Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “Protein domains”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Phosphatidylinositol phosphates modulate interactions between the StarD4 sterol trafficking protein and lipid membranes

There is substantial evidence for extensive nonvesicular sterol transport in cells. For example, lipid transfer by the steroidogenic acute regulator-related proteins (StarD) containing a StarT domain has been shown to involve several pathways of nonvesicular trafficking. Among the soluble StarT domain–containing proteins, StarD4 is expressed in most tissues and has been shown to be an effective sterol transfer protein. However, it was unclear whether the lipid composition of donor or acceptor membranes played a role in modulating StarD4-mediated transport. Here, we used fluorescence-based assays to demonstrate a phosphatidylinositol phosphate (PIP)-selective mechanism by which StarD4 can preferentially extract sterol from liposome membranes containing certain PIPs (especially, PI(4,5)P 2 and to a lesser degree PI(3,5)P2). Monophosphorylated PIPs and other anionic lipids had a smaller effect on sterol transport. This enhancement of transport was less effective when the same PIPs were present in the acceptor membranes. Furthermore, using molecular dynamics (MD) simulations, we mapped the key interaction sites of StarD4 with PIP-containing membranes and identified residues that are important for this interaction and for accelerated sterol transport activity. We show that StarD4 recognizes membrane-specific PIPs through specific interaction with the geometry of the PIP headgroup as well as the surrounding membrane environment. Finally, we also observed that StarD4 can deform membranes upon longer incubations. Taken together, these results suggest a mechanism by which PIPs modulate cholesterol transfer activity via StarD4.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

In Situ Proteolysis Condition-Induced Crystallization of the XcpVWX Complex in Different Lattices

Although prevalent in the determination of protein structures; crystallography always has the bottleneck of obtaining high-quality protein crystals for characterizing a wide range of proteins; especially large protein complexes. Stable fragments or domains of proteins are more readily to crystallize; which prompts the use of in situ proteolysis to remove flexible or unstable structures for improving crystallization and crystal quality. In this work; we investigated the effects of in situ proteolysis by chymotrypsin on the crystallization of the XcpVWX complex from the Type II secretion system of Pseudomonas aeruginosa. Different proteolysis conditions were found to result in two distinct lattices in the same crystallization solution. With a shorter chymotrypsin digestion at a lower concentration; the crystals exhibited a P3 hexagonal lattice that accommodates three complex molecules in one asymmetric unit. By contrast; a longer digestion with chymotrypsin of a 10-fold higher concentration facilitated the formation of a compact P212121 orthorhombic lattice with only one complex molecule in each asymmetric unit. The molecules in the hexagonal lattice have shown high atomic displacement parameter values compared with the ones in the orthorhombic lattice. Taken together; our results clearly demonstrate that different proteolysis conditions can result in the generation of distinct lattices in the same crystallization solution; which can be exploited in order to obtain different crystal forms of a better quality

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

N-Terminal domain homologs of the orange carotenoid protein increase quenching of cyanobacterial phycobilisomes

Stress exerted by excess captured light energy in cyanobacteria is prevented by the photoprotective activity of the orange carotenoid protein (OCP). Under high light, the OCP converts from an orange, inactive form (OCP O ) into the red form (OCP R ) that binds to and quenches the phycobilisome (PBS). Structurally, the OCP consists of 2 domains: the N-terminal effector domain and a C-terminal regulatory domain. Structural analysis of the OCP-PBS complex showed that the N-terminal domains of an OCP dimer interact with the PBS core. These N-terminal OCP domains have single-domain protein paralogs known as helical carotenoid proteins (HCPs). Using PBS quenching assays, we show that the HCP4 and HCP5 homologs efficiently quench PBS fluorescence in vitro, surpassing the quenching ability of the OCP. This is consistent with computational quantum mechanics/molecular mechanics results. Interestingly, when using a maximum quenching concentration of OCP with PBSs, HCP5 addition further increases PBS quenching. Our results provide mechanistic insight into the quenching capacity and roles of HCP4 and HCP5 in cyanobacteria, suggesting that they are more than simply functionally redundant to the OCP.

Sheppard, Damien I.↗
DOE OSTI2021

Structural and mechanistic basis for protein glutamylation by the kinase fold

The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold, yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases. The structural and mechanistic basis in which the kinase domain catalyzes protein glutamylation is unknown. Here, in this work, we present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase-like active site of SidJ adenylates an active-site Glu in SidE, resulting in the formation of a stable reaction intermediate complex. An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl-adenylate for peptide bond formation. Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE ligases during Legionella infection. Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non-ribosomal amino acid ligations and reveal an unappreciated level of SidE-family regulation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Evidence of the Involvement of a Cyclase Gene in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius

Ochratoxin A (OTA) is a well-known mycotoxin with wide distribution in food and feed. Fungal genome sequencing has great utility for identifying secondary metabolites gene clusters for known and novel compounds. A comparative analysis of the OTA-biosynthetic cluster in A. steynii, A. westerdijkiae, A. niger, A. carbonarius, and P. nordicum has revealed a high synteny in OTA cluster organization in five structural genes (otaA, otaB, ota, otaR1, and otaD). Moreover, a recent detailed comparative genome analysis of Aspergilli OTA producers led to the identification of a cyclase gene, otaY, located in the OTA cluster between the otaA and otaB genes, encoding for a predicted protein with high similarity to SnoaLs domain. These proteins have been shown to catalyze ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. In the present study, we demonstrated an upregulation of the cyclase gene in A. carbonarius under OTA permissive conditions, consistent with the expression trends of the other OTA cluster genes and their role in OTA biosynthesis by complete gene deletion. Our results pointed out the involvement of a cyclase gene in OTA biosynthetic pathway for the first time. They represent a step forward in the understanding of the molecular basis of OTA biosynthesis in A. carbonarius.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR

Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

NMPFamsDB: a database of novel protein families from microbial metagenomes and metatranscriptomes

Abstract The Novel Metagenome Protein Families Database (NMPFamsDB) is a database of metagenome- and metatranscriptome-derived protein families, whose members have no hits to proteins of reference genomes or Pfam domains. Each protein family is accompanied by multiple sequence alignments, Hidden Markov Models, taxonomic information, ecosystem and geolocation metadata, sequence and structure predictions, as well as 3D structure models predicted with AlphaFold2. In its current version, NMPFamsDB hosts over 100 000 protein families, each with at least 100 members. The reported protein families significantly expand (more than double) the number of known protein sequence clusters from reference genomes and reveal new insights into their habitat distribution, origins, functions and taxonomy. We expect NMPFamsDB to be a valuable resource for microbial proteome-wide analyses and for further discovery and characterization of novel functions. NMPFamsDB is publicly available in http://www.nmpfamsdb.org/ or https://bib.fleming.gr/NMPFamsDB.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

X-ray crystal structure of the Escherichia coli RadD DNA repair protein bound to ADP reveals a novel zinc ribbon domain

Genome maintenance is an essential process in all cells. In prokaryotes, the RadD protein is important for survival under conditions that include DNA-damaging radiation. Precisely how RadD participates in genome maintenance remains unclear. Here we present a high-resolution X-ray crystal structure of ADP-bound Escherichia coli RadD, revealing a zinc-ribbon element that was not modelled in a previous RadD crystal structure. Insights into the mode of nucleotide binding and additional structure refinement afforded by the new RadD model will help to drive investigations into the activity of RadD as a genome stability and repair factor.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

SPLIT: Stable Protein Coacervation Using a Light Induced Transition

Protein coacervates serve as hubs to concentrate and sequester proteins and nucleotides and thus function as membraneless organelles to manipulate cell physiology. We have engineered a coacervating protein to create tunable, synthetic membraneless organelles that assemble in response to a single pulse of light. Coacervation is driven by the intrinsically disordered RGG domain from the protein LAF-1, and opto-responsiveness is coded by the protein PhoCl, which cleaves in response to 405 nm light. We developed a fusion protein containing a solubilizing maltose-binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellularsized water-in-oil emulsions. An optimized version of this system displayed light-induced coacervation in Saccharomyces cerevisiae. Finally, the methods described here provide novel strategies for inducing protein phase separation using light.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2020

Structure of the Receptor Binding Domain of EnvP(b)1, an Endogenous Retroviral Envelope Protein Expressed in Human Tissues

EnvP(b)1 is an endogenous retroviral envelope gene found in human and other primate genomes. We report EnvP(b)1 sequences in primate genomes consistent with an integration event between 40 and 71 million years ago. Using a highly specific polyclonal antiserum raised against the putative receptor binding domain (RBD) of human EnvP(b)1, we detected expression in human placenta, ovaries, and thymus. We found that EnvP(b)1 is proteolytically processed, and using cell-cell fusion assays in multiple primate cell lines, we demonstrated that extant EnvP(b)1 proteins from a variety of primate genomes are fusogenic. This work supports the idea that EnvP(b)1 is under purifying selection and its fusogenic activity has been maintained for over 40 million years. We determined the structure of the RBD of human EnvP(b)1, which defines structural similarities with extant leukemia viruses, despite little sequence conservation. This structure highlights a common scaffold from which novel receptor binding specificities likely evolved. The evolutionary plasticity of this domain may underlie the diversity of related Envs in circulating viruses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Multiscale simulations reveal TDP-43 molecular-level interactions driving condensation

The RNA-binding protein TDP-43 is associated with mRNA processing and transport from the nucleus to the cytoplasm. TDP-43 localizes in the nucleus as well as accumulating in cytoplasmic condensates such as stress granules. Aggregation and formation of amyloid-like fibrils of cytoplasmic TDP-43 are hallmarks of numerous neurodegenerative diseases, most strikingly present in >90% of amyotrophic lateral sclerosis (ALS) patients. If excessive accumulation of cytoplasmic TDP-43 causes, or is caused by, neurodegeneration is presently not known. In this work, we use molecular dynamics simulations at multiple resolutions to explore TDP-43 self- and cross-interaction dynamics. A full-length molecular model of TDP-43, all 414 amino acids, was constructed from select structures of the protein functional domains (N-terminal domain, and two RNA recognition motifs, RRM1 and RRM2) and modeling of disordered connecting loops and the low complexity glycine-rich C-terminus domain. All-atom CHARMM36m simulations of single TDP-43 proteins served as guides to construct a coarse-grained Martini 3 model of TDP-43. The Martini model and a coarser implicit solvent Cα model, optimized for disordered proteins, were subsequently used to probe TDP-43 interactions; self-interactions from single-chain full-length TDP-43 simulations, cross-interactions from simulations with two proteins and simulations with assemblies of dozens to hundreds of proteins. Our findings illustrate the utility of different modeling scales for accessing TDP-43 molecular-level interactions and suggest that TDP-43 has numerous interaction preferences or patterns, exhibiting an overall strong, but dynamic, association and driving the formation of biomolecular condensates.

59 BASIC BIOLOGICAL SCIENCES↗