MutationExplorer: a webserver for mutation of proteins and 3D visualization of energetic impacts.

The possible effects of mutations on stability and function of a protein can only be understood in the context of protein 3D structure. The MutationExplorer webserver maps sequence changes onto protein structures and allows users to study variation by inputting sequence changes. As the user enters variants, the 3D model evolves, and estimated changes in energy are highlighted. In addition to a basic per-residue input format, MutationExplorer can also upload an entire replacement sequence. Previously the purview of desktop applications, such an upload can back-mutate PDB structures to wildtype sequence in a single step. Another supported variation source is human single nucelotide polymorphisms (SNPs), genomic coordinates input in VCF format. Structures are flexibly colorable, not only by energetic differences, but also by hydrophobicity, sequence conservation, or other biochemical profiling. Coloring by interface score reveals mutation impacts on binding surfaces. MutationExplorer strives for efficiency in user experience. For example, we have prepared 45 000 PDB depositions for instant retrieval and initial display. All modeling steps are performed by Rosetta. Visualizations leverage MDsrv/Mol*. MutationExplorer is available at: http://proteinformatics.org/mutation_explorer/.

Time-resolved cryo-EM of G-protein activation by a GPCR.

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit1. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory Gs protein in complex with the ß2-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein-receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events.

MUTATIONEXPLORER- A WEBSERVER FOR MUTATION OF PROTEINS AND 3D VISUALIZATION OF ENERGETIC IMPACTS.

The possible effects of mutations on stability and function of a protein can only be understood in the context of protein 3D structure. The MutationExplorer webserver maps sequence changes onto protein structures and allows users to study variation by inputting sequence changes. As the user enters variants, the 3D model evolves, and estimated changes in energy are highlighted. In addition to a basic per-residue input format, MutationExplorer can also upload an entire replacement sequence. Previously the purview of desktop applications, such an upload can back-mutate PDB structures to wildtype sequence in a single step. Another supported variation source is human single nucelotide polymorphisms (SNPs), genomic coordinates input in VCF format.

The adhesion G-protein-coupled receptor mayo/CG11318 controls midgut development in Drosophila.

Adhesion G-protein-coupled receptors (aGPCRs) form a large family of cell surface molecules with versatile tasks in organ development. Many aGPCRs still await their functional and pharmacological deorphanization. Here, we characterized the orphan aGPCR CG11318/mayo of Drosophila melanogaster and found it expressed in specific regions of the gastrointestinal canal and anal plates, epithelial specializations that control ion homeostasis. Genetic removal of mayo results in tachycardia, which is caused by hyperkalemia of the larval hemolymph. The hyperkalemic effect can be mimicked by a raise in ambient potassium concentration, while normal potassium levels in mayoKO mutants can be restored by pharmacological inhibition of potassium channels. Intriguingly, hyperkalemia and tachycardia are caused non-cell autonomously through mayo-dependent control of enterocyte proliferation in the larval midgut, which is the primary function of this aGPCR. These findings characterize the ancestral aGPCR Mayo as a homeostatic regulator of gut development.

7TM domain structures of adhesion GPCRs: what's new and what's missing?

Adhesion-type G protein-coupled receptors (aGPCRs) have long resisted approaches to resolve the structural details of their heptahelical transmembrane (7TM) domains. Single-particle cryogenic electron microscopy (cryo-EM) has recently produced aGPCR 7TM domain structures for ADGRD1, ADGRG1, ADGRG2, ADGRG3, ADGRG4, ADGRG5, ADGRF1, and ADGRL3. We review the unique properties, including the position and conformation of their activating tethered agonist (TA) and signaling motifs within the 7TM bundle, that the novel structures have helped to identify. We also discuss questions that the kaleidoscope of novel aGPCR 7TM domain structures have left unanswered. These concern the relative positions, orientations, and interactions of the 7TM and GPCR autoproteolysis-inducing (GAIN) domains with one another. Clarifying their interplay remains an important goal of future structural studies on aGPCRs.

Detecting Pre-Analytically Delayed Blood Samples for Laboratory Diagnostics Using Raman Spectroscopy.

In this proof-of-principle study, we systematically studied the potential of Raman spectroscopy for detecting pre-analytical delays in blood serum samples. Spectra from 330 samples from a liver cirrhosis cohort were acquired over the course of eight days, stored one day at room temperature, and stored subsequently at 4 °C. The spectra were then used to train Convolutional Neural Networks (CNN) to predict the delay to sample examination. We achieved 90% accuracy for binary classification of the serum samples in the groups "without delay" versus "delayed". Spectra recorded on the first day could be distinguished clearly from all subsequent measurements. Distinguishing between spectra taken in the range from the second to the last day seems to be possible as well, but currently, with an accuracy of approximately 70% only. Importantly, filtering out the fluorescent background significantly reduces the precision of detection.

Analysis of the Dynamics of the Human Growth Hormone Secretagogue Receptor Reveals Insights into the Energy Landscape of the Molecule.

G protein-coupled receptors initiate signal transduction in response to ligand binding. Growth hormone secretagogue receptor (GHSR), the focus of this study, binds the 28 residue peptide ghrelin. While structures of GHSR in different states of activation are available, dynamics within each state have not been investigated in depth. We analyze long molecular dynamics simulation trajectories using "detectors" to compare dynamics of the apo and ghrelin-bound states yielding timescale-specific amplitudes of motion. We identify differences in dynamics between apo and ghrelin-bound GHSR in the extracellular loop 2 and transmembrane helices 5-7. NMR of the GHSR histidine residues reveals chemical shift differences in these regions. We evaluate timescale specific correlation of motions between residues of ghrelin and GHSR, where binding yields a high degree of correlation for the first 8 ghrelin residues, but less correlation for the helical end. Finally, we investigate the traverse of GHSR over a rugged energy landscape via principal component analysis.

Function and dynamics of the intrinsically disordered carboxyl terminus of ß2 adrenergic receptor.

Advances in structural biology have provided important mechanistic insights into signaling by the transmembrane core of G-protein coupled receptors (GPCRs); however, much less is known about intrinsically disordered regions such as the carboxyl terminus (CT), which is highly flexible and not visible in GPCR structures. The ß2 adrenergic receptor's (ß2AR) 71 amino acid CT is a substrate for GPCR kinases and binds ß-arrestins to regulate signaling. Here we show that the ß2AR CT directly inhibits basal and agonist-stimulated signaling in cell lines lacking ß-arrestins. Combining single-molecule fluorescence resonance energy transfer (FRET), NMR spectroscopy, and molecular dynamics simulations, we reveal that the negatively charged ß2AR-CT serves as an autoinhibitory factor via interacting with the positively charged cytoplasmic surface of the receptor to limit access to G-proteins. The stability of this interaction is influenced by agonists and allosteric modulators, emphasizing that the CT plays important role in allosterically regulating GPCR activation.

SmoothT-a server constructing low-energy pathways from conformational ensembles for interactive visualization and enhanced sampling.

MOTIVATION: The SmoothT software and webservice offers the construction of pathways from an ensemble of conformations. The user provides an archive of molecule conformations in Protein Databank (PDB) format, from which a starting and a final conformation need to be selected. The individual PDB files need to contain an energy value or score, estimating the quality of the respective confirmation. Additionally, the user has to provide a root-mean-square deviation (RMSD) cut-off, below which conformations are considered neighboring. From this, SmoothT constructs a graph that connects similar conformations. RESULTS: SmoothT returns the energetically most favorable pathway within in this graph. This pathway is directly displayed as interactive animation using the NGL viewer. Simultaneously, the energy along the pathway is plotted, highlighting the conformation that is currently displayed in the 3D window. AVAILABILITY AND IMPLEMENTATION: SmoothT is available as webservice at: http://proteinformatics.org/smoothT. Examples, a tutorial, and FAQs can be found there. Ensembles up to 2 GB (compressed) can be uploaded. Results will be stored for 5 days. The server is completely free and requires no registration. The C++ source code is available at: https://github.com/starbeachlab/smoothT.

Biasing AlphaFold2 to predict GPCRs and kinases with user-defined functional or structural properties.

Determining the three-dimensional structure of proteins in their native functional states has been a longstanding challenge in structural biology. While integrative structural biology has been the most effective way to get a high-accuracy structure of different conformations and mechanistic insights for larger proteins, advances in deep machine-learning algorithms have paved the way to fully computational predictions. In this field, AlphaFold2 (AF2) pioneered ab initio high-accuracy single-chain modeling. Since then, different customizations have expanded the number of conformational states accessible through AF2. Here, we further expanded AF2 with the aim of enriching an ensemble of models with user-defined functional or structural features. We tackled two common protein families for drug discovery, G-protein-coupled receptors (GPCRs) and kinases. Our approach automatically identifies the best templates satisfying the specified features and combines those with genetic information. We also introduced the possibility of shuffling the selected templates to expand the space of solutions. In our benchmark, models showed the intended bias and great accuracy. Our protocol can thus be exploited for modeling user-defined conformational states in an automatic fashion.

Time-resolved cryo-EM of G protein activation by a GPCR.

G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating the exchange of guanine nucleotide in the Gα subunit. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G protein complex. Using variability analysis to monitor the transitions of the stimulatory Gs protein in complex with the ß 2 -adrenergic receptor (ß 2 AR) at short sequential time points after GTP addition, we identified the conformational trajectory underlying G protein activation and functional dissociation from the receptor. Twenty transition structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of events driving G protein activation upon GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα Switch regions and the α5 helix that weaken the G protein-receptor interface. Molecular dynamics (MD) simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP upon closure of the alpha-helical domain (AHD) against the nucleotide-bound Ras-homology domain (RHD) correlates with irreversible α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signaling events.

Towards Probing Conformational States of Y2 Receptor Using Hyperpolarized 129Xe NMR.

G protein-coupled receptors can adopt many different conformational states, each of them exhibiting different restraints towards downstream signaling pathways. One promising strategy to identify and quantify this conformational landscape is to introduce a cysteine at a receptor site sensitive to different states and label this cysteine with a probe for detection. Here, the application of NMR of hyperpolarized 129Xe for the detection of the conformational states of human neuropeptide Y2 receptor is introduced. The xenon trapping cage molecule cryptophane-A attached to a cysteine in extracellular loop 2 of the receptor facilitates chemical exchange saturation transfer experiments without and in the presence of native ligand neuropeptide Y. High-quality spectra indicative of structural states of the receptor-cage conjugate were obtained. Specifically, five signals could be assigned to the conjugate in the apo form. After the addition of NPY, one additional signal and subtle modifications in the persisting signals could be detected. The correlation of the spectroscopic signals and structural states was achieved with molecular dynamics simulations, suggesting frequent contact between the xenon trapping cage and the receptor surface but a preferred interaction with the bound ligand.

MDsrv: visual sharing and analysis of molecular dynamics simulations.

Molecular dynamics simulation is a proven technique for computing and visualizing the time-resolved motion of macromolecules at atomic resolution. The MDsrv is a tool that streams MD trajectories and displays them interactively in web browsers without requiring advanced skills, facilitating interactive exploration and collaborative visual analysis. We have now enhanced the MDsrv to further simplify the upload and sharing of MD trajectories and improve their online viewing and analysis. With the new instance, the MDsrv simplifies the creation of sessions, which allows the exchange of MD trajectories with preset representations and perspectives. An important innovation is that the MDsrv can now access and visualize trajectories from remote datasets, which greatly expands its applicability and use, as the data no longer needs to be accessible on a local server. In addition, initial analyses such as sequence or structure alignments, distance measurements, or RMSD calculations have been implemented, which optionally support visual analysis. Finally, based on Mol*, MDsrv now provides faster and more efficient visualization of even large trajectories compared to its predecessor tool NGL.

AlignMe: an update of the web server for alignment of membrane protein sequences.

The AlignMe web server is dedicated to accurately aligning sequences of membrane proteins, a particularly challenging task due to the strong evolutionary divergence and the low compositional complexity of hydrophobic membrane-spanning proteins. AlignMe can create pairwise alignments of either two primary amino acid sequences or two hydropathy profiles. The web server for AlignMe has been continuously available for >10 years, supporting 1000s of users per year. Recent improvements include anchoring, multiple submissions, and structure visualization. Anchoring is the ability to constrain a position in an alignment, which allows expert information about related residues in proteins to be incorporated into an alignment without manual modification. The original web interface to the server limited the user to one alignment per submission, hindering larger scale studies. Now, batches of alignments can be initiated with a single submission. Finally, to provide structural context for the relationship between proteins, sequence similarity can now be mapped onto one or more structures (or structural models) of the proteins being aligned, by links to MutationExplorer, a web-based visualization tool. Together with a refreshed user interface, these features further enhance an important resource in the membrane protein community. The AlignMe web server is freely available at https://www.bioinfo.mpg.de/AlignMe/.

Molecular Effects of Auto-Antibodies on Angiotensin II Type 1 Receptor Signaling and Cell Proliferation.

The angiotensin II (Ang II) type 1 receptor (AT1R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT1R can also be activated by auto-antibodies (AT1R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT1R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT1R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT1R signaling. AT1R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects Gq/11 and Gi activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT1R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT1R is impeded by binding of AT1R-Abs. Secondly, exclusive AT1R-Abs-induced Gq/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT1R activation of Gi signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of Gi basal activity potentiates proliferation triggered by AT1R-Abs. Finally, although AT1R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT1R and Abs. This current study thus provides extended insights into the molecular action of AT1R-Abs and associated mechanisms of interrelated pathogenesis.

A method to construct the dynamic landscape of a bio-membrane with experiment and simulation.

Biomolecular function is based on a complex hierarchy of molecular motions. While biophysical methods can reveal details of specific motions, a concept for the comprehensive description of molecular dynamics over a wide range of correlation times has been unattainable. Here, we report an approach to construct the dynamic landscape of biomolecules, which describes the aggregate influence of multiple motions acting on various timescales and on multiple positions in the molecule. To this end, we use 13C NMR relaxation and molecular dynamics simulation data for the characterization of fully hydrated palmitoyl-oleoyl-phosphatidylcholine bilayers. We combine dynamics detector methodology with a new frame analysis of motion that yields site-specific amplitudes of motion, separated both by type and timescale of motion. In this study, we show that this separation allows the detailed description of the dynamic landscape, which yields vast differences in motional amplitudes and correlation times depending on molecular position.

ProteinPrompt: a webserver for predicting protein-protein interactions.

Motivation: Protein-protein interactions (PPIs) play an essential role in a great variety of cellular processes and are therefore of significant interest for the design of new therapeutic compounds as well as the identification of side effects due to unexpected binding. Here, we present ProteinPrompt, a webserver that uses machine learning algorithms to calculate specific, currently unknown PPIs. Our tool is designed to quickly and reliably predict contact propensities based on an input sequence in order to scan large sequence libraries for potential binding partners, with the goal to accelerate and assure the quality of the laborious process of drug target identification. Results: We collected and thoroughly filtered a comprehensive database of known binders from several sources, which is available as download. ProteinPrompt provides two complementary search methods of similar accuracy for comparison and consensus building. The default method is a random forest (RF) algorithm that uses the auto-correlations of seven amino acid scales. Alternatively, a graph neural network (GNN) implementation can be selected. Additionally, a consensus prediction is available. For each query sequence, potential binding partners are identified from a protein sequence database. The proteom of several organisms are available and can be searched for binders. To evaluate the predictive power of the algorithms, we prepared a test dataset that was rigorously filtered for redundancy. No sequence pairs similar to the ones used for training were included in this dataset. With this challenging dataset, the RF method achieved an accuracy rate of 0.88 and an area under the curve of 0.95. The GNN achieved an accuracy rate of 0.86 using the same dataset. Since the underlying learning approaches are unrelated, comparing the results of RF and GNNs reduces the likelihood of errors. The consensus reached an accuracy of 0.89. Availability and implementation: ProteinPrompt is available online at: http://proteinformatics.org/ProteinPrompt, where training and test data used to optimize the methods are also available. The server makes it possible to scan the human proteome for potential binding partners of an input sequence within minutes. For local offline usage, we furthermore created a ProteinPrompt Docker image which allows for batch submission: https://gitlab.hzdr.de/proteinprompt/ProteinPrompt. In conclusion, we offer a fast, accurate, easy-to-use online service for predicting binding partners from an input sequence.

Structures of active melanocortin-4 receptor-Gs-protein complexes with NDP-α-MSH and setmelanotide.

The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R-Gs-protein complexes with two drugs recently approved by the FDA, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data from structure-derived MC4R mutants, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6-regulated receptor activation. The ligand-binding modes of NDP-α-MSH, a high-affinity linear variant of the endogenous agonist α-MSH, and setmelanotide, a cyclic anti-obesity drug with biased signaling toward Gq/11, underline the key role of TM3 in ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-specific TM3 interplay subsequently impacts receptor-Gs-protein interfaces at intracellular loop 2, which also regulates the G-protein coupling profile of this promiscuous receptor. Finally, our structures reveal mechanistic details of MC4R activation/inhibition, and provide important insights into the regulation of the receptor signaling profile which will facilitate the development of tailored anti-obesity drugs.

Voronoia 4-ever.

We present an updated version of the Voronoia service that enables fully automated analysis of the atomic packing density of macromolecules. Voronoia combines previous efforts to analyse 3D protein and RNA structures into a single service, combined with state-of-the-art online visualization. Voronoia uses the Voronoi cell method to calculate the free space between neighbouring atoms to estimate van der Waals interactions. Compared to other methods that derive van der Waals interactions by calculating solvent-free surfaces, it explicitly considers volume or packing defects. Large internal voids refer either to water molecules or ions unresolved by X-ray crystallography or cryo-EM, cryptic ligand binding pockets, or parts of a structural model that require further refinement. Voronoia is, therefore mainly used for functional analyses of 3D structures and quality assessments of structural models. Voronoia 4-ever updates the database of precomputed packing densities of PDB entries, allows uploading multiple structures, adds new filter options and facilitates direct access to the results through intuitive display with the NGL viewer. Voronoia is available at: htttp://proteinformatics.org/voronoia.

Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain.

Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.