Binding screen for cystic fibrosis transmembrane conductance regulator correctors finds new chemical
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Hall, Justin D.↗
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Contribution of buried distal amino acid residues in horse liver alcohol dehydrogenase to structure and catalysis: Distal Substitutions in Liver Alcohol Dehydrogenase
Abstract not provided
Shanmuganatham, Karthik K.↗
Insights revealed by the co-crystal structure of the Saccharomyces cerevisiae histidine phosphotransfer protein Ypd1 and the receiver domain of its downstream response regulator Ssk1
Two-component signaling systems are the primary means by which bacteria, archaea, and certain plants and fungi react to their environments. The model yeast, Saccharomyces cerevisiae, uses the Sln1 signaling pathway to respond to hyperosmotic stress. This pathway contains a hybrid histidine kinase (Sln1) that autophosphorylates and transfers a phosphoryl group to its own receiver domain (R1). The phosphoryl group is then transferred to a histidine phosphotransfer protein (Ypd1) that finally passes it to the receiver domain (R2) of a downstream response regulator (Ssk1). Under normal conditions, Ssk1 is constitutively and preferentially phosphorylated in the phosphorelay. Upon detecting hyperosmotic stress, Ssk1 rapidly dephosphorylates and activates the high-osmolarity glycerol (HOG) pathway, initiating a response. Despite their distinct physiological roles, both Sln1 and Ssk1 bind to Ypd1 at a common docking site. Co-crystal structures of response regulators in complex with their phosphorelay partners are scarce, leaving many mechanistic and structural details uncharacterized for systems like the Sln1 pathway. In this work, we present the co-crystal structure of Ypd1 and a near wild-type variant of the receiver domain of Ssk1 (Ssk1-R2-W638A) at a resolution of 2.80 Å. Our structural analyses of Ypd1-receiver domain complexes, biochemical determination of binding affinities for Ssk1-R2 variants, in silico free energy estimates, and sequence comparisons reveal distinctive electrostatic properties of the Ypd1/ Ssk1-R2-W638A complex that may provide insight into the regulation of the Sln1 pathway as a function of dynamic osmolyte concentration.
59 BASIC BIOLOGICAL SCIENCES↗
Understanding the stability of a plastic‐degrading Rieske iron oxidoreductase system
Abstract Rieske oxygenases (ROs) are a diverse metalloenzyme class with growing potential in bioconversion and synthetic applications. We postulated that ROs are nonetheless underutilized because they are unstable. Terephthalate dioxygenase (TPA DO PDB ID 7Q05 ) is a structurally characterized heterohexameric α 3 β 3 RO that, with its cognate reductase (TPA RED ), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPA DO /TPA RED system exhibits only ~300 total turnovers at its optimal pH and temperature. We investigated the thermal stability of the system and the unfolding pathway of TPA DO through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature ( T m ) of 39.9°C for the monomeric TPA RED , while the independent T m of TPA DO is 50.8°C. Differential scanning calorimetry revealed a two‐step thermal decomposition pathway for TPA DO with T m values of 47.6 and 58.0°C (Δ H = 210 and 509 kcal mol −1 , respectively) for each step. Temperature‐dependent small‐angle x‐ray scattering and dynamic light scattering both detected heat‐induced dissociation of TPA DO subunits at 53.8°C, followed by higher‐temperature loss of tertiary structure that coincided with protein aggregation. The computed enthalpies of dissociation for the monomer interfaces were most congruent with a decomposition pathway initiated by β‐β interface dissociation, a pattern predicted to be widespread in ROs. As a strategy for enhancing TPA DO stability, we propose prioritizing the re‐engineering of the β subunit interfaces, with subsequent targeted improvements of the subunits.
59 BASIC BIOLOGICAL SCIENCES↗
Ligand‐induced CaMKIIα hub Trp403 flip, hub domain stacking, and modulation of kinase activity
Abstract γ‐Hydroxybutyric acid (GHB) analogs are small molecules that bind competitively to a specific cavity in the oligomeric CaMKIIα hub domain. Binding affects conformation and stability of the hub domain, which may explain the neuroprotective action of some of these compounds. Here, we describe molecular details of interaction of the larger‐type GHB analog 2‐(6‐(4‐chlorophenyl)imidazo[1,2‐b]pyridazine‐2‐yl)acetic acid (PIPA). Like smaller‐type analogs, PIPA binding to the CaMKIIα hub domain promoted thermal stability. PIPA additionally modulated CaMKIIα activity under sub‐maximal CaM concentrations and ultimately led to reduced substrate phosphorylation. A high‐resolution X‐ray crystal structure of a stabilized CaMKIIα (6x mutant) hub construct revealed details of the binding mode of PIPA, which involved outward placement of tryptophan 403 (Trp403), a central residue in a flexible loop close to the upper hub cavity. Small‐angle X‐ray scattering (SAXS) solution structures and mass photometry of the CaMKIIα wild‐type hub domain in the presence of PIPA revealed a high degree of ordered self‐association (stacks of CaMKIIα hub domains). This stacking neither occurred with the smaller compound 3‐hydroxycyclopent‐1‐enecarboxylic acid (HOCPCA), nor when Trp403 was replaced with leucine (W403L). Additionally, CaMKIIα W403L hub was stabilized to a larger extent by PIPA compared to CaMKIIα hub wild type, indicating that loop flexibility is important for holoenzyme stability. Thus, we propose that ligand‐induced outward placement of Trp403 by PIPA, which promotes an unforeseen mechanism of hub domain stacking, may be involved in the observed reduction in CaMKIIα kinase activity. Altogether, this sheds new light on allosteric regulation of CaMKIIα activity via the hub domain.
Biochemistry & Molecular Biology↗
Structure analysis reveals the flexibility of the ADAMTS-5 active site
A ((1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl) succinamide derivative (here referred to as Compound 12) shows significant activity toward many matrix metalloproteinases (MMPs), including MMP-2, MMP-8, MMP-9, and MMP-13. Modeling studies had predicted that this compound would not bind to ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs-5) due to its shallow S1' pocket. However, inhibition analysis revealed it to be a nanomolar inhibitor of both ADAMTS-4 and -5. The observed inconsistency was explained by analysis of crystallographic structures, which showed that Compound 12 in complex with the catalytic domain of ADAMTS-5 (cataTS5) exhibits an unusual conformation in the S1' pocket of the protein. This first demonstration that cataTS5 can undergo an induced conformational change in its active site pocket by a molecule like Compound 12 should enable the design of new aggrecanase inhibitors with better potency and selectivity profiles.
59 BASIC BIOLOGICAL SCIENCES↗
Structures of the autoproteolytic domain from the Saccharomyces cerevisiae nuclear pore complex component, Nup145
Nuclear pore complexes (NPCs) are large, octagonally symmetric dynamic macromolecular assemblies responsible for exchange of proteins and RNAs between the nucleus and cytoplasm. NPCs are made up of at least 456 polypeptides from {approx}30 distinct nucleoporins. Several of these components, sharing similar structural motifs, form stable subcomplexes that form a coaxial structure containing two outer rings (the nuclear and cytoplasmic rings), two inner rings, and a membrane ring. The yeast (Saccharomyces cerevisiae) Nup145 and its human counterpart are unique among the nucleoporins, in that they undergo autoproteolysis to generate functionally distinct proteins. The human counterpart of Nup145 is expressed as two alternatively spliced mRNA transcripts. The larger 190 kDa precursor undergoes post-translational autoproteolysis at the Phe863-Ser864 peptide bond yielding the 92 kDa Nup98 and the 96 kDa Nup96. The smaller 98 kDa precursor is also autoproteolysed at an analogous site giving 92 kDa Nup98-N and a 6 kDa C-terminal fragment, which may form a noncovalent complex. The yeast Nup145 precursor [Fig. 1(A)] contains twelve repeats of a 'GLFG' peptide motif (FG repeats) at its N-terminus, an internal autoproteolytic domain (a region of high conservation with the homologous yeast nucleoporins Nup110 and Nup116, neither of which undergo autoproteolysis), followed by the C-terminal domain. Various forms of the FG repeats are present in nearly half of all nucleoporins; they form intrinsically disordered regions implicated in gating mechanisms that control passage of macromolecules through NPCs. Nup145 undergoes autoproteolysis at the Phe605-Ser606 peptide bond to generate two functionally distinct proteins, Nup145N and Nup145C. Subsequently, Nup145C associates with six other proteins to form the heptameric Y-complex, a component of the outer rings of the NPC. Nup145N, on the other hand, can shuttle between the NPC and the nuclear interior. It has been suggested that Nup145N, by analogy with Nup98, carries RNA between the nucleus and the NPC. Nup145 belongs to a highly conserved family of homologs found throughout eukarya. Curiously, the Phe-Ser autoproteolytic site is not always conserved, resulting in the absence of autoproteolysis for some of these closely related proteins. Our structural understanding of NPC function has benefited recently from X-ray crystal structures of segments of individual proteins. As a part of ongoing efforts at the New York SGX Research Center for Structural Genomics (www.nysgxrc.org) to determine crystal structures of distinct components of the yeast NPC, we report herein structures of Nup145N residues 443-605 [Nup145N(443-605)] from the larger autoproteolytic segment. The monomeric architecture of Nup145N(443-605) is similar to that of the autoproteolytic domain of human Nup98-N and the homologous domain of Nup116. In contrast, Nup145N(443-605) and Nup98-N show different modes of association in the crystalline state. Results of Small Angle X-ray Scattering (SAXS) in solution are consistent with the head-to-tail dimer of Nup145N(443-605), observed in two different crystal forms.
59 BASIC BIOLOGICAL SCIENCES↗
Structure of the C-terminal domain of Saccharomyces cerevisiae Nup133, a component of the nuclear pore complex
Nuclear pore complexes (NPCs), responsible for the nucleo-cytoplasmic exchange of proteins and nucleic acids, are dynamic macromolecular assemblies forming an eight-fold symmetric co-axial ring structure. Yeast (Saccharomyces cerevisiae) NPCs are made up of at least 456 polypeptide chains of {approx}30 distinct sequences. Many of these components (nucleoporins, Nups) share similar structural motifs and form stable subcomplexes. We have determined a high-resolution crystal structure of the C-terminal domain of yeast Nup133 (ScNup133), a component of the heptameric Nup84 subcomplex. Expression tests yielded ScNup133(944-1157) that produced crystals diffracting to 1.9{angstrom} resolution. ScNup133(944-1157) adopts essentially an all {alpha}-helical fold, with a short two stranded {beta}-sheet at the C-terminus. The 11 {alpha}-helices of ScNup133(944-1157) form a compact fold. In contrast, the previously determined structure of human Nup133(934-1156) bound to a fragment of human Nup107 has its constituent {alpha}-helices are arranged in two globular blocks. These differences may reflect structural divergence among homologous nucleoporins.
59 BASIC BIOLOGICAL SCIENCES↗
Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa
Abstract not provided
Kaucikas, Marius↗
The N‐terminal intrinsically disordered region of Ncb5or docks with the cytochrome b 5 core to form a helical motif that is of ancient origin
Abstract NADH cytochromeb 5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochromeb 5 (b 5 ) and cytochromeb 5 reductase (b 5 R) domains separated by a CHORD‐Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter‐domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full‐length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high‐resolution atomic structures of theb 5 domain and a construct containing the CS andb 5 R domains (CS/b 5 R). Ncb5or also contains an N‐terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L 34 MDWIRL 40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to ab 5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N‐terminal region naturally fused to theb 5 domain, we were able to obtain a high‐resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25–129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp 120 (corresponding to invariant Trp 37 in Ncb5or) to be part of an 11‐residue α‐helix (S 116 QMDWLKLTRT 126 ) packing against two of the four helices in theb 5 domain that surround heme (α2 and α5). The Trp 120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr 85 and Tyr 88 (α2), Cys 124 (α5), and Leu 47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp 37 in nucleating the formation of the N‐terminal helix, whose location in the N/b 5 module suggests a role in regulating the function of this multi‐domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b 5 module as reflected by its existence in a class of cytochromeb 5 proteins from three kingdoms among eukaryotes.
Biochemistry & Molecular Biology↗
HSV ‐1 ICP0 dimer domain adopts a novel β‐barrel fold
Abstract Infected cell protein 0 (ICP0) is an immediate‐early regulatory protein of herpes simplex virus 1 (HSV‐1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C‐terminal dimer domain (residues 555–767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0's dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C‐terminus of ICP0 was purified from bacteria and analyzed by X‐ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine β‐strands and two α‐helices. Interestingly, two adjacent β‐strands from one monomer “reach” into the adjacent subunit during dimer formation, generating two β‐barrel‐like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two β‐strands of each dimer, creating a “stacking” of the β‐barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C‐terminal SUMO‐interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV‐1 life cycle.
Biochemistry & Molecular Biology↗
Geometric analysis of enhanced thermal conductivity in epoxy composites: A comparison of graphite and carbon nanofiber fillers
We analyze the geometric effects of two different carbon fillers on the enhancement of the thermal conductivity of carbon‐epoxy composites. This study compares the thermal properties of composites containing graphite powder (2‐dimensional) and carbon nanofibers (1‐dimensional) incorporated in an industrial epoxy. Calculations using the generalized effective medium model were also used to examine the effect of the geometry and aspect ratio of the carbon filler. Experiments show that at a filler volume fraction loading of 0.10, the effective thermal conductivity of the composites was improved up to eightfold for carbon nanofiber and threefold for graphite in comparison to the neat epoxy. The superior performance of the carbon nanofiber composite is due to the larger aspect ratio of nanofiber which allows greater overlap between neighboring particles. However, this greater overlap also results in the composite becoming prohibitively viscous at low filler volume fractions. In graphite composite at the maximum filler volume fraction of 0.3, the resulting thermal conductivity improvement was 14‐fold over the neat epoxy. Calculations indicated that the improved thermal conductivity was primarily due to the filler particle geometry. Additionally, calculations suggest the wider distribution of graphite particle aspect ratio could have a positive influence on enhancing composite thermal conductivity.
Ruminski, Anne M.↗
The Characterization and Optimization of GaN Cap Layers and SiN Cap Layers on AlGaN/GaN HEMT Structures Grown on 200 mm GaN on Silicon
We have studied capping layers for AlGaN‐based high electron mobility transistor (HEMT) structures, looking at different thicknesses of GaN, SiN, and GaN + SiN caps. SiN capping has no effect on the sheet resistance of the layers, as expected from a high quality amorphous passivation layer. GaN cap layers increase the sheet resistance whether combined with a SiN cap or not, a consequence of the polarisation charge present at the GaN/AlGaN interface. For both GaN and SiN caps on their own, we get excellent morphology from only 2 nm thickness, with no degradation for layers up to 10 nm thick. For GaN + SiN caps, we have a speckled morphology with lots of small holes, which transmission electron microscopy (TEM) analysis showed to be linked to holes created in the GaN layers before the deposition of SiN layers. We attribute this to the roughening effect of silane on GaN layers, known in the literature. Upon processing SiN capped and GaN + SiN capped layers into HEMT based heterojunction diode devices, the two have equivalent forward current, but GaN + SiN capping has greatly increased reverse current. We conclude that GaN + SiN is not an appropriate capping layer for AlGaN HEMT based diodes, compared with high quality in situ SiN passivation directly grown on the AlGaN.
Charles, Matthew↗
Density functional theory for d- and f-electron materials and compounds
Here, the fundamental requirements for a computationally tractable Density Functional Theory-based method for relativistic f- and (nonrelativistic) d-electron materials and compounds are presented. The need for basing the Kohn–Sham equations on the Dirac equation is discussed. The full Dirac scheme needs exchange-correlation functionals in terms of four-currents, but ordinary functionals, using charge density and spin-magnetization, can be used in an approximate Dirac treatment. The construction of a functional that includes the additional confinement physics needed for these materials is illustrated using the subsystem-functional scheme. If future studies show that a full Dirac, four-current based, exchange-correlation functional is needed, the subsystem functional scheme is one of the few schemes that can still be used for constructing functional approximations.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) peak intensity normalization for complex mixture analyses
Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is a preferred technique for analyzing complex organic mixtures. Currently, there is no consensus normalization approach, nor an objective method for selecting one, for quantitative analyses of FT-ICR-MS data. We investigate a method to evaluate and score the amount of bias various normalization approaches introduce into the data. We evaluate the ability of the Statistical Procedure for the Analysis of Normalization Strategies (SPANS) to guide selection of appropriate normalization approaches for two different FT-ICR-MS datasets. Further, we test the robustness of SPANS results to changes in SPANS parameter values and assess the impact of using various normalization approaches on downstream statistical analyses. The normalization approach identified by SPANS differed for the two datasets. Normalization methods impacted the statistical significance of peaks differently, underscoring the importance of carefully evaluating potential methods. More consistent SPANS scores resulted when at least 120 significant peaks are used, where larger sets of peaks were obtained by increasing the p-value threshold. Interestingly, we show that Total Sum Scaling and highest peak normalization, used in previous studies, underperformed relative to SPANS-recommended normalization approaches. While there is no single, best normalization method for all datasets, SPANS provides a mechanism to identify an appropriate normalization method for analyzing FT-ICR-MS data quantitatively. As a result, the number of peaks used in the background distributions of SPANS contributes more significantly to the reproducibility of results than the p-value thresholds used to obtain those peaks.
47 OTHER INSTRUMENTATION↗
FREDA: A Web Application for the Processing, Analysis, and Visualization of Fourier‐Transform Mass Spectrometry Data
The high-resolution measurement capability of Fourier-transform mass spectrometry (FT-MS) has made it a necessity for exploring the molecular composition of complex organic mixtures, like soil, plant, aquatic, and petroleum samples. This demand has driven a need for informatics tools to explore and analyze FT-MS data in a robust and reproducible manner. FREDA is an interactive web application developed to enable spectrometrists to format, process, and explore their FT-MS data without the need for statistical programming expertise. FREDA was built to explore outputs from a molecular identification tool, like CoreMS, and provide a suite of methods to filter data, compute chemical properties of peaks, statistically compare samples and groups of samples, conduct exploratory data analysis, and download the results with a report detailing all steps conducted. To demonstrate the utility of FREDA, an example analysis was conducted using FT-MS data from a soil microbiology study of samples collected in two different soil depths at the Sphagnum bog forest north of Grand Rapids, Minnesota. Differences between the two depths are observed using Kendrick, Gibbs free energy, and van Krevelen plots. G-tests are used to quantify a significant difference between the groups. All analyses and plotting are conducted using only the FREDA application. FREDA is an open-source and readily available web application that allows users to explore and make statistically valid conclusions about their FT-MS data. The application is available online (https://map.emsl.pnnl.gov/app/freda) with a tutorial web series (https://youtu.be/k5HLE2kNSBY?si=yB6sGoyvzxrFf5MP) and freely accessible code on Github (https://github.com/EMSL-Computing/FREDA).
47 OTHER INSTRUMENTATION↗
Computing quality scores and uncertainty for approximate pattern matching in geospatial semantic graphs
Geospatial semantic graphs provide a robust foundation for representing and analyzing remote sensor data. In particular, they support a variety of pattern search operations that capture the spatial and temporal relationships among the objects and events in the data. However, in the presence of large data corpora, even a carefully constructed search query may return a large number of unintended matches. This work considers the problem of calculating a quality score for each match to the query, given that the underlying data are uncertain. As a result, we present a preliminary evaluation of three methods for determining both match quality scores and associated uncertainty bounds, illustrated in the context of an example based on overhead imagery data.
97 MATHEMATICS AND COMPUTING↗