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Results for “Protein domains”

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At least 775 records · Page 43

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NASA NTRS1999

Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

NASA Discipline Plant Biology↗

DOE OSTI2021

Structural insight into host plasma membrane association and assembly of HIV-1 matrix protein

Oligomerization of Pr55 Gag is a critical step of the late stage of the HIV life cycle. It has been known that the binding of IP6, an abundant endogenous cyclitol molecule at the MA domain, has been linked to the oligomerization of Pr55 Gag . However, the exact binding site of IP6 on MA remains unknown and the structural details of this interaction are missing. Here, we present three high-resolution crystal structures of the MA domain in complex with IP6 molecules to reveal its binding mode. Additionally, extensive Differential Scanning Fluorimetry analysis combined with cryo- and ambient-temperature X-ray crystallography and GNM-based transfer entropy calculations identify the key residues that participate in IP6 binding. Our data provide novel insights about the multilayered HIV-1 virion assembly process that involves the interplay of IP6 with PIP2, a phosphoinositide essential for the binding of Pr55 Gag to membrane. IP6 and PIP2 have neighboring alternate binding sites within the same highly basic region (residues 18–33). This indicates that IP6 and PIP2 bindings are not mutually exclusive and may play a key role in coordinating virion particles’ membrane localization. Based on our three different IP6-MA complex crystal structures, we propose a new model that involves IP6 coordination of the oligomerization of outer MA and inner CA domain’s 2D layers during assembly and budding.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Comprehensive Genome-Wide Natural Variation and Expression Analysis of Tubby-like Proteins Gene Family in Brachypodium distachyon

The Tubby-like proteins (TLPs) gene family is a group of transcription factors found in both animals and plants. In this study, we identified twelve B. distachyon TLPs, divided into six groups based on conserved domains and evolutionary relationships. We predicted cis-regulatory elements involved in light, hormone, and biotic and abiotic stresses. The expression patterns in response to light and hormones revealed that BdTLP3, 4, 7, and 14 are involved in light responses, and BdTLP1 is involved in ABA responses. Furthermore, BdTLP2, 7, 9, and 13 are expressed throughout vegetative and reproductive stages, whereas BdTLP1, 3, 5, and 14 are expressed at germinating grains and early vegetative development, and BdTLP4, 6, 8, and 10 are expressed at the early reproduction stage. The natural variation in the eleven most diverged B. distachyon lines revealed high conservation levels of BdTLP1-6 to high variation in BdTLP7-14 proteins. Based on diversifying selection, we identified amino acids in BdTLP1, 3, 8, and 13, potentially substantially affecting protein functions. This analysis provided valuable information for further functional studies to understand the regulation, pathways involved, and mechanism of BdTLPs.

Plant Sciences↗

DOE OSTI2025

Correction to “COCOMO2: A Coarse-Grained Model for Interacting Folded and Disordered Proteins”

Biomolecular interactions are essential in many biological processes, including complex formation and phase separation processes. Coarse-grained computational models are especially valuable for studying such processes via simulation. Here, we present COCOMO2, an updated residue-based coarse-grained model that extends its applicability from intrinsically disordered peptides to folded proteins. This is accomplished with the introduction of a surface exposure scaling factor, which adjusts interaction strengths based on solvent accessibility, to enable the more realistic modeling of interactions involving folded domains without additional computational costs. COCOMO2 was parametrized directly with solubility and phase separation data to improve its performance on predicting concentration-dependent phase separation for a broader range of biomolecular systems compared to the original version. COCOMO2 enables new applications including the study of condensates that involve IDPs together with folded domains and the study of complex assembly processes. COCOMO2 also provides an expanded foundation for the development of multiscale approaches for modeling biomolecular interactions that span from residue-level to atomistic resolution.

Molecular interactions↗

NASA NTRS1992

Early events of polyoma infection: adsorption, penetration and nuclear transport

Polyoma virions have different attachment proteins which are responsible for hemagglutination of erythrocytes and attachment to cultured mouse kidney cells (MKC). Virion binding studies demonstrated that MKC possess specific (productive infection) and nonspecific (nonproductive) receptors. Empty polyoma capsids have hemagglutination activity and bind to non-specific MKC receptors, but they are not capable of competing for specific virion cell receptors or preventing productive infection. Isoelectric focusing of the virion major capsid protein, VP1, separated this protein into six species (A through F). These species had identical amino acid sequences, but differed in degree of modification (phosphorylation, acetylation, sulfation and hydroxylation). Evidence based upon precipitation with specific antisera supports the view that VP1 species E is required for specific adsorption and that D and F are required for hemagglutination. The virion attachment domain has been localized to an 18 kilodalton fragment of the C-terminal region of VP1. Monopinocytotic vesicles containing 125I-labeled polyoma virions were isolated from infected MKC. A crosslinker was used to bind the MKC cell receptor(s) covalently to VP1 attachment protein, and a new 120 kilodalton band was identified by SDS-PAGE. An anti-idiotype antibody prepared against a neutralizing polyoma monoclonal antiody was used to identify a putative 50 kilodalton receptor protein from a detergent extract of MKC, as well as from MKC membrane preparation.

NASA Discipline Developmental Biology↗

NASA NTRS1998

Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions

The extracellular matrix (ECM) activates signalling pathways that control cell behaviour by binding to cell-surface integrin receptors and inducing the formation of focal adhesion complexes (FACs). In addition to clustered integrins, FACs contain proteins that mechanically couple the integrins to the cytoskeleton and to immobilized signal-transducing molecules. Cell adhesion to the ECM also induces a rapid increase in the translation of preexisting messenger RNAs. Gene expression can be controlled locally by targeting mRNAs to specialized cytoskeletal domains. Here we investigate whether cell binding to the ECM promotes formation of a cytoskeletal microcompartment specialized for translational control at the site of integrin binding. High-resolution in situ hybridization revealed that mRNA and ribosomes rapidly and specifically localized to FACs that form when cells bind to ECM-coated microbeads. Relocation of these protein synthesis components to the FAC depended on the ability of integrins to mechanically couple the ECM to the contractile cytoskeleton and on associated tension-moulding of the actin lattice. Our results suggest a new type of gene regulation by integrins and by mechanical stress which may involve translation of mRNAs into proteins near the sites of signal reception.

Non-NASA Center↗

NASA NTRS2000

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

NASA Discipline Plant Biology↗

DOE OSTI2024

Unraveling the Molecular Origin of Prey-Wrapping Spider Silk's Unique Mechanical Properties and Assembly Process Using NMR

Prey wrapping spider silk's unique mechanical properties are investigated confirming the silk's high degree of extensibility and superior toughness compared to other types of spider silk. For the first time, the pre-spinning dope phase is studied in isotope-enriched intact aciniform (AC) silk glands using solution NMR that reveals a combination of α-helical domains linked by disordered random coil chains consistent with previously proposed “beads-on-a-string” models. The model is further refined through the AlphaFold2 protein structure prediction tool. Finally, extensive magic angle spinning (MAS) solid-state (SS) NMR data for isotopically-enriched fibers is used to refine the structural model for AC silk from two species, A. aurantia and A. argentata. The SSNMR data shows that the AC silk fibers are highly α-helical, coiled-coil in structure but, also exhibit significant β-sheet components that can be traced back to the Gly-rich disordered linker regions in the pre-spinning dope phase that are converted to β-sheet structures during fiber formation. This combination of mechanical and structural characterization enhances the understanding of AC silk's liquid-to-solid transition and structure-mechanics relationship. In conclusion, these prey wrap silk results and models will provide the basis for the design of biomimetic materials inspired by the AC spider silk system.

36 MATERIALS SCIENCE↗

DOE OSTI2026

The distinct trimeric structure of the immunodominant chlamydial antigen Major Outer Membrane Protein

The chlamydial Major Outer Membrane Protein (MOMP) is a promising subunit vaccine candidate due to its abundance in the outer membrane (OM), the presence of four surface-exposed variable domains (VDs) containing neutralizing and serotyping epitopes, and confirmed T-cell epitopes located in its constant domains (CDs). However, recombinant and denatured forms of MOMP have failed to elicit immune responses comparable to native preparations, indicating that conformation is critical for immunogenicity. Here, we present two cryo-EM structures of native Chlamydia muridarum MOMP, isolated from infectious elementary bodies (EBs). EB MOMP forms a distinct trimer with a stem of three narrow, non-permeable β-barrels and an extracellular, folded antigenic cap that displays VDs. In complex with a neutralizing Fab fragment of a conformational antibody, the cap undergoes structural reorganization that shows how epitope presentation is modulated by immune engagement. These structures reveal the molecular basis of MOMP’s serovar specificity and provide a template for structure-based vaccine design.

Guo, Yirui↗

DOE OSTI2023

Structural insights into plasmalemma vesicle-associated protein (PLVAP): Implications for vascular endothelial diaphragms and fenestrae

In many organs, small openings across capillary endothelial cells (ECs) allow the diffusion of low–molecular weight compounds and small proteins between the blood and tissue spaces. These openings contain a diaphragm composed of radially arranged fibers, and current evidence suggests that a single-span type II transmembrane protein, plasmalemma vesicle-associated protein-1 (PLVAP), constitutes these fibers. Here, we present the three-dimensional crystal structure of an 89-amino acid segment of the PLVAP extracellular domain (ECD) and show that it adopts a parallel dimeric alpha-helical coiled-coil configuration with five interchain disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing residues (sulfur SAD) to generate phase information. Biochemical and circular dichroism (CD) experiments show that a second PLVAP ECD segment also has a parallel dimeric alpha-helical configuration—presumably a coiled coil—held together with interchain disulfide bonds. Overall, ~2/3 of the ~390 amino acids within the PLVAP ECD adopt a helical configuration, as determined by CD. We also determined the sequence and epitope of MECA-32, an anti-PLVAP antibody. Taken together, these data lend strong support to the model of capillary diaphragms formulated by Tse and Stan in which approximately ten PLVAP dimers are arranged within each 60- to 80-nm-diameter opening like the spokes of a bicycle wheel. Passage of molecules through the wedge-shaped pores is presumably determined both by the length of PLVAP—i.e., the long dimension of the pore—and by the chemical properties of amino acid side chains and N-linked glycans on the solvent-accessible faces of PLVAP.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2000

Molecular and biochemical evidence for the involvement of calcium/calmodulin in auxin action

The use of (35)S-labeled calmodulin (CaM) to screen a corn root cDNA expression library has led to the isolation of a CaM-binding protein, encoded by a cDNA with sequence similarity to small auxin up RNAs (SAURs), a class of early auxin-responsive genes. The cDNA designated as ZmSAUR1 (Zea mays SAURs) was expressed in Escherichia coli, and the recombinant protein was purified by CaM affinity chromatography. The CaM binding assay revealed that the recombinant protein binds to CaM in a calcium-dependent manner. Deletion analysis revealed that the CaM binding site was located at the NH(2)-terminal domain. A synthetic peptide of amino acids 20-45, corresponding to the potential CaM binding region, was used for calcium-dependent mobility shift assays. The synthetic peptide formed a stable complex with CaM only in the presence of calcium. The CaM affinity assay indicated that ZmSAUR1 binds to CaM with high affinity (K(d) approximately 15 nM) in a calcium-dependent manner. Comparison of the NH(2)-terminal portions of all of the characterized SAURs revealed that they all contain a stretch of the basic alpha-amphiphilic helix similar to the CaM binding region of ZmSAUR1. CaM binds to the two synthetic peptides from the NH(2)-terminal regions of Arabidopsis SAUR-AC1 and soybean 10A5, suggesting that this is a general phenomenon for all SAURs. Northern analysis was carried out using the total RNA isolated from auxin-treated corn coleoptile segments. ZmSAUR1 gene expression began within 10 min, increased rapidly between 10 and 60 min, and peaked around 60 min after 10 microM alpha-naphthaleneacetic acid treatment. These results indicate that ZmSAUR1 is an early auxin-responsive gene. The CaM antagonist N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride inhibited the auxin-induced cell elongation but not the auxin-induced expression of ZmSAUR1. This suggests that calcium/CaM do not regulate ZmSAUR1 at the transcriptional level. CaM binding to ZmSAUR1 in a calcium-dependent manner suggests that calcium/CaM regulate ZmSAUR1 at the post-translational level. Our data provide the first direct evidence for the involvement of calcium/CaM-mediated signaling in auxin-mediated signal transduction.

NASA Program Fundamental Space Biology↗

DOE OSTI2022

Spatial Graph Attention and Curiosity-driven Policy for Antiviral Drug Discovery

We developed Distilled Graph Attention Policy Network (DGAPN), a reinforcement learning model to generate novel graph-structured chemical representations that optimize user-defined objectives by efficiently navigating a physically constrained domain. The framework is examined on the task of generating molecules that are designed to bind, noncovalently, to functional sites of SARS-CoV-2 proteins. We present a spatial Graph Attention (sGAT) mechanism that leverages self-attention over both node and edge attributes as well as encoding the spatial structure --- this capability is of considerable interest in synthetic biology and drug discovery. An attentional policy network is introduced to learn the decision rules for a dynamic, fragment-based chemical environment, and state-of-the-art policy gradient techniques are employed to train the network with stability. Exploration is driven by the stochasticity of the action space design and the innovation reward bonuses learned and proposed by random network distillation. In experiments, our framework achieved outstanding results compared to state-of-the-art algorithms, while reducing the complexity of paths to chemical synthesis.

Wu, Yulun↗

DOE OSTI2022

Spatial Graph Attention and Curiosity-driven Policy for Antiviral Drug Discovery

We developed Distilled Graph Attention Policy Networks (DGAPNs), a curiosity-driven reinforcement learning model to generate novel graph-structured chemical representations that optimize user-defined objectives by efficiently navigating a physically constrained domain. The framework is examined on the task of generating molecules that are designed to bind, noncovalently, to functional sites of SARS-CoV-2 proteins. We present a spatial Graph Attention Network (sGAT) that leverages self-attention over both node and edge attributes as well as encoding spatial structure -- this capability is of considerable interest in areas such as molecular and synthetic biology and drug discovery. An attentional policy network is then introduced to learn decision rules for a dynamic, fragment-based chemical environment, and state-of-the-art policy gradient techniques are employed to train the network with enhanced stability. Exploration is efficiently encouraged by incorporating innovation reward bonuses learned and proposed by random network distillation. In experiments, our framework achieved outstanding results compared to state-of-the-art algorithms, while increasing the diversity of proposed molecules and reducing the complexity of paths to chemical synthesis.

Wu, Yulun↗

DOE OSTI2022

SPATIAL GRAPH ATTENTION AND CURIOSITY-DRIVEN POLICY FOR ANTIVIRAL DRUG DISCOVERY

We developed Distilled Graph Attention Policy Network (DGAPN), a reinforcement learning model to generate novel graph-structured chemical representations that optimize user-defined objectives by efficiently navigating a physically constrained domain. The framework is examined on the task of generating molecules that are designed to bind, noncovalently, to functional sites of SARS-CoV-2 proteins. We present a spatial Graph Attention (sGAT) mechanism that leverages self-attention over both node and edge attributes as well as encoding the spatial structure - this capability is of considerable interest in synthetic biology and drug discovery. An attentional policy network is introduced to learn the decision rules for a dynamic, fragment-based chemical environment, and state-of-the-art policy gradient techniques are employed to train the network with stability. Exploration is driven by the stochasticity of the action space design and the innovation reward bonuses learned and proposed by random network distillation. In experiments, our framework achieved outstanding results compared to state-of-the-art algorithms, while reducing the complexity of paths to chemical synthesis.

Wu, Y↗

DOE OSTI2024

Osteocalcin binds to a GPRC6A Venus fly trap allosteric site to positively modulate GPRC6A signaling

GPRC6A, a member of the Family C G-protein coupled receptors, regulates energy metabolism and sex hormone production and is activated by diverse ligands, including cations, L-amino acids, the osteocalcin (Ocn) peptide and the steroid hormone testosterone. We sought a structural framework for the ability of multiple distinct classes of ligands to active GPRC6A. We created a structural model of GPRC6A using Alphafold2. Using this model we explored a putative orthosteric ligand binding site in the bilobed Venus fly trap (VFT) domain of GPRC6A and two positive allosteric modulator (PAM) sites, one in the VFT and the other in the 7 transmembrane (7TM) domain. We provide evidence that Ocn peptides act as a PAM for GPRC6A by binding to a site in the VFT that is distinct from the orthosteric site for calcium and L-amino acids. In agreement with this prediction, alternatively spliced GPRC6A isoforms 2 and 3, which lack regions of the VFT, and mutations in the computationally predicted Ocn binding site, K352E and H355P, prevent Ocn activation of GPRC6A. These observations explain how dissimilar ligands activate GPRC6A and set the stage to develop novel molecules to activate and inhibit this previously poorly understood receptor.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2000

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

Hong, Y. S.↗

DOE OSTI2025

COCOMO2: A Coarse-Grained Model for Interacting Folded and Disordered Proteins

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2022

Rational inhibitor design for Pseudomonas aeruginosa salicylate adenylation enzyme PchD

Pseudomonas aeruginosa is an increasingly antibiotic-resistant pathogen that causes severe lung infections, burn wound infections, and diabetic foot infections. P. aeruginosaproduces the siderophore pyochelin through the use of a non-ribosomal peptide synthetase (NRPS) biosynthetic pathway. Targeting members of siderophore NRPS proteins is one avenue currently under investigation for the development of new antibiotics against antibiotic-resistant organisms. Here, the crystal structure of the pyochelin adenylation domain PchD is reported. The structure was solved to 2.11 Å when co-crystallized with the adenylation inhibitor 5'-O-(N-salicylsulfamoyl)adenosine (salicyl-AMS) and to 1.69 Å with a modified version of salicyl-AMS designed to target an active site cysteine (4-cyano-salicyl-AMS). In the structures, PchD adopts the adenylation conformation, similar to that reported for AB3403 from Acinetobacter baumannii.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗