Computationally derived structural insights into Rare Earth selectivity in lanmodulin and its variants
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59 BASIC BIOLOGICAL SCIENCES↗
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Developing a Peptide That Inhibits DNA Repair by Blocking the Binding of Artemis and DNA Ligase IV to Enhance Tumor Radiosensitivity
Artemis and DNA Ligase IV are 2 critical elements in the nonhomologous end joining pathway of DNA repair, acting as the nuclease and DNA ligase, respectively. Enhanced cellular radiosensitivity by inhibition of either protein contributes to a promising approach to develop molecular targeted radiosensitizers. The interaction between Artemis and DNA Ligase IV is required for the activation of Artemis as nuclease at 3’overhang DNA; thus, we aim to generate an inhibitory peptide targeting the interaction between Artemis and DNA Ligase IV for novel radiosensitizer development.
62 RADIOLOGY AND NUCLEAR MEDICINE↗
3D-Scaffold: A Deep Learning Framework to Generate 3D Coordinates of Drug-like Molecules with Desired Scaffolds
The prerequisite of therapeutic drug design is to identify novel molecules with desired biophysical and biochemical properties. Deep generative models have demonstrated their ability to find such molecules by exploring a huge chemical space efficiently. An effective way to obtain molecules with desired target properties is the preservation of critical scaffolds in the generation process. To this end, we propose a domain aware generative framework called 3D-Scaffold that takes 3D coordinates of a desired scaffold as an input and generates 3D coordinates of novel therapeutic candidates as an output while always preserving the desired scaffolds in generated structures. We show that our framework generates predominantly valid, unique, novel, and experimentally synthesizable molecules that have drug-like properties similar to the molecules in the training set. Using domain specific datasets, we generate covalent and non-covalent antiviral inhibitors. Therefore, to measure the success of our framework in generating therapeutic candidates, generated structures were subjected to high throughput virtual screening via docking simulations, which shows favorable interaction against SARS-CoV-2 main protease and non-structural protein endoribonuclease (NSP15) targets. Most importantly, our model performs well with relatively small volumes of training data and generalizes to new scaffolds, making it applicable to other domain.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
The Mycobacterium tuberculosis protein O -phosphorylation landscape
Bacterial phosphosignaling has long been synonymous with the histidine kinases of the two component systems (TCSs), but many bacteria, including Mycobacterium tuberculosis (Mtb), also code for Ser/Thr protein kinases (STPKs). STPKs are the main phosphosignaling enzymes in eukaryotes, but the full extent of phosphorylation on protein Ser/Thr and Tyr (O-phosphorylation) in bacteria is unclear. Here, we explored the global signaling capacity of the STPKs in Mtb. We generated a panel of STPK loss- and gain-of-function strains and measured the resulting O-phosphorylation and transcriptional changes. This deep phosphoproteome shows that O-phosphorylation in Mtb is a vastly underexplored protein modification that affects >70% of the proteome. The substrate-kinase interactions of STPKs show an extensive interface with the transcriptional machinery, resulting in STPK regulation of gene expression of over 30% of Mtb genes. Finally, Mtb O-phosphorylation gives rise to an expansive, distributed, and cooperative network of a complexity that has previously only been associated with eukaryotic phosphosignaling networks.
59 BASIC BIOLOGICAL SCIENCES↗
Quaternary structure independent folding of voltage-gated ion channel pore domain subunits
Every voltage-gated ion channel (VGIC) has a pore domain (PD) made from four subunits, each comprising an antiparallel transmembrane helix pair bridged by a loop. The extent to which PD subunit structure requires quaternary interactions is unclear. In this report we present crystal structures of a set of bacterial voltage-gated sodium channel (BacNa V ) 'pore only' proteins that reveal a surprising collection of non-canonical quaternary arrangements in which the PD tertiary structure is maintained. This context-independent structural robustness, supported by molecular dynamics simulations, indicates that VGIC-PD tertiary structure is independent of quaternary interactions. This fold occurs throughout the VGIC superfamily and in diverse transmembrane and soluble proteins. Strikingly, characterization of PD subunit-binding Fabs indicates that non-canonical quaternary PD conformations can occur in full-length VGICs. Together, our data demonstrate that the VGIC-PD is an autonomously folded unit. This property has implications for VGIC biogenesis, understanding functional states, de novo channel design, and VGIC structural origins.
59 BASIC BIOLOGICAL SCIENCES↗
Nanobodies against the metal binding domains of ATP7B as tools to study copper transport in the cell
Abstract Nanobodies are genetically engineered single domain antibodies derived from the unusual heavy-chain only antibodies found in llamas and camels. The small size of the nanobodies and flexible selection schemes make them uniquely versatile tools for protein biochemistry and cell biology. We have developed a panel of nanobodies against the metal binding domains of the human copper transporter ATP7B, a multidomain membrane protein with a complex regulation of enzymatic activity and intracellular localization. To enable the use of the nanobodies as tools to investigate copper transport in the cell, we characterized their binding sites and affinity by isothermal titration calorimetry and NMR. We have identified nanobodies against each of the first four metal binding domains of ATP7B, with a wide affinity range, as evidenced by dissociation constants from below 10−9 to 10−6 M. We found both the inhibitory and activating nanobodies among those tested. The diverse properties of the nanobodies make the panel useful for the structural studies of ATP7B, immunoaffinity purification of the protein, modulation of its activity in the cell, protein dynamics studies, and as mimics of copper chaperone ATOX1, the natural interaction partner of ATP7B.
Uhlemann, Eva-Maria E.↗
Fully synthetic platform to rapidly generate tetravalent bispecific nanobody–based immunoglobulins
Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human V H 3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor–binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the V H and V L domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.
60 APPLIED LIFE SCIENCES↗
Drug Target and Chemotype Identification Pipeline
The TargetID pipeline enables rapid identification and characterization of binding sites in SARS-CoV-2 proteins as well as the core chemical components with which these sites interact.
Sandholtz, SarahH↗
Phytochrome-Dependent Regulation of ZFP6 and ZFPH Impacts Photomorphogenesis in Arabidopsis thaliana
Phytochromes (phy) are key regulators of photomorphogenesis in plants. Among the different phys characterized in higher plants (i.e., phyA to phyE), phyA and phyB primarily regulate phenotypic responses in plants under far-red (FR) and red (R) conditions, respectively. Recent findings suggest that some zinc finger proteins (ZFPs) are involved in plant light-modulated morphogenesis. However, the interaction(s) between phyA, phyB and ZFP homologs potentially involved in photomorphogenesis, as well as their phenotypic and molecular effects in Arabidopsis seedlings exposed to R and FR light remain to be elucidated fully. Prior analyses with phytochrome chromophore deficient lines indicated that ZFP6 expression is misregulated compared to levels in Col-0 wild type (WT). Here, we used plants with phytochrome chromophore or apoprotein (specifically phyA and phyB) deficiencies, lines with mutations in ZFP6 and ZFP6 HOMOLOG ( ZFPH ) genes, and plants overexpressing ZFP6 to examine regulatory interactions between phytochromes, ZFP6, and ZFPH. Our results indicate that phytochromes are required for downregulation of ZFP6 and ZFPH and suggest a role for light-regulated control of ZFP levels in phytochrome-dependent photomorphogenesis. Conversely, PHYB is downregulated in zfp6 mutants under R light. Analyses of a zfp6zfph double mutant confirmed disruption in photomorphogenic phenotypes, including the regulation of hypocotyl elongation in seedlings grown under FR light. In addition, PIF3 and PIF4 levels are transcriptionally regulated by ZFP6 and ZFPH in a gibberellic acid-dependent manner. ZFP6 overexpression resulted in opposite phenotypic responses to those observed in the zfp6 and zfph mutants grown in FR and R light, as well as a reduction in the rosette size of mature ZFP6 OX plants relative to WT under white light. Based on these observations, we provide insight into how phy and ZFPs interact to regulate specific aspects of light-dependent processes in Arabidopsis.
59 BASIC BIOLOGICAL SCIENCES↗
Phenotypic characterization of xpr, a global regulator of extracellular virulence factors in Staphylococcus aureus
We recently described a Tn551 insertion in the chromosome of Staphylococcus aureus S6C that resulted in drastically reduced expression of extracellular lipase (M. S. Smeltzer, S. R. Gill, and J. J. Iandolo, J. Bacteriol. 174:4000-4006, 1992). The insertion was localized to a chromosomal site (designated omega 1058) distinct from the lipase structural gene (geh) and the accessory gene regulator (agr), both of which were structurally intact in the lipase-negative (Lip-) mutants. In this report, we describe a phenotypic comparison between strains S6C, a hyperproducer of enterotoxin B; KSI9051, a derivative of S6C carrying the Tn551 insertion at omega 1058; ISP546, an 8325-4 strain that carries a Tn551 insertion in the agr locus; and ISP479C, the parent strain of ISP546 cured of the Tn551 delivery plasmid pI258repA36. Compared with their respective parent strains, ISP546 and KSI9051 produced greatly reduced amounts of lipase, alpha-toxin, delta-toxin, protease, and nuclease. KSI9051 also produced reduced amounts of staphylococcal enterotoxin B. Coagulase production was increased in ISP546 but not in KSI9051. Using a mouse model, we also demonstrated that ISP546 and KSI9051 were far less virulent than ISP479C and S6C. We have designated the genetic element defined by the Tn551 insertion at omega 1058 xpr to denote its role as a regulator of extracellular protein synthesis. We conclude that xpr and agr are similar and possibly interactive regulatory genes that play an important role in pathogenesis of staphylococcal disease.
NASA Discipline Cell Biology↗
LncRNA embryonic stem cells expressed 1 (Lncenc1) is identified as a novel regulator in neuropathic pain by interacting with EZH2 and downregulating the expression of Bai1 in mouse microglia
LncRNA embryonic stem cells expressed 1 (Lncenc1), named after its high expression in naïve embryonic stem cells (nESCs), has been rarely studied in almost all pathological processes. Evidences suggest that Lncenc1 is likely to work in the form of RNA-protein complex. Here, we found that Lncenc1 in dorsal root ganglion (DRG) was significantly upregulated in response to mouse nerve injury caused by partial sciatic nerve ligation (pSNL). Overexpression of Lncenc1 mediated by adenoviral expression vector promoted the activation of microglia and the production of inflammatory cytokines including TNF-α, IL-1β and MCP-1. In contrast, knockdown of Lncenc1 suppressed activation of microglia and production of inflammatory cytokines. In the mechanism exploration, we found that Lncenc1 could bind with the RNA binding protein (RBP) enhancer of zeste homologue 2 (EZH2), an identified contributor in microglial activation and neuropathic pain. Lncenc1 interacted with EZH2 and downregulated the expression of brain-specific angiogenesis inhibitor 1 (BAI1). Either inhibition of EZH2 or overexpression of BAI1 could reverse the effects of Lncenc1 overexpression on microglial activation and neuroinflammation. Finally, the Lncenc1-siRNA was intrathecally injected into pSNL mice, and its effects on neuropathic pain were evaluated. Knockdown of Lncenc1 attenuated the development and maintenance of mechanical and thermal hyperalgesia of pSNL mice, accompanied by an increase in BAI1 expression and decrease in inflammatory cytokines. In conclusion, Lncenc1 contributes to neuropathic pain by interacting with EZH2 and downregulating the BAI1 gene in mouse microglia.
60 APPLIED LIFE SCIENCES↗
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.↗
The Antioxidant Enzyme Methionine Sulfoxide Reductase A (MsrA) Interacts with Jab1/CSN5 and Regulates Its Function
Methionine sulfoxide (MetO) is an oxidative posttranslational modification that primarily occurs under oxidative stress conditions, leading to alteration of protein structure and function. This modification is regulated by MetO reduction through the evolutionarily conserved methionine sulfoxide reductase (Msr) system. The Msr type A enzyme (MsrA) plays an important role as a cellular antioxidant and promotes cell survival. The ubiquitin- (Ub) like neddylation pathway, which is controlled by the c-Jun activation domain-binding protein-1 (Jab1), also affects cell survival. Jab1 negatively regulates expression of the cell cycle inhibitor cyclin-dependent kinase inhibitor 1B (P27) through binding and targeting P27 for ubiquitination and degradation. Here we report the finding that MsrA interacts with Jab1 and enhances Jab1's deneddylase activity (removal of Nedd8). In turn, an increase is observed in the level of deneddylated Cullin-1 (Cul-1, a component of E3 Ub ligase complexes). Furthermore, the action of MsrA increases the binding affinity of Jab1 to P27, while MsrA ablation causes a dramatic increase in P27 expression. Thus, an interaction between MsrA and Jab1 is proposed to have a positive effect on the function of Jab1 and to serve as a means to regulate cellular resistance to oxidative stress and to enhance cell survival.
59 BASIC BIOLOGICAL SCIENCES↗
Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris
The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.
Karr, Laurel↗
A plant-specific cytochrome b 5 –like protein is essential for phytosterol biosynthesis
Sterols are essential isoprenoid derivatives that contribute to membrane structure and function. In plants, they also serve as precursors to phytohormones and specialized metabolites important for development, defense, and health. Although the sterol biosynthetic pathway is considered well-characterized, we report the discovery of a plant-specific cytochrome b 5 –like protein, CB5LP, as a critical component of phytosterol biosynthesis. Loss of CB5LP in Arabidopsis causes embryonic defects, seedling lethality, and accumulation of 14α-methyl-sterols, with reduced levels of sitosterol and stigmasterol—indicating a defect in sterol 14α-demethylation. TurboID-based proximity labeling and in vitro assays show that CB5LP physically and functionally interacts with CYP51, a cytochrome P450 enzyme catalyzing this demethylation step. Unlike canonical cytochrome b 5 proteins, CB5LP has a reversed topology and is exclusive to plants, acting as an evolutionarily distinct electron donor. This discovery reveals an uncharacterized redox partnership essential for sterol biosynthesis and highlights a promising target for the development of selective herbicide.
59 BASIC BIOLOGICAL SCIENCES↗
Development of $\mathrm{AMOEBA}$ Polarizable Force Field for Rare-Earth La 3+ Interaction with Bioinspired Ligands
Rare-earth metals (REMs) are crucial for many important industries, such as power generation and storage, in addition to cancer treatment and medical imaging. One promising new REM refinement approach involves mimicking the highly selective and efficient binding of REMs observed in relatively recently discovered proteins. However, realizing any such bioinspired approach requires an understanding of the biological recognition mechanisms. In this report we developed a new classical polarizable force field based on the AMOEBA framework for modeling a lanthanum ion (La 3+ ) interacting with water, acetate, and acetamide, which have been found to coordinate the ion in proteins. The parameters were derived by comparing to high-level ab initio quantum mechanical (QM) calculations that include relativistic effects. The AMOEBA model, with advanced atomic multipoles and electronic polarization, is successful in capturing both the QM distance-dependent La 3+ –ligand interaction energies and experimental hydration free energy. A new scheme for pairwise polarization damping (POLPAIR) was developed to describe the polarization energy in La 3+ interactions with both charged and neutral ligands. Simulations of La3+ in water showed water coordination numbers and ion–water distances consistent with previous experimental and theoretical findings. Water residence time analysis revealed both fast and slow kinetics in water exchange around the ion. This new model will allow investigation of fully solvated lanthanum ion–protein systems using GPU-accelerated dynamics simulations to gain insights on binding selectivity, which may be applied to the design of synthetic analogues.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Lignin-enhanced reduction of structural Fe(III) in nontronite: Dual roles of lignin as electron shuttle and donor
Lignin is a major component of plant-derived soil organic matter (SOM) in soils and sediments. Fe-bearing clay minerals are widely distributed in these environments and often co-exist with lignin. While previous studies have reported the electron shuttling and donating roles of certain redox-active SOM in the dissimilatory reduction of structural Fe(III) in Fe-bearing clay minerals, the role of lignin in this process remains unknown. Here we studied this role by incubating an Fe-rich smectite (nontronite NAu-2) with two types of lignin (soluble and insoluble) in the absence and presence of an Fe(III)-reducing bacterium Shewanella putrefaciens CN32 under anaerobic condition. Labile carbon source (i.e., lactate) was added in some experiments as an extra electron donor. The results demonstrated that both soluble and insoluble lignins abiotically reduced structural Fe(III) in NAu-2. The reduction extent was proportional to lignin concentration. After abiotic reaction, lignin served as either electron shuttle or electron donor in the presence of CN32: (1) When lactate was present, lignin served as an electron shuttle to enhance the rate of Fe(III) reduction; (2) When lactate was absent, lignin served as an electron donor for Fe(III) reduction. Although the biotic Fe(III) reduction extents were similar in the presence of either soluble or insoluble lignin, the reduction rates with soluble lignin were always higher than those with insoluble lignin, likely owing to their different electron transfer mechanisms. After interaction with NAu-2 and/or CN32, soluble lignin structure largely remained intact, but with some decreases of humic/fulvic acid-like and protein-like compounds, aromatic functional groups (e.g., C-H, C=O, COOH), and aliphatic/aromatic compounds. An increase of semiquinone-like organic radicals was observed after lignin interaction with NAu-2. These chemical changes of lignin were likely coupled with reduction of structural Fe(III) in nontronite. The nontronite structure remained largely unchanged without much dissolution and mineral transformation. The findings of this study provide insights into the role of lignin in promoting mineral-microbe interactions and have significant implications for coupled Fe and C biogeochemical cycles in soils and sediments.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
The HHV-6B U20 glycoprotein binds ULBP1, masking it from recognition by NKG2D and interfering with natural killer cell activation
Human Herpesvirus 6B (HHV-6B) impedes host immune responses by downregulating class I MHC molecules (MHC-I), hindering antigen presentation to CD8+ T cells. Downregulation of MHC-I disengages inhibitory receptors on natural killer (NK) cells, resulting in activation and killing of the target cell if NK cell activating receptors such as NKG2D have engaged stress ligands upregulated on the target cells. Previous work has shown that HHV-6B downregulates three MHC-like stress ligands MICB, ULBP1, and ULBP3, which are recognized by NKG2D. The U20 glycoprotein of the related virus HHV-6A has been implicated in the downregulation of ULBP1, but the precise mechanism remains undetermined. We set out to investigate the role of HHV-6B U20 in modulating NK cell activity. We used HHV-6B U20 expressed as a recombinant protein or transduced into target cells, as well as HHV-6B infection, to investigate binding interactions with NK cell ligands and receptors and to assess effects on NK cell activation. Small-angle X-ray scattering was used to align molecular models derived from machine-learning approaches. We demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity. Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels, either at the cell surface or in toto. HHV-6B infection and soluble U20 have the same effect. Transduction of U20 blocks NK cell activation in response to cell-surface ULBP1. Structural modeling of the U20 – ULBP1 complex indicates some similarities to the m152-RAE1γ complex.
60 APPLIED LIFE SCIENCES↗