Structural determinants of substrate specificity in family 1 beta-glucosidases: novel insights from the crystal structure of sorghum dhurrinase-1, a plant beta-glucosidase with strict specificity, in complex with its natural substrate

Plant beta-glucosidases play a crucial role in defense against pests. They cleave, with variable specificity, beta-glucosides to release toxic aglycone moieties. The Sorghum bicolor beta-glucosidase isoenzyme Dhr1 has a strict specificity for its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-D-glucoside), whereas its close homolog, the maize beta-glucosidase isoenzyme Glu1, which shares 72% sequence identity, hydrolyzes a broad spectrum of substrates in addition to its natural substrate 2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one. Structural data from enzyme.substrate complexes of Dhr1 show that the mode of aglycone binding differs from that previously observed in the homologous maize enzyme. Specifically, the data suggest that Asn(259), Phe(261), and Ser(462), located in the aglycone-binding site of S. bicolor Dhr1, are crucial for aglycone recognition and binding. The tight binding of the aglycone moiety of dhurrin promotes the stabilization of the reaction intermediate in which the glycone moiety is in a deformed (1)S(3) conformation within the glycone-binding site, ready for nucleophilic attack to occur. Compared with the broad specificity maize beta-glucosidase, this different binding mode explains the narrow specificity of sorghum dhurrinase-1.     

Penicillin-binding protein PBP2 of Escherichia coli localizes preferentially in the lateral wall and at mid-cell in comparison with the old cell pole

The localization of penicillin-binding protein 2 (PBP2) in Escherichia coli has been studied using a functional green fluorescent protein (GFP)-PBP2 fusion protein. PBP2 localized in the bacterial envelope in a spot-like pattern and also at mid-cell during cell division. PBP2 disappeared from mid-cell just before separation of the two daughter cells. It localized with a preference for the cylindrical part of the bacterium in comparison with the old cell poles, which are known to be inert with respect to peptidoglycan synthesis. In contrast to subunits of the divisome, PBP2 failed to localize at mid-cell when PBP3 was inhibited by the specific antibiotic aztreonam. Therefore, despite its dependency on active PBP3 for localization at mid-cell, it seems not to be an integral part of the divisome. Cells grown for approximately half a mass doubling time in the presence of the PBP2 inhibitor mecillinam synthesized nascent cell poles with an increased diameter, indicating that PBP2 is required for the maintenance of the correct diameter of the new cell pole.     

Effects of incomplete inter-hospital network data on the assessment of transmission dynamics of hospital-acquired infections

In the year 2020, there were 105 different statutory insurance companies in Germany with heterogeneous regional coverage. Obtaining data from all insurance companies is challenging, so that it is likely that projects will have to rely on data not covering the whole population. Consequently, the study of epidemic spread in hospital referral networks using data-driven models may be biased. We studied this bias using data from three German regional insurance companies covering four federal states: AOK (historically “general local health insurance company”, but currently only the abbreviation is used) Lower Saxony (in Federal State of Lower Saxony), AOK Bavaria (in Bavaria), and AOK PLUS (in Thuringia and Saxony). To understand how incomplete data influence network characteristics and related epidemic simulations, we created sampled datasets by randomly dropping a proportion of patients from the full datasets and replacing them with random copies of the remaining patients to obtain scale-up datasets to the original size. For the sampled and scale-up datasets, we calculated several commonly used network measures, and compared them to those derived from the original data. We found that the network measures (degree, strength and closeness) were rather sensitive to incompleteness. Infection prevalence as an outcome from the applied susceptible-infectious-susceptible (SIS) model was fairly robust against incompleteness. At incompleteness levels as high as 90% of the original datasets the prevalence estimation bias was below 5% in scale-up datasets. Consequently, a coverage as low as 10% of the local population of the federal state population was sufficient to maintain the relative bias in prevalence below 10% for a wide range of transmission parameters as encountered in clinical settings. Our findings are reassuring that despite incomplete coverage of the population, German health insurance data can be used to study effects of patient traffic between institutions on the spread of pathogens within healthcare networks.     

Strongly altered receptor binding properties in PP and NPY chimeras are accompanied by changes in structure and membrane binding

Neuropeptide Y (NPY) and the pancreatic polypeptide (PP) are members of the neuropeptide Y family of hormones. They bind to the Y receptors with very different affinities: Whereas PP is highly selective for the Y(4) receptor, NPY displays highest affinites for Y(1), Y(2), and Y(5) receptor subtypes. Introducing the NPY segment 19-23 into PP leads to an increase in affinity at the Y(1) and Y(2) receptor subtypes whereas the exchange of this segment from PP into NPY leads to a large decrease in affinity at all receptor subtypes. PP displays a very stable structure in solution, with the N terminus being back-folded onto the C-terminal alpha-helix (the so-called PP-fold). The helix of NPY is less stable and the N terminus is freely diffusing in solution. The exchange of this segment, however, does not alter the PP-fold propensities of the chimeric peptides in solution. The structures of the phospholipid micelle-bound peptides serving to mimic the membrane-bound species display segregation into a more flexible N-terminal region and a well-defined alpha-helical region. The introduction of the [19-23]-pNPY segment into hPP leads to an N-terminal extension of the alpha-helix, now starting at Pro(14) instead of Met(17). In contrast, a truncated helix is observed in [(19)(-)(23)hPP]-pNPY, starting at Leu(17) instead of Ala(14). All peptides display moderate binding affinities to neutral membranes (K(assoc) in the range of 1.7 to 6.8 x 10(4) mol(-)(1) as determined by surface plasmon resonance) with the differences in binding being most probably related to the exchange of Arg-19 (pNPY) by Glu-23 (hPP). Differences in receptor binding properties between the chimeras and their parental peptides are therefore most likely due to changes in the conformation of the micelle-bound peptides.     

Structural similarities of micelle-bound peptide YY (PYY) and neuropeptide Y (NPY) are related to their affinity profiles at the Y receptors

Here, we investigate the structure of porcine peptide YY (pPYY) both when unligated in solution at pH 4.2 and when bound to dodecylphosphocholine (DPC) micelles at pH 5.5. pPYY in solution displays the PP-fold, with the N-terminal segment being back-folded onto the C-terminal alpha-helix, which extends from residue 17 to 31. In contrast to the solution structure of Keire et al. published in the year 2000 the C-terminal helix does not display a kink around residue 23-25. The root mean square deviation (RMSD) for backbone atoms of the NMR ensemble of conformers to the mean structure is 0.99(+/-0.35) Angstrom for residues 14-31. The back-fold is supported by values of 0.60+/-0.1 for the (15)N(1)H-NOE and by generalized order parameters S(2) of 0.74+/-0.1 for residues 5-31 which indicate that the peptide is folded in that segment. We have additionally used DPC micelles as a membrane model and determined the structure of pPYY when bound to it. Therein, an alpha-helix occurs in the segment comprising residues 17-31 and the N terminus freely diffuses in solution. The hydrophobic side of the amphipathic helix forms the micelle-binding interface and hydrophobic side-chains extend into the micelle interior. A significant stabilization of helical conformation occurs in the C-terminal pentapeptide, which is important for receptor binding. The latter is supported by positive values of the heteronuclear NOE in that segment (0.52+/-0.1 compared to 0.08+/-0.4 for the unligated form) and by values of S(2) of 0.6+/-0.2 (versus 0.38+/-0.2 for the unligated form). The structures of micelle-bound pPYY and pNPY are much more similar than those of pPYY and bPP with pairwise RMSDs of 1.23(+/-0.21)A or 3.21(+/-0.39) Angstrom, respectively. In contrast to the conformational similarities in the DPC-bound state their structures in solution are very different. In fact pPYY is more similar to bPP, which with its strong preference for the Y(4) receptor displays a completely different binding profile. Considering the high degree of sequence homology of pNPY and pPYY (>80%) and the fact, that their binding affinities at all receptor subtypes are high and, more importantly, rather similar, it is much more likely that PYY and NPY are recognized by the Y receptors from the membrane-bound state. As a consequence of the latter the PP-fold is not important for recognition of PYY or NPY at the Y receptors. To our knowledge this work provides for the first time strong arguments derived from structural data that support a membrane-bound receptor recognition pathway.     

Clinical Manifestation of Depression after Stroke: Is It Different from Depression in Other Patient Populations?

Background

Despite ample research on depression after stroke, the debate continues regarding whether symptoms such as sleep disturbances, loss of energy, changes in appetite and diminished concentration should be considered to be consequences of stroke or general symptoms of depression. By comparing symptoms in depressed and non-depressed stroke patients with patients in general practice and patients with symptomatic atherosclerotic diseases, we aim to further clarify similarities and distinctions of depression after stroke and depression in other patient populations. Based on this, it is possible to determine if somatic symptoms should be evaluated in stroke patients in diagnosing depression after stroke.

Methods

An observational multicenter study is conducted in three hospitals and seven general practices including 382 stroke patients admitted to hospital with a clinical diagnosis of intracerebral hemorrhage or ischemic infarction, 1160 patients in general practice (PREDICT-NL), and 530 patients with symptomatic atherosclerotic diseases (SMART-Medea).

Results

The prevalence of major depressive disorder according to DSM-IV criteria was 14.1% (95% CI 11.0%-18.0%) in the stroke cohort, 5.4% (95% CI 3.8%-7.9%) in the symptomatic atherosclerotic diseases cohort and 12.9% (95% CI 11.1%-15.0%) in the general practice cohorts. Comparing depressed patients of the three cohorts demonstrated broadly similar symptom profiles, as well as comparable levels of individual symptom prevalence. However, the stroke patients suffered more severely from these symptoms than patients in the other populations.

Conclusions

The findings suggest that depression after stroke is not a different type of depression. This finding indicates that all depressive symptoms should be evaluated in stroke patients, including somatic symptoms.     

Pak1 and PIX regulate contact inhibition during epithelial wound healing

Wound healing in epithelia requires coordinated cell migration and proliferation regulated by signaling mechanisms that are poorly understood. Here we show that epithelial cells expressing constitutively active or kinase-dead mutants of the Rac/Cdc42 effector Pak1 fail to undergo growth arrest upon wound closure. Strikingly, this phenotype is only observed when the Pak1 kinase mutants are expressed in cells possessing a free lateral surface, i.e. one that is not engaged in contact with neighboring cells. The Pak1 kinase mutants perturb contact inhibition by a mechanism that depends on the Pak-interacting Rac-GEF PIX. In control cells, endogenous activated Pak and PIX translocate from focal complexes to cell-cell contacts during wound closure. This process is abrogated in cells expressing Pak1 kinase mutants. In contrast, Pak1 mutants rendered defective in PIX binding do not impede translocation of activated Pak and PIX, and exhibit normal wound healing. Thus, recruitment of activated Pak and PIX to cell-cell contacts is pivotal to transduction of growth-inhibitory signals from neighboring cells in epithelial wound healing.     

The structure of the efflux pump AcrB in complex with bile acid

Gastrointestinal bacteria, like Escherichia coli, must remove bile acid to survive in the gut. Bile acid removal in E. coli is thought to be mediated primarily by the multidrug efflux pump, AcrB. Here, we present the structure of E. coli AcrB in complex with deoxycholate at 3.85 Å resolution. All evidence suggests that bile acid is transported out of the cell via the periplasmic vestibule of the AcrAB-TolC complex.     

Expression of members of the Fgf family and their receptors during midfacial development

Members of the FGF family play diverse roles in patterning, cell proliferation and differentiation during embryogenesis. To begin to address their function during craniofacial development we have analyzed the expression of 18 members of the Fgf family (Fgf1-15, -17, -18 and -20) and the four members of the FGF-receptor family in the prospective midfacial region between E9.5 and E11.5 by whole-mount in situ hybridization. We show that at E9.5, Fgf3, -8, -9, -10 and -17 are broadly expressed in midfacial ectoderm. Concomitant with the outgrowth of the nasal processes at E10.5, expression of Fgf3, -8, -9, -10, -15, -17 and -18 was detected in spatially restricted regions of ectoderm at the edge of the nasal pit and at the oral edge of the medial nasal process. Expression of Fgf8, Fgf9, Fgf10 and Fgf17 was still observed in these domains at E11.5. In contrast to the restricted expression patterns of the ligands, FgfR1 and FgfR2 were broadly expressed in facial mesenchyme and ectoderm, respectively, indicating a wide competence of midfacial tissue to respond to FGF signaling.