Structural basis for GPCR-independent activation of heterotrimeric Gi proteins.

Heterotrimeric G proteins are key molecular switches that control cell behavior. The canonical activation of G proteins by agonist-occupied G protein-coupled receptors (GPCRs) has recently been elucidated from the structural perspective. In contrast, the structural basis for GPCR-independent G protein activation by a novel family of guanine-nucleotide exchange modulators (GEMs) remains unknown. Here, we present a 2.0-Å crystal structure of Gαi in complex with the GEM motif of GIV/Girdin. Nucleotide exchange assays, molecular dynamics simulations, and hydrogen-deuterium exchange experiments demonstrate that GEM binding to the conformational switch II causes structural changes that allosterically propagate to the hydrophobic core of the Gαi GTPase domain. Rearrangement of the hydrophobic core appears to be a common mechanism by which GPCRs and GEMs activate G proteins, although with different efficiency. Atomic-level insights presented here will aid structure-based efforts to selectively target the noncanonical G protein activation.

A Modified Public Health Automated Case Event Reporting Platform for Enhancing Electronic Laboratory Reports With Clinical Data: Design and Implementation Study.

BACKGROUND: Public health reporting is the cornerstone of public health practices that inform prevention and control strategies. There is a need to leverage advances made in the past to implement an architecture that facilitates the timely and complete public health reporting of relevant case-related information that has previously not easily been available to the public health community. Electronic laboratory reporting (ELR) is a reliable method for reporting cases to public health authorities but contains very limited data. In an earlier pilot study, we designed the Public Health Automated Case Event Reporting (PACER) platform, which leverages existing ELR infrastructure as the trigger for creating an electronic case report. PACER is a FHIR (Fast Health Interoperability Resources)-based system that queries the electronic health record from where the laboratory test was requested to extract expanded additional information about a case. OBJECTIVE: This study aims to analyze the pilot implementation of a modified PACER system for electronic case reporting and describe how this FHIR-based, open-source, and interoperable system allows health systems to conduct public health reporting while maintaining the appropriate governance of the clinical data. METHODS: ELR to a simulated public health department was used as the trigger for a FHIR-based query. Predetermined queries were translated into Clinical Quality Language logics. Within the PACER environment, these Clinical Quality Language logical statements were managed and evaluated against the providers' FHIR servers. These predetermined logics were filtered, and only data relevant to that episode of the condition were extracted and sent to simulated public health agencies as an electronic case report. Design and testing were conducted at the Georgia Tech Research Institute, and the pilot was deployed at the Medical University of South Carolina. We evaluated this architecture by examining the completeness of additional information in the electronic case report, such as patient demographics, medications, symptoms, and diagnoses. This additional information is crucial for understanding disease epidemiology, but existing electronic case reporting and ELR architectures do not report them. Therefore, we used the completeness of these data fields as the metrics for enriching electronic case reports. RESULTS: During the 8-week study period, we identified 117 positive test results for chlamydia. PACER successfully created an electronic case report for all 117 patients. PACER extracted demographics, medications, symptoms, and diagnoses from 99.1% (116/117), 72.6% (85/117), 70.9% (83/117), and 65% (76/117) of the cases, respectively. CONCLUSIONS: PACER deployed in conjunction with electronic laboratory reports can enhance public health case reporting with additional relevant data. The architecture is modular in design, thereby allowing it to be used for any reportable condition, including evolving outbreaks. PACER allows for the creation of an enhanced and more complete case report that contains relevant case information that helps us to better understand the epidemiology of a disease.

Druggable exosites of the human kino-pocketome.

Small molecules binding at any of the multiple regulatory sites on the molecular surface of a protein kinase may stabilize or disrupt the corresponding interaction, leading to consequent modulation of the kinase cellular activity. As such, each of these sites represents a potential drug target. Even targeting sites outside the immediate ATP site, the so-called exosites, may cause desirable biological effects through an allosteric mechanism. Targeting exosites can alleviate adverse effects and toxicity that is common when ATP-site compounds bind promiscuously to many other types of kinases. In this study we have identified, catalogued, and annotated all potentially druggable exosites on the protein kinase domains within the existing structural human kinome. We then priority-ranked these exosites by those most amenable to drug design. In order to identify pockets that are either consistent across the kinome, or unique and specific to a particular structure, we have also implemented a normalized representation of all pockets, and displayed these graphically. Finally, we have built a database and designed a web-based interface for users interested in accessing the 3-dimensional representations of these pockets. We envision this information will assist drug discovery efforts searching for untargeted binding pockets in the human kinome.

Orphan receptor ligand discovery by pickpocketing pharmacological neighbors.

Understanding the pharmacological similarity of G protein-coupled receptors (GPCRs) is paramount for predicting ligand off-target effects, drug repurposing, and ligand discovery for orphan receptors. Phylogenetic relationships do not always correctly capture pharmacological similarity. Previous family-wide attempts to define pharmacological relationships were based on three-dimensional structures and/or known receptor-ligand pairings, both unavailable for orphan GPCRs. Here, we present GPCR-CoINPocket, a novel contact-informed neighboring pocket metric of GPCR binding-site similarity that is informed by patterns of ligand-residue interactions observed in crystallographically characterized GPCRs. GPCR-CoINPocket is applicable to receptors with unknown structure or ligands and accurately captures known pharmacological relationships between GPCRs, even those undetected by phylogeny. When applied to orphan receptor GPR37L1, GPCR-CoINPocket identified its pharmacological neighbors, and transfer of their pharmacology aided in discovery of the first surrogate ligands for this orphan with a 30% success rate. Although primarily designed for GPCRs, the method is easily transferable to other protein families.

HIV-1 Tat phosphorylation on Ser-16 residue modulates HIV-1 transcription.

BACKGROUND: HIV-1 transcription activator protein Tat is phosphorylated in vitro by CDK2 and DNA-PK on Ser-16 residue and by PKR on Tat Ser-46 residue. Here we analyzed Tat phosphorylation in cultured cells and its functionality. RESULTS: Mass spectrometry analysis showed primarily Tat Ser-16 phosphorylation in cultured cells. In vitro, CDK2/cyclin E predominantly phosphorylated Tat Ser-16 and PKR-Tat Ser-46. Alanine mutations of either Ser-16 or Ser-46 decreased overall Tat phosphorylation. Phosphorylation of Tat Ser-16 was reduced in cultured cells treated by a small molecule inhibitor of CDK2 and, to a lesser extent, an inhibitor of DNA-PK. Conditional knock-downs of CDK2 and PKR inhibited and induced one round HIV-1 replication respectively. HIV-1 proviral transcription was inhibited by Tat alanine mutants and partially restored by S16E mutation. Pseudotyped HIV-1 with Tat S16E mutation replicated well, and HIV-1 Tat S46E-poorly, but no live viruses were obtained with Tat S16A or Tat S46A mutations. TAR RNA binding was affected by Tat Ser-16 alanine mutation. Binding to cyclin T1 showed decreased binding of all Ser-16 and Ser-46 Tat mutants with S16D and Tat S46D mutationts showing the strongest effect. Molecular modelling and molecular dynamic analysis revealed significant structural changes in Tat/CDK9/cyclin T1 complex with phosphorylated Ser-16 residue, but not with phosphorylated Ser-46 residue. CONCLUSION: Phosphorylation of Tat Ser-16 induces HIV-1 transcription, facilitates binding to TAR RNA and rearranges CDK9/cyclin T1/Tat complex. Thus, phosphorylation of Tat Ser-16 regulates HIV-1 transcription and may serve as target for HIV-1 therapeutics.

Partially Assembled Nucleosome Structures at Atomic Detail.

The evidence is now overwhelming that partially assembled nucleosome states (PANS) are as important as the canonical nucleosome structure for the understanding of how accessibility to genomic DNA is regulated in cells. We use a combination of molecular dynamics simulation and atomic force microscopy to deliver, in atomic detail, structural models of three key PANS: the hexasome (H2A·H2B)·(H3·H4)2, the tetrasome (H3·H4)2, and the disome (H3·H4). Despite fluctuations of the conformation of the free DNA in these structures, regions of protected DNA in close contact with the histone core remain stable, thus establishing the basis for the understanding of the role of PANS in DNA accessibility regulation. On average, the length of protected DNA in each structure is roughly 18 basepairs per histone protein. Atomistically detailed PANS are used to explain experimental observations; specifically, we discuss interpretation of atomic force microscopy, Förster resonance energy transfer, and small-angle x-ray scattering data obtained under conditions when PANS are expected to exist. Further, we suggest an alternative interpretation of a recent genome-wide study of DNA protection in active chromatin of fruit fly, leading to a conclusion that the three PANS are present in actively transcribing regions in a substantial amount. The presence of PANS may not only be a consequence, but also a prerequisite for fast transcription in vivo.

Reactivation of latent HIV-1 provirus via targeting protein phosphatase-1.

BACKGROUND: HIV-1 escapes antiretroviral drugs by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus. This provirus presents the major hurdle in HIV-1 eradication and cure. Transcriptional activation, which is prerequisite for reactivation and the eradication of latent proviruses, is impaired in latently infected T cells due to the lack of host transcription factors, primarily NF-κB and P-TEFb (CDK9/cyclin T1). We and others previously showed that protein phosphatase-1 (PP1) regulates HIV-1 transcription by modulating CDK9 phosphorylation. Recently we have developed a panel of small molecular compounds targeting a non-catalytic site of PP1. RESULTS: Here we generated a new class of sulfonamide-containing compounds that activated HIV-1 in acute and latently infected cells. Among the tested molecules, a small molecule activator of PP1 (SMAPP1) induced both HIV-1 replication and reactivation of latent HIV-1 in chronically infected cultured and primary cells. In vitro, SMAPP1 interacted with PP1 and increased PP1 activity toward a recombinant substrate. Treatment with SMAPP1 increased phosphorylation of CDK9's Ser90 and Thr186 residues, but not Ser175. Proteomic analysis showed upregulation of P-TEFb and PP1 related proteins, including PP1 regulatory subunit Sds22 in SMAPP1-treated T cells. Docking analysis identified a PP1 binding site for SMAPP1 located within the C-terminal binding pocket of PP1. CONCLUSION: We identified a novel class of PP1-targeting compounds that reactivate latent HIV-1 provirus by targeting PP1, increasing CDK9 phosphorylation and enhancing HIV transcription. This compound represents a novel candidate for anti-HIV-1 therapeutics aiming at eradication of latent HIV-1 reservoirs.

PeptiSite: a structural database of peptide binding sites in 4D.

We developed PeptiSite, a comprehensive and reliable database of biologically and structurally characterized peptide-binding sites, in which each site is represented by an ensemble of its complexes with protein, peptide and small molecule partners. The unique features of the database include: (1) the ensemble site representation that provides a fourth dimension to the otherwise three dimensional data, (2) comprehensive characterization of the binding site architecture that may consist of a multimeric protein assembly with cofactors and metal ions and (3) analysis of consensus interaction motifs within the ensembles and identification of conserved determinants of these interactions. Currently the database contains 585 proteins with 650 peptide-binding sites. http://peptisite.ucsd.edu/ link allows searching for the sites of interest and interactive visualization of the ensembles using the ActiveICM web-browser plugin. This structural database for protein-peptide interactions enables understanding of structural principles of these interactions and may assist the development of an efficient peptide docking benchmark.

Pocketome: an encyclopedia of small-molecule binding sites in 4D.

The importance of binding site plasticity in protein-ligand interactions is well-recognized, and so are the difficulties in predicting the nature and the degree of this plasticity by computational means. To assist in understanding the flexible protein-ligand interactions, we constructed the Pocketome, an encyclopedia of about one thousand experimentally solved conformational ensembles of druggable binding sites in proteins, grouped by location and consistent chain/cofactor composition. The multiplicity of pockets within the ensembles adds an extra, fourth dimension to the Pocketome entry data. Within each ensemble, the pockets were carefully classified by the degree of their pairwise similarity and compatibility with different ligands. The core of the Pocketome is derived regularly and automatically from the current releases of the Protein Data Bank and the Uniprot Knowledgebase; this core is complemented by entries built from manually provided seed ligand locations. The Pocketome website (www.pocketome.org) allows searching for the sites of interest, analysis of conformational clusters, important residues, binding compatibility matrices and interactive visualization of the ensembles using the ActiveICM web browser plugin. The Pocketome collection can be used to build multi-conformational docking and 3D activity models as well as to design cross-docking and virtual ligand screening benchmarks.

CDK2 regulates HIV-1 transcription by phosphorylation of CDK9 on serine 90.

BACKGROUND: HIV-1 transcription is activated by the viral Tat protein that recruits host positive transcription elongation factor-b (P-TEFb) containing CDK9/cyclin T1 to the HIV-1 promoter. P-TEFb in the cells exists as a lower molecular weight CDK9/cyclin T1 dimer and a high molecular weight complex of 7SK RNA, CDK9/cyclin T1, HEXIM1 dimer and several additional proteins. Our previous studies implicated CDK2 in HIV-1 transcription regulation. We also found that inhibition of CDK2 by iron chelators leads to the inhibition of CDK9 activity, suggesting a functional link between CDK2 and CDK9. Here, we investigate whether CDK2 phosphorylates CDK9 and regulates its activity. RESULTS: The siRNA-mediated knockdown of CDK2 inhibited CDK9 kinase activity and reduced CDK9 phosphorylation. Stable shRNA-mediated CDK2 knockdown inhibited HIV-1 transcription, but also increased the overall level of 7SK RNA. CDK9 contains a motif (90SPYNR94) that is consensus CDK2 phosphorylation site. CDK9 was phosphorylated on Ser90 by CDK2 in vitro. In cultured cells, CDK9 phosphorylation was reduced when Ser90 was mutated to an Ala. Phosphorylation of CDK9 on Ser90 was also detected with phospho-specific antibodies and it was reduced after the knockdown of CDK2. CDK9 expression decreased in the large complex for the CDK9-S90A mutant and was correlated with a reduced activity and an inhibition of HIV-1 transcription. In contrast, the CDK9-S90D mutant showed a slight decrease in CDK9 expression in both the large and small complexes but induced Tat-dependent HIV-1 transcription. Molecular modeling showed that Ser 90 of CDK9 is located on a flexible loop exposed to solvent, suggesting its availability for phosphorylation. CONCLUSION: Our data indicate that CDK2 phosphorylates CDK9 on Ser 90 and thereby contributes to HIV-1 transcription. The phosphorylation of Ser90 by CDK2 represents a novel mechanism of HIV-1 regulated transcription and provides a new strategy for activation of latent HIV-1 provirus.

Automated Production of Research Data Marts from a Canonical Fast Healthcare Interoperability Resource (FHIR) Data Repository: Applications to COVID-19 Research.

OBJECTIVE: Objective: The COVID-19 pandemic has enhanced the need for timely real-world data (RWD) for research. To meet this need, several large clinical consortia have developed networks for access to RWD from electronic health records (EHR), each with its own common data model (CDM) and custom pipeline for extraction, transformation, and load operations for production and incremental updating. However, the demands of COVID-19 research for timely RWD (e.g., 2-week delay) make this less feasible. METHODS AND MATERIALS: We describe the use of the Fast Healthcare Interoperability Resource (FHIR) data model as a canonical model for representation of clinical data for automated transformation to the Patient-Centered Outcomes Research Network (PCORnet) and Observational Medical Outcomes Partnership (OMOP) CDMs and the near automated production of linked clinical data repositories (CDRs) for COVID-19 research using the FHIR subscription standard. The approach was applied to healthcare data from a large academic institution and was evaluated using published quality assessment tools. RESULTS: Six years of data (1.07M patients, 10.1M encounters, 137M laboratory results), were loaded into the FHIR CDR producing 3 linked real-time linked repositories: FHIR, PCORnet, and OMOP. PCORnet and OMOP databases were refined in subsequent post processing steps into production releases and met published quality standards. The approach greatly reduced CDM production efforts. CONCLUSIONS: FHIR and FHIR CDRs can play an important role in enhancing the availability of RWD from EHR systems. The above approach leverages 21 st Century Cures Act mandated standards and could greatly enhance the availability of datasets for research.

Automated production of research data marts from a canonical fast healthcare interoperability resource data repository: applications to COVID-19 research.

OBJECTIVE: The rapidly evolving COVID-19 pandemic has created a need for timely data from the healthcare systems for research. To meet this need, several large new data consortia have been developed that require frequent updating and sharing of electronic health record (EHR) data in different common data models (CDMs) to create multi-institutional databases for research. Traditionally, each CDM has had a custom pipeline for extract, transform, and load operations for production and incremental updates of data feeds to the networks from raw EHR data. However, the demands of COVID-19 research for timely data are far higher, and the requirements for updating faster than previous collaborative research using national data networks have increased. New approaches need to be developed to address these demands. METHODS: In this article, we describe the use of the Fast Healthcare Interoperability Resource (FHIR) data model as a canonical data model and the automated transformation of clinical data to the Patient-Centered Outcomes Research Network (PCORnet) and Observational Medical Outcomes Partnership (OMOP) CDMs for data sharing and research collaboration on COVID-19. RESULTS: FHIR data resources could be transformed to operational PCORnet and OMOP CDMs with minimal production delays through a combination of real-time and postprocessing steps, leveraging the FHIR data subscription feature. CONCLUSIONS: The approach leverages evolving standards for the availability of EHR data developed to facilitate data exchange under the 21st Century Cures Act and could greatly enhance the availability of standardized datasets for research.

AFM studies in diverse ionic environments of nucleosomes reconstituted on the 601 positioning sequence.

Atomic force microscopy (AFM) was used to study mononucleosomes reconstituted from a DNA duplex of 353 bp containing the strong 601 octamer positioning sequence, together with recombinant human core histone octamers. Three parameters were measured: 1) the length of DNA wrapped around the core histones; 2) the number of superhelical turns, calculated from the total angle through which the DNA is bent, and 3) the volume of the DNA-histone core. This approach allowed us to define in detail the structural diversity of nucleosomes caused by disassembly of the octasome to form subnucleosomal structures containing hexasomes, tetrasomes and disomes. At low ionic strength (TE buffer) and in the presence of physiological concentrations of monovalent cations, the majority of the particles were subnucleosomal, but physiological concentrations of bivalent cations resulted in about half of the nucleosomes being canonical octasomes in which the exiting DNA duplexes cross orthogonally. The dominance of this last species explains why bivalent but not monovalent cations can induce the initial step towards compaction and convergence of neighboring nucleosomes in nucleosomal arrays to form the chromatin fiber in the absence of linker histone. The observed nucleosome structural diversity may reflect the functional plasticity of nucleosomes under physiological conditions.

Quantum Mechanics Approaches to Drug Research in the Era of Structural Chemogenomics.

The rapid growth of the available crystallographic information about proteins and binding pockets creates remarkable opportunities for enriching the drug research pipelines with computational prediction of novel protein-ligand interactions. While ab initio quantum mechanical approaches are known to provide unprecedented accuracy in structure-based binding energy calculations, they are limited to only small systems of dozens of atoms. In the structural chemogenomics era, it is critical that new approaches are developed that enable application of QM methodologies to non-covalent interactions in systems as large as protein-ligand complexes and conformational ensembles. This perspective highlights recent advances towards bridging the gap between high accuracy and high volume computations in drug research.

Compound activity prediction using models of binding pockets or ligand properties in 3D.

Transient interactions of endogenous and exogenous small molecules with flexible binding sites in proteins or macromolecular assemblies play a critical role in all biological processes. Current advances in high-resolution protein structure determination, database development, and docking methodology make it possible to design three-dimensional models for prediction of such interactions with increasing accuracy and specificity. Using the data collected in the Pocketome encyclopedia, we here provide an overview of two types of the three-dimensional ligand activity models, pocketbased and ligand property-based, for two important classes of proteins, nuclear and G-protein coupled receptors. For half the targets, the pocket models discriminate actives from property matched decoys with acceptable accuracy (the area under ROC curve, AUC, exceeding 84%) and for about one fifth of the targets with high accuracy (AUC > 95%). The 3D ligand property field models performed better than 95% in half of the cases. The high performance models can already become a basis of activity predictions for new chemicals. Family-wide benchmarking of the models highlights strengths of both approaches and helps identify their inherent bottlenecks and challenges.