Structure-based mapping of the histone-binding pocket of KDM4D using functionalized tetrazole and pyridine core compounds.

KDM4 histone demethylases became an exciting target for inhibitor development as the evidence linking them directly to tumorigenesis mounts. In this study, we set out to better understand the binding cavity using an X-ray crystallographic approach to provide a detailed landscape of possible interactions within the under-investigated region of KDM4. Our design strategy was based on utilizing known KDM binding motifs, such as nicotinic acid and tetrazolylhydrazides, as core motifs that we decided to enrich with flexible tails to map the distal histone binding site. The resulting X-ray structures of the novel compounds bound to KDM4D, a representative of the KDM4 family, revealed the interaction pattern with distal residues in the histone-binding site. The most prominent protein rearrangement detected upon ligand binding is the loop movement that blocks the accessibility to the histone binding site. Apart from providing new sites that potential inhibitors can target, the novel compounds may prove helpful in exploring the capacity of ligands to bind in sites distal to the cofactor-binding site of other KDMs or 2-oxoglutarate (2OG)-dependent oxygenases. The case study proves that combining a strong small binding motif with flexible tails to probe the binding pocket will facilitate lead discovery in classical drug-discovery campaigns, given the ease of accessing X-ray quality crystals.

A small step towards an important goal: fragment screen of the c-di-AMP-synthesizing enzyme CdaA.

CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.

Optimization of the Lead Compound NVP-BHG712 as a Colorectal Cancer Inhibitor.

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.

Cytokine levels associated with favorable clinical outcome in the CAPSID randomized trial of convalescent plasma in patients with severe COVID-19.

Objectives: To determine the profile of cytokines in patients with severe COVID-19 who were enrolled in a trial of COVID-19 convalescent plasma (CCP). Methods: Patients were randomized to receive standard treatment and 3 CCP units or standard treatment alone (CAPSID trial, ClinicalTrials.gov NCT04433910). The primary outcome was a dichotomous composite outcome (survival and no longer severe COVID-19 on day 21). Time to clinical improvement was a key secondary endpoint. The concentrations of 27 cytokines were measured (baseline, day 7). We analyzed the change and the correlation between serum cytokine levels over time in different subgroups and the prediction of outcome in receiver operating characteristics (ROC) analyses and in multivariate models. Results: The majority of cytokines showed significant changes from baseline to day 7. Some were strongly correlated amongst each other (at baseline the cluster IL-1ß, IL-2, IL-6, IL-8, G-CSF, MIP-1α, the cluster PDGF-BB, RANTES or the cluster IL-4, IL-17, Eotaxin, bFGF, TNF-α). The correlation matrix substantially changed from baseline to day 7. The heatmaps of the absolute values of the correlation matrix indicated an association of CCP treatment and clinical outcome with the cytokine pattern. Low levels of IP-10, IFN-γ, MCP-1 and IL-1ß on day 0 were predictive of treatment success in a ROC analysis. In multivariate models, low levels of IL-1ß, IFN-γ and MCP-1 on day 0 were significantly associated with both treatment success and shorter time to clinical improvement. Low levels of IP-10, IL-1RA, IL-6, MCP-1 and IFN-γ on day 7 and high levels of IL-9, PDGF and RANTES on day 7 were predictive of treatment success in ROC analyses. Low levels of IP-10, MCP-1 and high levels of RANTES, on day 7 were associated with both treatment success and shorter time to clinical improvement in multivariate models. Conclusion: This analysis demonstrates a considerable dynamic of cytokines over time, which is influenced by both treatment and clinical course of COVID-19. Levels of IL-1ß and MCP-1 at baseline and MCP-1, IP-10 and RANTES on day 7 were associated with a favorable outcome across several endpoints. These cytokines should be included in future trials for further evaluation as predictive factors.

Binding Adaptation of GS-441524 Diversifies Macro Domains and Downregulates SARS-CoV-2 de-MARylation Capacity.

Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host-cell homoeostasis. One of these mechanisms is ADP-ribosylation, a fundamental post-translational modification (PTM) characterized by the addition of ADP-ribose (ADPr) on substrates. Poly(ADP-ribose) polymerases (PARPs) are implicated in this process and they perform ADP-ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains (MDs) are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro-D-type class because of their properties to recognize and revert the ADP-ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA-dependent RNA polymerase (RdRp). Herein, GS-441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS-441524 selectively modifies SARS-CoV-2 MD de-MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MD•GS-441524 complexes, using solution NMR and X-ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS-CoV-2 MD.

A beginner's view of end of life care on German intensive care units.

BACKGROUND: Little is known about importance and implementation of end-of-life care (EOLC) in German intensive care units (ICU). This survey analyses preferences and differences in training between "medical" (internal medicine, neurology) and "surgical" (surgery, anaesthesiology) residents during intensive care rotation. METHODS: This is a point-prevalence study, in which intensive care medicine course participants of one educational course were surveyed. Physicians from multiple ICU and university as well as non-university hospitals and all care levels were asked to participate. The questionnaire was composed of a paper and an electronic part. Demographic and structural data were prompted and EOLC data (48 questions) were grouped into six categories considering importance and implementation: category 1 (important, always implemented), 2 (important, sometimes implemented), 3 (important, never implemented) and 4-6 (unimportant, implementation always, sometimes, never). The trial is registered at the "Deutsches Register für klinische Studien (DRKS)", Study number DRKS00026619, registered on September 10th 2021, www.drks.de . RESULTS: Overall, 194/ 220 (88%) participants responded. Mean age was 29.7 years, 55% were female and 60% had scant ICU working experience. There were 64% medical and 35% surgical residents. Level of care and size of ICU differed significantly between medical and surgical (both p < 0.001). Sufficient implementation was stated for 66% of EOLC questions, room for improvement (category 2 and 3) was seen in 25, and 8% were classified as irrelevant (category 6). Areas with the most potential for improvement included prognosis and outcome and patient autonomy. There were no significant differences between medical and surgical residents. CONCLUSIONS: Even though EOLC is predominantly regarded as sufficiently implemented in German ICU of all specialties, our survey unveiled still 25% room for improvement for medical as well as surgical ICU residents. This is important, as areas of improvement potential may be addressed with reasonable effort, like individualizing EOLC procedures or setting up EOLC teams. Health care providers as well as medical societies should emphasize EOLC training in their curricula.

Enhanced Properties of a Benzimidazole Benzylpyrazole Lysine Demethylase Inhibitor: Mechanism-of-Action, Binding Site Analysis, and Activity in Cellular Models of Prostate Cancer.

Jumonji domain-containing lysine demethylase (KDM) enzymes are encoded by genes of the KDM superfamily. Activities of the KDM4 subfamily promote aggressive phenotypes associated with prostate cancer (PCa). Previously, we discovered a benzimidazole pyrazole molecule that inhibited KDM4 isoforms with properties tractable for development. Here, we demonstrate that a benzyl-substituted variant of this inhibitor exhibits improved potency in biochemical assays, is cell-permeable, and kills PCa cells at low micromolar concentrations. By X-ray crystallography and kinetics-based assays, we demonstrate that the mechanism of inhibition is complex, proceeding via competition with the enzyme for binding of active-site Fe2+ and by populating a distal site on the enzyme surface. Furthermore, we provide evidence that the inhibitor's cytostatic properties arise from direct intracellular inhibition of KDM4 enzymes. PCa cells treated with the inhibitor exhibit reduced expression of genes regulated by the androgen receptor, an outcome accompanied by epigenetic maintenance of a heterochromatic state.

Substrate binding modes of purine and pyrimidine nucleotides to human ecto-5'-nucleotidase (CD73) and inhibition by their bisphosphonic acid derivatives.

Human ecto-5-nucleotidase (CD73) is involved in purinergic signalling, which influences a diverse range of biological processes. CD73 hydrolyses AMP and is the major control point for the levels of extracellular adenosine. Inhibitors of CD73 thus block the immunosuppressive action of adenosine, a promising approach for cancer immunotherapy. Interestingly, ADP and ATP are competitive inhibitors of CD73, with the most potent small-molecule inhibitors to date being non-hydrolysable ADP analogues. While AMP is the major substrate of the enzyme, CD73 has been reported to hydrolyse other 5'-nucleoside monophosphates. Based on a fragment screening campaign at the BESSY II synchrotron, we present the binding modes of various deoxyribo- and ribonucleoside monophosphates and of four additional fragments binding to the nucleoside binding site of the open form of the enzyme. Kinetic analysis of monophosphate hydrolysis shows that ribonucleotide substrates are favoured over their deoxyribose equivalents with AMP being the best substrate. We characterised the initial step of AMP hydrolysis, the binding mode of AMP to the open conformation of CD73 and compared that to other monophosphate substrates. In addition, the inhibitory activity of various bisphosphonic acid derivatives of nucleoside diphosphates was determined. Although AMPCP remains the most potent inhibitor, replacement of the adenine base with other purines or with pyrimidines increases the Ki value only between twofold and sixfold. On the other hand, these nucleobases offer new opportunities to attach substituents for improved pharmacological properties.

Prolidase - A protein with many faces.

Prolidase is a metal-dependent peptidase specialized in the cleavage of dipeptides containing proline or hydroxyproline on their C-termini. Prolidase homologues are found in all kingdoms of life. The importance of prolidase in human health is underlined by a rare hereditary syndrome referred to as Prolidase Deficiency. A growing number of studies highlight the importance of prolidase in various other human conditions, including cancer. Some recent studies link prolidase's activity-independent regulatory role to tumorigenesis. Furthermore, the enzyme or engineered variants have some applications in biotechnology. In this short review, we aim to highlight different aspects of the protein the importance of which is increasingly recognized over the last years.

Insulin polymorphism induced by two polyphenols: new crystal forms and advances in macromolecular powder diffraction.

This study focuses on the polymorphism of human insulin (HI) upon the binding of the phenolic derivatives p-coumaric acid or trans-resveratrol over a wide pH range. The determination of the structural behaviour of HI via X-ray powder diffraction (XRPD) and single-crystal X-ray diffraction (SCXRD) is reported. Four distinct polymorphs were identified, two of which have not been reported previously. The intermediate phase transitions are discussed. One of the novel monoclinic polymorphs displays the highest molecular packing among insulin polymorphs of the same space group to date; its structure was elucidated by SCXRD. XRPD data collection was performed using a variety of instrumental setups and a systematic comparison of the acquired data is presented. A laboratory diffractometer was used for screening prior to high-resolution XRPD data collection on the ID22 beamline at the European Synchrotron Radiation Facility. Additional measurements for the most representative samples were performed on the X04SA beamline at the Swiss Light Source (SLS) using the MYTHEN II detector, which allowed the detection of minor previously untraceable impurities and dramatically improved the d-spacing resolution even for poorly diffracting samples.

The susceptibility of disulfide bonds towards radiation damage may be explained by S⋯O interactions.

Radiation-induced damage to protein crystals during X-ray diffraction data collection is a major impediment to obtaining accurate structural information on macromolecules. Some of the specific impairments that are inflicted upon highly brilliant X-ray irradiation are metal-ion reduction, disulfide-bond cleavage and a loss of the integrity of the carboxyl groups of acidic residues. With respect to disulfide-bond reduction, previous results have indicated that not all disulfide bridges are equally susceptible to damage. A careful analysis of the chemical environment of disulfide bonds in the structures of elastase, lysozyme, acetylcholinesterase and other proteins suggests that S-S bonds which engage in a close contact with a carbonyl O atom along the extension of the S-S bond vector are more susceptible to reduction than the others. Such an arrangement predisposes electron transfer to occur from the O atom to the disulfide bond, leading to its reduction. The interaction between a nucleophile and an electrophile, akin to hydrogen bonding, stabilizes protein structures, but it also provides a pathway of electron transfer to the S-S bond, leading to its reduction during exposure of the protein crystal to an intense X-ray beam. An otherwise stabilizing interaction can thus be the cause of destabilization under the condition of radiation exposure.

Engineering micromechanics of soft porous crystals for negative gas adsorption.

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 sp3 hybridized carbon atoms to soften the molecular building blocks, or stiffen them with sp2 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.

In Situ Imine-Based Linker Formation for the Synthesis of Zirconium MOFs: A Route to CO2 Capture Materials and Ethylene Oligomerization Catalysts.

In situ formation of imine-based organic linkers facilitates the formation of metal-organic frameworks (MOFs), in particular if linker solubility hampers the direct synthesis. The reaction of ZrCl4 with 4-formylbenzoic acid or 4-formyl-3-hydroxybenzoic acid as the aldehyde source and 4-aminobenzoic acid as the amine source is shown to produce zirconium MOFs isoreticular to UiO-66 (PCN-161 and a novel DUT-133, [Zr6O4(OH)4(C15H9NO5)6], respectively). A similar reaction with p-phenylenediamine as the amine-containing building block gave 2-fold interpenetrated framework (PCN-164). Detailed characterization, including single crystal and powder X-ray diffraction, water stability tests, thermal stability, and in situ 1H and 13C NMR were performed to elucidate the formation mechanism of zirconium MOFs containing imine-based linkers. The resulting zirconium MOFs were evaluated as potential materials for CO2 capture and as ethylene oligomerization catalysts with anchored nickel as the active species.

Bioassay for Endothelial Damage Mediators Retrieved by Hemoadsorption.

Hemoadsorption devices are used to treat septic shock by adsorbing inflammatory cytokines and as yet incompletely defined danger and pathogen associated molecular patterns. In an ideal case, hemoadsorption results in immediate recovery of microvascular endothelial cells' (mEC) function and rapid recovery from catecholamine-dependency and septic shock. We here tested a single device, which consists of polystyrene-divinylbenzene core particles of 450 µm diameter with a high affinity for hydrophobic compounds. The current study aimed at the proof of concept that endothelial-specific damage mediators are adsorbed and can be recovered from hemoadsorption devices. Because of excellent clinical experience, we tested protein fractions released from a hemoadsorber in a novel endothelial bioassay. Video-based, long-term imaging of mEC proliferation and cell death were evaluated and combined with apoptosis and ATP measurements. Out of a total of 39 fractions recovered from column fractionation, we identified 3 fractions that caused i) inhibition of mEC proliferation, ii) increased cell death and iii) induction of apoptosis in mEC. When adding these 3 fractions to mEC, their ATP contents were reduced. These fractions contained proteins of approximately 15 kDa, and high amounts of nucleic acid, which was at least in part oxidized. The efficacy for endothelial cell damage prevention by hemoadsorption can be addressed by a novel endothelial bioassay and long-term video observation procedures. Protein fractionation of the hemoadsorption devices used is feasible to study and define endothelial damage ligands on a molecular level. The results suggest a significant effect by circulating nucleic acids - bound to an as yet undefined protein, which may constitute a major danger-associated molecular pattern (DAMP) in the exacerbation of inflammation when patients experience septic shock. Hemoadsorption devices may thus limit endothelial damage, through the binding of nucleic acid-bearing aggregates and thus contribute to improved endothelial barrier function.

Structure-Based Screening of Tetrazolylhydrazide Inhibitors versus KDM4 Histone Demethylases.

Human histone demethylases are known to play an important role in the development of several tumor types. Consequently, they have emerged as important medical targets for the treatment of human cancer. Herein, structural studies on tetrazolylhydrazide inhibitors as a new scaffold for a certain class of histone demethylases, the JmjC proteins, are reported. A series of compounds are structurally described and their respective binding modes to the KDM4D protein, which serves as a high-resolution model to represent the KDM4 subfamily in crystallographic studies, are examined. Similar to previously reported inhibitors, the compounds described herein are competitors for the natural KDM4 cofactor, 2-oxoglutarate. The tetrazolylhydrazide scaffold fills an important gap in KDM4 inhibition and newly described, detailed interactions of inhibitor moieties pave the way to the development of compounds with high target-binding affinity and increased membrane permeability, at the same time.

End-of-life perceptions among physicians in intensive care units managed by anesthesiologists in Germany: a survey about structure, current implementation and deficits.

BACKGROUND: Structural aspects and current practice about end-of-life (EOL) decisions in German intensive care units (ICUs) managed by anesthesiologists are unknown. A survey among intensive care anesthesiologists has been conducted to explore current practice, barriers and opinions on EOL decisions in ICU. METHODS: In November 2015, all members of the German Society of Anesthesiology and Intensive Care Medicine (DGAI) and the Association of German Anesthesiologists (BDA) were asked to participate in an online survey to rate the presence or absence and the importance of 50 items. Answers were grouped into three categories considering implementation and relevance: Category 1 reflects high implementation and high relevance, Category 2 low and low, and Category 3 low and high. RESULTS: Five-hundred and forty-one anesthesiologists responded. Only four items reached ≥90% agreement as being performed "yes, always" or "mostly", and 29 items were rated "very" or "more important". A profound discrepancy between current practice and attributed importance was revealed. Twenty-eight items attributed to Category 1, six to Category 2 and sixteen to Category 3. Items characterizing the most urgent need for improvement (Category 3) referred to patient outcome data, preparation of health care directives and interdisciplinary discussion, standard operating procedures, implementation of practical instructions and inclusion of nursing staff and families in the process. CONCLUSION: The present survey affirms an urgent need for improvement in EOL practice in German ICUs focusing on advanced care planning, distinct aspects of changing goals of care, implementation of standard operating procedures, continuing education and reporting of outcome data.

The Role of Troponin in Blunt Cardiac Injury After Multiple Trauma in Humans.

BACKGROUND: The incidence of cardiac injury in immediate fatalities after blunt trauma remains underestimated, and reliable diagnostic strategies are still missing. Furthermore, clinical data concerning heart-specific troponin serum levels, injury severity score (ISS), catecholamine treatment and survival of patients on admission to the hospital have rarely been interrelated so far. Therefore, the object of the present study was to identify predictive parameters for mortality in the context of blunt cardiac injury. METHODS: This retrospective observational study included 173 severely injured patients with an ISS ≥25 admitted to the University Hospital of Ulm, a level 1 trauma center, during 2009-2013 . Furthermore, 83 blunt trauma victims who died before hospital admission were subjected to postmortem examination at the Institute of Legal Medicine, University of Ulm, during 2009-2014. ISS, cardiac injury and associated thoracic injuries were determined in both groups. Furthermore, in the hospitalized patients, serum troponin and IL-6 levels were measured. RESULTS: Macroscopic heart injury was observed in 18 % of the patients who died at the scene and only in 1 % of the patients admitted to the hospital, indicating that macroscopic heart injury is associated with an immediate life-threatening condition. Troponin levels were elevated in 43 % of the patients after admission to the hospital. Moreover, troponin serum concentrations were significantly higher in patients treated with norepinephrine (26.4 ± 4 ng/l) and in non-survivors (84.9 ± 22.8 ng/l) compared to patients without catecholamines and survivors, respectively. CONCLUSIONS: Macroscopic heart injury was 20 times more frequent in non-survivors than in survivors. Serum troponin levels correlated with mortality after multiple injury and therefore may represent a valuable prognostic marker in trauma patients.

Circulating adipokine levels and prognostic value in septic patients.

BACKGROUND: Adipokines have a wide range of effects and are linked to sepsis and septic shock. The aim of the present study was to describe the changes in adipokine levels in septic patients in relation to patients' preseptic adipokine levels. Furthermore, we examined adipokines as prognostic markers. METHODS: Fourteen consecutive critically ill patients meeting the clinical criteria for severe sepsis or septic shock 3 days up to 1 month after major visceral surgery were enrolled prospectively. Plasma adipokines were measured preoperatively, 1 and 4 days after diagnosis of severe sepsis or septic shock following elective surgery. RESULTS: Median plasma adiponectin levels were lowered and resistin and leptin levels elevated in sepsis compared with preseptic plasma levels. MCP-1, C-reactive protein and white blood cell count were higher in septic compared with preseptic patients. Survivors had significantly higher preseptic adipokine levels than non-survivors. Adiponectin levels of survivors decreased significant (on average by 33 %) at day one after onset of sepsis compared with preseptic levels. In contrast, median adiponectin levels of patients dying during sepsis showed a slight increase (11 %). Median BMI of survivors was 30 kg/m(2), median BMI of non-survivors was 25, respectively. CONCLUSIONS: Adipokine levels change during the course of sepsis. Higher preseptic adiponectin levels and decreasing adiponectin levels after onset of sepsis are associated with survival of sepsis. Survival of overweight and obese patients was higher than in normal weight patients. Changes in adiponektin levels could be a prognostic marker for outcome of severe sepsis/septic shock following surgery.

Against the odds? De novo structure determination of a pilin with two cysteine residues by sulfur SAD.

Exploiting the anomalous signal of the intrinsic S atoms to phase a protein structure is advantageous, as ideally only a single well diffracting native crystal is required. However, sulfur is a weak anomalous scatterer at the typical wavelengths used for X-ray diffraction experiments, and therefore sulfur SAD data sets need to be recorded with a high multiplicity. In this study, the structure of a small pilin protein was determined by sulfur SAD despite several obstacles such as a low anomalous signal (a theoretical Bijvoet ratio of 0.9% at a wavelength of 1.8 Å), radiation damage-induced reduction of the cysteines and a multiplicity of only 5.5. The anomalous signal was improved by merging three data sets from different volumes of a single crystal, yielding a multiplicity of 17.5, and a sodium ion was added to the substructure of anomalous scatterers. In general, all data sets were balanced around the threshold values for a successful phasing strategy. In addition, a collection of statistics on structures from the PDB that were solved by sulfur SAD are presented and compared with the data. Looking at the quality indicator R(anom)/R(p.i.m.), an inconsistency in the documentation of the anomalous R factor is noted and reported.

Crystal structures of SCP2-thiolases of Trypanosomatidae, human pathogens causing widespread tropical diseases: the importance for catalysis of the cysteine of the unique HDCF loop.

Thiolases are essential CoA-dependent enzymes in lipid metabolism. In the present study we report the crystal structures of trypanosomal and leishmanial SCP2 (sterol carrier protein, type-2)-thiolases. Trypanosomatidae cause various widespread devastating (sub)-tropical diseases, for which adequate treatment is lacking. The structures reveal the unique geometry of the active site of this poorly characterized subfamily of thiolases. The key catalytic residues of the classical thiolases are two cysteine residues, functioning as a nucleophile and an acid/base respectively. The latter cysteine residue is part of a CxG motif. Interestingly, this cysteine residue is not conserved in SCP2-thiolases. The structural comparisons now show that in SCP2-thiolases the catalytic acid/base is provided by the cysteine residue of the HDCF motif, which is unique for this thiolase subfamily. This HDCF cysteine residue is spatially equivalent to the CxG cysteine residue of classical thiolases. The HDCF cysteine residue is activated for acid/base catalysis by two main chain NH-atoms, instead of two water molecules, as present in the CxG active site. The structural results have been complemented with enzyme activity data, confirming the importance of the HDCF cysteine residue for catalysis. The data obtained suggest that these trypanosomatid SCP2-thiolases are biosynthetic thiolases. These findings provide promise for drug discovery as biosynthetic thiolases catalyse the first step of the sterol biosynthesis pathway that is essential in several of these parasites.