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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 343 records · Page 19
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Prediction of inter-chain distance maps of protein complexes with 2D attention-based deep neural networks

Residue-residue distance information is useful for predicting tertiary structures of protein monomers or quaternary structures of protein complexes. Many deep learning methods have been developed to predict intra-chain residue-residue distances of monomers accurately, but few methods can accurately predict inter-chain residue-residue distances of complexes. We develop a deep learning method CDPred (i.e., Complex Distance Prediction) based on the 2D attention-powered residual network to address the gap. Tested on two homodimer datasets, CDPred achieves the precision of 60.94% and 42.93% for top L/5 inter-chain contact predictions (L: length of the monomer in homodimer), respectively, substantially higher than DeepHomo’s 37.40% and 23.08% and GLINTER’s 48.09% and 36.74%. Tested on the two heterodimer datasets, the top Ls/5 inter-chain contact prediction precision (Ls: length of the shorter monomer in heterodimer) of CDPred is 47.59% and 22.87% respectively, surpassing GLINTER’s 23.24% and 13.49%. Moreover, the prediction of CDPred is complementary with that of AlphaFold2-multimer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Real-time structural motif searching in proteins using an inverted index strategy

Biochemical and biological functions of proteins are the product of both the overall fold of the polypeptide chain, and, typically, structural motifs made up of smaller numbers of amino acids constituting a catalytic center or a binding site that may be remote from one another in amino acid sequence. Detection of such structural motifs can provide valuable insights into the function(s) of previously uncharacterized proteins. Technically, this remains an extremely challenging problem because of the size of the Protein Data Bank (PDB) archive. Existing methods depend on a clustering by sequence similarity and can be computationally slow. We have developed a new approach that uses an inverted index strategy capable of analyzing >170,000 PDB structures with unmatched speed. The efficiency of the inverted index method depends critically on identifying the small number of structures containing the query motif and ignoring most of the structures that are irrelevant. Our approach (implemented at motif.rcsb.org ) enables real-time retrieval and superposition of structural motifs, either extracted from a reference structure or uploaded by the user. Herein, we describe the method and present five case studies that exemplify its efficacy and speed for analyzing 3D structures of both proteins and nucleic acids.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Thermodynamic Basis for the Stabilization of Helical Peptoids by Chiral Sidechains

Peptoids are a class of highly customizable biomimetic foldamers that retain properties from both proteins and polymers. It has been shown that peptoids can adopt peptide-like secondary structures through the careful selection of sidechain chemistries, but the underlying conformational landscapes that drive these assemblies at the molecular level remain poorly understood. Given the high flexibility of the peptoid backbone, it is essential that methods applied to study peptoid secondary structure formation possess the requisite sensitivity to discriminate between structurally similar yet energetically distinct microstates. In this work, a generalizable simulation scheme is used to robustly sample the complex folding landscape of various 12mer polypeptoids, resulting in a predictive model that links sidechain chemistry with preferential assembly into one of 12 accessible backbone motifs. Using a variant of the metadynamics sampling method, four peptoid dodecamers are simulated in water: sarcosine, N-(1-phenylmethyl)glycine (Npm), (S)-N-(1-phenylethyl)glycine (Nspe), and (R)-N-(1-phenylethyl)glycine (Nrpe)–to determine the underlying entropic and energetic impacts of hydrophobic and chiral peptoid sidechains on secondary structure formation. Our results indicate that the driving forces to assemble Nrpe and Nspe sequences into polyproline type-I helices in water are found to be enthalpically driven, with small benefits from an entropic gain for isomerization and steric strain due to the presence of the chiral center. The minor entropic gains from bulky chiral sidechains in Nrpe- and Nspe-containing peptoids can be explained through increased configurational entropy in the cis state. However, overall assembly into a helix is found to be overall entropically unfavorable. Furthermore, these results highlight the importance of considering the many various competing interactions in the rational design of peptoid secondary structure building blocks.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

SEC ‐ SAXS / MC Ensemble Structural Studies of the Microtubule Binding Protein Cdt1 Show Monomeric, Folded‐Over Conformations

ABSTRACT Cdt1 is a mixed folded protein critical for DNA replication licensing and it also has a “moonlighting” role at the kinetochore via direct binding to microtubules and the Ndc80 complex. However, it is unknown how the structure and conformations of Cdt1 could allow it to participate in these multiple, unique sets of protein complexes. While robust methods exist to study entirely folded or unfolded proteins, structure–function studies of combined, mixed folded/disordered proteins remain challenging. In this work, we employ orthogonal biophysical and computational techniques to provide structural characterization of mitosis‐competent human Cdt1. Thermal stability analyses shows that both folded winged helix domains1 are unstable. CD and NMR show that the N‐terminal and linker regions are intrinsically disordered. DLS shows that Cdt1 is monomeric and polydisperse, while SEC‐MALS confirms that it is monomeric at high concentrations, but without any apparent inter‐molecular self‐association. SEC‐SAXS enabled computational modeling of the protein structures. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble of structures. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with SAXS. The best‐fit models have the N‐terminal and linker disordered regions extended into the solution and the two folded domains close to each other in apparent “folded over” conformations. We hypothesize the best‐fit Cdt1 conformations could be consistent with a function as a scaffold protein that may be sterically blocked without binding partners. Our study also provides a template for combining experimental and computational techniques to study mixed‐folded proteins.

Cell Biology↗
DOE OSTI2021

Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies)

We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of ~120 kDa, called Legobody, does not perturb the nanobody–target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Å overall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Å resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The Nucleoid-Associated Protein GapR Uses Conserved Structural Elements To Oligomerize and Bind DNA

Nucleoid-associated proteins (NAPs) are DNA binding proteins critical for the organization and function of the bacterial chromosome. A newly discovered NAP in Caulobacter crescentus, GapR, is thought to facilitate the movement of the replication and transcription machines along the chromosome by stimulating type II topoisomerases to remove positive supercoiling. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recently published DNA-bound crystal structure of GapR, we identified the structural elements involved in oligomerization and DNA binding. Moreover, we show that GapR is maintained as a tetramer upon its dissociation from DNA and that tetrameric GapR is capable of binding DNA molecules in vitro. Analysis of protein chimeras revealed that two helices of GapR are functionally conserved in H-NS, demonstrating that two evolutionarily distant NAPs with distinct mechanisms of action utilize conserved structural elements to oligomerize and bind DNA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Cryo-EM structure determination of small therapeutic protein targets at 3 Å-resolution using a rigid imaging scaffold

Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller proteins. Here, we obtain structures of small proteins by binding them to a rigid molecular scaffold based on a designed protein cage, revealing atomic details at resolutions reaching 2.9 Å. We apply this system to the key cancer signaling protein KRAS (19 kDa in size), obtaining four structures of oncogenic mutational variants by cryo-EM. Importantly, a structure for the key G12C mutant bound to an inhibitor drug (AMG510) reveals significant conformational differences compared to prior data in the crystalline state. The findings highlight the promise of cryo-EM scaffolds for advancing the design of drug molecules against small therapeutic protein targets in cancer and other human diseases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Frameshifting Stimulatory Sequence Induces Large Structural Change of Ribosomal Proteins When Bound to E. coli Ribosomes

Biological macromolecular machines occupy a continuum of structural conformations to perform cellular tasks. Mapping this conformational space provides an insight into its functionality. While the cryo-electron microscopy resolution revolution has expanded our ability to characterize the conformational continuums, there are obstacles in structurally characterizing regions of high flexibility. These technical barriers have impeded characterization of flexible ribosomal proteins when the ribosome is interacting with mRNA stem-loop structures such as a frameshifting stimulatory sequence (FSS). Small-angle neutron/X-ray scattering and electron microscopy were used to study ribosomal samples and compared structural differences between a ribosome that is bound to an FSS stem-loop compared to a ribosome bound to linear mRNA. This comparison shows that a large protein stalk elongates by 22% when the 70S interacts with an mRNA stem-loop. Finally, our results suggest that ribosomal proteins have extensive flexibility and may influence important ribosomal mechanisms, such as those that involve FSS.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Structure of a tripartite protein complex that targets toxins to the type VII secretion system

Type VII secretion systems are membrane-embedded nanomachines used by Gram-positive bacteria to export effector proteins from the cytoplasm to the extracellular environment. Many of these effectors are polymorphic toxins comprised of an N-terminal Leu-x-Gly (LXG) domain of unknown function and a C-terminal toxin domain that inhibits the growth of bacterial competitors. In recent work, it was shown that LXG effectors require two cognate Lap proteins for T7SS-dependent export. Here, we present the 2.6 Å structure of the LXG domain of the TelA toxin from the opportunistic pathogen Streptococcus intermedius in complex with both of its cognate Lap targeting factors. The structure reveals an elongated α-helical bundle within which each Lap protein makes extensive hydrophobic contacts with either end of the LXG domain. Remarkably, despite low overall sequence identity, we identify striking structural similarity between our LXG complex and PE–PPE heterodimers exported by the distantly related ESX type VII secretion systems of Mycobacteria implying a conserved mechanism of effector export among diverse Gram-positive bacteria. Overall, our findings demonstrate that LXG domains, in conjunction with their cognate Lap targeting factors, represent a tripartite secretion signal for a widespread family of T7SS toxins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

CryoTransformer: a transformer model for picking protein particles from Cryo-EM micrographs

Cryo-electron microscopy (cryo-EM) is a powerful technique for determining the structures of large protein complexes. Picking single protein particles from cryo-EM micrographs (images) is a crucial step in reconstructing protein structures from them. However, the widely used template-based particle picking process requires some manual particle picking and is labor-intensive and time-consuming. Though machine learning and artificial intelligence (AI) can potentially automate particle picking, the current AI methods pick particles with low precision or low recall. The erroneously picked particles can severely reduce the quality of reconstructed protein structures, especially for the micrographs with low signal-to-noise-ratio (SNR).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Classical Swine Fever Virus Structural Glycoprotein E2 Interacts with Host Protein ACADM during the Virus Infectious Cycle

The E2 glycoprotein is one of the four structural proteins of the classical swine fever virus (CSFV) particle. E2 has been shown to be involved in many virus functions, including adsorption to host cells, virus virulence and interaction with several host proteins. Using a yeast two-hybrid screen, we have previously shown that the CSFV E2 specifically interacts with swine host protein medium-chain-specific acyl-Coenzyme A dehydrogenase (ACADM), an enzyme that catalyzes the initial step of the mitochondrial fatty acid beta-oxidation pathway. Here, we show that interaction between ACADM and E2 also happens in swine cells infected with CSFV using two different procedures: coimmunoprecipitation and a proximity ligation assay (PLA). In addition, the amino acid residues in E2 critically mediating the interaction with ACADM, M49 and P130 were identified via a reverse yeast two-hybrid screen using an expression library composed of randomly mutated versions of E2. A recombinant CSFV, E2ΔACADMv, harboring substitutions at residues M49I and P130Q in E2, was developed via reverse genomics from the highly virulent Brescia isolate. E2ΔACADMv was shown to have the same kinetics growth in swine primary macrophages and SK6 cell cultures as the parental Brescia strain. Similarly, E2ΔACADMv demonstrated a similar level of virulence when inoculated to domestic pigs as the parental Brescia. Animals intranasally inoculated with 10 5 TCID 50 developed a lethal form of clinical disease with virological and hematological kinetics changes undistinguishable from those produced by the parental strain. Therefore, interaction between CSFV E2 and host ACADM is not critically involved in the processes of virus replication and disease production.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Artificial intelligence techniques for integrative structural biology of intrinsically disordered proteins

Here, we outline recent developments in artificial intelligence (AI) and machine learning (ML) techniques for integrative structural biology of intrinsically disordered proteins (IDP) ensembles. IDPs challenge the traditional protein structure-function paradigm by adapting their conformations in response to specific binding partners leading them to mediate diverse, and often complex cellular functions such as biological signaling, self-organization and compartmentalization. Obtaining mechanistic insights into their function can therefore be challenging for traditional structural determination techniques. Often, scientists have to rely on piecemeal evidence drawn from diverse experimental techniques to characterize their functional mechanisms. Multiscale simulations can help bridge critical knowledge gaps about IDP structure-function relationships - however, these techniques also face challenges in resolving emergent phenomena within IDP conformational ensembles. We posit that scalable statistical inference techniques can effectively integrate information gleaned from multiple experimental techniques as well as from simulations, thus providing access to atomistic details of these emergent phenomena.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Computational design of closely related proteins that adopt two well-defined but structurally divergent folds

The plasticity of naturally occurring protein structures, which can change shape considerably in response to changes in environmental conditions, is critical to biological function. While computational methods have been used for de novo design of proteins that fold to a single state with a deep free-energy minimum [P.-S. Huang, S. E. Boyken, D. Baker, Nature 537, 320–327 (2016)], and to reengineer natural proteins to alter their dynamics [J. A. Davey, A. M. Damry, N. K. Goto, R. A. Chica, Nat. Chem. Biol.13, 1280–1285 (2017)] or fold [P. A. Alexander, Y. He, Y. Chen, J. Orban, P. N. Bryan, Proc. Natl. Acad. Sci. U.S.A. 106, 21149–21154 (2009)], the de novo design of closely related sequences which adopt well-defined but structurally divergent structures remains an outstanding challenge. We designed closely related sequences (over 94% identity) that can adopt two very different homotrimeric helical bundle conformations—one short (~66 Å height) and the other long (~100 Å height)—reminiscent of the conformational transition of viral fusion proteins. Crystallographic and NMR spectroscopic characterization shows that both the short- and long-state sequences fold as designed. We sought to design bistable sequences for which both states are accessible, and obtained a single designed protein sequence that populates either the short state or the long state depending on the measurement conditions. The design of sequences which are poised to adopt two very different conformations sets the stage for creating large-scale conformational switches between structurally divergent forms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural delineation and phase-dependent activation of the costimulatory CD27:CD70 complex

CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive. Here we report the crystal structure of human CD27 in complex with human CD70. Analysis of our structure shows that CD70 adopts a classical TNF ligand homotrimeric assembly to engage CD27 receptors in a 3:3 stoichiometry. By combining structural and rational mutagenesis data with reported disease-correlated mutations, we identified the key amino acid residues of CD27 and CD70 that control this interaction. We also report increased potency for plate-bound CD70 constructs compared with solution-phase ligand in a functional activity to stimulate T-cells in vitro. These findings offer new mechanistic insight into this critical costimulatory interaction.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗