An environment for sustainable research software in Germany and beyond: current state, open challenges, and call for action.

Research software has become a central asset in academic research. It optimizes existing and enables new research methods, implements and embeds research knowledge, and constitutes an essential research product in itself. Research software must be sustainable in order to understand, replicate, reproduce, and build upon existing research or conduct new research effectively. In other words, software must be available, discoverable, usable, and adaptable to new needs, both now and in the future. Research software therefore requires an environment that supports sustainability. Hence, a change is needed in the way research software development and maintenance are currently motivated, incentivized, funded, structurally and infrastructurally supported, and legally treated. Failing to do so will threaten the quality and validity of research. In this paper, we identify challenges for research software sustainability in Germany and beyond, in terms of motivation, selection, research software engineering personnel, funding, infrastructure, and legal aspects. Besides researchers, we specifically address political and academic decision-makers to increase awareness of the importance and needs of sustainable research software practices. In particular, we recommend strategies and measures to create an environment for sustainable research software, with the ultimate goal to ensure that software-driven research is valid, reproducible and sustainable, and that software is recognized as a first class citizen in research. This paper is the outcome of two workshops run in Germany in 2019, at deRSE19 - the first International Conference of Research Software Engineers in Germany - and a dedicated DFG-supported follow-up workshop in Berlin.

Large-scale prediction of binding affinity in protein-small ligand complexes: the PRODIGY-LIG web server.

SUMMARY: Recently we published PROtein binDIng enerGY (PRODIGY), a web-server for the prediction of binding affinity in protein-protein complexes. By using a combination of simple structural properties, such as the residue-contacts made at the interface, PRODIGY has demonstrated a top performance compared with other state-of-the-art predictors in the literature. Here we present an extension of it, named PRODIGY-LIG, aimed at the prediction of affinity in protein-small ligand complexes. The predictive method, properly readapted for small ligand by making use of atomic instead of residue contacts, has been successfully applied for the blind prediction of 102 protein-ligand complexes during the D3R Grand Challenge 2. PRODIGY-LIG has the advantage of being simple, generic and applicable to any kind of protein-ligand complex. It provides an automatic, fast and user-friendly tool ensuring broad accessibility. AVAILABILITY AND IMPLEMENTATION: PRODIGY-LIG is freely available without registration requirements at http://milou.science.uu.nl/services/PRODIGY-LIG.

Substrate specificity of the pyrophosphohydrolase LpxH determines the asymmetry of Bordetella pertussis lipid A.

Lipopolysaccharides are anchored to the outer membrane of Gram-negative bacteria by a hydrophobic moiety known as lipid A, which potently activates the host innate immune response. Lipid A of Bordetella pertussis, the causative agent of whooping cough, displays unusual structural asymmetry with respect to the length of the acyl chains at the 3 and 3' positions, which are 3OH-C10 and 3OH-C14 chains, respectively. Both chains are attached by the acyltransferase LpxA, the first enzyme in the lipid A biosynthesis pathway, which, in B. pertussis, has limited chain length specificity. However, this only partially explains the strict asymmetry of lipid A. In attempts to modulate the endotoxicity of B. pertussis lipid A, here we expressed the gene encoding LpxA from Neisseria meningitidis, which specifically attaches 3OH-C12 chains, in B. pertussis This expression was lethal, suggesting that one of the downstream enzymes in the lipid A biosynthesis pathway in B. pertussis cannot handle precursors with a 3OH-C12 chain. We considered that the UDP-diacylglucosamine pyrophosphohydrolase LpxH could be responsible for this defect as well as for the asymmetry of B. pertussis lipid A. Expression of meningococcal LpxH in B. pertussis indeed resulted in new symmetric lipid A species with 3OH-C10 or 3OH-C14 chains at both the 3 and 3' positions, as revealed by MS analysis. Furthermore, co-expression of meningococcal lpxH and lpxA resulted in viable cells that incorporated 3OH-C12 chains in B. pertussis lipid A. We conclude that the asymmetry of B. pertussis lipid A is determined by the acyl chain length specificity of LpxH.

Natural helix 9 mutants of PPARγ differently affect its transcriptional activity.

OBJECTIVE: The nuclear receptor PPARγ is the master regulator of adipocyte differentiation, distribution, and function. In addition, PPARγ induces terminal differentiation of several epithelial cell lineages, including colon epithelia. Loss-of-function mutations in PPARG result in familial partial lipodystrophy subtype 3 (FPDL3), a rare condition characterized by aberrant adipose tissue distribution and severe metabolic complications, including diabetes. Mutations in PPARG have also been reported in sporadic colorectal cancers, but the significance of these mutations is unclear. Studying these natural PPARG mutations provides valuable insights into structure-function relationships in the PPARγ protein. We functionally characterized a novel FPLD3-associated PPARγ L451P mutation in helix 9 of the ligand binding domain (LBD). Interestingly, substitution of the adjacent amino acid K450 was previously reported in a human colon carcinoma cell line. METHODS: We performed a detailed side-by-side functional comparison of these two PPARγ mutants. RESULTS: PPARγ L451P shows multiple intermolecular defects, including impaired cofactor binding and reduced RXRα heterodimerisation and subsequent DNA binding, but not in DBD-LBD interdomain communication. The K450Q mutant displays none of these functional defects. Other colon cancer-associated PPARγ mutants displayed diverse phenotypes, ranging from complete loss of activity to wildtype activity. CONCLUSIONS: Amino acid changes in helix 9 can differently affect LBD integrity and function. In addition, FPLD3-associated PPARγ mutations consistently cause intra- and/or intermolecular defects; colon cancer-associated PPARγ mutations on the other hand may play a role in colon cancer onset and progression, but this is not due to their effects on the most well-studied functional characteristics of PPARγ.

West-Life: A Virtual Research Environment for structural biology.

The West-Life project (https://about.west-life.eu/) is a Horizon 2020 project funded by the European Commission to provide data processing and data management services for the international community of structural biologists, and in particular to support integrative experimental approaches within the field of structural biology. It has developed enhancements to existing web services for structure solution and analysis, created new pipelines to link these services into more complex higher-level workflows, and added new data management facilities. Through this work it has striven to make the benefits of European e-Infrastructures more accessible to life-science researchers in general and structural biologists in particular.

Assessment of contact predictions in CASP12: Co-evolution and deep learning coming of age.

Following up on the encouraging results of residue-residue contact prediction in the CASP11 experiment, we present the analysis of predictions submitted for CASP12. The submissions include predictions of 34 groups for 38 domains classified as free modeling targets which are not accessible to homology-based modeling due to a lack of structural templates. CASP11 saw a rise of coevolution-based methods outperforming other approaches. The improvement of these methods coupled to machine learning and sequence database growth are most likely the main driver for a significant improvement in average precision from 27% in CASP11 to 47% in CASP12. In more than half of the targets, especially those with many homologous sequences accessible, precisions above 90% were achieved with the best predictors reaching a precision of 100% in some cases. We furthermore tested the impact of using these contacts as restraints in ab initio modeling of 14 single-domain free modeling targets using Rosetta. Adding contacts to the Rosetta calculations resulted in improvements of up to 26% in GDT_TS within the top five structures.

SpotOn: High Accuracy Identification of Protein-Protein Interface Hot-Spots.

We present SpotOn, a web server to identify and classify interfacial residues as Hot-Spots (HS) and Null-Spots (NS). SpotON implements a robust algorithm with a demonstrated accuracy of 0.95 and sensitivity of 0.98 on an independent test set. The predictor was developed using an ensemble machine learning approach with up-sampling of the minor class. It was trained on 53 complexes using various features, based on both protein 3D structure and sequence. The SpotOn web interface is freely available at: http://milou.science.uu.nl/services/SPOTON/ .

Rearrangement of the Extracellular Domain/Extracellular Loop 1 Interface Is Critical for Thyrotropin Receptor Activation.

The thyroid stimulating hormone receptor (TSHR) is a G protein-coupled receptor (GPCR) with a characteristic large extracellular domain (ECD). TSHR activation is initiated by binding of the hormone ligand TSH to the ECD. How the extracellular binding event triggers the conformational changes in the transmembrane domain (TMD) necessary for intracellular G protein activation is poorly understood. To gain insight in this process, the knowledge on the relative positioning of ECD and TMD and the conformation of the linker region at the interface of ECD and TMD are of particular importance. To generate a structural model for the TSHR we applied an integrated structural biology approach combining computational techniques with experimental data. Chemical cross-linking followed by mass spectrometry yielded 17 unique distance restraints within the ECD of the TSHR, its ligand TSH, and the hormone-receptor complex. These structural restraints generally confirm the expected binding mode of TSH to the ECD as well as the general fold of the domains and were used to guide homology modeling of the ECD. Functional characterization of TSHR mutants confirms the previously suggested close proximity of Ser-281 and Ile-486 within the TSHR. Rigidifying this contact permanently with a disulfide bridge disrupts ligand-induced receptor activation and indicates that rearrangement of the ECD/extracellular loop 1 (ECL1) interface is a critical step in receptor activation. The experimentally verified contact of Ser-281 (ECD) and Ile-486 (TMD) was subsequently utilized in docking homology models of the ECD and the TMD to create a full-length model of a glycoprotein hormone receptor.

A newly discovered TSHR variant (L665F) associated with nonautoimmune hyperthyroidism in an Austrian family induces constitutive TSHR activation by steric repulsion between TM1 and TM7.

OBJECTIVE: New in vivo mutations in G protein-coupled receptors open opportunities for insights into the mechanism of receptor activation. Here we describe the molecular mechanism of constitutive TSH receptor (TSHR) activation in an Austrian family with three generations of familial nonautoimmune hyperthyroidism. PATIENTS: The index patient was diagnosed with hyperthyroidism during her first pregnancy. Her first two children were diagnosed with hyperthyroidism at the age of 11 and 10 years, respectively. TSH suppression was also observed in the third child at the age of 8 years, who has normal free T4 levels until now. TSH suppression in infancy was observed in the fourth child. The mother of the index patient was diagnosed with toxic multinodular goiter at the age of 36 years. METHODS: DNA was extracted from blood samples from the index patient, her mother, and her four children. Screening for TSHR mutations was performed by high-resolution melting assays and subsequent sequencing. Elucidation of the underlying mechanism of TSHR activation was carried out by generation and structural analysis of TSHR transmembrane homology models and verification of model predictions by functional characterization of receptor mutations. RESULTS AND CONCLUSIONS: A newly discovered TSHR mutation L665F in transmembrane helix 7 of the receptor was detected in six members of this family. Functional characterization of L665F revealed constitutive activation for the Gs pathway and thus represents the molecular cause for hyperthyroidism in this family. The constitutive activation is possibly linked to a steric clash introduced by the L665F mutation between transmembrane helices 1 and 7.