In their recent commentary in Protein Science, Jaskolski et al. analyzed three randomly picked diffraction data sets from fragment‐screening group depositions from the PDB and, based on that, they ...claimed that such data are principally problematic. We demonstrate here that if such data are treated properly, none of the proclaimed criticisms persist.
Human dihydrolipoamide dehydrogenase (hLADH, hE3) deficiency (OMIM# 246900) is an often prematurely lethal genetic disease usually caused by inactive or partially inactive hE3 variants. Here we ...report the crystal structure of wild-type hE3 at an unprecedented high resolution of 1.75 Å and the structures of six disease-causing hE3 variants at resolutions ranging from 1.44 to 2.34 Å. P453L proved to be the most deleterious substitution in structure as aberrations extensively compromised the active site. The most prevalent G194C-hE3 variant primarily exhibited structural alterations close to the substitution site, whereas the nearby cofactor-binding residues were left unperturbed. The G426E substitution mainly interfered with the local charge distribution introducing dynamics to the substitution site in the dimer interface; G194C and G426E both led to minor structural changes. The R460G, R447G and I445M substitutions all perturbed a solvent accessible channel, the so-called H+/H2O channel, leading to the active site. Molecular pathomechanisms of enhanced reactive oxygen species (ROS) generation and impaired binding to multienzyme complexes were also addressed according to the structural data for the relevant mutations. In summary, we present here for the first time a comprehensive study that links three-dimensional structures of disease-causing hE3 variants to residual hLADH activities, altered capacities for ROS generation, compromised affinities for multienzyme complexes and eventually clinical symptoms. Our results may serve as useful starting points for future therapeutic intervention approaches.
Current sepsis guidelines recommend antimicrobial treatment (AT) within one hour after onset of sepsis-related organ dysfunction (OD) and surgical source control within 12 hours. The objective of ...this study was to explore the association between initial infection management according to sepsis treatment recommendations and patient outcome.
In a prospective observational multi-center cohort study in 44 German ICUs, we studied 1,011 patients with severe sepsis or septic shock regarding times to AT, source control, and adequacy of AT. Primary outcome was 28-day mortality.
Median time to AT was 2.1 (IQR 0.8 - 6.0) hours and 3 hours (-0.1 - 13.7) to surgical source control. Only 370 (36.6%) patients received AT within one hour after OD in compliance with recommendation. Among 422 patients receiving surgical or interventional source control, those who received source control later than 6 hours after onset of OD had a significantly higher 28-day mortality than patients with earlier source control (42.9% versus 26.7%, P <0.001). Time to AT was significantly longer in ICU and hospital non-survivors; no linear relationship was found between time to AT and 28-day mortality. Regardless of timing, 28-day mortality rate was lower in patients with adequate than non-adequate AT (30.3% versus 40.9%, P < 0.001).
A delay in source control beyond 6 hours may have a major impact on patient mortality. Adequate AT is associated with improved patient outcome but compliance with guideline recommendation requires improvement. There was only indirect evidence about the impact of timing of AT on sepsis mortality.
The EMBL‐Hamburg Automated Crystal Structure Determination Platform is a system that combines a number of existing macromolecular crystallographic computer programs and several decision‐makers into a ...software pipeline for automated and efficient crystal structure determination. The pipeline can be invoked as soon as X‐ray data from derivatized protein crystals have been collected and processed. It is controlled by a web‐based graphical user interface for data and parameter input, and for monitoring the progress of structure determination. A large number of possible structure‐solution paths are encoded in the system and the optimal path is selected by the decision‐makers as the structure solution evolves. The processes have been optimized for speed so that the pipeline can be used effectively for validating the X‐ray experiment at a synchrotron beamline.
In the past two decades, most of the steps in a macromolecular crystallography experiment have undergone tremendous development with respect to speed, feasibility and increase of throughput. The part ...of the experimental workflow that is still a bottleneck, despite significant efforts, involves the manipulation and harvesting of the crystals for the diffraction experiment. Here, a novel low‐cost device is presented that functions as a cover for 96‐well crystallization plates. This device enables access to the individual experiments one at a time by its movable parts, while minimizing evaporation of all other experiments of the plate. In initial tests, drops of many typically used crystallization cocktails could be successfully protected for up to 6 h. Therefore, the manipulation and harvesting of crystals is straightforward for the experimenter, enabling significantly higher throughput. This is useful for many macromolecular crystallography experiments, especially multi‐crystal screening campaigns.
A simple and low‐cost device has been developed to minimize evaporation in microtiter plates for easy crystal handling and harvesting.
In recent years, crystallographic fragment screening has matured into an almost routine experiment at several modern synchrotron sites. The hits of the screening experiment, i.e. small molecules or ...fragments binding to the target protein, are revealed along with their 3D structural information. Therefore, they can serve as useful starting points for further structure‐based hit‐to‐lead development. However, the progression of fragment hits to tool compounds or even leads is often hampered by a lack of chemical feasibility. As an attractive alternative, compound analogs that embed the fragment hit structurally may be obtained from commercial catalogs. Here, a workflow is reported based on filtering and assessing such potential follow‐up compounds by template docking. This means that the crystallographic binding pose was integrated into the docking calculations as a central starting parameter. Subsequently, the candidates are scored on their interactions within the binding pocket. In an initial proof‐of‐concept study using five starting fragments known to bind to the aspartic protease endothiapepsin, 28 follow‐up compounds were selected using the designed workflow and their binding was assessed by crystallography. Ten of these compounds bound to the active site and five of them showed significantly increased affinity in isothermal titration calorimetry of up to single‐digit micromolar affinity. Taken together, this strategy is capable of efficiently evolving the initial fragment hits without major synthesis efforts and with full control by X‐ray crystallography.
A workflow to expand crystallographic fragment hits to higher affinity compounds using readily available analogs is described and successfully applied.
Crystallographic fragment screening (CFS) has become one of the major techniques for screening compounds in the early stages of drug‐discovery projects. Following the advances in automation and ...throughput at modern macromolecular crystallography beamlines, the bottleneck for CFS has shifted from collecting data to organizing and handling the analysis of such projects. The complexity that emerges from the use of multiple methods for processing and refinement and to search for ligands requires an equally sophisticated solution to summarize the output, allowing researchers to focus on the scientific questions instead of on software technicalities. FragMAXapp is the fragment‐screening project‐management tool designed to handle CFS projects at MAX IV Laboratory. It benefits from the powerful computing infrastructure of large‐scale facilities and, as a web application, it is accessible from everywhere.
The amount of data generated during crystallographic fragment‐screening projects requires sophisticated automated methods to analyse the data and to identify binders. FragMAXapp is the MAX IV Laboratory approach to managing fragment‐screening campaigns: a web application that provides scientists with access to the MAX IV computing cluster and visualization tools.
Framework materials at the molecular level, such as metal-organic frameworks (MOF), were recently found to exhibit exotic and counterintuitive micromechanical properties. Stimulated by host-guest ...interactions, these so-called soft porous crystals can display counterintuitive adsorption phenomena such as negative gas adsorption (NGA). NGA materials are bistable frameworks where the occurrence of a metastable overloaded state leads to pressure amplification upon a sudden framework contraction. How can we control activation barriers and energetics
via
functionalization of the molecular building blocks that dictate the frameworks' mechanical response? In this work we tune the elastic and inelastic properties of building blocks at the molecular level and analyze the mechanical response of the resulting frameworks. From a set of 11 frameworks, we demonstrate that widening of the backbone increases stiffness, while elongation of the building blocks results in a decrease in critical yield stress of buckling. We further functionalize the backbone by incorporation of sp
3
hybridized carbon atoms to soften the molecular building blocks, or stiffen them with sp
2
and sp carbons. Computational modeling shows how these modifications of the building blocks tune the activation barriers within the energy landscape of the guest-free bistable frameworks. Only frameworks with free energy barriers in the range of 800 to 1100 kJ mol
−1
per unit cell, and moderate yield stress of 0.6 to 1.2 nN for single ligand buckling, exhibit adsorption-induced contraction and negative gas adsorption. Advanced experimental
in situ
methodologies give detailed insights into the structural transitions and the adsorption behavior. The new framework DUT-160 shows the highest magnitude of NGA ever observed for nitrogen adsorption at 77 K. Our computational and experimental analysis of the energetics and mechanical response functions of porous frameworks is an important step towards tuning activation barriers in dynamic framework materials and provides critical design principles for molecular building blocks leading to pressure amplifying materials.
We characterise the elastic properties of molecular building blocks and how they impact the mechanical properties of soft porous crystals.
The ephrin type‐A receptor 2 (EPHA2) kinase belongs to the largest family of receptor tyrosine kinases. There are several indications of an involvement of EPHA2 in the development of infectious ...diseases and cancer. Despite pharmacological potential, EPHA2 is an under‐examined target protein. In this study, we synthesized a series of derivatives of the inhibitor NVP‐BHG712 and triazine‐based compounds. These compounds were evaluated to determine their potential as kinase inhibitors of EPHA2, including elucidation of their binding mode (X‐ray crystallography), affinity (microscale thermophoresis), and selectivity (Kinobeads assay). Eight inhibitors showed affinities in the low‐nanomolar regime (KD<10 nM). Testing in up to seven colon cancer cell lines that express EPHA2 reveals that several derivatives feature promising effects for the control of human colon carcinoma. Thus, we have developed a set of powerful tool compounds for fundamental new research on the interplay of EPH receptors in a cellular context.
Towards controlling colon carcinoma: Derivatives of the compound NVP‐BHG712 as well as triazine‐based molecules prove to be potent inhibitors of the receptor tyrosine kinase EPHA2. Several of these newly synthesized inhibitors showed highly promising effects in controlling human colon carcinoma. Furthermore, these molecules can act as tool compounds to gain a deeper understanding of the role of ephrin receptors in cellular context.
We report the crystal structures of the human (dihydro)lipoamide dehydrogenase (hLADH, hE3) and its disease-causing homodimer interface mutant D444V-hE3 at 2.27 and 1.84 Å resolution, respectively. ...The wild type structure is a unique uncomplexed, unliganded hE3 structure with the true canonical sequence. Based on the structural information a novel molecular pathomechanism is proposed for the impaired catalytic activity and enhanced capacity for reactive oxygen species generation of the pathogenic mutant. The mechanistic model involves a previously much ignored solvent accessible channel leading to the active site that might be perturbed also by other disease-causing homodimer interface substitutions of this enzyme.
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•X-ray study of lipoamide dehydrogenase (E3) and its disease-causing D444V mutant.•D444V stimulates ROS generation and abolishes harboring to multienzyme complexes.•Perturbation of a channel leading to the active site was identified.•Structural alterations account for enzyme activity, affinity, and ROS generation.•Generalized molecular pathomechanism for all dimer-interface mutants is suggested.