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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 415 records · Page 23
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Structures of the Mycobacterium tuberculosis GlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) from Mycobacterium tuberculosis at 2.6 Å resolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase from Synechocystis (strain 6803) as well as the equivalent enzyme from Thermosynechococcus elongatus . This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII from Escherichia coli and is probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII from M. tuberculosis ( Mt FBPaseII) is conserved and is analogous to that described previously for the E. coli enzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures of Mt FBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for the Synechocystis enzyme. The structural and functional insights derived from the structure of Mt FBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.

Wolf, Nina M.↗
DOE OSTI2020

NAPAbench 2: A network synthesis algorithm for generating realistic protein-protein interaction (PPI) network families

Comparative network analysis provides effective computational means for gaining novel insights into the structural and functional compositions of biological networks. In recent years, various methods have been developed for biological network alignment, whose main goal is to identify important similarities and critical differences between networks in terms of their topology and composition. A major impediment to advancing network alignment techniques has been the lack of gold-standard benchmarks that can be used for accurate and comprehensive performance assessment of such algorithms. The original NAPAbench (network alignment performance assessment benchmark) was developed to address this problem, and it has been widely utilized by many researchers for the development, evaluation, and comparison of novel network alignment techniques. In this work, we introduce NAPAbench 2—a major update of the original NAPAbench that was introduced in 2012. NAPAbench 2 includes a completely redesigned network synthesis algorithm that can generate protein-protein interaction (PPI) network families whose characteristics closely match those of the latest real PPI networks. Furthermore, the network synthesis algorithm comes with an intuitive GUI that allows users to easily generate PPI network families with an arbitrary number of networks of any size, according to a flexible user-defined phylogeny. In addition, NAPAbench 2 provides updated benchmark datasets—created using the redesigned network synthesis algorithm—which can be used for comprehensive performance assessment of network alignment algorithms and their scalability.

42 ENGINEERING↗
DOE OSTI2020

In Silico Insights into Protein–Protein Interaction Disruptive Mutations in the PCSK9-LDLR Complex

Gain-of-function mutations in PCSK9 (proprotein convertase subtilisin/kexin type 9) lead to reduced uptake of LDL (low density lipoprotein) cholesterol and, therefore, increased plasma LDL levels. However, the mechanism by which these mutants reduce LDL reuptake is not fully understood. Here, we have used molecular dynamics simulations, MM/PBSA (Molecular Mechanics/Poisson–Boltzmann Surface Area) binding affinity calculations, and residue interaction networks, to investigate the protein–protein interaction (PPI) disruptive effects of two of PCSK9's gain-of-function mutations, Ser127Arg and Asp374Tyr on the PCSK9 and LDL receptor complex. In addition to these PPI disruptive mutants, a third, non-interface mutation (Arg496Trp) is included as a positive control. Our results indicate that Ser127Arg and Asp374Tyr confer significantly improved binding affinity, as well as different binding modes, when compared to the wild-type. These PPI disruptive mutations lie between the EGF(A) (epidermal growth factor precursor homology domain A) of the LDL receptor and the catalytic domain of PCSK9 (Asp374Tyr) and between the prodomain of PCSK9 and the β-propeller of the LDL receptor (Ser127Arg). The interactions involved in these two interfaces result in an LDL receptor that is sterically inhibited from entering its closed conformation. This could potentially implicate the prodomain as a target for small molecule inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Uncovering the basis of protein-protein interaction specificity with a combinatorially complete library

Protein-protein interaction specificity is often encoded at the primary sequence level. However, the contributions of individual residues to specificity are usually poorly understood and often obscured by mutational robustness, sequence degeneracy, and epistasis. Using bacterial toxin-antitoxin systems as a model, we screened a combinatorially complete library of antitoxin variants at three key positions against two toxins. This library enabled us to measure the effect of individual substitutions on specificity in hundreds of genetic backgrounds. These distributions allow inferences about the general nature of interface residues in promoting specificity. We find that positive and negative contributions to specificity are neither inherently coupled nor mutually exclusive. Further, a wild-type antitoxin appears optimized for specificity as no substitutions improve discrimination between cognate and non-cognate partners. By comparing crystal structures of paralogous complexes, we provide a rationale for our observations. Collectively, this work provides a generalizable approach to understanding the logic of molecular recognition.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1972

Detection of non-protein amino acids in the presence of protein amino acids. II.

Studies conducted with the JEOL 5AH amino acid analyzer are described. This instrument makes possible the programming of the chromatographic process. Data are presented showing the separations of seventeen non-protein amino acids in the presence of eighteen protein amino acids. It is pointed out that distinct separations could be obtained in the case of a number of chemically similar compounds, such as ornithine and lysine, N-amidino alanine and arginine, and iminodiacetic acid and S-carboxymethyl cysteine and aspartic acid.

Shapshak, P.↗
NASA NTRS1977

The evolution of the protein synthesis system. I - A model of a primitive protein synthesis system

A model is developed to describe the evolution of the protein synthesis system. The model is comprised of two independent autocatalytic systems, one including one gene (A-gene) and two activated amino acid polymerases (O and A-polymerases), and the other including the addition of another gene (N-gene) and a nucleotide polymerase. Simulation results have suggested that even a small enzymic activity and polymerase specificity could lead the system to the most accurate protein synthesis, as far as permitted by transitions to systems with higher accuracy.

Mizutani, H.↗
NASA NTRS1998

Green Fluorescent Protein as a Model for Protein Crystal Growth Studies

Green fluorescent protein (GFP) from jellyfish Aequorea Victoria has become a popular marker for e.g. mutagenesis work. Its fluorescent property, which originates from a chromophore located in the center of the molecule, makes it widely applicable as a research too]. GFP clones have been produced with a variety of spectral properties, such as blue and yellow emitting species. The protein is a single chain of molecular weight 27 kDa and its structure has been determined at 1.9 Angstrom resolution. The combination of GFP's fluorescent property, the knowledge of its several crystallization conditions, and its increasing use in biophysical and biochemical studies, all led us to consider it as a model material for macromolecular crystal growth studies. Initial preparations of GFP were from E.coli with yields of approximately 5 mg/L of culture media. Current yields are now in the 50 - 120 mg/L range, and we hope to further increase this by expression of the GFP gene in the Pichia system. The results of these efforts and of preliminary crystal growth studies will be presented.

Agena, Sabine↗
NASA NTRS2003

Website on Protein Interaction and Protein Structure Related Work

In today's world, three seemingly diverse fields - computer information technology, nanotechnology and biotechnology are joining forces to enlarge our scientific knowledge and solve complex technological problems. Our group is dedicated to conduct theoretical research exploring the challenges in this area. The major areas of research include: 1) Yeast Protein Interactions; 2) Protein Structures; and 3) Current Transport through Small Molecules.

Samanta, Manoj↗
DOE OSTI2017

Design of the First-in-Class, Highly Potent Irreversible Inhibitor Targeting the Menin-MLL Protein–Protein Interaction

The structure-based design of M-525 as the first-in-class, highly potent, irreversible small-molecule inhibitor of the menin-MLL interaction is presented. M-525 targets cellular menin protein at sub-nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition and in the suppression of MLL-regulated gene expression in MLL leukemia cells. M-525 demonstrates high cellular specificity over non-MLL leukemia cells and is more than 30 times more potent than its corresponding reversible inhibitors. Mass spectrometric analysis and co-crystal structure of M-525 in complex with menin firmly establish its mode of action. A single administration of M-525 effectively suppresses MLL-regulated gene expression in tumor tissue. An efficient procedure was developed to synthesize M-525. This study demonstrates that irreversible inhibition of menin may be a promising therapeutic strategy for MLL leukemia.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Insights from the Structure of Mycobacterium tuberculosis Topoisomerase I with a Novel Protein Fold

The DNA topoisomerase I enzyme of Mycobacterium tuberculosis (MtTOP1) is essential for the viability of the organism and survival in a murine model. This topoisomerase is being pursued as a novel target for the discovery of new therapeutic agents for the treatment of drug-resistant tuberculosis. In this study, we succeeded in obtaining a structure of MtTOP1 by first predicting that the C-terminal region of MtTOP1 contains four repeated domains that do not involve the Zn-binding tetracysteine motifs seen in the C-terminal domains of Escherichia coli topoisomerase I. A construct (amino acids A2-T704), MtTOP1-704t, that includes the N-terminal domains (D1-D4) and the first predicted C-terminal domain (D5) of MtTOP1 was expressed and found to retain DNA cleavage-religation activity and catalyze single-stranded DNA catenation. MtTOP1-704t was crystallized, and a structure of 2.52 angstrom resolution limit was obtained. The structure of the MtTOP1 N-terminal domains has features that have not been observed in other previously available bacterial topoisomerase I crystal structures. The first C-terminal domain D5 forms a novel protein fold of a four-stranded antiparallel beta-sheet stabilized by a crossing-over alpha-helix. Since there is only one type IA topoisomerase present in Mycobacteriaceae and related Actinobacteria, this subfamily of type IA topoisomerase may be required for multiple functions in DNA replication, transcription, recombination, and repair. The unique structural features observed for MtTOP1 may allow these topoisomerase I enzymes to carry out physiological functions associated with topoisomerase III enzyme in other bacteria.

crystallization↗
DOE OSTI2018

Synthesis and cytotoxicity of novel dispiro derivatives of 5-arylidenoxazolones, potential inhibitors of p53—MDM2 protein-protein interaction

Regioselective synthesis of new dispiro indolinones combining both an indolinone and an oxazolone fragment in their structure comprised the 1,3-dipolar cycloaddition of azomethine ylides, generated in situ from isatin and sarcosine, at 2-aryl-5-arylmethylidene-substituted 1,3-oxazol-5(4H)-ones. When ortho and para halogen atoms were present in the aromatic substituents of the starting oxazolones, complex mixtures containing large amounts of oxazoline ring opening products and their dispiro derivatives were formed. The cytotoxicity of compounds was tested by MTT on LNCaP, PC3, HCT116, MCF7, A549, HEK, and VA13 cell lines. The compound possessing the best cytotoxicity revealed the IC{sub 50} = 1.08±0.96 μM towards the p53- expressing LNCaP cells and lower activity (IC50 = 3.21±1.45 μM) towards the non-expressing p53 protein PC3 cells, however, it has proved inactive towards the HCT cells, both expressing (HCТ+/+) and non-expressing (HCT–/–) p53.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2013

Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor

The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3 with Raf-1 and proline-rich AKT substrate, 40 kD{sub a} and neutralized the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransferase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal structure of 14-3-3{zeta} in complex with FOBISIN101 was solved. Unexpectedly, the double bond linking the pyridoxal-phosphate and benzoate moieties was reduced by X-rays to create a covalent linkage of the pyridoxal-phosphate moiety to lysine 120 in the binding groove of 14-3-3, leading to persistent 14-3-3 inactivation. We suggest that FOBISIN101-like molecules could be developed as an entirely unique class of 14-3-3 inhibitors, which may serve as radiation-triggered therapeutic agents for the treatment of 14-3-3-mediated diseases, such as cancer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

The structures of penicillin-binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into β-lactam resistance

The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the cross-links in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of β-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly understood. PBP4 and PBP5 are low-affinity, class B transpeptidases that confer antibiotic resistance to Enterococcus faecalis and Enterococcus faecium, respectively. We report the crystal structures of PBP4 (1.8 Å) and PBP5 (2.7 Å) in their apo and acyl-enzyme complexes with the β-lactams benzylpenicillin, imipenem, and ceftaroline. We found that, although these three β-lactams adopt geometries similar to those observed in other class B PBP structures, there are small, but significant, differences that likely decrease antibiotic efficacy. Further, we also discovered that the N-terminal domain extensions in this class of PBPs undergo large rigid-body rotations without impacting the structure of the catalytic transpeptidase domain. Together, our findings are defining the subtle functional and structural differences in the Enterococcus PBPs that allow them to support transpeptidase activity while also conferring bacterial resistance to antibiotics that function as substrate mimics.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗