Structural and biochemical characterization of a nucleotide hydrolase from Streptococcus pneumonia.

In this report, we structurally and biochemically characterized the unknown gene product SP1746 from Streptococcus pneumoniae serotype 4. Various crystal structures of SP1746 in the apo form and in complex with different nucleotides were determined. SP1746 is a globular protein, which belongs to the histidine-aspartate (HD) domain superfamily with two Fe3+ ions in the active site that are coordinated by key active site residues and water molecules. All nucleotides bind in a similar orientation in the active site with their phosphate groups anchored to the diiron cluster. Biochemically, SP1746 hydrolyzes different nucleotide substrates. SP1746 most effectively hydrolyzes diadenosine tetraphosphate (Ap4A) to two ADPs. Based on the aforementioned data, we annotated SP1746 as an Ap4A hydrolase, belonging to the YqeK family. Our in vitro data indicate a potential role for SP1746 in regulating Ap4A homeostasis, which requires validation with in vivo experiments in bacteria in the future.

Using Protein Design and Directed Evolution to Monomerize a Bright Near-Infrared Fluorescent Protein.

The small ultrared fluorescent protein (smURFP) is a bright near-infrared (NIR) fluorescent protein (FP) that forms a dimer and binds its fluorescence chromophore, biliverdin, at its dimer interface. To engineer a monomeric NIR FP based on smURFP potentially more suitable for bioimaging, we employed protein design to extend the protein backbone with a new segment of two helices that shield the original dimer interface while covering the biliverdin binding pocket in place of the second chain in the original dimer. We experimentally characterized 13 designs and obtained a monomeric protein with a weak fluorescence. We enhanced the fluorescence of this designed protein through two rounds of directed evolution and obtained designed monomeric smURFP (DMsmURFP), a bright, stable, and monomeric NIR FP with a molecular weight of 19.6 kDa. We determined the crystal structures of DMsmURFP both in the apo state and in complex with biliverdin, which confirmed the designed structure. The use of DMsmURFP in in vivo imaging of mammalian systems was demonstrated. The backbone design-based strategy used here can also be applied to monomerize other naturally multimeric proteins with intersubunit functional sites.

Effect of icariin on depressive behaviour in rat pups. Evidences for its mechanism of action by integrating network pharmacology, metabolomics and gut microbiota composition.

BACKGROUND: Prenatal stress (PS) can cause cognitive disorder and a range of psychological illnesses, including anxiety and depression. Icariin (ICA) has shown promising effects in improving PS-induced depressive behaviour. However, its mechanism of action remains unclear. PURPOSE: This study was performed to reveal the key targets, metabolites and gut microbiota for ICA in improving depressive behaviour in PS rat pups. METHODS: A prenatal restraint stress animal model was established for Sprague-Dawley (SD) rats in late pregnancy. Male pups were randomly divided into six groups: no stress group (NS), PS group, PS + saline group (PS_S), PS + high-dose ICA group (ICAH, 80 mg/kg*day), PS + low-dose ICA group (ICAL, 40 mg/kg*day) and PS + fluoxetine group (FLU, 10 mg/kg*day). The depressive behaviour of each group of rat pups was evaluated using open field test, forced swimming test and sucrose preference test. Different metabolites were identified using untargeted metabolomics of serum and faeces, and metabolic pathways were analyzed through MetaboAnalyst. Targets for ICA acting on depression were determined after network pharmacology was applied. An integrated network of network pharmacology and metabolomics were constructed using Cytoscape software, and molecular docking were performed to verify the interactions between ICA and key targets. Finally, gut microbiota of rat pups in each group were analyzed after 16S rDNA sequencing. RESULTS: PS could cause rat pups to exhibit depressive behaviour, and ICA could significantly improve this depressive behaviour. A total of 49 differential metabolites were found in serum and 23 differential metabolites were found in faeces, and 24 metabolites in serum and 6 metabolites in faeces could be reversed following ICA administration. Integrated analysis focused on five key targets (i.e. adenosyl homocysteinase; medium-chain specific acyl-CoA dehydrogenase, mitochondrial; thymidine phosphorylase; cGMP-specific 3',5'-cyclic phosphodiesterase and xanthine dehydrogenase/oxidase) and three metabolites (i.e. palmitoylcarnitine, methionine and hypoxanthine). Molecular docking indicated that ICA combined well with key targets. Gut microbiota analysis showed that g_Bacteroides, f_Bacteroidaceae and s_Lactobacillus reuteri were required for ICA to improve depressive behaviour. CONCLUSION: In this study, the antidepressant mechanism of ICA was clarified with a strategy of integrating metabolomics, network pharmacology and gut microbiota. ICA has a good effect on improving metabolism and increasing the abundance of probiotics in the intestine. The present research provided new insights into the anti-depressant mechanism of ICA.

Structural basis for evolutionarily conserved interactions between TFIIS and Paf1C.

The elongation factor TFIIS interacts with Paf1C complex to facilitate processive transcription by Pol II. We here determined the crystal structure of the trypanosoma TFIIS LW domain in a complex with the LFG motif of Leo1, as well as the structures of apo-form TFIIS LW domains from trypanosoma, yeast and human. We revealed that all three TFIIS LW domains possess a conserved hydrophobic core that mediates their interactions with Leo1. Intriguingly, the structural study revealed that trypanosoma Leo1 binding induces the TFIIS LW domain to undergo a conformational change reflected in the length and orientation of α6 helix that is absent in the yeast and human counterparts. These differences explain the higher binding affinity of the TFIIS LW domain interacting with Leo1 in trypanosoma than in yeast and human, and indicate species-specific variations in the interactions. Importantly, the interactions between the TFIIS LW domain and an LFG motif of Leo1 were found to be critical for TFIIS to anchor the entire Paf1C complex. Thus, in addition to revealing a detailed structural basis for the TFIIS-Paf1C interaction, our studies also shed light on the origin and evolution of the roles of TFIIS and Paf1C complex in regulation of transcription elongation.

The role of m6A methylation in prenatal maternal psychological distress and birth outcome.

BACKGROUND: Prenatal maternal psychological distress (PMPD) is a known risk factor for adverse birth outcomes. N6-methyladenosine RNA (m6A) methylation is crucial in moderating RNA biology. This study aimed to evaluate the relationships between PMPD, birth outcomes, and placental m6A methylation. METHODS: This was a prospective cohort study. PMPD exposure was assessed by questionnaires about prenatal stress, depression, and anxiety. Placental m6A methylation was measured using a colorimetric assay. The relationships between PMPD, m6A methylation, gestational age (GA), and birth weight (BW) were analyzed using structural equation models (SEMs). Maternal weight gain during pregnancy and infant sex were included as covariables. RESULTS: The study included 209 mother-infant dyads. In an adjusted SEM, PMPD was associated with BW (B = -26.034; 95 % CI: -47.123, -4.868) and GA (B = -0.603; 95 % CI: -1.102, -0.154). M6A methylation was associated with PMPD (B = 0.055; 95 % CI: 0.040,0.073) and BW (B = -305.799; 95 % CI: -520.164, -86.460) but not GA. The effect of PMPD on BW was partially mediated by m6A methylation (B = -16.817; 95 % CI: -31.348, -4.638) and GA (B = -12.280; 95 % CI: -23.612, -3.079). Maternal weight gain was associated with BW (B = 5.113; 95 % CI: 0.229,10.438). LIMITATIONS: The study sample size was small, and the specific mechanism of m6A methylation on birth outcomes needs to be further explored. CONCLUSIONS: In this study, PMPD exposure negatively affected BW and GA. Placental m6A methylation was associated with PMPD and BW and partially mediated the effect of PMPD on BW. Our findings highlight the importance of perinatal psychological evaluation and intervention.

An Fc-muted bispecific antibody targeting PD-L1 and 4-1BB induces antitumor immune activity in colorectal cancer without systemic toxicity.

BACKGROUND: Resistance to immune checkpoint inhibitor (ICI) therapy narrows the efficacy of cancer immunotherapy. Although 4-1BB is a promising drug target as a costimulatory molecule of immune cells, no 4-1BB agonist has been given clinical approval because of severe liver toxicity or limited efficacy. Therefore, a safe and efficient immunostimulatory molecule is urgently needed for cancer immunotherapy. METHODS: HK010 was generated by antibody engineering, and the Fab/antigen complex structure was analyzed using crystallography. The affinity and activity of HK010 were detected by multiple in vitro bioassays, including enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), flow cytometry, and luciferase-reporter assays. Humanized mice bearing human PD-L1-expressing MC38 (MC38/hPDL1) or CT26 (CT26/hPDL1) tumor transplants were established to assess the in vivo antitumor activity of HK010. The pharmacokinetics (PK) and toxicity of HK010 were evaluated in cynomolgus monkeys. RESULTS: HK010 was generated as an Fc-muted immunoglobulin (Ig)G4 PD-L1x4-1BB bispecific antibody (BsAb) with a distinguished Fab/antigen complex structure, and maintained a high affinity for human PD-L1 (KD: 2.27 nM) and low affinity for human 4-1BB (KD: 493 nM) to achieve potent PD-1/PD-L1 blockade and appropriate 4-1BB agonism. HK010 exhibited synergistic antitumor activity by blocking the PD-1/PD-L1 signaling pathway and stimulating the 4-1BB signaling pathway simultaneously, and being strictly dependent on the PD-L1 receptor in vitro and in vivo. In particular, when the dose was decreased to 0.3 mg/kg, HK010 still showed a strong antitumor effect in a humanized mouse model bearing MC38/hPDL1 tumors. Strikingly, HK010 treatment enhanced antitumor immunity and induced durable antigen-specific immune memory to prevent rechallenged tumor growth by recruiting CD8+ T cells and other lymphocytes into tumor tissue and activating tumor-infiltrating lymphocytes. Moreover, HK010 not only did not induce nonspecific production of proinflammatory cytokines but was also observed to be well tolerated in cynomolgus monkeys in 5 week repeated-dose (5, 15, or 50 mg/kg) and single-dose (75 or 150 mg/kg) toxicity studies. CONCLUSION: We generated an Fc-muted anti-PD-L1x4-1BB BsAb, HK010, with a distinguished structural interaction with PD-L1 and 4-1BB that exhibits a synergistic antitumor effect by blocking the PD-1/PD-L1 signaling pathway and stimulating the 4-1BB signaling pathway simultaneously. It is strictly dependent on the PD-L1 receptor with no systemic toxicity, which may offer a new option for cancer immunotherapy.

Prolactin and glucocorticoid receptors in the prefrontal cortex are associated with anxiety-like behavior in prenatally stressed adolescent offspring rats.

Prenatal stress (PS) causes anxiety in mothers and their offspring and chewing is a commonly observed behavior during maternal stress. Prolactin (PRL) is an anti-anxiety factor that suppresses the hypothalamic-pituitary-adrenal axis. Here, we studied the roles of PRL, corticosterone (CORT), and their receptors in PS-induced anxiety-like behavior in dams and their offspring. We further investigated whether chewing during maternal stress could prevent PS-induced harmful consequences. Pregnant rats were randomly divided into PS, PS + chewing, and control groups. Anxiety-like behaviors of dams and their adolescent offspring were assessed using the open field test and elevated plus maze. Serum levels of PRL and CORT were measured by ELISA. Expression of mRNA and protein of PRLR and glucocorticoid receptor (GR) in the prefrontal cortex (PFC) were evaluated by qRT-PCR and western blotting, respectively. Compared to the control rats, dams and their female offspring, but not male offspring, in the PS group showed increased anxiety-like behaviors. The PS-affected rats had a lower serum PRL level and increased PRLR expression in the PFC. In contrast, these rats had a higher serum CORT level and decreased GR expression in the PFC. Chewing ameliorated anxiety-like behaviors and counteracted stress-induced changes in serum PRL and CORT, as well as the expression of their receptors in the PFC. Conclusion: PS-induced anxiety-like behavior is associated with changes in the serum levels of PRL and CORT and expression of their receptors in the PFC. Moreover, chewing blunts the hormonal and receptor changes and may serve as an effective stress-coping method for preventing PS-induced anxiety-like behavior.

Molecular basis for the recognition of CIZ1 by ERH.

Enhancer of rudimentary homologue (ERH), a small protein conserved in eukaryotes, is involved in a wide spectrum of cellular events, including cell cycle progression, piRNA biogenesis, miRNA maturation and gene expression. Human ERH is recruited to replication foci by CDKN1A-interacting zinc finger protein 1 (CIZ1), and plays an important role in cell growth control. However, the molecular basis for CIZ1 recognition by ERH remains unknown. By using GST pull-down experiment, we found that a fragment within CIZ1, upstream of its first zinc finger, is sufficient for binding to ERH. We solved the structure of CIZ1-bound ERH, in which the ERH dimer binds to two CIZ1 fragments to form a 2 : 2 heterotetramer. CIZ1 forms intermolecular antiparallel ß-strands with ERH, and its binding surface on ERH is distinct from those of other known ERH-binding ligands. The ERH-CIZ1 interface was further validated by mutagenesis and binding experiments. Our structural study complemented by biochemistry experiments not only provides insights into a previously unidentified ligand-binding mode for ERH but also sheds light on the understanding of evolutionarily conserved roles for ERH orthologs.

Associations of mobile phone addiction with suicide ideation and suicide attempt: findings from six universities in China.

Background: Mobile phones are becoming indispensable for life and have changed various aspects of people's lives. The psychological impacts of excessive mobile phone use have emerged as an impressive problem among college students. However, little is known about the associations of mobile phone addiction with suicide ideation and suicide attempt. Methods: A cross-sectional study was conducted with students from six universities in 2022. We collected the socio-demographic characteristics, suicide ideation, suicide attempt, psychosocial factors (depressive symptoms, social support, sleep quality), and health-related characteristics (smoking, drinking, body mass index). Mobile phone addiction was ascertained by the Mobile Phone Addiction Tendency Scale (MPATS). The associations of mobile phone addiction with suicide ideation and suicide attempt were estimated using binary logistic regression and restricted cubic splines regression. Results: A total of 18,723 college students [6,531 males (34.9%) and 12,192 females (65.1%)] were included in the final analysis. Eleven percent of participants had a history of suicide ideation, and 1.8% of participants had engaged in suicide attempt. A total of 5,553 students (29.7%) met the criteria of mobile phone addiction (MPATS score ≥48), and the average score on the MPATS was 39.5 ± 13.0. After adjustment for potential covariates, mobile phone addiction was significantly associated with increased odds of suicide ideation (OR, 1.70; 95% CI, 1.53-1.88) and suicide attempt (OR, 1.48; 95% CI, 1.18-1.86). Gender did not affect the associations of mobile phone addiction with suicide ideation and suicide attempt (P for interaction > 0.05). The restricted cubic splines regression displayed a nonlinear dose-response association between MPATS score and risk of suicide ideation (P for non-linearity < 0.001), while a monotonically increasing risk of suicide attempt was found to be associated with an increasing MPATS score (P for non-linearity = 0.420). Conclusions: Mobile phone addiction is associated with suicide ideation and suicide attempt among college students. The findings indicate that early examination, prevention, and intervention for mobile phone addiction may benefit the prevent and control of suicide.

Structural insights into the PrpTA toxin-antitoxin system in Pseudoalteromonas rubra.

Bacteria could survive stresses by a poorly understood mechanism that contributes to the emergence of bacterial persisters exhibiting multidrug tolerance (MDT). Recently, Pseudoalteromonas rubra prpAT module was found to encode a toxin PrpT and corresponding cognate antidote PrpA. In this study, we first reported multiple individual and complex structures of PrpA and PrpT, which uncovered the high-resolution three-dimensional structure of the PrpT:PrpA2:PrpT heterotetramer with the aid of size exclusion chromatography-multi-angle light scattering experiments (SEC-MALS). PrpT:PrpA2:PrpT is composed of a PrpA homodimer and two PrpT monomers which are relatively isolated from each other and from ParE family. The superposition of antitoxin monomer structures from these structures highlighted the flexible C-terminal domain (CTD). A striking conformational change in the CTDs of PrpA homodimer depolymerized from homotetramer was provoked upon PrpT binding, which accounts for the unique PrpT-PrpARHH mutual interactions and further neutralizes the toxin PrpT. PrpA2-54-form I and II crystal structures both contain a doughnut-shaped hexadecamer formed by eight homodimers organized in a cogwheel-like form via inter-dimer interface dominated by salt bridges and hydrogen bonds. Moreover, PrpA tends to exist in solution as a homodimer other than a homotetramer (SEC-MALS) in the absence of flexible CTD. Multiple multi-dimers, tetramer and hexamer included, of PrpA2-54 mediated by the symmetric homodimer interface and the complicated inter-dimer interface could be observed in the solution. SEC-MALS assays highlighted that phosphate buffer (PB) and the increase in the concentration appear to be favorable for the PrpA2-54 oligomerization in the solution. Taken together with previous research, a model of PrpA2-54 homotetramer in complex with prpAT promoter and the improved mechanism underlying how PrpTA controls the plasmid replication were proposed here.

Structural studies of SALL family protein zinc finger cluster domains in complex with DNA reveal preferential binding to an AATA tetranucleotide motif.

The Spalt-like 4 transcription factor (SALL4) plays an essential role in controlling the pluripotent property of embryonic stem cells via binding to AT-rich regions of genomic DNA, but structural details on this binding interaction have not been fully characterized. Here, we present crystal structures of the zinc finger cluster 4 (ZFC4) domain of SALL4 (SALL4ZFC4) bound with different dsDNAs containing a conserved AT-rich motif. In the structures, two zinc fingers of SALL4ZFC4 recognize an AATA tetranucleotide. We also solved the DNA-bound structures of SALL3ZFC4 and SALL4ZFC1. These structures illuminate a common preference for the AATA tetranucleotide shared by ZFC4 of SALL1, SALL3, and SALL4. Furthermore, our cell biology experiments demonstrate that the DNA-binding activity is essential for SALL4 function as DNA-binding defective mutants of mouse Sall4 failed to repress aberrant gene expression in Sall4-/- mESCs. Thus, these analyses provide new insights into the mechanisms of action underlying SALL family proteins in controlling cell fate via preferential targeting to AT-rich sites within genomic DNA during cell differentiation.

Structures of the ADGRG2-Gs complex in apo and ligand-bound forms.

Adhesion G protein-coupled receptors are elusive in terms of their structural information and ligands. Here, we solved the cryogenic-electron microscopy (cryo-EM) structure of apo-ADGRG2, an essential membrane receptor for maintaining male fertility, in complex with a Gs trimer. Whereas the formations of two kinks were determinants of the active state, identification of a potential ligand-binding pocket in ADGRG2 facilitated the screening and identification of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate and deoxycorticosterone as potential ligands of ADGRG2. The cryo-EM structures of DHEA-ADGRG2-Gs provided interaction details for DHEA within the seven transmembrane domains of ADGRG2. Collectively, our data provide a structural basis for the activation and signaling of ADGRG2, as well as characterization of steroid hormones as ADGRG2 ligands, which might be used as useful tools for further functional studies of the orphan ADGRG2.

Associations between maternal prenatal depression and neonatal behavior and brain function - Evidence from the functional near-infrared spectroscopy.

BACKGROUND: Maternal prenatal depression is a significant public health issue associated with mental disorders of offspring. This study aimed to determine if maternal prenatal depressive symptoms are associated with changes in neonatal behaviors and brain function at the resting state. METHODS: A total of 204 pregnant women were recruited during the third trimester and were evaluated by Edinburgh Postpartum Depression Scale (EPDS). The mother-infant pairs were divided into the depressed group (n = 75) and control group (n = 129) based on the EPDS, using a cut-off value of 10. Cortisol levels in the cord blood and maternal blood collected on admission for delivery were measured. On day three of life, all study newborns were evaluated by the Neonatal Behavior Assessment Scale (NBAS) and 165 infants were evaluated by resting-state functional near-infrared spectroscopy (rs-fNIRS). To minimize the influences of potential bias on the rs-fNIRS results, we used a binary logistic regression model to carry out propensity score matching between the depressed group and the control group. Rs-fNIRS data from 21 pairs of propensity score-matched newborns were used for analysis. The associations between maternal EPDS scores, neonatal NBAS scores, and cortisol levels were analyzed using linear regressions and the mediation analysis models. RESULTS: Compared to the control group, the newborns in the depressed group had lower scores in the social-interaction and autonomic system dimensions of NBAS (P < 0.01). Maternal and umbilical cord plasma cortisol levels in the depressed group were higher (P < 0.01) than in the control group. However, only umbilical cord plasma cortisol played a negative mediating role in the relationship between maternal EPDS and NBAS in the social-interaction and autonomic system (ß med = -0.054 [-0.115,-0.018] and -0.052 [-0.105,-0.019]. Proportional mediation was 13.57 % and 12.33 for social-interaction and autonomic systems, respectively. The newborns in the depressed group showed decreases in the strength of rs-fNIRS functional connections, primarily the connectivity of the left frontal-parietal and temporal-parietal regions. However, infants in the depressed and control groups showed no differences in topological characteristics of the brain network, including standardized clustering coefficient, characteristic path length, small-world property, global efficiency, and local efficiency (P > 0.05). The social-interaction Z-scores had positive correlations with functional connectivity strength of left prefrontal cortex-left parietal lobe (r = 0.57, p < 0.01)，prefrontal cortex-left parietal lobe - left temporal lobe (r = 0.593, p < 0.01) and left parietal lobe - left temporal lobe (r = 0.498, p < 0.01). Autonomic system Z-scores were also significantly positive correlation with prefrontal cortex-left parietal lobe (r = 0.509, p < 0.01)，prefrontal cortex-left parietal lobe - left temporal lobe (r = 0.464, p < 0.01), left parietal lobe - left temporal lobe (r = 0.381, p < 0.05), and right temporal lobe and left temporal lobe (r = 0.310, p < 0.05). CONCLUSION: This study shows that maternal prenatal depression may affect the development of neonatal social-interaction and autonomic system and the strength of neonatal brain functional connectivity. The fetal cortisol may play a role in behavioral development in infants exposed to maternal prenatal depression. Our findings highlight the importance of prenatal screening for maternal depression and early postnatal behavioral evaluation that provide the opportunity for early diagnosis and intervention to improve neurodevelopmental outcomes.

Trends in Research of Prenatal Stress From 2011 to 2021: A Bibliometric Study.

Background: Maternal stress during pregnancy can raise the risk of mental disorders in offspring. The continuous emergence of clinical concepts and the introduction of new technologies are great challenges. In this study, through bibliometric analysis, the research trends and hotspots on prenatal stress (PS) were explored to comprehend clinical treatments and recommend future scientific research directions. Methods: Studies on PS published on the Web of Science Core Collection (WoSCC) database between 2011 and 2021 were reviewed. Bibliometric analysis was conducted according to the number of publications, keywords, journals, citations, affiliations, and countries. With the data collected from the WoSCC, visualization of geographic distribution; clustering analysis of keywords, affiliations, and authors; and descriptive analysis and review of PS were carried out. Results: A total of 7,087 articles published in 2011-2021 were retrieved. During this period, the number of publications increased. Psychoneuroendocrinology is the leading journal on PS. The largest contributor was the United States. The University of California system was leading among institutions conducting relevant research. Wang H, King S, and Tain YL were scholars with significant contributions. Hotspots were classified into four clusters, namely, pregnancy, prenatal stress, oxidative stress, and growth. Conclusion: The number of studies on PS increased. Journals, countries, institutions, researchers with the most contributions, and most cited articles worldwide were extracted. Studies have mostly concentrated on treating diseases, the application of new technologies, and the analysis of epidemiological characteristics. Multidisciplinary integration is becoming the focus of current development. Epigenetics is increasingly used in studies on PS. Thus, it constitutes a solid foundation for future clinical medical and scientific research.

Structural Analysis of Ino2p/Ino4p Mutual Interactions and Their Binding Interface with Promoter DNA.

Gene expression is mediated by a series of regulatory proteins, i.e., transcription factors. Under different growth conditions, the transcriptional regulation of structural genes is associated with the recognition of specific regulatory elements (REs) in promoter DNA. The manner by which transcription factors recognize distinctive REs is a key question in structural biology. Previous research has demonstrated that Ino2p/Ino4p heterodimer is associated with the transcriptional regulation of phospholipid biosynthetic genes. Mechanistically, Ino2p/Ino4p could specifically recognize the inositol/choline-responsive element (ICRE), followed by the transcription activation of the phospholipid biosynthetic gene. While the promoter DNA sequence for Ino2p has already been characterized, the structural basis for the mutual interaction between Ino2p/Ino4p and their binding interface with promoter DNA remain relatively unexplored. Here, we have determined the crystalline structure of the Ino2pDBD/Ino4pDBD/DNA ternary complex, which highlights some residues (Ino2pHis12/Glu16/Arg20/Arg44 and Ino4pHis12/Glu16/Arg19/Arg20) associated with the sequence-specific recognition of promoter DNA. Our biochemical analysis showed that mutating these residues could completely abolish protein-DNA interaction. Despite the requirement of Ino2p and Ino4p for interprotein-DNA interaction, both proteins can still interact-even in the absence of DNA. Combined with the structural analysis, our in vitro binding analysis demonstrated that residues (Arg35, Asn65, and Gln69 of Ino2pDBD and Leu59 of Ino4pDBD) are critical for interprotein interactions. Together, these results have led to the conclusion that these residues are critical to establishing interprotein-DNA and protein-DNA mutual interactions.

AutoPX: a new software package to process X-ray diffraction data from biomacromolecular crystals.

A new software package, autoPX, for processing X-ray diffraction data from biomacromolecular crystals is reported. This processing software package is designed on the basis of novel methods such as the location of diffraction spots by an improved Canny operator, indexing by a modified Fourier transform, a novel definition of mosaicity that expresses the dispersion state of reciprocal diffraction spots, and the correction of predicted diffraction spot coordinates by homography transform. New programming of some traditional algorithms necessary for integration and scaling is also included. Several examples of crystal structure determination using data from the SSRF beamlines reduced using autoPX, HKL-2000, DIALS and XDS are also demonstrated, and indicate that autoPX is capable of processing diffraction data from biomacromolecular crystals and providing adequate solutions to problems encountered at the SSRF beamlines.