Experimental-theoretical study of laccase as a detoxifier of aflatoxins.

We investigate laccase-mediated detoxification of aflatoxins, fungal carcinogenic food contaminants. Our experimental comparison between two aflatoxins with similar structures (AFB1 and AFG2) shows significant differences in laccase-mediated detoxification. A multi-scale modeling approach (Docking, Molecular Dynamics, and Density Functional Theory) identifies the highly substrate-specific changes required to improve laccase detoxifying performance. We employ a large-scale density functional theory-based approach, involving more than 7000 atoms, to identify the amino acid residues that determine the affinity of laccase for aflatoxins. From this study we conclude: (1) AFB1 is more challenging to degrade, to the point of complete degradation stalling; (2) AFG2 is easier to degrade by laccase due to its lack of side products and favorable binding dynamics; and (3) ample opportunities to optimize laccase for aflatoxin degradation exist, especially via mutations leading to π-π stacking. This study identifies a way to optimize laccase for aflatoxin bioremediation and, more generally, contributes to the research efforts aimed at rational enzyme optimization.

Structure and dynamics of the quaternary hunchback mRNA translation repression complex.

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.

Observing Protein Degradation by the PAN-20S Proteasome by Time-Resolved Neutron Scattering.

The proteasome is a key player of regulated protein degradation in all kingdoms of life. Although recent atomic structures have provided snapshots on a number of conformations, data on substrate states and populations during the active degradation process in solution remain scarce. Here, we use time-resolved small-angle neutron scattering of a deuterium-labeled GFPssrA substrate and an unlabeled archaeal PAN-20S system to obtain direct structural information on substrate states during ATP-driven unfolding and subsequent proteolysis in solution. We find that native GFPssrA structures are degraded in a biexponential process, which correlates strongly with ATP hydrolysis, the loss of fluorescence, and the buildup of small oligopeptide products. Our solution structural data support a model in which the substrate is directly translocated from PAN into the 20S proteolytic chamber, after a first, to our knowledge, successful unfolding process that represents a point of no return and thus prevents dissociation of the complex and the release of harmful, aggregation-prone products.

A Structurally Simple Vaccine Candidate Reduces Progression and Dissemination of Triple-Negative Breast Cancer.

The Tn antigen is a well-known tumor-associated carbohydrate determinant, often incorporated in glycopeptides to develop cancer vaccines. Herein, four copies of a conformationally constrained mimetic of the antigen TnThr (GalNAc-Thr) were conjugated to the adjuvant CRM197, a protein licensed for human use. The resulting vaccine candidate, mime[4]CRM elicited a robust immune response in a triple-negative breast cancer mouse model, correlated with high frequency of CD4+ T cells and low frequency of M2-type macrophages, which reduces tumor progression and lung metastasis growth. Mime[4]CRM-mediated activation of human dendritic cells is reported, and the proliferation of mime[4]CRM-specific T cells, in cancer tissue and peripheral blood of patients with breast cancer, is demonstrated. The locked conformation of the TnThr mimetic and a proper presentation on the surface of CRM197 may explain the binding of the conjugate to the anti-Tn antibody Tn218 and its efficacy to fight cancer cells in mice.

Implementation of altered provider incentives for a more individual-risk-based assignment of dental recall intervals: evidence from a health systems reform in Denmark.

Equipping health systems with suitable incentives for efficient resource allocation remains a major health policy challenge. This study examines the impacts of 2015 regulatory changes in Danish dental care which aimed at effectuating a transition from six-to-twelve-monthly dental recall intervals, for every patient, towards a model where patients with higher need receive dental recalls systematically more frequently than patients with lower need. Exploiting administrative data from the years 2012-2016 from the Danish National Health Insurance database containing 72,155,539 treatment claims for 3,759,721 unique patients, we estimated a series of interrupted time-series regression models with patient-level fixed-effects. In comparison to the pre-reform period, the proportion of patients with recall intervals of up to 6 months was by 1.2%-points larger post-implementation; that of patients with 6-12-monthly recalls increased by 0.7%-points; that of patients with more than 12-monthly dental recalls decreased by 1.9%-points. The composition of care shifted more substantially: the proportion of treatment sessions including preventive care increased by 31.5%-points (95%-CI: 31.4;31.6); that of sessions including scaling increased by 24.1%-points (24.0;24.2); that of sessions including diagnostics decreased by 34.5%-points (34.4;34.6). These findings suggest that dental care providers may have responded differently to regulatory changes than intended by the health policy.

TET peptidases: A family of tetrahedral complexes conserved in prokaryotes.

The TET peptidases are large polypeptide destruction machines present among prokaryotes. They form 12-subunits hollow tetrahedral particles, and belong to the family of M42 metallo-peptidases. Structural characterization of various archaeal and bacterial complexes has revealed a unique mechanism of internal compartmentalization and peptide trafficking that distinguishes them from the other oligomeric peptidases. Different versions of the TET complex often co-exist in the cytosol of microorganisms. In depth enzymatic studies have revealed that they are non-processive cobalt-activated aminopeptidases and display contrasting substrate specificities based on the properties of the catalytic chambers. Recent studies have shed light on the assembly mechanism of homo and hetero-dodecameric TET complexes and shown that the activity of TET aminopeptidase towards polypeptides is coupled with its assembly process. These findings suggested a functional regulation based on oligomerization control in vivo. This review describes a current knowledge on M42 TET peptidases biochemistry and discuss their possible physiological roles. This article is a part of the Special Issue entitled: «A potpourri of proteases and inhibitors: from molecular toolboxes to signalling scissors¼.

Transient electrostatic interactions dominate the conformational equilibrium sampled by multidomain splicing factor U2AF65: a combined NMR and SAXS study.

Multidomain proteins containing intrinsically disordered linkers exhibit large-scale dynamic modes that play key roles in a multitude of molecular recognition and signaling processes. Here, we determine the conformational space sampled by the multidomain splicing factor U2AF65 using complementary nuclear magnetic resonance spectroscopy and small-angle scattering data. Available degrees of conformational freedom are initially stochastically sampled and experimental data then used to delineate the potential energy landscape in terms of statistical probability. The spatial distribution of U2AF65 conformations is found to be highly anisotropic, comprising significantly populated interdomain contacts that appear to be electrostatic in origin. This hypothesis is supported by the reduction of signature PREs reporting on expected interfaces with increasing salt concentration. The described spatial distribution reveals the complete spectrum of the unbound forms of U2AF65 that coexist with the small percentage of a preformed RNA-bound domain arrangement required for polypyrimidine-tract recognition by conformational selection. More generally, the proposed approach to describing conformational equilibria of multidomain proteins can be further combined with other experimental data that are sensitive to domain dynamics.

Assessing the conformational changes of pb5, the receptor-binding protein of phage T5, upon binding to its Escherichia coli receptor FhuA.

Within tailed bacteriophages, interaction of the receptor-binding protein (RBP) with the target cell triggers viral DNA ejection into the host cytoplasm. In the case of phage T5, the RBP pb5 and the receptor FhuA, an outer membrane protein of Escherichia coli, have been identified. Here, we use small angle neutron scattering and electron microscopy to investigate the FhuA-pb5 complex. Specific deuteration of one of the partners allows the complete masking in small angle neutron scattering of the surfactant and unlabeled proteins when the complex is solubilized in the fluorinated surfactant F6-DigluM. Thus, individual structures within a membrane protein complex can be described. The solution structure of FhuA agrees with its crystal structure; that of pb5 shows an elongated shape. Neither displays significant conformational changes upon interaction. The mechanism of signal transduction within phage T5 thus appears different from that of phages binding cell wall saccharides, for which structural information is available.

Pyrococcus horikoshii TET2 peptidase assembling process and associated functional regulation.

Tetrahedral (TET) aminopeptidases are large polypeptide destruction machines present in prokaryotes and eukaryotes. Here, the rules governing their assembly into hollow 12-subunit tetrahedrons are addressed by using TET2 from Pyrococcus horikoshii (PhTET2) as a model. Point mutations allowed the capture of a stable, catalytically active precursor. Small angle x-ray scattering revealed that it is a dimer whose architecture in solution is identical to that determined by x-ray crystallography within the fully assembled TET particle. Small angle x-ray scattering also showed that the reconstituted PhTET2 dodecameric particle displayed the same quaternary structure and thermal stability as the wild-type complex. The PhTET2 assembly intermediates were characterized by analytical ultracentrifugation, native gel electrophoresis, and electron microscopy. They revealed that PhTET2 assembling is a highly ordered process in which hexamers represent the main intermediate. Peptide degradation assays demonstrated that oligomerization triggers the activity of the TET enzyme toward large polypeptidic substrates. Fractionation experiments in Pyrococcus and Halobacterium cells revealed that, in vivo, the dimeric precursor co-exists together with assembled TET complexes. Taken together, our observations explain the biological significance of TET oligomerization and suggest the existence of a functional regulation of the dimer-dodecamer equilibrium in vivo.

Modulation of structure and dynamics by disulfide bond formation in unfolded states.

During oxidative folding, the formation of disulfide bonds has profound effects on guiding the protein folding pathway. Until now, comparatively little is known about the changes in the conformational dynamics in folding intermediates of proteins that contain only a subset of their native disulfide bonds. In this comprehensive study, we probe the conformational landscape of non-native states of lysozyme containing a single native disulfide bond utilizing nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), circular dichroism (CD) data, and modeling approaches. The impact on conformational dynamics varies widely depending on the loop size of the single disulfide variants and deviates significantly from random coil predictions for both NMR and SAXS data. From these experiments, we conclude that the introduction of single disulfides spanning a large portion of the polypeptide chain shifts the structure and dynamics of hydrophobic core residues of the protein so that these regions exhibit levels of order comparable to the native state on the nanosecond time scale.