Structure–function studies of the RNA polymerase II elongation complex

X-ray crystallographic and complementary functional studies have contributed significantly to the current understanding of gene transcription. Here, recent structure–function studies on various aspects of the elongation phase of transcription are summarized

Molecular mechanism of ligand recognition by membrane transport protein, Mhp1

The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1

Antiproliferative Effects of DNA Methyltransferase 3B Depletion Are Not Associated with DNA Demethylation

Silencing of genes by hypermethylation contributes to cancer progression and has been shown to occur with increased frequency at specific genomic loci. However, the precise mechanisms underlying the establishment and maintenance of aberrant methylation marks are still elusive. The de novo DNA methyltransferase 3B (DNMT3B) has been suggested to play an important role in the generation of cancer-specific methylation patterns. Previous studies have shown that a reduction of DNMT3B protein levels induces antiproliferative effects in cancer cells that were attributed to the demethylation and reactivation of tumor suppressor genes. However, methylation changes have not been analyzed in detail yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer cell lines. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. However, genome-scale DNA methylation profiling failed to reveal methylation changes at putative DNMT3B target genes, even in the complete absence of DNMT3B. These results show that DNMT3B is dispensable for the maintenance of aberrant DNA methylation patterns in human colon cancer cells and they have important implications for the development of targeted DNA methyltransferase inhibitors as epigenetic cancer drugs

Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor

G protein-coupled receptors (GPCRs) are physiologically important transmembrane signalling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography1–3, the details of ligand induced signal transduction are not well understood owing to missing dynamical information. In principle, such information can be provided by NMR4, but so far only limited data of functional relevance on few side-chain sites of eukaryotic GPCRs have been obtained 5–9. Here we show that receptor motions can be followed at virtually any backbone site in a thermostabilized mutant of the turkey β1-adrenergic receptor (β1AR) 10–12. Labelling with [15N] valine in a eukaryotic expression system provides over twenty resolved resonances that report on structure and dynamics in six ligand complexes and the apo form. The response to the various ligands is heterogeneous in the vicinity of the binding pocket, but gets transformed into a homogeneous readout at the intracellular side of helix 5 (TM5), which correlates linearly with ligand efficacy for the G protein pathway. The effect of several pertinent, thermostabilizing point mutations was assessed by reverting them to the native sequence. Whereas the response to ligands remains largely unchanged, binding of the G protein mimetic nanobody NB80 and G protein activation are only observed when two conserved tyrosines (Y227 and Y343) are restored. Binding of NB80 leads to very strong spectral changes throughout the receptor, including the extracellular ligand entrance pocket. This indicates that even the fully thermostabilized receptor undergoes activating motions in TM5, but that the fully active state is only reached in presence of Y227 and Y343 by stabilization with a G protein-like partner. The combined analysis of chemical shift changes from the point mutations and ligand responses identifies crucial connections in the allosteric activation pathway, and presents a general experimental method to delineate signal transmission networks at high resolution in GPCRs

Validity and Reliability of an Electronic Contact Mat for Drop Jump Assessment in Physically Active Adults

The aim of the present study was to investigate the concurrent validity and the test–retest reliability of an electronic contact mat for drop jump assessment in physically active adults. Seventy-nine young, physically active adults participated in the validity study, and 49 subjects were recruited for the reliability study. The motor task required subjects to perform two-legged drop jumps using drop heights of 24, 43, and 62 cm as well as one-legged drop jumps with the left and right leg using a drop height of 24 cm. Ground contact times were simultaneously quantified with an electronic contact mat, a force plate (i.e., gold standard), and a light-barrier system (another criterion device). Concurrent validity was assessed using intraclass correlation coefficient (ICC), systematic bias, limits of agreement, and linear regression analysis. Test–retest reliability (one week apart) was determined by calculating the ICC, the standard error of measurement (SEM), the coefficient of variation (CV), and Lin´s concordance correlation coefficient (рc). Further, we determined the minimal detectable change (MDC95%). Irrespective of drop height and jump condition, good agreements between testing devices (ICC ≥ 0.95) were shown. Compared to the force plate (−0.6 to 3.1 ms) but not to the light-barrier system (31.4 to 41.7 ms), the contact mat showed low systematic bias values. In terms of test–retest reliability, our analyses showed that the measuring devices are in agreement (ICC: 0.70–0.92; SEM: 8.5–18.4 ms; CV: 3.6–6.4%). Depending on the measurement device, drop height, and jump condition, a MDC95% value ranging from 23.6 to 50.9 ms represents the minimum amount of change needed to identify practical relevant effects in repeated measurements of drop jump performance. Our findings indicate that the electronic contact mat is a valid and reliable testing device for drop jump assessment from different drop heights in young physically active adults

Grain size manipulation by wire laser direct energy deposition of 316L with ultrasonic assistance

The epitaxial growth of coarse and columnar grain structures along the build direction of additive manufactured metals is a usual phenomenon. As a result, as-built components often exhibit pronounced anisotropic mechanical properties, reduced ductility, and, hence, a high cracking susceptibility. To enhance the mechanical properties and processability of additive manufactured parts, the formation of equiaxed and fine grained structures is thought to be most beneficial. In this study, the potential of grain refinement by ultrasonic excitation of the melt pool during laser wire additive manufacturing has been investigated. An ultrasound system was developed and integrated in a laser wire deposition machine. AISI 316L steel was used as a substrate and feedstock material. A conversion of coarse, columnar grains (d(m) = 284.5 mu m) into fine, equiaxed grains (d(m) = 130.4 mu m) and a weakening of typical -fiber texture with increasing amplitude were verified by means of light microscopy, scanning electron microscopy, and electron backscatter diffraction analysis. It was demonstrated that the degree of grain refinement could be controlled by the regulation of ultrasound amplitude. No significant changes in the dendritic structure have been observed. The combination of sonotrode/melt pool direct coupling and the laser wire deposition process represents a pioneering approach and promising strategy to investigate the influence of ultrasound on grain refinement and microstructural tailoring.Godkänd;2023;Nivå 0;2023-09-05 (hanlid);Konferensartikel i tidskrift</p