-α3) of the N-domain involved in Csm2 binding exhibit a rotational shift. The altered architecture of the Csm5 subunit demonstrates remarkable versatility of the ferredoxin-like fold in the RNA binding protein and provides a structural basis for the mechanism for crRNA and target RNA binding in the type III-A Crispr-Cas system.

Carboxy-Terminal Region of a Thermostable CITase from Thermoanaerobacter thermocopriae Has the Ability to Produce Long Isomaltooligosaccharides.

Isomaltooligosaccharides (IMOs) have good prebiotic effects, and long IMOs (LIMOs) with a degree of polymerization (DP) of 7 or above show improved effects. However, they are not yet commercially available, and require costly enzymes and processes for production. The Nterminal region of the thermostable Thermoanaerobacter thermocopriae cycloisomaltooligosaccharide glucanotransferase (TtCITase) shows cyclic isomaltooligosaccharide (CI)-producing activity owing to a catalytic domain of glycoside hydrolase (GH) family 66 and carbohydrate-binding module (CBM) 35. In the present study, we elucidated the activity of the C-terminal region of TtCITase (TtCITase-C; Met740-Phe1,559), including a CBM35-like region and the GH family 15 domain. The domain was successfully cloned, expressed, and purified as a single protein with a molecular mass of 115 kDa. TtCITase-C exhibited optimal activity at 40°C and pH 5.5, and retained 100% activity at pH 5.5 after 18-h incubation. TtCITase-C synthesized α-1,6 glucosyl products with over seven degrees of polymerization (DP) by an α-1,6 glucosyl transfer reaction from maltopentaose, isomaltopentaose, or commercialized maltodextrins as substrates. These results indicate that TtCITase-C could be used for the production of α-1,6 glucosyl oligosaccharides with over DP7 (LIMOs) in a more cost-effective manner, without requiring cyclodextran.

In vivo genome editing using the Cpf1 ortholog derived from Eubacterium eligens.

Cpf1 is an RNA-guided endonuclease that can be programmed to cleave DNA targets. Specific features, such as containing a short crRNA, creating a staggered cleavage pattern and having a low off-target rate, render Cpf1 a promising gene-editing tool. Here, we present a new Cpf1 ortholog, EeCpf1, as a genome-editing tool; this ortholog is derived from the gut bacterial species Eubacterium eligens. EeCpf1 exhibits a higher cleavage activity with the Mn2+ metal cofactor and efficiently cuts the target DNA with an engineered, nucleotide extended crRNA at the 5' target site. When mouse blastocysts were injected with multitargeting crRNAs against the IL2R-γ gene, an essential gene for immunodeficient mouse model production, EeCpf1 efficiently generated IL2R-γ knockout mice. For the first time, these results demonstrate that EeCpf1 can be used as an in vivo gene-editing tool for the production of knockout mice. The utilization of engineered crRNA with multiple target sites will help to explore the in vivo DNA cleavage activities of Cpf1 orthologs from other species that have not been demonstrated.