Homogeneous Oligomers of Pro-apoptotic BAX Reveal Structural Determinants of Mitochondrial Membrane Permeabilization.

BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.

Characterization of the Zinc Uptake Repressor (Zur) from Acinetobacter baumannii.

Bacterial cells tightly regulate the intracellular concentrations of essential transition metal ions by deploying a panel of metal-regulated transcriptional repressors and activators that bind to operator-promoter regions upstream of regulated genes. Like other zinc uptake regulator (Zur) proteins, Acinetobacter baumannii Zur represses transcription of its regulon when ZnII is replete and binds more weakly to DNA when ZnII is limiting. Previous studies established that Zur proteins are homodimeric and harbor at least two metal sites per protomer or four per dimer. CdII X-ray absorption spectroscopy (XAS) of the Cd2Zn2 AbZur metalloderivative with CdII bound to the allosteric sites reveals a S(N/O)3 first coordination shell. Site-directed mutagenesis suggests that H89 and C100 from the N-terminal DNA binding domain and H107 and E122 from the C-terminal dimerization domain comprise the regulatory metal site. KZn for this allosteric site is 6.0 (±2.2) × 1012 M-1 with a functional "division of labor" among the four metal ligands. N-terminal domain ligands H89 and C100 contribute far more to KZn than H107 and E122, while C100S AbZur uniquely fails to bind to DNA tightly as measured by an in vitro transcription assay. The heterotropic allosteric coupling free energy, ΔGc, is negative, consistent with a higher KZn for the AbZur-DNA complex and defining a bioavailable ZnII set-point of ≈6 × 10-14 M. Small-angle X-ray scattering (SAXS) experiments reveal that only the wild-type Zn homodimer undergoes allosteric switching, while the C100S AbZur fails to switch. These data collectively suggest that switching to a high affinity DNA-binding conformation involves a rotation/translation of one protomer relative to the other in a way that is dependent on the integrity of C100. We place these findings in the context of other Zur proteins and Fur family repressors more broadly.

Changes in the shape and function of the fetal heart of pre- and gestational diabetes mothers.

BACKGROUND: Hyperglycemia during pregnancy can affect fetal heart in many ways, including causing cardiac malformation, leading to hypertrophic cardiomyopathy and cardiac dysfunction. Echocardiographic evaluation can assist identify alterations in heart structure, morphology and function, enabling prompt monitoring and management. However, according to earlier research, the cardiac alterations are modest in hyperglycemic mothers' fetuses, and might not be detectable using conventional methods and it is also unclear whether these changes are related to the metabolism of mothers. Fetal Heart Quantification (Fetal HQ) can assess ventricular geometry and function more sensitively and thoroughly, and identify sub-clinical cardiac dysfunction. The purpose of this study was to evaluate fetal heart by Fetal HQ in fetuses of hyperglycemic mothers who either had pre-gestational or gestational diabetes and to correlate them with maternal metabolic indices. METHODS: The fetuses of 25 gestational age-matched control mothers, 48 women with gestational diabetes mellitus (GDM), and 11 women with diabetes mellitus (DM) were included in the prospective case-control research. Using fetal echocardiography and speckle tracking echocardiography (STE), the heart of the fetus was evaluated. Differences in the groups' anthropometric, metabolic, and cardiac parameters were examined. It was assessed whether maternal features, prenatal glucose, lipids, and maternal hemoglobin A1c (HbA1c) correlated with fetal cardiac parameters. RESULTS: The LV EDV and ESV were significantly higher in the GDM group as compared to the DM group (p < 0.05). The GSI% was significantly lower in the GDM group compared with the control (p < 0.05). The LV SV and CO of the GDM group were both significantly higher compared with the DM group (p < 0.05). There was a significant decrease in RV FS for segments 1-7 in GDM fetuses compared to the control (p < 0.05) and for segments 5-10 compared to DM (p < 0.05). Fetal cardiac morphology and function indices correlate with maternal pregestational weight, BMI, early pregnancy fast glucose, lipids, and glycemic control levels. CONCLUSIONS: Fetuses exposed to gestational diabetes have altered heart morphology and function that is linked to maternal metabolic parameters, which presents a special indication for performing geometry and function cardiac assessment. Fetal HQ can be employed to evaluate the fetal cardiac shape and function in fetuses exposed to gestational diabetes.

The low-resolution structural models of hepatitis C virus RNA subdomain 5BSL3.2 and its distal complex with domain 3'X point to conserved regulatory mechanisms within the Flaviviridae family.

Subdomain 5BSL3.2 of hepatitis C virus RNA lies at the core of a network of distal RNA-RNA contacts that connect the 5' and 3' regions of the viral genome and regulate the translation and replication stages of the viral cycle. Using small-angle X-ray scattering and NMR spectroscopy experiments, we have determined at low resolution the structural models of this subdomain and its distal complex with domain 3'X, located at the 3'-terminus of the viral RNA chain. 5BSL3.2 adopts a characteristic 'L' shape in solution, whereas the 5BSL3.2-3'X distal complex forms a highly unusual 'Y'-shaped kissing junction that blocks the dimer linkage sequence of domain 3'X and promotes translation. The structure of this complex may impede an effective association of the viral polymerase with 5BSL3.2 and 3'X to start negative-strand RNA synthesis, contributing to explain the likely mechanism used by these sequences to regulate viral replication and translation. In addition, sequence and shape features of 5BSL3.2 are present in functional RNA motifs of flaviviruses, suggesting conserved regulatory processes within the Flaviviridae family.

Analysis of Potential Risks in Abnormal Physical Development Due to Dietary Habits in Preschool Children.

Background: The prevalence of abnormal physical development in preschool children is often linked to their dietary habits, necessitating a comprehensive investigation. Understanding the intricacies of these habits is crucial for formulating targeted interventions to enhance the overall health and well-being of this vulnerable population. Objective: This study aims to explore the dietary habits of preschool children in Shijiazhuang and evaluate their impact on abnormal physical development. The primary objective is to identify key dietary issues, particularly focusing on picky eating, and assess their association with undernutrition and obesity in this age group. Methods: Utilizing a stratified sampling approach, the study involves preschool children and their caregivers from various kindergartens in Shijiazhuang. On-site medical examinations are conducted to measure height and weight and calculate body mass index (BMI). Additionally, parents were surveyed to gather information on the general aspects and dietary habits of their children. Binary logistic regression analysis was employed to ascertain the correlation between picky eating and the risk of undernutrition and obesity. Results: The findings indicate that approximately 70% of preschool children maintain a normal BMI, while 16.67% experience undernutrition, and 13.33% face issues of being overweight or obese. Picky eating emerges as the predominant dietary habit issue, affecting 51.33% of the participants. Binary logistic regression analysis identifies picky eating as a significant risk factor for undernutrition and obesity among children. Conclusions: Picky eating stands out as the primary dietary habit concern for preschool children, concurrently posing a substantial risk for abnormal physical development. Urgent measures are warranted to rectify children's suboptimal dietary habits, elevate nutritional standards, and foster their overall health and development. These findings underscore the imperative need for interventions targeting dietary improvement in preschoolers, contributing to improving their well-being and long-term health outcomes.

The DNA binding domain of the Vibrio vulnificus SmcR transcription factor is flexible and binds diverse DNA sequences.

Quorum sensing gene expression in vibrios is regulated by the LuxR/HapR family of transcriptional factors, which includes Vibrio vulnificus SmcR. The consensus binding site of Vibrio LuxR/HapR/SmcR proteins is palindromic but highly degenerate with sequence variations at each promoter. To examine the mechanism by which SmcR recognizes diverse DNA sites, we generated SmcR separation-of-function mutants that either repress or activate transcription but not both. SmcR N55I is restricted in recognition of single base-pair variations in DNA binding site sequences and thus is defective at transcription activation but retains interaction with RNA polymerase (RNAP) alpha. SmcR S76A, L139R and N142D substitutions disrupt the interaction with RNAP alpha but retain functional DNA binding activity. X-ray crystallography and small angle X-ray scattering data show that the SmcR DNA binding domain exists in two conformations (wide and narrow), and the protein complex forms a mixture of dimers and tetramers in solution. The three RNAP interaction-deficient variants also have two DNA binding domain conformations, whereas SmcR N55I exhibits only the wide conformation. These data support a model in which two mechanisms drive SmcR transcriptional activation: interaction with RNAP and a multi-conformational DNA binding domain that permits recognition of variable DNA sites.

Developmental Process and Symmetry of Fetal Sulci in the Mesial Area Assessed by Ultrasound: A Cohort Study in Chinese Population.

OBJECTIVE: We aimed to investigate the progression of cortical development in Chinese population and to determine the rate of isolated asymmetric cortical development. We also explored the outcomes of these fetuses and determined whether cortical asymmetry represents normal individual physiological variation. METHODS: Our observational cohort study included 456 healthy singleton pregnant women who visited Peking University First Hospital between September 2020 and December 2021. We evaluated the progression and symmetry of the parieto-occipital sulcus, calcarine sulcus, and cingulate sulcus using a scoring system during routine fetal ultrasound examinations. The outcomes of the included fetuses after birth were assessed using the Ages and Stages Questionnaire, Third Edition (ASQ-3). RESULTS: The median gestational ages at which the parieto-occipital, calcarine, and cingulate sulci reached grade 1 were 22, 22, and 26 weeks, respectively. Among 456 included fetuses, 426 showed symmetric cortical development and 30 showed asymmetric cortical development during ultrasound examination. Fetuses with asymmetric cortical development underwent 'catch-up growth' and developed to the same grade in 2-6 weeks. All fetuses with symmetric or asymmetric cortical development had normal neurodevelopment after birth according to ASQ-3 assessment. CONCLUSION: The gestational age at which the parieto-occipital, calcarine, and cingulate sulci can be detected using ultrasound varies in different studies. Racial differences may be present in cortical development. Normal fetuses may physiologically have mildly asymmetric cortical development in the mesial area.

Structural basis of peptidoglycan endopeptidase regulation.

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., ß-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.

Associations of ApoE Polymorphisms with Postoperative Atrial Fibrillation and Cardiac Injury in Patients with Coronary Artery Bypass Graft Surgery.

Genetic factors may be involved in postoperative atrial fibrillation (PoAF) development and cardiac injury. However, the associations of the apolipoprotein E (ApoE) gene polymorphisms with PoAF and cardiac injury after coronary artery bypass graft surgery (CABG) remain unclear.We recruited 150 patients with CABG, comprising 92 and 58 cases for the ApoE4 and ApoE3 groups, respectively, and analyzed PoAF incidence and the levels of cardiac biomarkers, including N-terminal prohormone of brain natriuretic peptide, cardiac troponin T (cTnT), and cardiac troponin I (cTnI). The linear regression model or logistic regression analysis was applied to investigate the associations of ApoE gene polymorphisms with PoAF and biomarkers for cardiac injury.A total of 58 (38.7%) patients with CABG developed PoAF, with 40 and 18 cases in the ApoE4 and ApoE3 groups (43.5% versus 31.0%, P < 0.05), respectively. Logistic regression analysis revealed that the ApoE4 allele was an independent risk factor for PoAF (OR = 3.340, P = 0.001), while the ApoE3 allele was a protective factor for the PoAF (OR = 0.841, P = 0.043). Patients carrying the ApoE4 allele had higher levels of cTnT and cTnI than those carrying the ApoE3 allele. ApoE3 was a protective factor for cardiac injury (ß = -0.220, P = 0.001), whereas ApoE4 was a risk factor for cTnI (ß = 0.335, P = 0.015).Our study reveals that the ApoE allele contributes to the occurrence of PoAF and severity of cardiac injury in an allele-dependent manner, with the ApoE4 allele increasing the risk and the ApoE3 allele reducing the risk.

Simpson-Golabi-Behmel syndrome in one of the Dichorionic-diamniotic twin: a case report and literature review.

BACKGROUND: Simpson-Golabi-Behmel syndrome (SGBS) is a rare X-linked overgrowth syndrome. The main clinical manifestations are overgrowth and multiple malformations. CASE PRESENTATION: A 38-year-old Chinese woman was pregnant with dichorionic-diamniotic (DCDA) twins after in-vitro fertilization. Series of ultrasound examinations indicated that the measurements (abdominal circumference and estimated foetal weight) of one twin were significantly greater than those of the other one. The genetic testing results of the larger baby indicated of Simpson-Golabi-Behmel syndrome. CONCLUSION: SGBS is difficult to diagnose due to different clinical manifestations. Clinicians need to be more aware of typical SGBS's clinical findings and choose genetic testing methods individually to improve its prenatal diagnosis.

A hypothyroid mother after subtotal thyroidectomy delivered a newborn with hyperthyroidism from fetal stage: a case report.

BACKGROUND: Neonatal hyperthyroidism is an extension of fetal disease. Most cases of neonatal hyperthyroidism are transient but may excessively harm multiple organ functions through the actions of maternal thyroid-stimulating hormone receptor antibodies on the neonatal thyroid gland. CASE PRESENTATION: The hyperthyroid mother underwent subtotal thyroidectomy before pregnancy and regularly took levothyroxine to avoid hypothyroidism, but still had a high-level thyroid-stimulating hormone receptor antibody (TRAb). The neonate suffered from hyperthyroidism due to the transplacental TRAb. After a regular medication schedule of an antithyroid drug, combined with a ß-blocker to control the ventricular rate, the infant gradually recovered, allowing normal motor and intellectual development. CONCLUSIONS: Maternal subtotal thyroidectomy cannot prevent the secretion of thyroid receptor antibodies, which may cause either hypothyroidism or hyperthyroidism. The balance between antithyroid drugs and levothyroxine is critical in clinical practice.

Alternative splicing and allosteric regulation modulate the chromatin binding of UHRF1.

UHRF1 is an important epigenetic regulator associated with apoptosis and tumour development. It is a multidomain protein that integrates readout of different histone modification states and DNA methylation with enzymatic histone ubiquitylation activity. Emerging evidence indicates that the chromatin-binding and enzymatic modules of UHRF1 do not act in isolation but interplay in a coordinated and regulated manner. Here, we compared two splicing variants (V1, V2) of murine UHRF1 (mUHRF1) with human UHRF1 (hUHRF1). We show that insertion of nine amino acids in a linker region connecting the different TTD and PHD histone modification-binding domains causes distinct H3K9me3-binding behaviour of mUHRF1 V1. Structural analysis suggests that in mUHRF1 V1, in contrast to V2 and hUHRF1, the linker is anchored in a surface groove of the TTD domain, resulting in creation of a coupled TTD-PHD module. This establishes multivalent, synergistic H3-tail binding causing distinct cellular localization and enhanced H3K9me3-nucleosome ubiquitylation activity. In contrast to hUHRF1, H3K9me3-binding of the murine proteins is not allosterically regulated by phosphatidylinositol 5-phosphate that interacts with a separate less-conserved polybasic linker region of the protein. Our results highlight the importance of flexible linkers in regulating multidomain chromatin binding proteins and point to divergent evolution of their regulation.

A case of fetal isolated ventricular noncompaction with TNNT2 gene mutation and literature review.

BACKGROUND: Ventricular noncompaction (VNC) is a cardiomyopathy characterized by overdeveloped ventricular trabeculaes and deep recess, which has been rarely reported. CASE PRESENTATION: A 29-year-old Chinese pregnant woman with no obvious fetal abnormality in regular prenatal examination during first and second trimester. However, at 32 weeks of gestation, both obstetric growth scan and fetal echocardiogram revealed an enlarged heart with grid-like changes at the apical region. Eventually, the genetic and autopsy findings indicated the deceased infant with VNC. CONCLUSION: Isolated VNC could be detected prenatally, even during the late pregnancy. Fetuses suspected of VNC should be offered genetic tests.

Microvascular Rarefaction and Myocardial Fibrosis in Hypertrophic Obstructive Cardiomyopathy: A Histopathological Comparison of Pediatric and Adult Patients.

BACKGROUND: Hypertrophic obstructive cardiomyopathy (HOCM) is a genetic cardiomyopathy characterized by microvascular ischemia and myocardial fibrosis. Microvessels play an important role in myocardial fibrosis in HOCM. However, the changes of myocardial microvessels and myocardial fibrosis in pediatric and adult patients with HOCM remain unclear. This study was to investigate the changes in myocardial microvessel density (MVD) and myocardial fibrosis in pediatric and adult patients with HOCM. METHODS: We analyzed the changes in MVD and myocardial fibrosis in myectomy left ventricular (LV) septal wall specimens in 12 adult patients and five pediatric patients with HOCM. Control myocardium from the LV septal wall was collected at autopsy of 5 adults and 4 pediatric individuals, who died of non-cardiac causes. RESULTS: There was no significant difference in MVD between pediatric HOCM patients and control subjects (706.4±187.5 vs. 940.2±491.1, P > 0.05), but the myocardial fibrosis area ratio was significantly increased in HOCM than in control subjects (10.6±3.5 vs. 4.9±1.2, P < 0.01). MVD was significantly reduced, and myocardial fibrosis area ratio was significantly higher in adult HOCM patients than in control subjects (i.e. 523.3± 209.4 vs. 845.7±260.7, P < 0.05; 12.8±5.1 vs. 4.4±1.3, P < 0.05). There was no significant difference in MVD and myocardial fibrosis between pediatric and adult HOCM patients (706.4±187.5 vs. 523.3±209.4, P > 0.05; 10.6±3.5 vs. 12.8±5.1, P > 0.05).  Conclusions: Pediatric and adult patients with HOCM have high myocardial fibrosis. The present findings suggest that myocardial microvascular density lesions contribute to myocardial fibrosis during childhood.

Coronary artery bypass grafting in patients with malignancy: a single-institute case series of eight patients.

BACKGROUND: The surgical strategy among patients with malignancy and coronary artery disease (CAD) remains controversial. In this study, we present the experiences of coronary artery bypass grafting (CABG) in patients with malignancy and analyzed the treatment outcomes. METHODS: From January 2011 to October 2021, eight patients combined with coronary artery disease and malignancy, six of them with three-vessel disease and two with anterior descending branch lesions on coronary angiography. The age ranged from 54 to 73 years (61.8 ± 7.7years). Four patients underwent CABG and staging for surgical oncology, and 2 patients underwent CABG and surgical oncology simultaneously. Four patients underwent CABG procedure with cardiopulmonary bypass (on-pump CABG), and the other patients underwent the procedure without cardiopulmonary bypass (off-pump CABG). All patients were followed up for 3 to 96 months (40.4 ± 31.5 months) postoperatively. RESULTS: The mean number of grafts was 2.6 ± 1.1, there was no in-hospital death, postoperative myocardial infarction, and stroke. Among the eight patients, one patient received chemotherapy and radiation before bypass surgery, which occurred postoperatively pulmonary infection, and the rest of 7 patients had no major adverse cardiovascular events during follow-up periods. CONCLUSION: Based on the results of the present study, simultaneous or staged CABG and oncologic surgery according to the TNM stage of the tumor and cardiac assessment is an effective treatment for patients with severe CAD combined with malignancy.

The MLL1 trimeric catalytic complex is a dynamic conformational ensemble stabilized by multiple weak interactions.

Histone H3K4 methylation is an epigenetic mark associated with actively transcribed genes. This modification is catalyzed by the mixed lineage leukaemia (MLL) family of histone methyltransferases including MLL1, MLL2, MLL3, MLL4, SET1A and SET1B. The catalytic activity of this family is dependent on interactions with additional conserved proteins, but the structural basis for subunit assembly and the mechanism of regulation is not well understood. We used a hybrid methods approach to study the assembly and biochemical function of the minimally active MLL1 complex (MLL1, WDR5 and RbBP5). A combination of small angle X-ray scattering, cross-linking mass spectrometry, nuclear magnetic resonance spectroscopy and computational modeling were used to generate a dynamic ensemble model in which subunits are assembled via multiple weak interaction sites. We identified a new interaction site between the MLL1 SET domain and the WD40 ß-propeller domain of RbBP5, and demonstrate the susceptibility of the catalytic function of the complex to disruption of individual interaction sites.

Design and characterization of mutant and wildtype huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems.

The gene mutated in individuals with Huntington's disease (HD) encodes the 348-kDa huntingtin (HTT) protein. Pathogenic HD CAG-expansion mutations create a polyglutamine (polyQ) tract at the N terminus of HTT that expands above a critical threshold of ∼35 glutamine residues. The effect of these HD mutations on HTT is not well understood, in part because it is difficult to carry out biochemical, biophysical, and structural studies of this large protein. To facilitate such studies, here we have generated expression constructs for the scalable production of HTT in multiple eukaryotic expression systems. Our set of HTT expression clones comprised both N- and C-terminally FLAG-tagged HTT constructs with polyQ lengths representative of the general population, HD patients, and juvenile HD patients, as well as the more extreme polyQ expansions used in some HD tissue and animal models. Our expression system yielded milligram quantities of pure recombinant HTT protein, including many of the previously mapped post-translational modifications. We characterized both apo and HTT-HTT-associated protein 40 (HAP40) complex samples produced with this HD resource, demonstrating that this toolkit can be used to generate physiologically meaningful HTT complexes. We further demonstrate that these resources can produce sufficient material for protein-intensive experiments, such as small-angle X-ray scattering, providing biochemical insight into full-length HTT protein structure. The work outlined and the tools generated here lay a foundation for further biochemical and structural work on the HTT protein and for studying its functional interactions with other biomolecules.

Multiphase Assembly of Small Molecule Microcrystalline Peptide Hydrogel Allows Immunomodulatory Combination Therapy for Long-Term Heart Transplant Survival.

Combination therapies that target multiple pathways involved in immune rejection of transplants hold promise for patients in need of restorative surgery. Herein, a noninteracting multiphase molecular assembly approach is developed to crystallize tofacitinib, a potent JAK1/3 inhibitor, within a shear-thinning self-assembled fibrillar peptide hydrogel network. The resulting microcrystalline tofacitinib hydrogel (MTH) can be syringe-injected directly to the grafting site during surgery to locally deliver the small molecule. The rate of drug delivered from MTH is largely controlled by the dissolution of the encapsulated microcrystals. A single application of MTH, in combination with systemically delivered CTLA4-Ig, a co-stimulation inhibitor, affords significant graft survival in mice receiving heterotopic heart transplants. Locoregional studies indicate that the local delivery of tofacitinib at the graft site enabled by MTH is required for the observed enhanced graft survival.

Three-dimensional structure of the 3'X-tail of hepatitis C virus RNA in monomeric and dimeric states.

The 3'X domain is a 98-nt region located at the 3' end of hepatitis C virus genomic RNA that plays essential functions in the viral life cycle. It contains an absolutely conserved, 16-base palindromic sequence that promotes viral RNA dimerization, overlapped with a 7-nt tract implicated in a distal contact with a nearby functional sequence. Using small angle X-ray scattering measurements combined with model building guided by NMR spectroscopy, we have studied the stoichiometry, structure, and flexibility of domain 3'X and two smaller subdomain sequences as a function of ionic strength, and obtained a three-dimensional view of the full-length domain in its monomeric and dimeric states. In the monomeric form, the 3'X domain adopted an elongated conformation containing two SL1' and SL2' double-helical stems stabilized by coaxial stacking. This structure was significantly less flexible than that of isolated subdomain SL2' monomers. At higher ionic strength, the 3'X scattering envelope nearly doubled its size, reflecting the formation of extended homodimers containing an antiparallel SL2' duplex flanked by coaxially stacked SL1' helices. Formation of these dimers could initialize and/or regulate the packaging of viral RNA genomes into virions.

Structural analyses of the Haemophilus influenzae peptidoglycan synthase activator LpoA suggest multiple conformations in solution.

In many Gram-negative bacteria, the peptidoglycan synthase PBP1A requires the outer membrane lipoprotein LpoA for constructing a functional peptidoglycan required for bacterial viability. Previously, we have shown that the C-terminal domain of Haemophilus influenzae LpoA (HiLpoA) has a highly conserved, putative substrate-binding cleft between two α/ß lobes. Here, we report a 2.0 Å resolution crystal structure of the HiLpoA N-terminal domain. Two subdomains contain tetratricopeptide-like motifs that form a concave groove, but their relative orientation differs by ∼45° from that observed in an NMR structure of the Escherichia coli LpoA N domain. We also determined three 2.0-2.8 Å resolution crystal structures containing four independent full-length HiLpoA molecules. In contrast to an elongated model previously suggested for E. coli LpoA, each HiLpoA formed a U-shaped structure with a different C-domain orientation. This resulted from both N-domain twisting and rotation of the C domain (up to 30°) at the end of the relatively immobile interdomain linker. Moreover, a previously predicted hinge between the lobes of the LpoA C domain exhibited variations of up to 12°. Small-angle X-ray scattering data revealed excellent agreement with a model calculated by normal mode analysis from one of the full-length HiLpoA molecules but even better agreement with an ensemble of this molecule and two of the partially extended normal mode analysis-predicted models. The different LpoA structures helped explain how an outer membrane-anchored LpoA can either withdraw from or extend toward the inner membrane-bound PBP1A through peptidoglycan gaps and hence regulate the synthesis of peptidoglycan necessary for bacterial viability.