Biology subjects

Phillips, G.

Publications and source records attributed to Phillips, G..

11 recordsLinked to original sources

bioRxiv2024

Heterotrimeric Collagen Helix with High Specificity of Assembly Results in a Rapid Rate of Folding

The most abundant natural collagens form heterotrimeric triple helices. Synthetic mimics of collagen heterotrimers have been found to fold slowly, even compared to the already slow rates of homotrimeric helices. These prolonged folding rates are not understood and have not been studied. This work compares three heterotrimeric collagen mimics stabilities, specificities and folding rates. One of these was designed through a computational-assisted approach, resulting in a well-controlled composition and register, in addition to providing increased amino acid diversity and excellent specificity. The crystal structure of this heterotrimer elucidates the composition, register and geometry of pairwise cation-{pi} and axial and lateral salt bridges. Complementary experimental methods of circular dichroism and NMR suggest the folding paradigm is frustrated by unproductive, competing heterotrimer species and these species must completely unwind to the monomeric state before refolding into the thermodynamically favored assembly. This collagen heterotrimer, which displays the best reported thermal specificity, was also found to fold much faster (hours vs days) than comparable, well-designed systems. The heterotrimeric collagen folding rate was observed to be both concentration and temperature-independent, suggesting a complex, multi-step mechanism. These results suggest heterotrimer folding kinetics are dominated by frustration of the energy landscape caused by competing triple helices.

biophysics↗

bioRxiv2024

Post-translational Modification of α-Synuclein Modifies Monomer Dynamics and Aggregation Kinetics

The intrinsically disordered protein -Synuclein is identified as a major toxic aggregate in Parkinsons as well as several other neurodegenerative diseases. Recent work on this protein has focused on the effects of posttranslational modifications on aggregation kinetics. Among these, O-GlcNAcylation of -Synuclein has been observed to inhibit the aggregation propensity of the protein. Here we investigate the monomer dynamics of two O-GlcNAcylated -Synucleins, -Syn(gT72) and -Syn(gS87) and correlate them with the aggregation kinetics. We find that, compared to the unmodified protein, glycosylation at T72 makes the protein less compact and more diffusive while glycosylation at S87 makes the protein more compact and less diffusive. Based on a model of the earliest steps in aggregation, we predict that T72 should aggregate slower than unmodified protein, which is confirmed by ThT fluorescence measurements. In contrast, S87 should aggregate faster, which is not mirrored in ThT kinetics of later fibril formation but does not rule out a higher rate of formation of small oligomers. Together, these results show that posttranslational modifications do not uniformly affect aggregation propensity.

biophysics↗

bioRxiv2022

Large-bodied ornithomimosaurs inhabited Appalachia during the Late Cretaceous of North America

Reconstructing the evolution, diversity, and paleobiogeography of North Americas Late Cretaceous dinosaur assemblages requires spatiotemporally contiguous data; however, there remains a spatial and temporal disparity in dinosaur data on the continent. The rarity of vertebrate-bearing sedimentary deposits representing Turonian-Santonian ecosystems, and the relatively sparse record of dinosaurs from the eastern portion of the continent, present persistent challenges for studies of North American dinosaur evolution. Here we describe an assemblage of ornithomimosaurian materials from the Santonian Eutaw Formation of Mississippi. Morphological data coupled with osteohistological growth markers suggest the presence of two taxa of different body sizes, including one of the largest ornithomimosaurians known worldwide. The regression predicts a femoral circumference and a body mass of the Eutaw individuals similar to or greater than that of large-bodied ornithomimosaurs, Beishanlong grandis and Gallimimus bullatus. The paleohistology of MMNS VP-6332 demonstrates that the individual was at least 11 years of age (similar to B. grandis [[~]375 kg, 13-14 years old at death]). Additional pedal elements share some intriguing features with ornithomimosaurs yet suggest a larger-body size closer to Deinocheirus mirificus. The presence of a large-bodied ornithomimosaur in this region during this time is consistent with the relatively recent discoveries of early-diverging, large-bodied ornithomimosaurs from mid-Cretaceous strata of Laurasia (Arkansaurus fridayi and B. grandis). The smaller Eutaw taxon is represented by a tibia preserving seven growth cycles, with osteohistological indicators of decreasing growth, yet belongs to an individual with near reaching somatic maturity of the larger taxon, suggesting the co-existence of medium- and large-bodied ornithomimosaur taxa during the Late Cretaceous Santonian of North America. The Eutaw ornithomimosaur materials provide key information on the diversity and distribution of North American ornithomimosaurs and Appalachian dinosaurs and fit with broader evidence of multiple cohabiting species of ornithomimosaurian dinosaurs in Late Cretaceous ecosystems of Laurasia.

paleontology↗

bioRxiv2022

The mechanisms of catalysis and ligand binding for the SARS-CoV-2 NSP3 macrodomain from neutron and X-ray diffraction at room temperature

The NSP3 macrodomain of SARS CoV 2 (Mac1) removes ADP-ribosylation post-translational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the COVID-19 pandemic. Here, we determined neutron and X-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose-bound states. We characterize extensive solvation in the Mac1 active site, and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a re-evaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.

biochemistry↗

bioRxiv2021

RTK-dependent inducible degradation of mutant PI3K alpha drives GDC-0077 (Inavolisib) efficacy

PIK3CA is one of the most frequently mutated oncogenes; the p110 protein it encodes plays a central role in tumor cell proliferation and survival. Small molecule inhibitors targeting the PI3K p110 catalytic subunit have entered clinical trials, with early-phase GDC-0077 (Inavolisib) studies showing anti-tumor activity and a manageable safety profile in patients with PIK3CA-mutant, hormone receptor-positive breast cancer as a single agent or in combination therapy. Despite this, preclinical studies have shown that PI3K pathway inhibition releases negative feedback and activates receptor tyrosine kinase signaling, reengaging the pathway and attenuating drug activity. Here we discover that GDC-0077 and taselisib more potently inhibit mutant PI3K pathway signaling and cell viability through unique HER2-dependent degradation. Both are more effective than other PI3K inhibitors at maintaining prolonged pathway suppression, resulting in enhanced apoptosis and greater efficacy. This unique mechanism against mutant p110 reveals a new strategy for creating inhibitors that specifically target mutant tumors with selective degradation of the mutant oncoprotein and also provide a strong rationale for pursuing PI3K degraders in patients with HER2-positive breast cancer.

cancer biology↗

bioRxiv2021

Inhibitor Binding Modulates Protonation States in the Active Site of SARS-CoV-2 Main Protease

The main protease (3CL Mpro) from SARS-CoV-2, the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target. Although drugs have been developed to inhibit the proteases from HIV, hepatitis C and other viruses, no such therapeutic is available to inhibit the main protease of SARS-CoV-2. To directly observe the protonation states in SARS-CoV-2 Mpro and to elucidate their importance in inhibitor binding, we determined the structure of the enzyme in complex with the -ketoamide inhibitor telaprevir using neutron protein crystallography at near-physiological temperature. We compared protonation states in the inhibitor complex with those determined for a ligand-free neutron structure of Mpro. This comparison revealed that three active-site histidine residues (His41, His163 and His164) adapt to ligand binding, altering their protonation states to accommodate binding of telaprevir. We suggest that binding of other -ketoamide inhibitors can lead to the same protonation state changes of the active site histidine residues. Thus, by studying the role of active site protonation changes induced by inhibitors we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 Mpro.

biophysics↗

bioRxiv2020

Direct Observation of the Mechanism of Antibiotic Resistance by Mix-and-Inject at the European XFEL

In this study, we follow the diffusion and buildup of occupancy of the substrate ceftriaxone in M. tuberculosis {beta}-lactamase BlaC microcrystals by structural analysis of the enzyme substrate complex at single millisecond time resolution. We also show the binding and the reaction of an inhibitor, sulbactam, on a slower millisecond time scale. We use the mix-and-inject technique to initiate these reactions by diffusion, and determine the resulting structures by serial crystallography using ultrafast, intense X-ray pulses from the European XFEL (EuXFEL) arriving at MHz repetition rates. Here, we show how to use the EuXFEL pulse structure to dramatically increase the size of the data set and thereby the quality and time resolution of "molecular movies" which unravel ligand binding and enzymatically catalyzed reactions. This shows the great potential for the EuXFEL as a tool for biomedically relevant research, particularly, as shown here, for investigating bacterial antibiotic resistance. One Sentence SummaryDirect observation of fast ligand binding in a biomedically relevant enzyme at near atomic resolution with MHz X-ray pulses at the European XFEL.

biophysics↗

bioRxiv2020

Protonation states in SARS-CoV-2 main protease mapped by neutron crystallography

The main protease (3CL Mpro) from SARS-CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication, possessing an unusual catalytic dyad composed of His41 and Cys145. A long-standing question in the field has been what the protonation states of the ionizable residues in the substrate-binding active site cavity are. Here, we present the room-temperature neutron structure of 3CL Mpro from SARS-CoV-2, which allows direct determination of hydrogen atom positions and, hence, protonation states. The catalytic site natively adopts a zwitterionic reactive state where His41 is doubly protonated and positively charged, and Cys145 is in the negatively charged thiolate state. The neutron structure also identified the protonation states of other amino acid residues, mapping electrical charges and intricate hydrogen bonding networks in the SARS-CoV-2 3CL Mpro active site cavity and dimer interface. This structure highlights the ability of neutron protein crystallography for experimentally determining protonation states at near-physiological temperature - the critical information for structure-assisted and computational drug design.

biochemistry↗

bioRxiv2020

Ebselen Reacts with SARS Coronavirus-2 Main Protease Crystals

The SARS coronavirus 2 main protease 3CLpro tailor cuts various essential virus proteins out of long poly-protein translated from the virus RNA. If the 3CLpro is inhibited, the functional virus proteins cannot form and the virus cannot replicate and assemble. Any compound that inhibits the 3CLpro is therefore a potential drug to end the pandemic. Here we show that the diffraction power of 3CLpro crystals is effectively destroyed by Ebselen. It appears that Ebselen may be a widely available, relatively cost effective way to eliminate the SARS coronavirus 2.

biophysics↗

bioRxiv2017

Compositional shifts in the root microbiota track the life-cycle of field-grown rice plants

Bacterial communities associated with roots impact the health and nutrition of the host plant. While a multitude of static factors are known to influence the composition of the root-associated microbiota, the dynamics of these microbial assemblies over the plant life cycle are poorly understood. Here, we use dense temporal sampling of spatial compartments to characterize the root-associated microbiota of field grown rice (Oryza sativa) over the course of three consecutive growing seasons and two sites in diverse geographic regions. The root microbiota was found to be highly dynamic during the vegetative phase of plant growth, then stabilizes compositionally for the remainder of the life cycle. Bacterial taxa conserved between field sites can be used as predictive features of rice plant age by modeling using a random forests approach. The age-prediction models were used to reveal that drought stressed plants have developmentally delayed microbiota compared to unstressed plants. Further, by using genotypes with varying developmental rates, we show that shifts in the microbiome are correlated with rates of developmental transitions rather than age alone, such that different microbiota compositions reflect juvenile and adult life stages. These results suggest a model for successional dynamics of the root-associated microbiota over the plant life cycle.

plant biology↗

bioRxiv2017

Differential Mast Cell Outcomes Are Sensitive to FcϵRI-Syk Binding Kinetics

Crosslinking of IgE-bound Fc{varepsilon}RI triggers multiple cellular responses, including degranulation and cytokine production. Signaling is dependent on recruitment of Syk via docking of its dual SH2 domains to phosphorylated tyrosines within the Fc{varepsilon}RI immunoreceptor tyrosine-based activation motifs. Using single molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after Fc{varepsilon}RI activation. We found that Syk colocalizes transiently to Fc{varepsilon}RI and that Syk-Fc{varepsilon}RI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (SykY130E) led to an increased Syk-Fc{varepsilon}RI off-rate, loss of site-specific Syk autophosphorylation, and impaired downstream signaling. CRISPR-Cas9 engineered cells expressing only SykY130E were deficient in antigen-stimulated calcium release, degranulation and production of some cytokines (TNF-a, IL-3) but not others (MCP-1, IL-4). We propose that kinetic discrimination along the Fc{varepsilon}RI signaling pathway occurs at the level of Syk-Fc{varepsilon}RI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.\n\nSummarySchwartz et al. use single molecule imaging to quantify the transient nature of Fc{varepsilon}RI-Syk interactions in live mast cells. A functional mutation that increases Syk off-rate leads to loss of site-specific Syk phosphorylation and impaired signaling, highlighting the importance of finely tuned protein interactions in directing cellular outcomes.

cell biology↗