Mucin adhesion of serial cystic fibrosis airways Pseudomonas aeruginosa isolates.

The chronic airway infections with Pseudomonas aeruginosa are the major co-morbidity in people with cystic fibrosis (CF). Within CF lungs, P. aeruginosa persists in the conducting airways together with human mucins as the most abundant structural component of its microenvironment. We investigated the adhesion of 41 serial CF airway P. aeruginosa isolates to airway mucin preparations from CF sputa. Mucins and bacteria were retrieved from five modulator-naïve patients with advanced CF lung disease. The P. aeruginosa isolates from CF airways and non-CF reference strains showed a strain-specific signature in their adhesion to ovine, porcine and bovine submaxillary mucins and CF airway mucins ranging from no or low to moderate and strong binding. Serial CF clonal isolates and colony morphotypes from the same sputum sample were as heterogeneous in their affinity to mucin as representatives of other clones thus making 'mucin binding' one of the most variable intraclonal phenotypic traits of P. aeruginosa known to date. Most P. aeruginosa CF airway isolates did not adhere more strongly to CF airway mucins than to plastic surfaces. The strong binders, however, exhibited a strain-specific affinity gradient to O-glycans, CF airway and mammalian submaxillary mucins.

Fluorescence changes in carbon nanotube sensors correlate with THz absorption of hydration.

Single wall carbon nanotubes (SWCNTs) functionalized with (bio-)polymers such as DNA are soluble in water and sense analytes by analyte-specific changes of their intrinsic fluorescence. Such SWCNT-based (bio-)sensors translate the binding of a molecule (molecular recognition) into a measurable optical signal. This signal transduction is crucial for all types of molecular sensors to achieve high sensitivities. Although there is an increasing number of SWCNT-based sensors, there is yet no molecular understanding of the observed changes in the SWCNT's fluorescence. Here, we report THz experiments that map changes in the local hydration of the solvated SWCNT upon binding of analytes such as the neurotransmitter dopamine or the vitamin riboflavin. The THz amplitude signal serves as a measure of the coupling of charge fluctuations in the SWCNTs to the charge density fluctuations in the hydration layer. We find a linear (inverse) correlation between changes in THz amplitude and the intensity of the change in fluorescence induced by the analytes. Simulations show that the organic corona shapes the local water, which determines the exciton dynamics. Thus, THz signals are a quantitative predictor for signal transduction strength and can be used as a guiding chemical design principle for optimizing fluorescent biosensors.

STIC2 selectively binds ribosome-nascent chain complexes in the cotranslational sorting of Arabidopsis thylakoid proteins.

Chloroplast-encoded multi-span thylakoid membrane proteins are crucial for photosynthetic complexes, yet the coordination of their biogenesis remains poorly understood. To identify factors that specifically support the cotranslational biogenesis of the reaction center protein D1 of photosystem (PS) II, we generated and affinity-purified stalled ribosome-nascent chain complexes (RNCs) bearing D1 nascent chains. Stalled RNCs translating the soluble ribosomal subunit uS2c were used for comparison. Quantitative tandem-mass spectrometry of the purified RNCs identified around 140 proteins specifically associated with D1 RNCs, mainly involved in protein and cofactor biogenesis, including chlorophyll biosynthesis, and other metabolic pathways. Functional analysis of STIC2, a newly identified D1 RNC interactor, revealed its cooperation with chloroplast protein SRP54 in the de novo biogenesis and repair of D1, and potentially other cotranslationally-targeted reaction center subunits of PSII and PSI. The primary binding interface between STIC2 and the thylakoid insertase Alb3 and its homolog Alb4 was mapped to STIC2's ß-sheet region, and the conserved Motif III in the C-terminal regions of Alb3/4.

Development and validation of a test instrument for the assessment of perceived basic motor competencies in first and second graders: the SEMOK-1-2 instrument.

Both actual motor competencies (AMC) and perceived motor competencies (PMC) play an important role in motor development research and children's physical and psychological development. PMC refer to children's perceptions of their motor competencies. To assess the PMC of first and second grade children (aged 6-9 years), the SEMOK-1-2 instrument was developed. The instrument is aligned to the validated MOBAK-1-2 instrument which assesses AMC in the competence areas "object movement" and "self-movement" Accounting for possible reading difficulties in younger children, the motor tasks and answer options were illustrated and explained verbally. The purpose of this study was to test and validate the SEMOK-1-2 instrument and investigate the associations between the constructs AMC, PMC and physical activity (PA), whereby PA was measured by the participation in team and individual sports. Data from N = 404 pupils in the German-speaking part of Switzerland from first and second grades (M = 7.8 years, SD = 0.69, 49% boys) were analyzed. Confirmatory factor analyses were conducted to test the factorial validity of the SEMOK-1-2 instrument. Structural equation models were used to investigate the association between the constructs. The analyses confirmed a two-factor structure with the factors PMC "object movement" and PMC "self-movement", corresponding to the factors existing in the MOBAK-1-2 instrument. Latent correlations between AMC factors and the corresponding PMC factors were r = 0.79 for "object movement" and r = 0.76 for "self-movement". Associations with external criteria and covariates, such as sex, were associated with both AMC and PMC. Analyses also revealed that children who participated more often in individual and team sports showed higher levels in both AMC and PMC. The confirmation of the two-factorial structure of the SEMOK-1-2 instrument and the associations between AMC and PMC as well as external criteria indicate construct and criterion validity. The SEMOK-1-2 instrument can be economically utilized for assessing PMC and is also suitable for the monitoring of PMC in the context of Physical Education.

Structural insights into the activation mechanism of antimicrobial GBP1.

The dynamin-related human guanylate-binding protein 1 (GBP1) mediates host defenses against microbial pathogens. Upon GTP binding and hydrolysis, auto-inhibited GBP1 monomers dimerize and assemble into soluble and membrane-bound oligomers, which are crucial for innate immune responses. How higher-order GBP1 oligomers are built from dimers, and how assembly is coordinated with nucleotide-dependent conformational changes, has remained elusive. Here, we present cryo-electron microscopy-based structural data of soluble and membrane-bound GBP1 oligomers, which show that GBP1 assembles in an outstretched dimeric conformation. We identify a surface-exposed helix in the large GTPase domain that contributes to the oligomerization interface, and we probe its nucleotide- and dimerization-dependent movements that facilitate the formation of an antimicrobial protein coat on a gram-negative bacterial pathogen. Our results reveal a sophisticated activation mechanism for GBP1, in which nucleotide-dependent structural changes coordinate dimerization, oligomerization, and membrane binding to allow encapsulation of pathogens within an antimicrobial protein coat.

Integrative dynamic structural biology unveils conformers essential for the oligomerization of a large GTPase.

Guanylate binding proteins (GBPs) are soluble dynamin-like proteins that undergo a conformational transition for GTP-controlled oligomerization and disrupt membranes of intracellular parasites to exert their function as part of the innate immune system of mammalian cells. We apply neutron spin echo, X-ray scattering, fluorescence, and EPR spectroscopy as techniques for integrative dynamic structural biology to study the structural basis and mechanism of conformational transitions in the human GBP1 (hGBP1). We mapped hGBP1's essential dynamics from nanoseconds to milliseconds by motional spectra of sub-domains. We find a GTP-independent flexibility of the C-terminal effector domain in the µs-regime and resolve structures of two distinct conformers essential for an opening of hGBP1 like a pocket knife and for oligomerization. Our results on hGBP1's conformational heterogeneity and dynamics (intrinsic flexibility) deepen our molecular understanding relevant for its reversible oligomerization, GTP-triggered association of the GTPase-domains and assembly-dependent GTP-hydrolysis.

The large GTPase AtGBPL3 links nuclear envelope formation and morphogenesis to transcriptional repression.

Guanylate binding proteins (GBPs) are prominent regulators of immunity not known to be required for nuclear envelope formation and morphogenesis. Here we identify the Arabidopsis GBP orthologue AtGBPL3 as a lamina component with essential functions in mitotic nuclear envelope reformation, nuclear morphogenesis and transcriptional repression during interphase. AtGBPL3 is preferentially expressed in mitotically active root tips, accumulates at the nuclear envelope and interacts with centromeric chromatin as well as with lamina components transcriptionally repressing pericentromeric chromatin. Reduced expression of AtGBPL3 or associated lamina components similarly altered nuclear morphology and caused overlapping transcriptional deregulation. Investigating the dynamics of AtGBPL3-GFP and other nuclear markers during mitosis (1) revealed that AtGBPL3 accumulation on the surface of daughter nuclei precedes nuclear envelope reformation and (2) uncovered defects in this process in roots of AtGBPL3 mutants, which cause programmed cell death and impair growth. AtGBPL3 functions established by these observations are unique among dynamin-family large GTPases.

LPS-aggregating proteins GBP1 and GBP2 are each sufficient to enhance caspase-4 activation both in cellulo and in vitro.

The gamma-interferon (IFNγ)-inducible guanylate-binding proteins (GBPs) promote host defense against gram-negative cytosolic bacteria in part through the induction of an inflammatory cell death pathway called pyroptosis. To activate pyroptosis, GBPs facilitate sensing of the gram-negative bacterial outer membrane component lipopolysaccharide (LPS) by the noncanonical caspase-4 inflammasome. There are seven human GBP paralogs, and it is unclear how each GBP contributes to LPS sensing and pyroptosis induction. GBP1 forms a multimeric microcapsule on the surface of cytosolic bacteria through direct interactions with LPS. The GBP1 microcapsule recruits caspase-4 to bacteria, a process deemed essential for caspase-4 activation. In contrast to GBP1, closely related paralog GBP2 is unable to bind bacteria on its own but requires GBP1 for direct bacterial binding. Unexpectedly, we find that GBP2 overexpression can restore gram-negative-induced pyroptosis in GBP1KO cells, without GBP2 binding to the bacterial surface. A mutant of GBP1 that lacks the triple arginine motif required for microcapsule formation also rescues pyroptosis in GBP1KO cells, showing that binding to bacteria is dispensable for GBPs to promote pyroptosis. Instead, we find that GBP2, like GBP1, directly binds and aggregates "free" LPS through protein polymerization. We demonstrate that supplementation of either recombinant polymerized GBP1 or GBP2 to an in vitro reaction is sufficient to enhance LPS-induced caspase-4 activation. This provides a revised mechanistic framework for noncanonical inflammasome activation where GBP1 or GBP2 assembles cytosol-contaminating LPS into a protein-LPS interface for caspase-4 activation as part of a coordinated host response to gram-negative bacterial infections.

ATP-binding and hydrolysis of human NLRP3.

The innate immune system uses inflammasomal proteins to recognize danger signals and fight invading pathogens. NLRP3, a multidomain protein belonging to the family of STAND ATPases, is characterized by its central nucleotide-binding NACHT domain. The incorporation of ATP is thought to correlate with large conformational changes in NLRP3, leading to an active state of the sensory protein. Here we analyze the intrinsic ATP hydrolysis activity of recombinant NLRP3 by reverse phase HPLC. Wild-type NLRP3 appears in two different conformational states that exhibit an approximately fourteen-fold different hydrolysis activity in accordance with an inactive, autoinhibited state and an open, active state. The impact of canonical residues in the nucleotide binding site as the Walker A and B motifs and sensor 1 and 2 is analyzed by site directed mutagenesis. Cellular experiments show that reduced NLRP3 hydrolysis activity correlates with higher ASC specking after inflammation stimulation. Addition of the kinase NEK7 does not change the hydrolysis activity of NLRP3. Our data provide a comprehensive view on the function of conserved residues in the nucleotide-binding site of NLRP3 and the correlation of ATP hydrolysis with inflammasome activity.

Basic Motor Competencies of (Pre)School Children: The Role of Social Integration and Health-Related Quality of Life.

In (pre)school, children acquire and deepen their basic motor competencies (BMCs) and interact with peers and friends. BMCs are a central developmental goal in childhood and the prerequisite for participation in sportive aspects of social life. Both motor competencies and social integration are linked to children's health-related quality of life (HRQoL). The aim of the present study was to describe the connection between BMCs, social relationships, and aspects of HRQoL in (pre)school children. In this study, the BMCs of N = 1163 preschool children (M = 5.7 years, SD = 0.57, 52% boys) and N = 880 first and second graders (M = 7.5 years, SD = 0.58, 51% boys) were tested. The children's social integration was assessed by the teachers; the HRQoL was recorded from the parents' perspective. In both preschool and primary school, children with better BMCs also showed higher values in their social integration. Moreover, the results indicated a connection between BMCs and general HRQoL in primary school and BMCs and physical well-being in preschool. As BMCs, social integration, and HRQoL seem to be connected in (pre)school, this should be considered both from developmental and health-oriented perspectives, as well as for physical education (PE) lessons.

Basic Motor Competencies of 6- to 8-Year-Old Primary School Children in 10 European Countries: A Cross-Sectional Study on Associations With Age, Sex, Body Mass Index, and Physical Activity.

Basic motor competencies (BMC) are a prerequisite for children to be physically active, participate in sports and thus develop a healthy, active lifestyle. The present study provides a broad screening of BMC and associations with age, sex, body mass index (BMI) and extracurricular physical activity (PA) in 10 different European countries. The different country and regional contexts within Europe will offer a novel view on already established BMC associations. The cross-sectional study was conducted in 11 regions in 10 European countries in 2018. The motor competence areas, object movement (OM) and self-movement (SM), were assessed using the MOBAK-1-2 test instrument in 3758 first and second graders (age: M = 6.86 ± 0.60 years; 50% girls) during Physical Education classes. Children were questioned about their extracurricular PA and age. Their body weight and height were measured in order to calculate BMI. Statistical analyses included variances and correlations. The results showed significant differences in BMC levels between countries (OM: F = 18.74, p < 0.001, η2 = 0.048; SM: F = 73.10, p < 0.001, η2 = 0.163) whereas associations between BMC and correlates were similar. Boys performed significantly better in OM while girls performed better in SM. Age was consistently positively related to OM and SM with older children reaching higher levels of BMC than younger ones. While participation rates for extracurricular PA differed widely, participation in ball sports was correlated with OM and SM. Participation in individual sports showed a significant association with SM. In summary, BMC levels of children seem to depend on where they live and are strongly related to their participation in extracurricular PA. Therefore, education and health policies, in order to enhance motor competence development and PA participation, are recommended. Further research on country-specific Physical Education frameworks and their influence on BMC will provide more insights into structural factors and cultural characteristics of BMC development. On a school level, support tools and educational materials for teachers about BMC may enable children to achieve a basic level of motor competencies through Physical Education, contributing to lifelong participation in PA.

Disordered Regions Flanking the Binding Interface Modulate Affinity between CBP and NCOA.

Recognition motifs that mediate protein-protein interactions are usually embedded within longer intrinsically disordered regions. While binding interfaces involving the recognition motif in such interactions are well studied, less is known about the role of disordered regions flanking the motifs. The interaction between the transcriptional co-activators NCOA3 (ACTR) and CBP is mediated by coupled binding and folding of the two domains CID and NCBD. Here, we used circular dichroism and kinetics to directly quantify the contribution of the adjacent flanking regions of CID to its interaction with NCBD. Using N- and C-terminal combinatorial variants we found that the flanking regions promote binding in an additive fashion while retaining a large degree of disorder in the complex. Experiments at different ionic strengths demonstrated that the increase in affinity is not mediated by electrostatic interactions from the flanking regions. Instead, site-directed mutagenesis and molecular dynamics simulations suggest that binding is promoted by short-lived non-specific hydrophobic contacts between the flanking regions and NCBD. Our findings are consistent with highly frustrated interactions outside of the canonical binding interface resulting in a slightly energetically favorable fuzzy binding. Modulation of affinity via flanking regions could represent a general mechanism for functional regulation by intrinsically disordered protein regions.

De Novo Missense Mutations in TNNC1 and TNNI3 Causing Severe Infantile Cardiomyopathy Affect Myofilament Structure and Function and Are Modulated by Troponin Targeting Agents.

Rare pediatric non-compaction and restrictive cardiomyopathy are usually associated with a rapid and severe disease progression. While the non-compaction phenotype is characterized by structural defects and is correlated with systolic dysfunction, the restrictive phenotype exhibits diastolic dysfunction. The molecular mechanisms are poorly understood. Target genes encode among others, the cardiac troponin subunits forming the main regulatory protein complex of the thin filament for muscle contraction. Here, we compare the molecular effects of two infantile de novo point mutations in TNNC1 (p.cTnC-G34S) and TNNI3 (p.cTnI-D127Y) leading to severe non-compaction and restrictive phenotypes, respectively. We used skinned cardiomyocytes, skinned fibers, and reconstituted thin filaments to measure the impact of the mutations on contractile function. We investigated the interaction of these troponin variants with actin and their inter-subunit interactions, as well as the structural integrity of reconstituted thin filaments. Both mutations exhibited similar functional and structural impairments, though the patients developed different phenotypes. Furthermore, the protein quality control system was affected, as shown for TnC-G34S using patient's myocardial tissue samples. The two troponin targeting agents levosimendan and green tea extract (-)-epigallocatechin-3-gallate (EGCg) stabilized the structural integrity of reconstituted thin filaments and ameliorated contractile function in vitro in some, but not all, aspects to a similar degree for both mutations.

Effects of an Intervention for Promoting Basic Motor Competencies in Middle Childhood.

The development of motor competencies is necessary for participation in the culture of sport, exercise, and physical activity, which in turn supports the development of a healthy lifestyle. A lack of physical activity in childhood and deficits in motor performance emphasize the relevance of interventions for promoting basic motor competencies. However, there are research desiderata with regard to such interventions. This article describes an intervention program for promoting basic motor competencies in middle childhood (around 6 to 10 years of age). The intervention was investigated in a longitudinal study from June 2019 to January 2020 (n = 200; 58% girls, M = 8.84 years, SD = 0.63) at three primary schools. The intervention was conducted once a week in physical education (PE). The comparison group participated in regular PE. The intervention showed significant effects on basic motor competencies in object movement but not in self-movement. The results demonstrate that positive effects on basic motor competencies can be achieved with the help of a relatively simple intervention. Further longitudinal studies are desirable as a means of substantiating the results and developing evidence-based concepts to support children in their development in the best possible way.

The intramolecular allostery of GRB2 governing its interaction with SOS1 is modulated by phosphotyrosine ligands.

Growth factor receptor-bound protein 2 (GRB2) is a trivalent adaptor protein and a key element in signal transduction. It interacts via its flanking nSH3 and cSH3 domains with the proline-rich domain (PRD) of the RAS activator SOS1 and via its central SH2 domain with phosphorylated tyrosine residues of receptor tyrosine kinases (RTKs; e.g. HER2). The elucidation of structural organization and mechanistic insights into GRB2 interactions, however, remain challenging due to their inherent flexibility. This study represents an important advance in our mechanistic understanding of how GRB2 links RTKs to SOS1. Accordingly, it can be proposed that (1) HER2 pYP-bound SH2 potentiates GRB2 SH3 domain interactions with SOS1 (an allosteric mechanism); (2) the SH2 domain blocks cSH3, enabling nSH3 to bind SOS1 first before cSH3 follows (an avidity-based mechanism); and (3) the allosteric behavior of cSH3 to other domains appears to be unidirectional, although there is an allosteric effect between the SH2 and SH3 domains.

Identifying classes of the pain, fatigue, and depression symptom cluster in long-term prostate cancer survivors-results from the multi-regional Prostate Cancer Survivorship Study in Switzerland (PROCAS).

PURPOSE: Aside from urological and sexual problems, long-term (≥5 years after initial diagnosis) prostate cancer (PC) survivors might suffer from pain, fatigue, and depression. These concurrent symptoms can form a cluster. In this study, we aimed to investigate classes of this symptom cluster in long-term PC survivors, to classify PC survivors accordingly, and to explore associations between classes of this cluster and health-related quality of life (HRQoL). METHODS: Six hundred fifty-three stage T1-T3N0M0 survivors were identified from the Prostate Cancer Survivorship in Switzerland (PROCAS) study. Fatigue was assessed with the EORTC QLQ-FA12, depressive symptoms with the MHI-5, and pain with the EORTC QLQ-C30 questionnaire. Latent class analysis was used to derive cluster classes. Factors associated with the derived classes were determined using multinomial logistic regression analysis. RESULTS: Three classes were identified: class 1 (61.4%) - "low pain, low physical and emotional fatigue, moderate depressive symptoms"; class 2 (15.1%) - "low physical fatigue and pain, moderate emotional fatigue, high depressive symptoms"; class 3 (23.5%) - high scores for all symptoms. Survivors in classes 2 and 3 were more likely to be physically inactive, report a history of depression or some other specific comorbidity, be treated with radiation therapy, and have worse HRQoL outcomes compared to class 1. CONCLUSION: Three distinct classes of the pain, fatigue, and depression cluster were identified, which are associated with treatment, comorbidities, lifestyle factors, and HRQoL outcomes. Improving classification of PC survivors according to severity of multiple symptoms could assist in developing interventions tailored to survivors' needs.

Associations between physical activity, basic motor competencies and automatic evaluations of exercise.

Dual process theories suggest that the decision to be physically active is influenced by reflective and automatic processes. However, associations of automatic (affective) evaluations of exercise with physical activity and underlying basic motor competencies have not yet been investigated in children and young adolescents. Ninety-one participants (52 male; age: 10-14 years) were recruited from academic high schools in Germany and Switzerland. Automatic evaluations of exercise were measured with the Single-Target Implicit Association Test (ST-IAT) and a D-score was calculated. Moderate-to-vigorous physical activity (MVPA) and vigorous physical activity (VPA) per day were determined via wrist-worn actigraphy over the course of seven days. Basic motor competencies were measured using the MOBAK-5 test battery. Pearson correlations showed non-significant associations of automatic evaluations of exercise with MVPA, but significant associations with VPA. Basic motor competencies were associated with automatic evaluations of exercise, and the MOBAK subscale of object movement was associated with both MVPA and VPA. Our results underscore the relevance of affective processes for physical activity behaviour. This could potentially be relevant for interventions targeting physical activity promotion. Longitudinal investigations and intervention studies are necessary to verify causal relationships and potential underlying mechanisms.

The histology of brain tumors for 67 331 children and 671 085 adults diagnosed in 60 countries during 2000-2014: a global, population-based study (CONCORD-3).

BACKGROUND: Global variations in survival for brain tumors are very wide when all histological types are considered together. Appraisal of international differences should be informed by the distribution of histology, but little is known beyond Europe and North America. METHODS: The source for the analysis was the CONCORD database, a program of global surveillance of cancer survival trends, which includes the tumor records of individual patients from more than 300 population-based cancer registries. We considered all patients aged 0-99 years who were diagnosed with a primary brain tumor during 2000-2014, whether malignant or nonmalignant. We presented the histology distribution of these tumors, for patients diagnosed during 2000-2004, 2005-2009, and 2010-2014. RESULTS: Records were submitted from 60 countries on 5 continents, 67 331 for children and 671 085 for adults. After exclusion of irrelevant morphology codes, the final study population comprised 60 783 children and 602 112 adults. Only 59 of 60 countries covered in CONCORD-3 were included because none of the Mexican records were eligible. We defined 12 histology groups for children, and 11 for adults. In children (0-14 years), the proportion of low-grade astrocytomas ranged between 6% and 50%. Medulloblastoma was the most common subtype in countries where low-grade astrocytoma was less commonly reported. In adults (15-99 years), the proportion of glioblastomas varied between 9% and 69%. International comparisons were made difficult by wide differences in the proportion of tumors with unspecified histology, which accounted for up to 52% of diagnoses in children and up to 65% in adults. CONCLUSIONS: To our knowledge, this is the first account of the global histology distribution of brain tumors, in children and adults. Our findings provide insights into the practices and the quality of cancer registration worldwide.

Screening is associated with lower mastectomy rates in eastern Switzerland beyond stage effects.

BACKGROUND: A recent study found an influence of organized mammography screening programmes (MSPs) on geographical and temporal variation of mastectomy rates. We aimed to quantify the effect on the example of one of the cantonal programmes in Switzerland. METHODS: We used incidence data for the years 2010-2017 from the cancer registry of Eastern Switzerland. We included women with invasive-non-metastatic breast cancer (BC) in the screening age group 50-69-year-olds in the canton of St.Gallen. We compared mastectomy rates among cancer patients detected through the organised screening programme (MSP) vs. otherwise detected by stage. RESULTS: MSP-detected patients in St.Gallen presented with lower stages. 95% of MSP-detected had stages I-II vs 76% of Non-MSP-detected. Within all non-metastatic stage, tumour size and nodal status groups, MSP-detected patients had lower mastectomy rates, overall 10% vs 24% in 50-69-year-old non-participants. Their odds of receiving a mastectomy are about half of the Non-MSP-detected (OR = 0.48, p = 0.002). CONCLUSIONS: Our study showed that MSPs have a positive effect on lowering mastectomy rates. Screening participants are significantly less likely to receive a mastectomy compared to non-participants, which must be attributed to additional factors than just lower stages. Lower mastectomy rates lead to a higher quality of life for many patients.

Structural requirements for membrane binding of human guanylate-binding protein 1.

Human guanylate-binding protein 1 (hGBP1) is a key player in innate immunity and fights diverse intracellular microbial pathogens. Its antimicrobial functions depend on hGBP1's GTP binding- and hydrolysis-induced abilities to form large, structured polymers and to attach to lipid membranes. Crucial for both of these biochemical features is the nucleotide-controlled release of the C terminally located farnesyl moiety. Here, we address molecular details of the hGBP1 membrane binding mechanism by employing recombinant, fluorescently labeled hGBP1, and artificial membranes. We demonstrate the importance of the GTPase activity and the resulting structural rearrangement of the hGBP1 molecule, which we term the open state. This open state is supported and stabilized by homodimer contacts involving the middle domain of the protein and is further stabilized by binding to the lipid bilayer surface. We show that on the surface of the lipid bilayer a hGBP1 monolayer is built in a pins in a pincushion-like arrangement with the farnesyl tail integrated in the membrane and the N-terminal GTPase domain facing outwards. We suggest that similar intramolecular contacts between neighboring hGBP1 molecules are responsible for both polymer formation and monolayer formation on lipid membranes. Finally, we show that tethering of large unilamellar vesicles occurs after the vesicle surface is fully covered by the monolayer. Both hGBP1 polymer formation and hGBP1-induced vesicle tethering have implications for understanding the molecular mechanism of combating bacterial pathogens. DATABASES: Structural data are available in RCSB Protein Data Bank under the accession numbers: 6K1Z, 2D4H.