A Frequent Diagnostic Pitfall: Dermatological Clinical Features and Pathological Results in Diagnosing Pagetoid Squamous Cell Carcinoma in situ (Case Series).

Squamous cell carcinoma (SCC) in situ can occur on any skin or mucus surface and is more commonly found in elderly patients on areas of skin that have been sunburnt. Most previous case reports are from dermatologists, with few published reports from pathologists. In this study, three patients underwent pathological routine and auxiliary immunohistochemical (IHC) examination and were ultimately diagnosed with pagetoid SCC in situ - a different diagnosis from the initial clinical assessment. All three patients received a complete resection of the skin mass. After follow-up, as of June 2023, the patients had no tumour recurrence or metastasis. Pagetoid SCC in situ is a particular type of SCC in situ that has no specific features in clinical manifestations, gross diagnosis or histopathological sections. The final diagnosis depends on IHC staining. Pagetoid SCC in situ expresses EMA, CK5/6 and p63 but not CEA, CK8 or S-100, which are expressed in extramammary Paget's disease. Pagetoid SCC in situ is usually only locally invasive, and the main treatment is complete surgical resection. The prognosis is related to human papillomavirus infection, surgical margin closure, disease location, tumour thickness and other factors.

Genomic characterization and outbreak investigations of methicillin-resistant Staphylococcus aureus in a county-level hospital in China.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common pathogen contributing to healthcare-associated infections, which can result in multiple sites infections. The epidemiological characteristics of MRSA exhibit variability among distinct regions and healthcare facilities. The aim of this study was to investigate the molecular epidemiology and nosocomial outbreak characteristics of MRSA in a county-level hospital in China. A total of 130 non-repetitive MRSA strains were collected from December 2020 to November 2021. Whole-genome sequencing (WGS) was performed to identify antimicrobial resistance and virulence factors. Phylogenetic analysis was conducted to ascertain genetic diversity and phylogenetic relationships. Independent transmission scenarios were determined by the phylogeny derived from single nucleotide polymorphisms (SNPs) within the core genome. All the MRSA isolates were collected from the intensive care unit (30.00%, 39/130), the department of otorhinolaryngology (10.00%, 13/130) and the department of burn unit (9.23%, 12/130). The clinical samples mainly included phlegm (53.85%, 70/130), purulent fluid (24.62%, 32/130), and secretions (8.46%, 11/130). The resistance rates to erythromycin, clindamycin and ciprofloxacin were 75.38, 40.00, and 39.23%, respectively. All the isolates belonged to 11 clonal complexes (CCs), with the major prevalent types were CC5, CC59, and CC398, accounting for 30.00% (39/130), 29.23% (38/130), and 16.92% (22/130), respectively. Twenty sequence types (STs) were identified, and ST59 (25.38%, 33/130) was the dominant lineage, followed by ST5 (23.84%, 31/130) and ST398 (16.92%, 22/130). Three different SCCmec types were investigated, most of isolates were type IV (33.85%, 44/130), followed by type II (27.69%, 36/130) and type III (0.77%, 1/130). The common clonal structures included CC5-ST5-t2460-SCCmec IIa, CC59-ST59-t437-SCCmec IV and CC398-ST398-t034-SCCmec (-), with rates of 16.92% (22/130), 14.62% (19/130), and 13.84% (18/130), respectively. Only 12 panton-valentine leucocidin (PVL) positive strains were identified. Two independent clonal outbreaks were detected, one consisting of 22 PVL-negative strains belongs to CC5-ST5-t2460-SCCmec IIa and the other consisting of 8 PVL-negative strains belongs to CC5-ST5-t311-SCCmec IIa. Overall, our study indicated that the CC5 lineage emerged as the predominant epidemic clone of MRSA, responsible for nosocomial outbreaks and transmission within a county-level hospital in China, highlighting the necessity to strengthen infection control measures for MRSA in such healthcare facilities.

Exploring the association between selective serotonin reuptake inhibitors and rhabdomyolysis risk based on the FDA pharmacovigilance database.

Rhabdomyolysis is a syndrome potentially fatal and has been associated with selective serotonin reuptake inhibitors (SSRIs) treatment in a few case reports. Herein, we purpose to establish the correlation between SSRIs use and rhabdomyolysis using the U.S. Food and Drug Administration Adverse Event Reporting System (FAERS) database. We conducted an analysis on reports that were submitted to the FAERS database during the period between January 1, 2004, and December 31, 2022. Four algorithms, including reporting odds ratio (ROR), proportional reporting ratio (PRR), Bayesian confidence propagation neural network (BCPNN), and empirical Bayes geometric mean (EBGM), were employed to quantify the signals of rhabdomyolysis associated with SSRIs. In total, 16,011,277 non-duplicated reports were obtained and analyzed. Among 33,574 reports related to rhabdomyolysis, SSRIs were classified as primary suspected drug in 889 cases. Disproportionality analysis identified a positive signal between rhabdomyolysis and SSRIs (ROR: 2.86, 95% CI 2.67-3.05; PRR: 2.84, χ2: 1037.16; IC0.25 = 1.39; EBGM0.5 = 2.64). Among six SSRIs, fluvoxamine had the strongest signal (ROR: 11.64, 95% CI 8.00-16.93; PRR: 11.38, χ2: 265.51; IC0.25 = 2.41; EBGM0.5 = 8.31), whereas no significant signal of rhabdomyolysis was detected for paroxetine (ROR: 1.83, 95% CI 1.55-2.15; PRR: 1.82, χ2: 53.82; IC0.25 = 0.73; EBGM0.5 = 1.59). After excluding cases co-administered with statins, the signal of rhabdomyolysis associated with SSRIs remains significant. Our analysis reveals that there are differences in safety signals among six SSRIs in respect to the risk of rhabdomyolysis, with fluvoxamine displaying the highest risk signal, while paroxetine did not show a significant signal. Given the potentially lethal nature of rhabdomyolysis, healthcare professionals should inform patients of the potential risk of rhabdomyolysis associated with SSRIs prior to initiating treatment.

MicroRNA-324-3p Plays A Protective Role Against Coxsackievirus B3-Induced Viral Myocarditis.

Viral myocarditis (VM) is an inflammatory disease of the myocardium associated with heart failure, which is caused by common viral infections. A majority of the infections are initiated by coxsackievirus B3 (CVB3). MicroRNAs (miRNAs) have a major role in various biological processes, including gene expression, cell growth, proliferation, and apoptosis, as well as viral infection and antiviral immune responses. Although, miRNAs have been found to regulate viral infections, their role in CVB3 infection remains poorly understood. In the previous study, miRNA microarray results showed that miR-324-3p expression levels were significantly increased when cells and mice were infected with CVB3. It was also found that miR-324-3p downregulated TRIM27 and decreased CVB3 replication in vitro and in vivo. In vitro, analysis of downstream signaling of TRIM27 revealed that, miR-324-3p inhibited CVB3 infection, and reduced cytopathic effect and viral plaque formation by reducing the expression of TRIM27. In vivo, miR-324-3p decreased the expression of TRIM27, reduced cardiac viral replication and load, thereby strongly attenuating cardiac injury and inflammation. Taken together, this study suggests that miR-324-3p targets TRIM27 to inhibit CVB3 replication and viral load, thereby reducing the cardiac injury associated with VM.

Nuclear Pore Complex 62 Promotes Metastasis of Gastric Cancer by Regulating Wnt/ß-Catenin and TGF-ß Signaling Pathways.

Gastric cancer (GC) is the third leading cause of cancer-related deaths in the world. Tumor metastasis is considered one of the main factors for GC development. Nup62 is a member of the nuclear pore complex (NPC). It bridges the nuclear envelope, is important in nucleocytoplasmic exchange, and is associated with cancer. This study aimed to investigate the role of Nup62 in GC metastasis. The relationship between the expression level of Nup62 in GC and patient survival was evaluated using Kaplan-Meier analysis. Then Nup62 expression in GC tissues and matched normal gastric tissues was analyzed by immunohistochemistry and that in cell lines by Western blot analysis. Furthermore, clonogenic and Transwell migration assays were performed, and the expression of epithelial-mesenchymal transition (EMT) proteins was detected to determine the metastatic functional roles of Nup62 in GC. Compared with the adjacent normal tissues, Nup62 was found to be upregulated in GC tissues using software prediction and detecting clinical specimens and cell lines. Moreover, the downregulation of Nup62 suppressed colony formation and decreased the number of migrated cells. In contrast, Nup62 overexpression promoted colony formation and increased the number of migrated cells. Further functional studies showed that the abnormal expression of Nup62 influenced cell migration and EMT through wingless/ß-catenin (Wnt/ß-catenin) and transforming growth factor (TGF)-ß signaling pathways. In summary, the findings indicate that Nup62 regulates cell migration by interfering with Wnt/ß-catenin and TGF-ß signaling pathways in GC.

N6-methyladenosine modification of MALAT1 promotes metastasis via reshaping nuclear speckles.

N6-methyladenosine (m6A), one of the most prevalent RNA post-transcriptional modifications, is involved in numerous biological processes. In previous studies, the functions of m6A were typically identified by perturbing the activity of the methyltransferase complex. Here, we dissect the contribution of m6A to an individual-long noncoding RNA-metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The mutant MALAT1 lacking m6A-motifs significantly suppressed the metastatic potential of cancer cells both in vitro and in vivo in mouse. Super-resolution imaging showed that the concatenated m6A residues on MALAT1 acted as a scaffold for recruiting YTH-domain-containing protein 1 (YTHDC1) to nuclear speckles. We further reveal that the recognition of MALAT1-m6A by YTHDC1 played a critical role in maintaining the composition and genomic binding sites of nuclear speckles, which regulate the expression of several key oncogenes. Furthermore, artificially tethering YTHDC1 onto m6A-deficient MALAT1 largely rescues the metastatic potential of cancer cells.

Structure of tRNA-Modifying Enzyme TiaS and Motions of Its Substrate Binding Zinc Ribbon.

The accurate modification of the tRNAIle anticodon wobble cytosine 34 is critical for AUA decoding in protein synthesis. Archaeal tRNAIle2 cytosine 34 is modified with agmatine in the presence of ATP by TiaS (tRNAIle2 agmatidine synthetase). However, no structure of apo-form full-length TiaS is available currently. Here, the crystal structures of apo TiaS and a complex of TiaS-agmatine-AMPPCP-Mg are presented, with properly folded zinc ribbon and Cys4-zinc coordination identified. Compared with tRNAIle2-bound form, the architecture of apo TiaS shows a totally different conformation of zinc ribbon. Molecular dynamics simulations of the docking complex between free-state TiaS and tRNAIle2 suggest that zinc ribbon domain is capable of performing large-scale motions to sample substrate binding-competent conformation. Principle component analysis and normal mode analysis show consistent results about the relative directionality of functionally correlated zinc ribbon motions. Apo TiaS and TiaS-agmatine-AMPPCP-Mg/TiaS-AMPCPP-Mg complex structures capture two snapshots of the flexible ATP-Mg binding p2loop step-by-step stabilization. Research from this study provides new insight into TiaS functional mechanism and the dynamic feature of zinc ribbons.

High Expression of ABL2 Suppresses Apoptosis in Gastric Cancer.

BACKGROUND: Diseases associated with Abelson-related gene (also called ABL2) include leukemia; furthermore, previous researches have studied the expressions and functions of ABL2 in different types of malignancies and found that it plays an important role in almost all kinds of cancers. AIMS: Nevertheless, the mechanism of ABL2 in gastric cancer (GC) remains vague. METHODS: In the present study, the level of ABL2 in human GC tissues was detected by immunohistochemistry. Also, the GC cell lines MGC-803 and BGC-823 were selected to stably knock down and overexpress the level of ABL2 by corresponding lentiviral vectors. Puromycin was used to maintain the stable low expression of ABL2 MGC-803 cells compared with control cells; what is more, the high expression of ABL2 BGC-823 cells was also obtained. Based on it, we detected the proteins associated with apoptosis, such as Bcl-2 family and caspase family by western blotting. RESULTS: The most appropriate concentration of puromycin to kill GC cells is 1 µg/mL; then, we obtained the corresponding stable cell lines. Furthermore, we found that high level of ABL2 in BGC-823 cells increased the expression of Bcl-XL, total PARP, and caspase3, while decreased the level of cleaved caspase3 and cleaved caspase9. Consistent results are received in MGC-803 cells. In addition, ABL2 overexpression led to the protein related with Ras/Erk and PI3K/AKT signaling pathway increased; also, we found that the major proteins play a significant role in it. CONCLUSION: All the data showed that high expression of ABL2 suppresses apoptosis through Ras/Erk and PI3K/AKT signaling pathway in GC cell lines.

Tetrandrine Induces Apoptosis in Human Nasopharyngeal Carcinoma NPC-TW 039 Cells by Endoplasmic Reticulum Stress and Ca2+/Calpain Pathways.

Tetrandrine is an alkaloid extracted from a traditional China medicine plant, and is considered part of food therapy as well. In addition, it has been widely reported to induce apoptotic cell death in many human cancer cells. However, the mechanism of Tetrandrine on human nasopharyngeal carcinoma cells (NPC) is still questioned. In our study, we examined whether Tetrandrine can induce apoptosis of NPC-TW 039 cells. We found that cell morphology was changed after treatment with different concentrations of Tetrandrine. Further, we indicated that the NPC-TW 039 cells viability decreased in a Tetrandrine dose-dependent manner. We also found that tetrandrine induced cell cycle arrest in G0/G1 phase. Tetrandrine induced DNA condensation by DAPI staining as well. In addition, we found that Tetrandrine induced Ca2+ release in the cytosol. At the same time, endoplasmic reticulum (ER) stress occurred. Then we used western blotting to examine the protein expression which is associated with mitochondria-mediated apoptotic pathways and caspase-dependent pathways. To further examine whether Ca2+ was released or not with Tetrandrine induced-apoptosis, we used the chelator of Ca2+ and showed that cell viability increased. At the same time, caspase-3 expression was decreased. Furthermore, confocal microscopy examination revealed that Tetrandrine induced expression of ER stress-related proteins GADD153 and GRP78. Our results indicate that Tetrandrine induces apoptosis through calcium-mediated ER stress and caspase pathway in NPC-TW 039 cells. In conclusion, Tetrandrine may could be used for treatment of human nasopharyngeal carcinoma in future.

Tetrandrine Induces Apoptosis of Human Nasopharyngeal Carcinoma NPC-TW 076 Cells through Reactive Oxygen Species Accompanied by an Endoplasmic Reticulum Stress Signaling Pathway.

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy of the head and neck and the incidence is higher in Southeast Asia. Tetrandrine (TET) is a bisbenzylisoquinoline alkaloid, a natural product, and exhibits biological activities including action against many human cancer cell lines. However, the molecular mechanism of TET-induced cell apoptosis in human NPC cells is still unclear. In the present study, we investigated TET-induced apoptotic cell death and associated possible signal pathways on human nasopharyngeal carcinoma NPC-TW 076 cells in vitro. Phase contrast microscopy was used to examine cell morphology and DAPI staining was used to examine chromatin condensation. Flow cytometry assay was used to measure total viable cells, cell cycle and sub-G1 phase distribution, reactive oxygen species (ROS), Ca2+, and mitochondria membrane potential (ΔΨm) in NPC-TW 076 cells. Results indicate that TET induced cell death through the cell morphological changes, caused G0/G1 phase arrest, increased ROS and Ca2+ production, and finally caused apoptotic cell death in NPC-TW 076 cells. There was no influence on the level of ΔΨm after TET treatment. Western blotting indicated that TET increased endoplasmic reticulum (ER) stress associated protein expression such as GADD153, GRP78, ATF-6α and ATF-6 ßwhich indicated that TET induced cell death through ER stress. ER stress is a potential target in cancer treatment, so the ability of TET to induce ER stress response and to activate programming cell death in NPC-TW 076 cells make this molecule become a promising anticancer agent.

Structural basis for copper/silver binding by the Synechocystis metallochaperone CopM.

Copper homeostasis integrates multiple processes from sensing to storage and efflux out of the cell. CopM is a cyanobacterial metallochaperone, the gene for which is located upstream of a two-component system for copper resistance, but the molecular basis for copper recognition by this four-helical bundle protein is unknown. Here, crystal structures of CopM in apo, copper-bound and silver-bound forms are reported. Monovalent copper/silver ions are buried within the bundle core; divalent copper ions are found on the surface of the bundle. The monovalent copper/silver-binding site is constituted by two consecutive histidines and is conserved in a previously functionally unknown protein family. The structural analyses show two conformational states and suggest that flexibility in the first α-helix is related to the metallochaperone function. These results also reveal functional diversity from a protein family with a simple four-helical fold.

Crystal structure of the catalytic subunit of magnesium chelatase.

Tetrapyrroles, including haem and chlorophyll, play vital roles for various biological processes, such as respiration and photosynthesis, and their biosynthesis is critical for virtually all organisms. In photosynthetic organisms, magnesium chelatase (MgCh) catalyses insertion of magnesium into the centre of protoporphyrin IX, the branch-point precursor for both haem and chlorophyll, leading tetrapyrrole biosynthesis into the magnesium branch(1,2). This reaction needs a cooperated action of the three subunits of MgCh: the catalytic subunit ChlH and two AAA(+) subunits, ChlI and ChlD (refs 3-5). To date, the mechanism of MgCh awaits further elucidation due to a lack of high-resolution structures, especially for the ∼150 kDa catalytic subunit. Here we report the crystal structure of ChlH from the photosynthetic cyanobacterium Synechocystis PCC 6803, solved at 2.5 Šresolution. The active site is buried deeply inside the protein interior, and the surrounding residues are conserved throughout evolution. This structure helps to explain the loss of function reported for the cch and gun5 mutations of the ChlH subunit, and to provide the molecular basis of substrate channelling during the magnesium-chelating process. The structure advances our understanding of the holoenzyme of MgCh, a metal chelating enzyme other than ferrochelatase.

Crystal structure of Arabidopsis glutamyl-tRNA reductase in complex with its stimulator protein.

Tetrapyrrole biosynthesis in plants, algae, and most bacteria starts from the NADPH-dependent reduction of glutamyl-tRNA by glutamyl-tRNA reductase (GluTR). The GluTR-catalyzed reaction is the rate-limiting step, and GluTR is the target of multiple posttranslational regulations, such as heme feedback inhibition, for the tetrapyrrole biosynthetic pathway. A recently identified GluTR regulator, GluTR binding protein (GluBP), has been shown to spatially organize tetrapyrrole synthesis by distributing GluTR into different suborganellar locations. Here we report the complex structure of GluTR-GluBP from Arabidopsis thaliana. The dimeric GluBP binds symmetrically to the catalytic domains of the V-shaped GluTR dimer via its C-terminal domain. A substantial conformational change of the GluTR NADPH-binding domain is observed, confirming the postulated rotation of the NADPH-binding domain for hydride transfer from NADPH to the substrate. Arg146, "guarding the door" for metabolic channeling, adopts alternative conformations, which may represent steps involved in substrate recognition and product release. A coupled enzyme assay shows that GluBP stimulates GluTR catalytic efficiency with an approximate threefold increase of the 5-aminolevulinic acid formation rate. In addition, the GluTR activity can be inhibited by heme in a concentration-dependent way regardless of the presence of GluBP. A structural alignment indicates that GluBP belongs to a heme-binding family involved in heme metabolism. We propose a catalytic mechanism model for GluTR, through which photosynthetic organisms can achieve precise regulation of tetrapyrrole biosynthesis.

Crystal structure of Arabidopsis Deg2 protein reveals an internal PDZ ligand locking the hexameric resting state.

Eukaryotic organelles have developed elaborate protein quality control systems to ensure their normal activity, among which Deg/HtrA proteases play an essential role. Plant Deg2 protease is a homologue of prokaryotic DegQ/DegP proteases and is located in the chloroplast stroma, where its proteolytic activity is required to maintain the efficiency of photosynthetic machinery during stress. Here, we demonstrate that Deg2 exhibits dual protease-chaperone activities, and we present the hexameric structure of Deg2 complexed with co-purified peptides. The structure shows that Deg2 contains a unique second PDZ domain (PDZ2) following a conventional PDZ domain (PDZ1), with PDZ2 orchestrating the cage assembly of Deg2. We discovered a conserved internal ligand for PDZ2 that mediates hexamer formation and thus locks the protease in the resting state. These findings provide insight into the diverse modes of PDZ domain-mediated regulation of Deg proteases.

[Rapid and high throughput measurement of lipase thermo-stability through ANS fluorescence signal assay].

We have developed a rapid and high throughput lipase-ANS (8-Anilino-l-naphthalenesulfonic acid) assay to evaluate the thermo-stability of lipases based on the ANS fluorescence signal's increasing and shifting when this small fluorescence probes binds to lipase. The testing lipase samples were incubated at a temperature range of 25 degrees C to 65 degrees C for 30 min before mixed with ANS solution (0.20 mg/mL lipase and 0.05 mmol/L ANS in the buffer of 20 mmol/L Tris-HCl, 100 mmol/L NaCl, pH 7.2) in a cuvette or microplate. Fluorescence signals of the samples were measured at EX 378 nm, EM 465 nm with a fluorescence photometer or a plate reader, and Tm was calculated with the software of GraphPad Prism5.0. The Tm values of several mutants of Penicillium expansum lipase (PEL) were measured with this ANS assay and conventional method simultaneously and the results show that Tm values are comparative and consistent between these methods, suggesting that the lipase-ANS assay is a reliable, rapid and high throughput method for lipase thermo-stability measurement.

Crystal structure of human adenylate kinase 4 (L171P) suggests the role of hinge region in protein domain motion.

It is well known that motion of LID and NMP-binding (NMP(bind)) domains in adenylate kinase (AK) is important in ligand binding and catalysis. However, the nature of such domain motions is poorly characterized. One of the critical hinge regions is hinge IV, which connects the CORE and LID domains. In addition, the hinge IV contains a strictly conserved residue, L171, in the AK family. To investigate the role of hinge IV, crystal structure of human adenylate kinase 4 (AK4) L171P mutant was determined. This mutation dramatically changes the orientation of the LID domain, which could be described as a novel twisted-and-closed conformation in contrast to the open and closed conformations in other AKs. This mutant provides a new example of domain motions in AK family.

Crystal structure of SAM-dependent O-methyltransferase from pathogenic bacterium Leptospira interrogans.

The S-adenosylmethionine (SAM)-dependent O-methyltransferase from Leptospira interrogans (LiOMT) expressed by gene LA0415 belongs to the Methyltransf_3 family (Pfam PF01596). In this family all of the five bacterial homologues with known function are reported as SAM-dependent O-methylstransferases involved in antibiotic production. The crystal structure of LiOMT in complex with S-adenosylhomocysteine reported here is the first bacterial protein structure in this family. The LiOMT structure shows a conserved SAM-binding region and a probable metal-dependent catalytic site. The molecules of LiOMT generate homodimers by N-terminal swapping, which assists the pre-organization of the substrate-binding site. Based on the sequence and structural analysis, it is implied by the catalytic and substrate-binding site that the substrate of LiOMT is a phenolic derivative, which probably has a large ring-shaped moiety.

Crystal structures of human IPP isomerase: new insights into the catalytic mechanism.

Type I isopentenyl diphosphate (IPP): dimethylally diphosphate (DMAPP) isomerase is an essential enzyme in human isoprenoid biosynthetic pathway. It catalyzes isomerization of the carbon-carbon double bonds in IPP and DMAPP, which are the basic building blocks for the subsequent biosynthesis. We have determined two crystal structures of human IPP isomerase I (hIPPI) under different crystallization conditions. High similarity between structures of human and Escherichia coli IPP isomerases proves the conserved catalytic mechanism. Unexpectedly, one of the hIPPI structures contains a natural substrate analog ethanol amine pyrophosphate (EAPP). Based on this structure, a water molecule is proposed to be the direct proton donor for IPP and different conformations of IPP and DMAPP bound in the enzyme are also proposed. In addition, structures of human IPPI show a flexible N-terminal alpha-helix covering the active pocket and blocking the entrance, which is absent in E. coli IPPI. Besides, the active site conformation is not the same in the two hIPPI structures. Such difference leads to a hypothesis that substrate binding induces conformational change in the active site. The inhibition mechanism of high Mn(2+) concentrations is also discussed.

Seeing the process of histidine phosphorylation in human bisphosphoglycerate mutase.

Bisphosphoglycerate mutase is an erythrocyte-specific enzyme catalyzing a series of intermolecular phosphoryl group transfer reactions. Its main function is to synthesize 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. In this paper, we directly observed real-time motion of the enzyme active site and the substrate during phosphoryl transfer. A series of high resolution crystal structures of human bisphosphoglycerate mutase co-crystallized with 2,3-bisphosphoglycerate, representing different time points in the phosphoryl transfer reaction, were solved. These structures not only clarify the argument concerning the substrate binding mode for this enzyme family but also depict the entire process of the key histidine phosphorylation as a "slow movie". It was observed that the enzyme conformation continuously changed during the different states of the reaction. These results provide direct evidence for an "in line" phosphoryl transfer mechanism, and the roles of some key residues in the phosphoryl transfer process are identified.

Crystal structure of human B-type phosphoglycerate mutase bound with citrate.

The B-type cofactor-dependent phosphoglycerate mutase (dPGM-B) catalyzes the interconversion of 2-phosphoglycerate and 3-phosphoglycerate in glycolysis and gluconeogenesis pathways using 2,3-bisphosphoglycerate as the cofactor. The crystal structures of human dPGM-B bound with citrate were determined in two crystal forms. These structures reveal a dimerization mode conserved in both of dPGM and BPGM (bisphosphoglycerate mutase), based on which a dPGM/BPGM heterodimer structure is proposed. Structural comparison supports that the conformational changes of residues 13-21 and 98-117 determine PGM/BPGM activity differences. The citrate-binding mode suggests a substrate-binding model, consistent with the structure of Escherichia coli dPGM/vanadate complex. A chloride ion was found in the center of the dimer, providing explanation for the contribution of chloride ion to dPGM activities. Based on the structural information, the possible reasons for the deficient human dPGM mutations found in some patients are also discussed.