NMR-Based and Automated Docking Characterization of Protein Structure, Dynamics, and Ligand Binding

NMR-based methods used in conjunction with a technique called docking are used to characterize ligand binding to proteins. Standard NMR methods were used to study the backbone dynamics of substrate binding to phosphomevalonate kinase (PMK) and it was observed that ligand binding caused PMK to undergo large conformational changes. These changes were reflected by the appearance of many chemical shift changes upon binding of the natural substrates of PMK (both the binary and ternary complexes) in 1H-15N HSQC NMR titration experiments. The same process was used to characterize the effect ligand binding has on the many arginines in the active site (and distal to the active site) to determine the effect of long-range coulombic interactions on ligand binding. While studying the backbone dynamics of PMK it was discovered that the N-terminal tail of PMK consisting of 10 residues was very disordered which is unlike every other monphosphate kinase. The function of this N-terminal tail was investigated by attempting to find other proteins in human liver cells that bind this peptide, monitored by ESI mass spectrometry. The thioredoxin system of Mycobacterium Tuberculosis consists of a thioredoxin reductase and three thioredoxins. To help facilitate the understanding of this mechanism the solution structures of the oxidized and the reduced forms of thioredoxin C (TrxC) were solved by NMR and modeled with the crystal structure of the thioredoxin reductase complex. The two redox states of TrxC are very similar to each other with most of the differences coming from subtle changes in the active site of TrxC. Automated docking is the process of computationally determining how a ligand binds to a protein and the correct orientation. A large scale docking study, termed virtual screening, was carried out by docking 10,590 compounds into three proteins to find inhibitors for each protein, and those predicted to bind best were tested experimentally. For each protein there were 3 compounds found to bind with reasonable affinity. When ligands bind to a protein they can undergo dynamic changes. To explore this phenomenon, 15N labeled NAD+ cofactor (and other derivatives) was synthesized and bound to oxidoreductases. Relevant binding motions were monitored using CPMG relaxation NMR experiments

Solution Structures of \u3cem\u3eMycobacterium tuberculosis\u3c/em\u3e Thioredoxin C and Models of Intact Thioredoxin System Suggest New Approaches to Inhibitor and Drug Design

Here, we report the NMR solution structures of Mycobacterium tuberculosis (M. tuberculosis) thioredoxin C in both oxidized and reduced states, with discussion of structural changes that occur in going between redox states. The NMR solution structure of the oxidized TrxC corresponds closely to that of the crystal structure, except in the C-terminal region. It appears that crystal packing effects have caused an artifactual shift in the α4 helix in the previously reported crystal structure, compared with the solution structure. On the basis of these TrxC structures, chemical shift mapping, a previously reported crystal structure of the M. tuberculosis thioredoxin reductase (not bound to a Trx) and structures for intermediates in the E. coli thioredoxin catalytic cycle, we have modeled the complete M. tuberculosis thioredoxin system for the various steps in the catalytic cycle. These structures and models reveal pockets at the TrxR/TrxC interface in various steps in the catalytic cycle, which can be targeted in the design of uncompetitive inhibitors as potential anti-mycobacterial agents, or as chemical genetic probes of function

Substrate Induced Structural and Dynamics Changes in Human Phosphomevalonate Iinase and Implications for Mechanism

Phosphomevalonate kinase (PMK) catalyzes an essential step in the mevalonate pathway, which is the only pathway for synthesis of isoprenoids and steroids in humans. PMK catalyzes transfer of the γ-phosphate of ATP to mevalonate 5-phosphate (M5P) to form mevalonate 5-diphosphate. Bringing these phosphate groups in proximity to react is especially challenging, given the high negative charge density on the four phosphate groups in the active site. As such, conformational and dynamics changes needed to form the Michaelis complex are of mechanistic interest. Herein, we report the characterization of substrate induced changes (Mg-ADP, M5P, and the ternary complex) in PMK using NMR-based dynamics and chemical shift perturbation measurements. Mg-ADP and M5P Kd\u27s were 6–60 μM in all complexes, consistent with there being little binding synergy. Binding of M5P causes the PMK structure to compress (τc = 13.5 nsec), whereas subsequent binding of Mg-ADP opens the structure up (τc = 15.6 nsec). The overall complex seems to stay very rigid on the psec-nsec timescale with an average NMR order parameter of S2 ∼0.88. Data are consistent with addition of M5P causing movement around a hinge region to permit domain closure, which would bring the M5P domain close to ATP to permit catalysis. Dynamics data identify potential hinge residues as H55 and R93, based on their low order parameters and their location in extended regions that connect the M5P and ATP domains in the PMK homology model. Likewise, D163 may be a hinge residue for the lid region that is homologous to the adenylate kinase lid, covering the “Walker-A” catalytic loop. Binding of ATP or ADP appears to cause similar conformational changes; however, these observations do not indicate an obvious role for γ-phosphate binding interactions. Indeed, the role of γ-phosphate interactions may be more subtle than suggested by ATP/ADP comparisons, because the conservative O to NH substitution in the β-γ bridge of ATP causes a dramatic decrease in affinity and induces few chemical shift perturbations. In terms of positioning of catalytic residues, binding of M5P induces a rigidification of Gly21 (adjacent to the catalytically important Lys22), although exchange broadening in the ternary complex suggests some motion on a slower timescale does still occur. Finally, the first nine residues of the N-terminus are highly disordered, suggesting that they may be part of a cleavable signal or regulatory peptide sequence. Proteins 2009. © 2008 Wiley-Liss, Inc

Molecular Docking and NMR Binding Studies to Identify Novel Inhibitors of Human Phosphomevalonate Kinase

Phosphomevalonate kinase (PMK) phosphorylates mevalonate-5-phosphate (M5P) in the mevalonate pathway, which is the sole source of isoprenoids and steroids in humans. We have identified new PMK inhibitors with virtual screening, using autodock. Promising hits were verified and their affinity measured using NMR-based 1H–15N heteronuclear single quantum coherence (HSQC) chemical shift perturbation and fluorescence titrations. Chemical shift changes were monitored, plotted, and fitted to obtain dissociation constants (Kd). Tight binding compounds with Kd’s ranging from 6–60 μM were identified. These compounds tended to have significant polarity and negative charge, similar to the natural substrates (M5P and ATP). HSQC cross peak changes suggest that binding induces a global conformational change, such as domain closure. Compounds identified in this study serve as chemical genetic probes of human PMK, to explore pharmacology of the mevalonate pathway, as well as starting points for further drug development

Microstructural Evolution in Thin Films of Electronic Materials

Contains reports on eight research projects.National Science Foundation (Grant ECS 85-06565)U.S. Air Force - Office of Scientific Research (Contract AFOSR 85-0154)National Science Foundation-Materials Research Laboratory(Grant DMR 81-19285)National Science Foundation (Grant DMR 85-06030)International Business Machines, Inc. Faculty Development AwardMitsui Career Development AwardInternational Business Machines, Inc.Semiconductor Research Corporation (Contract 86-05-080)Joint Services Electronics Program (Contract DAAG-29-83-K-0003)Charles Stark Draper LaboratoryDefense Advanced Research Projects Agency (DARPA)Nippon Telegraph and Telephone, Inc

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment

Comparative analysis of multiple inducible phages from Mannheimia haemolytica

© 2015 Niu et al. Background: Mannheimia haemolytica is a commensal bacterium that resides in the upper respiratory tract of cattle that can play a role in bovine respiratory disease. Prophages are common in the M. haemolytica genome and contribute significantly to host diversity. The objective of this research was to undertake comparative genomic analysis of phages induced from strains of M. haemolytica serotype A1 (535A and 2256A), A2 (587A and 1127A) and A6 (1152A and 3927A). Results: Overall, four P2-like (535AP1, 587AP1, 1127AP1 and 2256AP1; genomes: 34.9-35.7 kb; G+C content: 41.5-42.1 %; genes: 51-53 coding sequences, CDSs), four λ-like (535AP2, 587AP2, 1152AP2 and 3927AP1; genomes: 48.6-52.1 kb; 41.1-41.4 % mol G+C; genes: 77-83 CDSs and 2 tRNAs) and one Mu-like (3927AP2; genome: 33.8 kb; 43.1 % mol G+C; encoding 50 CDSs) phages were identified. All P2-like phages are collinear with the temperate phage φMhaA1-PHL101 with 535AP1, 2256AP1 and 1152AP1 being most closely related, followed by 587AP1 and 1127AP1. Lambdoid phages are not collinear with any other known λ-type phages, with 587AP2 being distinct from 535AP2, 3927AP1 and 1152AP2. All λ-like phages contain genes encoding a toxin-antitoxin (TA) system and cell-associated haemolysin XhlA. The Mu-like phage induced from 3927A is closely related to the phage remnant φMhaMu2 from M. haemolytica PHL21, with similar Mu-like phages existing in the genomes of M. haemolytica 535A and 587A. Conclusions: This is among the first reports of both λ- and Mu-type phages being induced from M. haemolytica. Compared to phages induced from commensal strains of M. haemolytica serotype A2, those induced from the more virulent A1 and A6 serotypes are more closely related. Moreover, when P2-, λ- and Mu-like phages co-existed in the M. haemolytica genome, only P2- and λ-like phages were detected upon induction, suggesting that Mu-type phages may be more resistant to induction. Toxin-antitoxin gene cassettes in λ-like phages may contribute to their genomic persistence or the establishment of persister subpopulations of M. haemolytica. Further work is required to determine if the cell-associated haemolysin XhlA encoded by λ-like phages contributes to the pathogenicity and ecological fitness of M. haemolytica