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At least 1,135 records · Page 63
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Structural analysis of the catalytic domain of Artemis endonuclease/SNM1C reveals distinct structural features

The endonuclease Artemis is responsible for opening DNA hairpins during V(D)J recombination and for processing a subset of pathological DNA double-strand breaks. Artemis is an attractive target for the development of therapeutics to manage various B cell and T cell tumors, because failure to open DNA hairpins and accumulation of chromosomal breaks may reduce the proliferation and viability of pre-T and pre-B cell derivatives. However, structure-based drug discovery of specific Artemis inhibitors has been hampered by a lack of crystal structures. Here, we report the structure of the catalytic domain of recombinant human Artemis. The catalytic domain displayed a polypeptide fold similar overall to those of other members in the DNA cross-link repair gene SNM1 family and in mRNA 3'-end-processing endonuclease CPSF-73, containing metallo-β-lactamase and β-CASP domains and a cluster of conserved histidine and aspartate residues capable of binding two metal atoms in the catalytic site. As in SNM1A, only one zinc ion was located in the Artemis active site. However, Artemis displayed several unique features. Unlike in other members of this enzyme class, a second zinc ion was present in the β-CASP domain that leads to structural reorientation of the putative DNA-binding surface and extends the substrate-binding pocket to a new pocket, pocket III. Moreover, the substrate-binding surface exhibited a dominant and extensive positive charge distribution compared with that in the structures of SNM1A and SNM1B, presumably because of the structurally distinct DNA substrate of Artemis. The structural features identified here may provide opportunities for designing selective Artemis inhibitors.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

ESnet Requirements Review Program Through the IRI Lens: A Meta-Analysis of Workflow Patterns Across DOE Office of Science Programs (Final Report)

The Department of Energy (DOE) ensures America’s security and prosperity by addressing its energy, environmental, and nuclear challenges through transformative science and technology solutions. The DOE’s Office of Science (SC) delivers groundbreaking scientific discoveries and major scientific tools that transform our understanding of nature and advance the energy, economic, and national security of the United States. The SC’s programs advance DOE mission science across a wide range of disciplines and have developed the research infrastructure needed to remain at the forefront of scientific discovery. The DOE SC’s world-class research infrastructure — exemplified by the 28 SC scientific user facilities — provides the research community with premier observational, experimental, computational, and network capabilities. Each user facility is designed to provide unique capabilities to advance core DOE mission science for its sponsor SC program and to stimulate a rich discovery and innovation ecosystem. Research communities gather and flourish around each user facility, bringing together diverse perspectives. A hallmark of many facilities is the large population of students, postdoctoral researchers, and early-career scientists who contribute as full-fledged users. These facility staff and users collaborate over years to devise new approaches to utilizing the user facility’s core capabilities. The history of the SC user facilities has many examples of wildly inventive researchers challenging operational orthodoxy to pioneer new vistas of discovery; for example, the use of the synchrotron X-ray light sources for study of proteins and other large biological molecules. This continual reinvention of the practice of science — as users and staff forge novel approaches expressed in research workflows — unlocks new discoveries and propels scientific progress. Within this research ecosystem, the high-performance computing (HPC) and networking user facilities stewarded by SC’s Advanced Scientific Computing Research (ASCR) program play a dynamic cross-cutting role, enabling complex workflows demanding high performance data, networking, and computing solutions. The DOE SC’s three HPC user facilities and the Energy Sciences Network (ESnet) high-performance research network serve all of the SC’s programs as well as the global research community. Argonne Leadership Computing Facility (ALCF), the National Energy Research Scientific Computing Center (NERSC), and Oak Ridge Leadership Computing Facility (OLCF) conceive, build, and provide access to a range of supercomputing, advanced computing, and large-scale data-infrastructure platforms, while ESnet interconnects DOE SC research infrastructure and enables seamless exchange of scientific data. All four facilities operate testbeds to expand the frontiers of computing and networking research. Together, the ASCR facilities enterprise seeks to understand and meet the needs and requirements across SC and DOE domain science programs and priority efforts, highlighted by the formal requirements reviews (RRs) methodology. In recent years, the research communities around the SC user facilities have begun experimenting with and demanding solutions integrated with HPC and data infrastructure. This rise of integrated-science approaches is documented in many community and high-level government reports. At the dawn of the era of exascale science and the acceleration of artificial intelligence (AI) innovation, there is a broad need for integrated computational, data, and networking solutions. In response to these drivers, DOE has developed a vision for an Integrated Research Infrastructure (IRI): To empower researchers to meld DOE’s world-class research tools, infrastructure, and user facilities seamlessly and securely in novel ways to radically accelerate discovery and innovation.

42 ENGINEERING↗
DOE OSTI2024

Putative rhamnogalacturonan-II glycosyltransferase identified through callus gene editing bypasses embryo lethality

Rhamnogalacturonan II (RG-II) is a structurally complex and conserved domain of the pectin present in the primary cell walls of vascular plants. Borate crosslinking of RG-II is required for plants to grow and develop normally. Mutations that alter RG-II structure also affect crosslinking and are lethal or severely impair growth. Thus, few genes involved in RG-II synthesis have been identified. Here we developed a method to generate viable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in callus tissue via CRISPR/Cas9-mediated gene editing. We combined this with a candidate gene approach to characterize the male gametophyte defective 2 (MPG2) gene that encodes a putative family GT29 glycosyltransferase. Plants homozygous for this mutation do not survive. We showed that in the callus mutant cell walls, RG-II does not crosslink normally because it lacks 3-deoxy-D-manno-octulosonic acid (Kdo) and thus cannot form the α-L-Rhap-(1→5)-α-D-kdop-(1→ sidechain. We suggest that MGP2 encodes an inverting RG-II CMP-β-Kdo transferase (RCKT1). Our discovery provides further insight into the role of sidechains in RG-II dimerization. Our method also provides a viable strategy for further identifying proteins involved in the biosynthesis of RG-II.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2000

Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain

The regulation of Ca(2+)-pumps is important for controlling [Ca(2+)] in the cytosol and organelles of all eukaryotes. Here, we report a genetic strategy to identify residues that function in autoinhibition of a novel calmodulin-activated Ca(2+)-pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by complementation of a yeast (K616) deficient in two Ca(2+)-pumps. Fifteen mutations were found that disrupted a segment of the N-terminal autoinhibitor located between Lys(23) and Arg(54). Three mutations (E167K, D219N, and E341K) were found associated with the stalk that connects the ATPase catalytic domain (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V(max), K(mATP), and K(mCa(2+)) similar to that of a pump in which the N-terminal autoinhibitor had been deleted. A highly conservative substitution at Asp(219) (D219E) still produced a deregulated pump, indicating that the autoinhibitory structure in the stalk is highly sensitive to perturbation. In plasma membrane H(+)-ATPases from yeast and plants, similarly positioned mutations resulted in hyperactive pumps. Together, these results suggest that a structural feature of the stalk is of general importance in regulating diverse P-type ATPases.

NASA Discipline Plant Biology↗
NASA NTRS1996

Positive selection moments identify potential functional residues in human olfactory receptors

Correlated mutation analysis and molecular models of olfactory receptors have provided evidence that residues in the transmembrane domains form a binding pocket for odor ligands. As an independent test of these results, we have calculated positive selection moments for the alpha-helical sixth transmembrane domain (TM6) of human olfactory receptors. The moments can be used to identify residues that have been preferentially affected by positive selection and are thus likely to interact with odor ligands. The results suggest that residue 622, which is commonly a serine or threonine, could form critical H-bonds. In some receptors a dual-serine subsite, formed by residues 622 and 625, could bind hydroxyl determinants on odor ligands. The potential importance of these residues is further supported by site-directed mutagenesis in the beta-adrenergic receptor. The findings should be of practical value for future physiological studies, binding assays, and site-directed mutagenesis.

Non-NASA Center↗
NASA NTRSDate not supplied

NSCOR for Evaluating Risk Factors and Biomarkers for Adaptation and Resilience to Spaceflight: Emotional Valence and Social Processes in ICC/ICE Environments

Space exploration class missions, such as a mission to Mars, will require optimization of human performance, adaptability, and resilience. This NASA Specialized Center of Research (NSCOR) utilizes the NIMH Research Domain Criteria (RDoC) framework to identify biological and behavioral markers of individual social adaptation and emotional resilience (as well as vulnerability) to spaceflight-relevant stressors such as living in extended isolation. The overarching goal of this NSCOR is to obtain novel information to help identify biomarkers of individuals who are resilient and/or adaptable to the stressors of isolated, confined, and controlled (ICC) and isolated, confined, and extreme (ICE) environments.A total of N=90 healthy adult astronaut surrogates are being studied in three spaceflight-analog environments: (1) n=40 healthy adults in the Isolation and Confinement Analog Research Unit (ICARUS), an ICC at the University of Pennsylvania, during 7-day missions, for a target total of 280 subject days; (2) n=32 healthy adult astronaut surrogates studied in NASA’s Human Exploration Research Analog (HERA), an ICC at Johnson Space Center during 45-day missions, for a target total of 2,112 subject days; and (3) n=18 healthy adults in the Alfred-Wegener-Institute’s Neumayer Station III, an ICE in Antarctica, during 14-month missions, for a target total of 7,560 subject days. Dr. Nindl’s Laboratory at the University of Pittsburgh is analyzing a priori selected protein biomarkers in blood, saliva, and urine. Complementary rodent models of exposure to early life stressors, confinement, and isolation are being evaluated at Dr. Hensch’s Laboratory to further validate the neurobehavioral and biological findings from the human studies.Given the inconsistency and varied definition of resilience/adaptation in the scientific literature, the NSCOR team developed a composite resilience/adaptation measure that reflects the most relevant outcomes to resilience/adaptation across psychosocial and neurobehavioral functions, as well as neurocognitive and spaceflight-relevant operational performance. To achieve this, group consensus was attained from subject matter experts to produce a rank-order of importance for each input variable. The final resilience/adaptation score included 36 variables that were collected across spaceflight analogs. As of 10/1/2021, the NSCOR project acquired data on n=27 subjects at ICARUS, n=16 at HERA, and n=18 at Neumayer. The COVID-19 pandemic delayed data acquisition at ICARUS and HERA.Among subjects studied to date, 99% of neural and neurobehavioral data (e.g., neuroimaging for structure and function, behavioral measures) as well as blood, saliva, and urine for biochemical assays have been acquired. For rodent models, Dr. Hensch’s laboratory has established biochemical and behavioral parameters reflecting confinement stress in social networks of mice for comparison to stress responses in the human spaceflight analog environments.Group social behaviors were measured with a Social Network Analysis (SNA) approach to define objective parameters associated with sociability and its plasticity by sex. This analytic approach may help identify a network of individuals who are more effective teammates or more likely to generate new social relationships. Data acquisition, biomarker assessment, and data quality control will continue through September 2022.

D F Dinges↗
DOE OSTI2021

KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

The first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Revisiting synthetic lethality of Gcn5-related N-acetyltransferase (GNAT) family mutations in Haloferax volcanii

ABSTRACT Lysine acetylation is a post-translational modification that occurs in all domains of life, highlighting its evolutionary significance. Previous genome comparison identified three Gcn5-related N-acetyltransferase (GNAT) family members as lysine acetyltransferase homologs (Pat1, Pat2, and Elp3) and two deacetylase homologs (Sir2 and HdaI) in the halophilic archaeonHaloferax volcanii, withelp3andpat2proposed as a synthetic lethal gene pair. Here, we advance these findings by performing single and double mutagenesis ofelp3with thepat1andpat2lysine acetyltransferase gene homologs. Genome sequencing and PCR screens of these strains reveal successful generation of Δelp3,Δpat1Δelp3, and Δpat2Δelp3mutant strains. Although these mutant strains exhibited a reduced growth rate compared to the parent, they remained viable. Overall, this study provides genetic evidence thatelp3andpat2, while impacting cell growth, are not a synthetic lethal gene pair as previously reported. IMPORTANCE Here, we reveal by whole-genome sequencing that the GNAT family gene homologselp3andpat2can be deleted in the sameHaloferax volcaniistrain. Beyond the targeted deletions, minimal differences between the parent and Δelp3Δpat2mutant were observed, suggesting that suppressor mutations are not responsible for our ability to generate this double mutant strain. Elp3 and Pat2, thus, may not share as close a functional relationship as implied by earlier study. Our finding is significant as Elp3 is thought to function in acetylation in tRNA modification, while Pat2 likely functions in the lysine acetylation of proteins.

Microbiology↗
DOE OSTI2019

Active site plasticity and possible modes of chemical inhibition of the human DNA deaminase APOBEC3B

The single-stranded DNA cytosine deaminase APOBEC3B (A3B) functions in innate immunity against viruses, but it is also strongly implicated in eliciting mutations in cancer genomes. Because of the critical role of A3B in promoting virus and tumor evolution, small molecule inhibitors are desirable. However, there is no reported structure for any of the APOBEC3-family enzymes in complex with a small molecule bound in the active site, which hampers the development of small molecules targeting A3B. Here we report high-resolution structures of an active A3B catalytic domain chimera with loop 7 residues exchanged with those from the corresponding region of APOBEC3G (A3G). The structures reveal novel open conformations lacking the catalytically essential zinc ion, with the highly conserved active site residues extensively rearranged. These inactive conformations are stabilized by 2-pyrimidone or an iodide ion bound in the active site. Molecular dynamics simulations corroborate the remarkable plasticity of the engineered active site and identify key interactions that stabilize the native A3B active site. These data provide insights into A3B active site dynamics and suggest possible modes of its inhibition by small molecules, which would aid in rational design of selective A3B inhibitors for constraining virus and tumor evolution.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1994

Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.

Schistosoma japonicum/enzymology/genetics/immunolo↗
DOE OSTI2021

Artificial Intelligence for Autonomous Molecular Design: A Perspective

Domain-aware artificial intelligence has been increasingly adopted in recent years to expedite molecular design in various applications, including drug design and discovery. Recent advances in areas such as physics-informed machine learning and reasoning, software engineering, high-end hardware development, and computing infrastructures are providing opportunities to build scalable and explainable AI molecular discovery systems. This could improve a design hypothesis through feedback analysis, data integration that can provide a basis for the introduction of end-to-end automation for compound discovery and optimization, and enable more intelligent searches of chemical space. Several state-of-the-art ML architectures are predominantly and independently used for predicting the properties of small molecules, their high throughput synthesis, and screening, iteratively identifying and optimizing lead therapeutic candidates. However, such deep learning and ML approaches also raise considerable conceptual, technical, scalability, and end-to-end error quantification challenges, as well as skepticism about the current AI hype to build automated tools. To this end, synergistically and intelligently using these individual components along with robust quantum physics-based molecular representation and data generation tools in a closed-loop holds enormous promise for accelerated therapeutic design to critically analyze the opportunities and challenges for their more widespread application. This article aims to identify the most recent technology and breakthrough achieved by each of the components and discusses how such autonomous AI and ML workflows can be integrated to radically accelerate the protein target or disease model-based probe design that can be iteratively validated experimentally. Taken together, this could significantly reduce the timeline for end-to-end therapeutic discovery and optimization upon the arrival of any novel zoonotic transmission event. Our article serves as a guide for medicinal, computational chemistry and biology, analytical chemistry, and the ML community to practice autonomous molecular design in precision medicine and drug discovery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The CopA2-Type P 1B -Type ATPase CcoI Serves as Central Hub for cbb 3 -Type Cytochrome Oxidase Biogenesis

Copper (Cu)-transporting P 1B -type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P 1B -type ATPase CopA is required for Cu-detoxification and exports excess Cu(I) in an ATP-dependent reaction from the cytosol into the periplasm. CopA is a member of the CopA1-type ATPase family and has been biochemically and structurally characterized in detail. In contrast, less is known about members of the CopA2-type ATPase family, which are predicted to transport Cu(I) into the periplasm for cuproprotein maturation. One example is CcoI, which is required for the maturation of cbb 3 -type cytochrome oxidase (cbb 3 -Cox) in different species. Here, we reconstituted purified CcoI of Rhodobacter capsulatus into liposomes and determined Cu transport using solid-supported membrane electrophysiology. The data demonstrate ATP-dependent Cu(I) translocation by CcoI, while no transport is observed in the presence of a non-hydrolysable ATP analog. CcoI contains two cytosolically exposed N-terminal metal binding sites (N-MBSs), which are both important, but not essential for Cu delivery to cbb 3 -Cox. CcoI and cbb 3 -Cox activity assays in the presence of different Cu concentrations suggest that the glutaredoxin-like N-MBS1 is primarily involved in regulating the ATPase activity of CcoI, while the CopZ-like N-MBS2 is involved in Cu(I) acquisition. The interaction of CcoI with periplasmic Cu chaperones was analyzed by genetically fusing CcoI to the chaperone SenC. The CcoI-SenC fusion protein was fully functional in vivo and sufficient to provide Cu for cbb 3 -Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during cbb 3 -Cox assembly.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Broken-Symmetry Density Functional Theory Analysis of the Ω Intermediate in Radical S -Adenosyl- l -methionine Enzymes: Evidence for a Near-Attack Conformer over an Organometallic Species

Radical S-adenosyl-L-methionine (SAM) enzymes are found in all domains of life and catalyze a wide range of biochemical reactions. Recently, an organometallic intermediate, Ω, has been experimentally implicated in the 5'-deoxyadenosyl radical generation mechanism of the radical SAM superfamily. In this work, we employ broken-symmetry density functional theory to evaluate several structural models of Ω. The results show that the calculated hyperfine coupling constants (HFCCs) for the proposed organometallic structure of Ω are inconsistent with the experiment. In contrast, a near-attack conformer of SAM bound to the catalytic [4Fe–4S] cluster, in which the distance between the unique iron and SAM sulfur is ~3 Å, yields HFCCs that are all within 1 MHz of the experimental values. Furthermore, these results clarify the structure of the ubiquitous Ω intermediate and suggest a paradigm shift reversal regarding the mechanism of SAM cleavage by members of the radical SAM superfamily.

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

Single-Molecule Detection of the Encounter and Productive Electron Transfer Complexes of a Photosynthetic Reaction Center

Small, diffusible redox proteins play an essential role in electron transfer (ET) in respiration and photosynthesis, sustaining life on Earth by shuttling electrons between membrane-bound complexes via finely tuned and reversible interactions. Ensemble kinetic studies show transient ET complexes form in two distinct stages: an “encounter” complex largely mediated by electrostatic interactions, which subsequently, through subtle reorganization of the binding interface, forms a “productive” ET complex stabilized by additional hydrophobic interactions around the redox-active cofactors. Here, using single-molecule force spectroscopy (SMFS) we dissected the transient ET complexes formed between the photosynthetic reaction center-light harvesting complex 1 (RC-LH1) of Rhodobacter sphaeroides and its native electron donor cytochrome c 2 (cyt c 2 ). Importantly, SMFS resolves the distribution of interaction forces into low (~150 pN) and high (~330 pN) components, with the former more susceptible to salt concentration and to alteration of key charged residues on the RC. Thus, the low force component is suggested to reflect the contribution of electrostatic interactions in forming the initial encounter complex, whereas the high force component reflects the additional stabilization provided by hydrophobic interactions to the productive ET complex. Employing molecular dynamics simulations, we resolve five intermediate states that comprise the encounter, productive ET and leaving complexes, predicting a weak interaction between cyt c 2 and the LH1 ring near the RC-L subunit that could lie along the exit path for oxidized cyt c 2 . The multimodal nature of the interactions of ET complexes captured here may have wider implications for ET in all domains of life.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1994

A full-coordinate model of the polymerase domain of HIV-1 reverse transcriptase and its interaction with a nucleic acid substrate

We present a full-coordinate model of residues 1-319 of the polymerase domain of HIV-I reverse transcriptase. This model was constructed from the x-ray crystallographic structure of Jacobo-Molina et al. (Jacobo-Molina et al., P.N.A.S. USA 90, 6320-6324 (1993)) which is currently available to the degree of C-coordinates. The backbone and side-chain atoms were constructed using the MAXSPROUT suite of programs (L. Holm and C. Sander, J. Mol. Biol. 218, 183-194 (1991)) and refined through molecular modeling. A seven base pair A-form dsDNA was positioned in the nucleic acid binding cleft to represent the template-primer complex. The orientation of the template-primer complex in the nucleic acid binding cleft was guided by the positions of phosphorus atoms in the crystal structure.

Non-NASA Center↗
DOE OSTI2023

Substrate binding-induced conformational transitions in the omega-3 fatty acid transporter MFSD2A

Major Facilitator Superfamily Domain containing 2 A (MFSD2A) is a transporter that is highly enriched at the blood-brain and blood-retinal barriers, where it mediates Na + -dependent uptake of ω-3 fatty acids in the form of lysolipids into the brain and eyes, respectively. Despite recent structural insights, it remains unclear how this process is initiated, and driven by Na + . Here, we perform Molecular Dynamics simulations which demonstrate that substrates enter outward facing MFSD2A from the outer leaflet of the membrane via lateral openings between transmembrane helices 5/8 and 2/11. The substrate headgroup enters first and engages in Na + -bridged interactions with a conserved glutamic acid, while the tail is surrounded by hydrophobic residues. This binding mode is consistent with a “trap-and-flip” mechanism and triggers transition to an occluded conformation. Furthermore, using machine learning analysis, we identify key elements that enable these transitions. These results advance our molecular understanding of the MFSD2A transport cycle.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Deficiency in PHD2-mediated hydroxylation of HIF2α underlies Pacak-Zhuang syndrome

Pacak-Zhuang syndrome is caused by mutations in the EPAS1 gene, which encodes for one of the three hypoxia-inducible factor alpha (HIFα) paralogs HIF2α and is associated with defined but varied phenotypic presentations including neuroendocrine tumors and polycythemia. However, the mechanisms underlying the complex genotype-phenotype correlations remain incompletely understood. Here, we devised a quantitative method for determining the dissociation constant (K d ) of the HIF2α peptides containing disease-associated mutations and the catalytic domain of prolyl-hydroxylase (PHD2) using microscale thermophoresis (MST) and showed that neuroendocrine-associated Class 1 HIF2α mutants have distinctly higher K d than the exclusively polycythemia-associated Class 2 HIF2α mutants. Based on the co-crystal structure of PHD2/HIF2α peptide complex at 1.8 Å resolution, we showed that the Class 1 mutated residues are localized to the critical interface between HIF2α and PHD2, adjacent to the PHD2 active catalytic site, while Class 2 mutated residues are localized to the more flexible region of HIF2α that makes less contact with PHD2. Concordantly, Class 1 mutations were found to significantly increase HIF2α-mediated transcriptional activation in cellulo compared to Class 2 counterparts. These results reveal a structural mechanism in which the strength of the interaction between HIF2α and PHD2 is at the root of the general genotype-phenotype correlations observed in Pacak-Zhuang syndrome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Maturation of Rhodobacter capsulatus Multicopper Oxidase CutO Depends on the CopA Copper Efflux Pathway and Requires the cutF Product

Copper (Cu) is an essential cofactor required for redox enzymes in all domains of life. Because of its toxicity, tightly controlled mechanisms ensure Cu delivery for cuproenzyme biogenesis and simultaneously protect cells against toxic Cu. Many Gram-negative bacteria contain extracytoplasmic multicopper oxidases (MCOs), which are involved in periplasmic Cu detoxification. MCOs are unique cuproenzymes because their catalytic center contains multiple Cu atoms, which are required for the oxidation of Cu 1+ to the less toxic Cu 2+ . Hence, Cu is both substrate and essential cofactor of MCOs. Here, we investigated the maturation of Rhodobacter capsulatus MCO CutO and its role in periplasmic Cu detoxification. A survey of CutO activity of R. capsulatus mutants with known defects in Cu homeostasis and in the maturation of the cuproprotein cbb 3 -type cytochrome oxidase ( cbb 3 -Cox) was performed. This revealed that CutO activity is largely independent of the Cu-delivery pathway for cbb 3 -Cox biogenesis, except for the cupric reductase CcoG, which is required for full CutO activity. The most pronounced decrease of CutO activity was observed with strains lacking the cytoplasmic Cu chaperone CopZ, or the Cu-exporting ATPase CopA, indicating that CutO maturation is linked to the CopZ-CopA mediated Cu-detoxification pathway. Our data demonstrate that CutO is important for cellular Cu resistance under both aerobic and anaerobic growth conditions. CutO is encoded in the cutFOG operon, but only CutF, and not CutG, is essential for CutO activity. No CutO activity is detectable when cutF or its putative Cu-binding motif are mutated, suggesting that the cutF product serves as a Cu-binding component required for active CutO production. Bioinformatic analyses of CutF-like proteins support their widespread roles as putative Cu-binding proteins for several Cu-relay pathways. Our overall findings show that the cytoplasmic CopZ-CopA dependent Cu detoxification pathway contributes to providing Cu to CutO maturation, a process that strictly relies on cutF .

59 BASIC BIOLOGICAL SCIENCES↗