Mechanistic analysis of carbon-carbon bond formation by deoxypodophyllotoxin synthase.

Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS's catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel-Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPS-catalyzed production of deoxypodophyllotoxin analogs.

Modular DNA-Origami-Based Nanoarrays Enhance Cell Binding Affinity through the "Lock-and-Key" Interaction.

Surface proteins of cells are generally recognized through receptor-ligand interactions (RLIs) in disease diagnosis, but their nonuniform spatial distribution and higher-order structure lead to low binding affinity. Constructing nanotopologies that match the spatial distribution of membrane proteins to improve the binding affinity remains a challenge. Inspired by the multiantigen recognition of immune synapses, we developed modular DNA-origami-based nanoarrays with multivalent aptamers. By adjusting the valency and interspacing of the aptamers, we constructed specific nanotopology to match the spatial distribution of target protein clusters and avoid potential steric hindrance. We found that the nanoarrays significantly enhanced the binding affinity of target cells and synergistically recognized low-affinity antigen-specific cells. In addition, DNA nanoarrays used for the clinical detection of circulating tumor cells successfully verified their precise recognition ability and high-affinity RLIs. Such nanoarrays will further promote the potential application of DNA materials in clinical detection and even cell membrane engineering.

Mild Photothermal-Stimulation Based on Injectable and Photocurable Hydrogels Orchestrates Immunomodulation and Osteogenesis for High-Performance Bone Regeneration.

A photoactivated bone scaffold integrated with minimally invasive implantation and mild thermal-stimulation capability shows great promise in the repair and regeneration of irregularly damaged bone tissues. Developing multifunctional photothermal biomaterials that can simultaneously serve as both controllable thermal stimulators and biodegradable engineering scaffolds for integrated immunomodulation, infection therapy, and impaired bone repair remains an enormous challenge. Herein, an injectable and photocurable hydrogel therapeutic platform (AMAD/MP) based on alginate methacrylate, alginate-graft-dopamine, and polydopamine (PDA)-functionalized Ti3C2 MXene (MXene@PDA) nanosheets is rationally designed for near-infrared (NIR)-mediated bone regeneration synergistic immunomodulation, osteogenesis, and bacterial elimination. The optimized AMAD/MP hydrogel exhibits favorable biocompatibility, osteogenic activity, and immunomodulatory functions in vitro. The proper immune microenvironment provided by AMAD/MP could further modulate the balance of M1/M2 phenotypes of macrophages, thereby suppressing reactive oxygen species-induced inflammatory status. Significantly, this multifunctional hydrogel platform with mild thermal stimulation efficiently attenuates local immune reactions and further promotes new bone formation without the addition of exogenous cells, cytokines, or growth factors. This work highlights the potential application of an advanced multifunctional hydrogel providing photoactivated on-demand thermal cues for bone tissue engineering and regenerative medicine.

TARM-1 Is Critical for Macrophage Activation and Th1 Response in Mycobacterium tuberculosis Infection.

T cell-interacting activating receptor on myeloid cells 1 (TARM-1) is a novel leukocyte receptor expressed in neutrophils and macrophages. It plays an important role in proinflammatory response in acute bacterial infection, but its immunomodulatory effects on chronic Mycobacterium tuberculosis infections remain unclear. TARM-1 expression was significantly upregulated on CD14high monocytes from patients with active pulmonary tuberculosis (TB) as compared that on cells from patients with latent TB or from healthy control subjects. Small interfering RNA knockdown of TARM-1 reduced expression levels of proinflammatory cytokines IL-12, IL-18, IL-1ß, and IL-8 in M. tuberculosis-infected macrophages, as well as that of HLA-DR and costimulatory molecules CD83, CD86, and CD40. Moreover, TARM-1 enhanced phagocytosis and intracellular killing of M. tuberculosis through upregulating reactive oxygen species. In an in vitro monocyte and T cell coculture system, blockade of TARM-1 activity by TARM-1 blocking peptide suppressed CD4+ T cell activation and proliferation. Finally, administration of TARM-1 blocking peptide in a mouse model of M. tuberculosis infection increased bacterial load and lung pathology, which was associated with decreased macrophage activation and IFN-γ production by T cell. Taken together, these results, to our knowledge, demonstrate a novel immune protective role of TARM-1 in M. tuberculosis infection and provide a potential therapeutic target for TB disease.

Identifying the Phenotypes of Tumor-Derived Extracellular Vesicles Using Size-Coded Affinity Microbeads.

Tumor-derived extracellular vesicle (tEV) biomarkers can reflect cancer cell phenotypes and have great potential for cancer diagnosis and treatment. However, tEVs display high heterogeneity, and rapid and sensitive identification of EV biomarkers remains challenging due to their low expression. Spectral overlap also significantly limits the multiplex analysis of EV biomarkers by fluorescent probes. Herein, we developed a method for highly sensitive tEV phenotyping that uses size-coded microbeads that carry hairpin probes that can bind to aptamers targeting distinct tEV biomarkers. We also designed a microfluidic chip containing spacer arrays that segregate these microbeads in distinct chip regions according to their size to generate location-specific signals indicating the level of different EV biomarkers. The EV biomarker signal on these microbeads was amplified by in situ rolling cyclic amplification (RCA). This strategy permits the simultaneous detection of multiple tEV phenotypes by fluorescence spectroscopy without the limitations of spectral overlap. This study demonstrates that this tEV phenotyping method can rapidly and simultaneously detect six different tEV phenotypes with high sensitivity. Due to the programmability of the sensing platform, this method can be rapidly adapted to detect different tEV phenotype substitutions of the detected biomarkers. Notably, clinical cohort studies show that this strategy may provide new ideas for the precise diagnosis and personalized treatment of cancer patients.

Centromere targeting of Mis18 requires the interaction with DNA and H2A-H2B in fission yeast.

Faithful chromosome segregation during mitosis requires the correct assembly of kinetochore on the centromere. CENP-A is a variant of histone H3, which specializes the centromere region on chromatin and mediates the kinetochore assembly. The Mis18 complex plays a critical role in initiating the centromere loading of the newly-synthesized CENP-A. However, it remains unclear how Mis18 complex (spMis18, spMis16 and spMis19) is located to the centromere to license the recruitment of Cnp1CENP-A in Schizosaccharomyces pombe. We found that spMis18 directly binds to nucleosomal DNA through its extreme C-terminus and interacts with H2A-H2B dimer via the acidic region on the surface of its Yippee-like domain. Live-cell imaging confirmed that mutation of the acidic region and deletion of the extreme C-terminus significantly impairs the localization of spMis18 and Cnp1 to the centromere and delays chromosome segregation during mitosis. Our findings illustrate that the interaction of spMis18 with histone H2A-H2B and DNA plays important roles in the recruitment of spMis18 and Cnp1 to the centromere in fission yeast.

Signaling Lymphocytic Activation Molecule Family-7 Alleviates Corneal Inflammation by Promoting M2 Polarization.

BACKGROUND: Signaling lymphocytic activation molecule family-7 (SLAMF7) functions as an immune checkpoint molecule on macrophages in antitumor immunity. However, its role in bacterial infection remains largely unknown. METHODS: Bone marrow-derived macrophages (BMDMs) isolated from wild-type (WT) or SLAMF7 knockout (KO) mice were infected with bacteria or treated with lipopolysaccharide/interferon-γ to investigate the expression and function of SLAMF7 in macrophage polarization. A Pseudomonas aeruginosa keratitis murine model was established to explore the effect of SLAMF7 on P. aeruginosa keratitis using WT vs SLAMF7 KO mice, or recombinant SLAMF7 vs phosphate-buffered saline-treated mice, respectively. RESULTS: SLAMF7 expression was enhanced on M1-polarized or bacterial-infected macrophages, and infiltrating macrophages in P. aeruginosa-infected mouse corneas. SLAMF7 promoted M2 polarization by inducing STAT6 activation. In vivo data showed that SLAMF7 KO aggravated, while treatment with recombinant SLAMF7 alleviated, corneal inflammation and disease severity. In addition, blockage of M2 polarization by Arg-1 inhibitor abrogated the effect of recombinant SLAMF7 in disease progression. CONCLUSIONS: SLAMF7 expression in macrophages was induced upon M1 polarization or bacterial infection and alleviated corneal inflammation and disease progression of P. aeruginosa keratitis by promoting M2 polarization. These findings may provide a potential strategy for the treatment of P. aeruginosa keratitis.

A Dual-Encoded Bead-Based Immunoassay with Tunable Detection Range for COVID-19 Serum Evaluation.

Serological assay for coronavirus 2019 (COVID-19) patients including asymptomatic cases can inform on disease progression and prognosis. A detection method taking into account multiplex, high sensitivity, and a wider detection range will help to identify and treat COVID-19. Here we integrated color-size dual-encoded beads and rolling circle amplification (RCA) into a bead-based fluorescence immunoassay implemented in a size sorting chip to achieve high-throughput and sensitive detection. We used the assay for quantifying COVID-19 antibodies against spike S1, nucleocapsid, the receptor binding domain antigens. It also detected inflammatory biomarkers including interleukin-6, interleukin-1ß, procalcitonin, C-reactive protein whose concentrations range from pg mL-1 to µg mL-1 . Use of different size beads integrating with RCA results in a tunable detection range. The assay can be readily modified to simultaneously measure more COVID-19 serological molecules differing by orders of magnitude.

High-affinity neoantigens correlate with better prognosis and trigger potent antihepatocellular carcinoma (HCC) activity by activating CD39+CD8+ T cells.

OBJECTIVE: It remains controversial whether tumour mutational burden (TMB) or neoantigens are prognostic markers in hepatocellular carcinoma (HCC). This study aimed to define the function of TMB or neoantigens in antitumour immunotherapy. DESIGN: Neoantigens of patients (n=56) were analysed by pVAC tools with major histocompatibility complex-1 (MHC-I) algorithms based on whole exome sequencing and neoantigens with mutant type IC50 <50 nM were defined as high-affinity neoantigens (HANs). Patients were segregated into HAN-high/low groups by median of HAN value, and overall survival (OS) was analysed. Autologous organoid killing model was developed to clarify the antitumour activity of HANs. RESULTS: The value of HAN showed a better correlation with OS (p=0.0199) than TMB (p=0.7505) or neoantigens (p=0.2297) in patients with HCC and positively correlated with the frequency of CD39+CD8+ tumour infiltrating lymphocytes (TILs). Furthermore, HAN-specific CD8+ T cells were identified in CD39+CD8+ TILs, which showed better antitumour activity in HAN-high versus HAN-low group. In addition, more effective HAN peptides were identified in HAN-high versus HAN-low group. Besides, flow cytometry data showed that in fresh tumour, CD39+PD-1intCD8+ TILs displayed an effector phenotype and stronger antitumour activity in HAN-high versus HAN-low group. More importantly, patients in HAN-high versus HAN-low group showed a better prognosis after anti-PD-1 therapy. CONCLUSIONS: Our study first demonstrates that HAN value positively correlates with better OS in patients with HCC. HANs trigger antitumour activity by activating tumour-reactive CD39+CD8+ T cells, and patients in HAN-high group benefited more from anti-PD-1 therapy than HAN-low group. These findings may provide a novel strategy for personalised antitumour therapies for HCC.

Crystal structures of NAD+-linked isocitrate dehydrogenase from the green alga Ostreococcus tauri and its evolutionary relationship with eukaryotic NADP+-linked homologs.

NAD+-linked isocitrate dehydrogenases (NAD-IDHs) catalyze the oxidative decarboxylation of isocitrate into α-ketoglutarate. Previously, we identified a novel phylogenetic clade including NAD-IDHs from several algae in the type II subfamily, represented by homodimeric NAD-IDH from Ostreococcus tauri (OtIDH). However, due to its lack of a crystalline structure, the molecular mechanisms of the ligand binding and catalysis of OtIDH are little known. Here, we elucidate four high-resolution crystal structures of OtIDH in a ligand-free and various ligand-bound forms that capture at least three states in the catalytic cycle: open, semi-closed, and fully closed. Our results indicate that OtIDH shows several novel interactions with NAD+, unlike type I NAD-IDHs, as well as a strictly conserved substrate binding mode that is similar to other homologs. The central roles of Lys283' in dual coenzyme recognition and Lys234 in catalysis were also revealed. In addition, the crystal structures obtained here also allow us to understand the catalytic mechanism. As expected, structural comparisons reveal that OtIDH has a very high structural similarity to eukaryotic NADP+-linked IDHs (NADP-IDHs) within the type II subfamily rather than with the previously reported NAD-IDHs within the type I subfamily. It has also been demonstrated that OtIDH exhibits substantial conformation changes upon ligand binding, similar to eukaryotic NADP-IDHs. These results unambiguously support our hypothesis that OtIDH and OtIDH-like homologs are possible evolutionary ancestors of eukaryotic NADP-IDHs in type II subfamily.

Activation-Induced Cell Death of Mucosal-Associated Invariant T Cells Is Amplified by OX40 in Type 2 Diabetic Patients.

Mucosal-associated invariant T (MAIT) cells play a key role in local and systemic immune responses. Studies suggest that type 2 diabetes (T2D) is associated with alterations in the human MAIT cell response. However, the mechanisms that regulate the survival and homeostasis of human MAIT cells are poorly defined. In this study, we demonstrate that the costimulatory TNF superfamily receptor OX40 was highly expressed in MAIT cells of patients with T2D. Compared with OX40-negative MAIT cells, OX40-positive MAIT cells showed a high activation and a memory phenotype. Surprisingly, OX40 expression was negatively correlated with the frequency of MAIT cells in the peripheral blood of T2D patients. Increased cleaved caspase-3 levels were observed in OX40+-expressing MAIT cells in T2D patients. In vitro, activated OX40 signaling by recombinant OX40L protein promoted caspase-3 activation and apoptosis of MAIT cells. Inhibition of caspase-3 restored apoptosis of MAIT cells induced by OX40 signaling. These results identify OX40 as an amplifier of activation-induced cell death of human blood MAIT cells and shed new light on the regulation of MAIT cells in the phase of immune responses in T2D.

Activation and Regulation of Blood Vδ2 T Cells Are Amplified by TREM-1+ during Active Pulmonary Tuberculosis.

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a receptor mainly expressed on myeloid cells, and it plays an important role in modulating immune response against infectious agents. The function of TREM-1 on nonmyeloid cells such as Vδ2 T cells has not been characterized, and their role in pulmonary tuberculosis (TB) remains unclear. To assess the expression of TREM-1 on blood Vδ2 T cells from pulmonary TB patients and investigate its mechanism of induction, we exploited flow cytometry analysis to study the expression of TREM-1 on Vδ2 T cells from active pulmonary TB patients and control subjects. In this study we demonstrate that TREM-1 (TREM-1+) is highly expressed on Vδ2 T cells of patients with active pulmonary TB. Unlike TREM-1--expressing Vδ2 T cells, TREM-1+-producing Vδ2 T cells display APC-like phenotypes. Surprisingly, TREM-1+ signaling promotes the Ag-presenting capability of Vδ2 T cells to induce the CD4+ T cell response. TREM-1+Vδ2 T cells induced the proliferation and differentiation of naive CD4+ T cells, as well as the elimination of intracellular mycobacteria. We identified TREM-1+ (but not TREM-1-) as an Ag-presentation amplifier on human blood Vδ2 T cells, and data shed new light on the regulation of Vδ2 T cells in the phase of innate and adaptive immune responses against Mycobacterium tuberculosis infection. Targeting TREM-1+Vδ2 T cells may be a promising approach for TB therapy.

Structural insights into the molecular mechanism underlying Sirt5-catalyzed desuccinylation of histone peptides.

Histone modification is a ubiquitous regulatory mechanism involved in a variety of biological processes, including gene expression, DNA damage repair, cell differentiation, and ontogenesis. Succinylation sites on histones have been identified and may have functional consequences. Here, we demonstrate that human sirtuin 5 (Sirt5) catalyzes the sequence-selective desuccinylation of numerous histone succinyl sites. Structural studies of Sirt5 in complex with four succinyl peptides indicate an essential role for the conserved main chain hydrogen bonds formed by the succinyl lysine (0), +1, and +3 sites for substrate-enzyme recognition. Furthermore, biochemical assays reveal that the proline residue at the +1 site of the histone succinylation substrate is unfavorable for Sirt5 interaction. Our findings illustrate the molecular mechanism underlying the sequence-selective desuccinylase activity of Sirt5 and provide insights for further studies of the biological functions associated with histone succinylation and Sirt5.

Structural insights into the conformational change of Staphylococcus aureus NreA at C-terminus.

Staphylococcus aureus is an anaerobic facultative microorganism that features the NreABC system for nitrate respiration. NreB is the sensor histidine kinase that phosphorylates the response regulator NreC to stimulate the expression of target genes. NreA is a nitrate sensor which dissociates from NreB in the present of nitrate and relieves its inhibition on NreB. However, the molecular basis of how NreA regulate NreB remains unknown. In this study, we determined the crystal structures of nitrate-bound NreA from S. aureus (SaNreA/NO3-) and its apoNreA-like mutant SaNreAY94A in complex with ethanediol (SaNreAY94A/EDO). Structural comparison reveals that the C-terminal loop in SaNreA/NO3- rearranges to an α-helix (α7) in SaNreAY94A/EDO, which converts an acidic pocket on the surface to a positively charged region. This conformational change of SaNreA C-terminus might play a role in SaNreB binding.

Contactless treatment for scoliosis by electromagnetically controlled shape-memory alloy rods: a preliminary study in rabbits.

PURPOSE: To evaluate the safety and efficacy of a system aiming to correct scoliosis called "electromagnetically controlled shape-memory alloy rods" (EC-SMAR) used in a rabbit model. METHODS: We heat-treated shape-memory alloy (SMA) rods to achieve a transition temperature between 34 and 47 °C and a C-shape austenite phase. We then developed a water-cooled generator capable of generating an alternating magnetic field (100 kHz) for induction heating. We next studied the efficacy of this system in vitro and determined some parameters prior to proceeding with animal experiments. We then employed a rabbit model, in which we fixed a straight rod along the spinous processes intraoperatively, and conducted induction heating postoperatively every 4 days for 1 month, while performing periodic X-ray assessments. RESULTS: Significant kyphotic deformations with Cobb angles of about 45° (p < 0.01) were created in five rabbits, and no complications occurred throughout the experiment. The rabbits are still very much alive and do not show any signs of discomfort. CONCLUSIONS: This is the first system that can modulate spinal deformation in a gradual, contactless, noninvasive manner through electromagnetic induction heating applied to SMA alloy rods. Although this study dealt with healthy spines, it provides promising evidence that this device also has the capacity to correct human kyphosis and even scoliosis in the future. These slides can be retrieved under Electronic Supplementary Material.

Phosphorylation of CENP-C by Aurora B facilitates kinetochore attachment error correction in mitosis.

Kinetochores are superprotein complexes that orchestrate chromosome segregation via a dynamic interaction with spindle microtubules. A physical connection between CENP-C and the Mis12-Ndc80-Knl1 (KMN) protein network is an important pathway that is used to assemble kinetochores on CENP-A nucleosomes. Multiple outer kinetochore components are phosphorylated by Aurora B kinase to activate the spindle assembly checkpoint (SAC) and to ensure accurate chromosome segregation. However, it is unknown whether Aurora B can phosphorylate inner kinetochore components to facilitate proper mitotic chromosome segregation. Here, we reported the structure of the fission yeast Schizosaccharomyces pombe Mis12-Nnf1 complex and showed that N-terminal residues 26-50 in Cnp3 (the CENP-C homolog of S. pombe) are responsible for interacting with the Mis12 complex. Interestingly, Thr28 of Cnp3 is a substrate of Ark1 (the Aurora B homolog of S. pombe), and phosphorylation impairs the interaction between the Cnp3 and Mis12 complex. The expression of a phosphorylation-mimicking Cnp3 mutant results in defective chromosome segregation due to improper kinetochore assembly. These results establish a previously uncharacterized regulatory mechanism involved in CENP-C-Mis12-facilitated kinetochore attachment error correction to ensure accurate chromosome segregation during mitosis.

Immunoglobulin-Like Transcript 5 Inhibits Macrophage-Mediated Bacterial Killing and Antigen Presentation During Sepsis.

BACKGROUND: Immunosuppression contributes to the mortality of sepsis. However, the underlying mechanism remains unclear. METHODS: In the present study, we investigated the role of inhibitory receptor immunoglobulin-like transcript 5 (ILT5) in sepsis. We first screened the expression of ILT family members, and we found that ILT5 was dramatically up-regulated in the peripheral blood mononuclear cells from sepsis patients versus healthy donors. RESULTS: Knockdown of ILT5 by small interfering ribonucleic acid increased bacterial killing and reactive oxygen species production in THP-1 and RAW264.7 cells. Moreover, ILT5-expressing monocytes/macrophages exhibited lower expression of antigen-presenting molecules including major histocompatibility complex-II and CD80. In the in vitro coculture system with monocytes/macrophages, blockage of ILT5 facilitated Th1 proliferation and differentiation of CD4+ T cells. Furthermore, in vivo experiments demonstrated that pretreatment with ILT5 blocking peptide improved the survival and pulmonary pathology of septic mice. CONCLUSIONS: Together, our study identified ILT5 as an immunosuppressive regulator during sepsis, which may provide potential therapeutic strategy for sepsis.

Diagnostic and Management Options of Osteoblastoma in the Spine.

BACKGROUND Osteoblastoma is a rare, benign, osteolytic neoplasm commonly found in the spine in early adulthood. Here, we review the clinical characteristics, radiographic findings, and surgical management of patients with spinal osteoblastoma. MATERIAL AND METHODS Thirteen patients with osteoblastoma who underwent surgery at our institute from June 2008 to November 2017 were enrolled in this study. The American Spinal Injury Association (ASIA) impairment scale was used to assess neurological function. All patients were treated with either total excision or intralesional piecemeal excision without postoperative radiotherapy. Clinical efficacy was evaluated by visual analog scale (VAS) scores, the Oswestry Disability Index (ODI) of nerve function, physical and radiographic examinations, bone fusion, and neurologic status. RESULTS The follow-up lasted 23-82 months (average, 43.8 months). The average surgical time was 178.1 minutes (range, 100-230 minutes), with an average intraoperative blood loss of 574 mL (range, 230-1100 mL). Postoperatively, VAS scores decreased from 6.2±1.7 to 0.5±0.7 (P<0.001). The preoperative and final ODI scores were 51.1±7.7 and 22.6±4.9, respectively, reflecting a significant decrease (P<0.001). According to the ASIA classification, 3 patients had grade C, 3 patients had grade D, and 7 patients had grade E disease. Three months postoperatively, 1 patient had grade D and 10 patients had grade E disease; ultimately, all cases were grade E disease. Only 1 patient experienced local recurrence and underwent en bloc marginal resection with postoperative radiotherapy. All patients remained neurologically stable without any major complications. CONCLUSIONS Accurate intraoperative localization with complete resection is the key to preventing recurrence. Aggressive surgical resection can achieve satisfactory clinical and radiographic outcomes.

Structural Insights into the Regulation of Staphylococcus aureus Phosphofructokinase by Tetramer-Dimer Conversion.

Most reported bacterial phosphofructokinases (Pfks) are tetramers that exhibit activity allosterically regulated via conformational changes between the R and T states. We report that the Pfk from Staphylococcus aureus NCTC 8325 ( SaPfk) exists as both an active tetramer and an inactive dimer in solution. Multiple effectors, including pH, ADP, ATP, and adenylyl-imidodiphosphate (AMP-PNP), cause equilibrium shifts from the tetramer to dimer, whereas the substrate F6P stabilizes SaPfk tetrameric assembly. Crystal structures of SaPfk in complex with different ligands and biochemical analysis reveal that the flexibility of the Gly150-Leu151 motif in helix α7 plays a role in tetramer-dimer conversion. Thus, we propose a molecular mechanism for allosteric regulation of bacterial Pfk via conversion between the tetramer and dimer in addition to the well-characterized R-state/T-state mechanism.

Structural insights into a novel functional dimer of Staphylococcus aureus RNase HII.

RNase HII exists ubiquitously in organisms and functions as a monomer in prokaryotes. We determined the crystal structure of Staphylococcus aureus RNase HII (Sa-RNase HII), which displays a novel dimer conformation, with the active site of each monomer covered by the other one. Both small-angle X-ray scattering and gel-filtration analysis confirmed that Sa-RNase HII exists as a homodimer in solution. Enzymatic analysis revealed that the "self-inhibited" dimeric form is catalytically active. Furthermore, continuous-wave electron paramagnetic resonance experiments clarified that the Sa-RNase HII dimer undergoes a large conformational change upon substrate binding, but remains a dimer to catalyze the reaction. Our structural and biochemical studies identified a novel functional dimer of Sa-RNase HII with distinct regulation mechanism for its catalytic activity.