Biology subjects

McDermott, A. E.

Publications and source records attributed to McDermott, A. E..

14 recordsLinked to original sources

bioRxiv2025

Structural Studies of an Anti-necroptosis Viral:Human Functional Hetero-amyloid M45:RIPK3 using SSNMR

The formation of RIP-homotypic interaction motif (RHIM)-based heteromeric amyloid assemblies between effector proteins such as RIPK1, ZBP1, or TRIF and the kinase RIPK3 serve as regulating signals for the necroptosis process, a key element of innate immune defense. Murine cytomegalovirus (MCMV) expresses the M45-encoded viral inhibitor of RIP activation (vIRA) which inhibits necroptosis in a RHIM-dependent manner. A pivotal question is how viral M45 forms hetero-amyloids with RIPK3 to effectively create an inhibitory assembly. We report a novel high-resolution structure of the M45:RIPK3 complex where M45 and RIPK3 alternately stack in an amyloid-state structure. Mutagenesis of the residues flanking the IQIG tetrad in M45 results in specific impacts on co-assembly with RIPK3, indicating an extended interface in the heteromeric fibrils. Other key interactions support the formation of stable viral:host fibrils. The M45: RIPK3 hetero-amyloid is likely to act as an anti-necroptotic signal by competing with formation of other pro-necroptotic species and introducing a barrier to RIPK3 autophosphorylation. Significance StatementThis study investigates the structural biology of the necroptotic pathway, an understudied programmed cell death mechanism that plays a crucial role in innate immunity and has implications for infectious diseases, cell cycle regulation, and cancer. We present the high-resolution structure of a cross-species hetero-amyloid in which M45, a murine cytomegalovirus (MCMV) protein, co-assembles with human RIPK3 to inhibit necroptosis by competing with pro-necroptotic amyloids. Using solid-state NMR, cryo-EM, mutagenesis, and biophysical analyses, we uncover a novel structural paradigm for cross-species hetero-amyloids, shedding light on viral strategies to manipulate host immunity and protein interactions.

biophysics↗

bioRxiv2024

Allosteric regulation of the tyrosine phosphatase PTP1B by a protein-protein interaction

The rapid identification of protein-protein interactions has been significantly enabled by mass spectrometry (MS) proteomics-based methods, including affinity purification-MS, crosslinking-MS, and proximity-labeling proteomics. While these methods can reveal networks of interacting proteins, they cannot reveal how specific protein-protein interactions alter protein function or cell signaling. For instance, when two proteins interact, there can be emergent signaling processes driven purely by the individual activities of those proteins being co-localized. Alternatively, protein-protein interactions can allosterically regulate function, enhancing or suppressing activity in response to binding. In this work, we investigate the interaction between the tyrosine phosphatase PTP1B and the adaptor protein Grb2, which have been annotated as binding partners in a number of proteomics studies. This interaction has been postulated to co-localize PTP1B with its substrate IRS-1 by forming a ternary complex, thereby enhancing the dephosphorylation of IRS-1 to suppress insulin signaling. Here, we report that Grb2 binding to PTP1B also allosterically enhances PTP1B catalytic activity. We show that this interaction is dependent on the proline-rich region of PTP1B, which interacts with the C-terminal SH3 domain of Grb2. Using NMR spectroscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) we show that Grb2 binding alters PTP1B structure and/or dynamics. Finally, we use MS proteomics to identify other interactors of the PTP1B proline-rich region that may also regulate PTP1B function similarly to Grb2. This work presents one of the first examples of a protein allosterically regulating the enzymatic activity of PTP1B and lays the foundation for discovering new mechanisms of PTP1B regulation in cell signaling. Significance StatementProtein-protein interactions are critical for cell signaling. The interaction between the phosphatase PTP1B and adaptor protein Grb2 co-localizes PTP1B with its substrates, thereby enhancing their dephosphorylation. We show that Grb2 binding also directly modulates PTP1B activity through an allosteric mechanism involving the proline-rich region of PTP1B. Our study reveals a novel mode of PTP1B regulation through a protein-protein interaction that is likely to be exploited by other cellular interactors of this important signaling enzyme.

biochemistry↗

bioRxiv2023

Contribution of protein conformational heterogeneity to NMR lineshapes at cryogenic temperatures

While low temperature NMR holds great promise for the analysis of unstable samples and for sensitizing NMR detection, spectral broadening in frozen protein samples is a common experimental challenge. One hypothesis explaining the additional linewidth is that a variety of conformations are in rapid equilibrium at room temperature and become frozen, creating an inhomogeneous distribution at cryogenic temperatures. Here we investigate conformational heterogeneity by measuring the backbone torsion angle ({Psi}) in E. coli DHFR at 105K. Motivated by the particularly broad N chemical shift distribution in this and other examples, we modified an established NCCN {Psi} experiment to correlate the chemical shift of Ni+1 to {Psi}i. With selective 15N and 13C enrichment of Ile, only the unique I60-I61 pair was expected to be detected in 13C-15N correlation spectrum. For this unique amide we detected three different conformation basins based on dispersed chemical shifts. Backbone torsion angles {Psi} were determined for each basin 114 {+/-} 7 for the major peak, and 150 {+/-} 8 and 164 {+/-} 16{degrees} for the minor peak as contrasted with 118 for the X-ray crystal structure (and 118-130 for various previously reported structures). These studies support the hypothesis that inhomogeneous distributions of protein backbone torsion angles contribute to the lineshape broadening in low temperature NMR spectra. Significance StatementUnderstanding protein conformational flexibility is essential for insights into the molecular basis of protein function and the thermodynamics of proteins. Here we investigate the ensemble of protein backbone conformations in a frozen protein freezing, which is likely a close representation for the ensemble in rapid equilibrium at room temperature. Various conformers are spectrally resolved due to the exquisite sensitivity of NMR shifts to local conformations, and NMR methods allow us to directly probe the torsion angles corresponding to each band of chemical shifts.

biophysics↗

bioRxiv2023

Predicted and Experimental NMR Chemical Shifts at Variable Temperatures: The Effect of Protein Conformational Dynamics

NMR chemical shifts provide a sensitive probe of protein structure and dynamics. Prediction of shifts, and therefore interpretation of shifts, particularly for the frequently measured amidic 15N sites, remains a tall challenge. We demonstrate that protein 15N chemical shift prediction from QM/MM predictions can be improved if conformational variation is included via MD sampling, focusing on the antibiotic target, E. coli Dihydrofolate reductase (DHFR). Variations of up to 25 ppm in predicted 15N chemical shifts are observed over the trajectory. For solution shifts the average of fluctuations on the low picosecond timescale results in a superior prediction to a single optimal conformation. For low temperature solid state measurements, the histogram of predicted shifts for locally minimized snapshots with specific solvent arrangements sampled from the trajectory explains the heterogeneous linewidths; in other words, the conformations and associated solvent are frozen out at low temperatures and result in inhomogeneously broadened NMR peaks. We identified conformational degrees of freedom that contribute to chemical shift variation. Backbone torsion angles show high amplitude fluctuations during the trajectory on the low picosecond timescale. For a number of residues, including I60, {psi} varies by up to 60{o} within a conformational basin during the MD simulations, despite the fact that I60 (and other sites studied) are in a secondary structure element and remain well folded during the trajectory. Fluctuations in {psi} appear to be compensated by other degrees of freedom in the protein, including {varphi} of the succeeding residue, resulting in "rocking" of the amide plane with changes in hydrogen bonding interactions. Good agreement for both room temperature and low temperature NMR spectra provides strong support for the specific approach to conformational averaging of computed chemical shifts.

biophysics↗

bioRxiv2023

Zinc Alters the Supramolecular Organization of Nucleic Acid Complexes with Full-Length TIA1

T-Cell Intracellular Antigen-1 (TIA1) is a 43 kDa multi-domain RNA-binding protein involved in stress granule formation during eukaryotic stress response, and has been implicated in neurodegenerative diseases including Welander distal myopathy and amyotrophic lateral sclerosis. TIA1 contains three RNA recognition motifs (RRMs), which are capable of binding nucleic acids and a C-terminal Q/N-rich prion-related domain (PRD) which has been variously described as intrinsically disordered or prion inducing and is believed to play a role in promoting liquid-liquid phase separation connected with the assembly of stress granule formation. Motivated by the fact that our prior work shows RRMs 2 and 3 are well-ordered in an oligomeric full-length form, while RRM1 and the PRD appear to phase separate, the present work addresses whether the oligomeric form is functional and competent for binding, and probes the consequences of nucleic acid binding for oligomerization and protein conformation change. New SSNMR data show that ssDNA binds to full-length oligomeric TIA1 primarily at the RRM2 domain, but also weakly at the RRM3 domain, and Zn2+ binds primarily to RRM3. Binding of Zn2+ and DNA was reversible for the full-length wild type oligomeric form, and did not lead to formation of amyloid fibrils, despite the presence of the C-terminal prion-related domain. While TIA1:DNA complexes appear as long "daisy chained" structures, the addition of Zn2+ caused the structures to collapse. We surmise that this points to a regulatory role for Zn2+. By occupying various "half" binding sites on RRM3 Zn2+ may shift the nucleic acid binding off RRM3 and onto RRM2. More importantly, the use of different half sites on different monomers may introduce a mesh of crosslinks in the supramolecular complex rendering it compact and markedly reducing the access to the nucleic acids (including transcripts) from solution.

biophysics↗

bioRxiv2022

Rotating Frame Relaxation for Magic Angle Spinning Solid State NMR, A Promising Tool for Characterizing Biopolymer Motion

Magic angle spinning NMR rotating frame relaxation measurements provide a powerful experimental strategy to probe biomolecules dynamics, as is illustrated by numerous recent applications. We discuss experimental strategies for this class of experiments, with a particular focus on systems where motion-driven modulation of the chemical shift interaction is the main mechanism for relaxation. We also explore and describe common strategies for interpreting the data sets and extracting motion timescale, activation energy, and angle or order parameters from rotating frame relaxation data. Using model free analysis, and numerical simulations, including time domain treatment, we explore conditions under which it is possible to obtain accurate and precise information about the timescales of motions. Overall, with rapid technical advances in solid state NMR, there is a bright future for this class of studies.

biophysics↗

bioRxiv2022

High-resolution magic angle spinning NMR of KcsA in liposomes: the highly mobile C-terminus

The structure of the transmembrane domain of bacterial potassium channel KcsA has been extensively characterized, yet little information is available on the structure of its cytosolic N- and C-termini. This study presents high-resolution magic angle spinning (HR-MAS) and fractional deuteration as tools to study these poorly resolved regions for proteoliposome-embedded KcsA. Using 1H-detected HR-MAS NMR, we show that the C-terminus transitions from a rigid structure to a more dynamic structure as the solution is rendered acidic. We make previously unreported assignments of residues in the C-terminus of lipid embedded channels. Further, we also show evidence for hydrolysis of lipid head groups in proteoliposome samples during typical experimental timeframes.

biophysics↗

bioRxiv2022

Hox11-expressing interstitial cells contribute to adult skeletal muscle at homeostasis

Adult skeletal muscle possesses remarkable regenerative capacity. This is attributed to tissue-specific stem cells, satellite cells. Interstitial stromal cells also play critical roles in muscle, and we have previously reported that Hoxa11 and Hoxd11, expressed in the interstitial cells of muscles that attach to the zeugopod (radius and ulna), are critical for the proper patterning and development of these muscles during embryogenesis. Using a Hoxa11eGFP knock-in reporter, we show that expression continues in a subset of muscle interstitial cells through adult stages. Using Hoxa11-CreERT2 mediated lineage reporting induced at adult stages, we observe lineage initiation only in the interstitial cells of muscle, as expected. However, this Hoxa11-expressing interstitial cell lineage progressively contributes to muscle fibers at postnatal and adult stages. The contribution to these muscles at adult homeostasis significantly exceeds parallel Pax7-CreERT2 mediated lineage labeling performed in parallel. To confirm that interstitial cell nuclear contents are contributed to muscle fibers, we additionally used the nuclear specific lineage reporter, ROSA-LSL-H2BmCherry with Hoxa11-CreERT2 and observe that Hoxa11-expressing interstitial cells contribute their nuclei to myofibers. Hox lineage contribution is observed into all four muscle sub-types over months of lineage labeling. At no point after Hoxa11-mediated lineage induction do we observe lineage labeling into Pax7-expressing satellite cells. This adds to a small but growing body of evidence that supports a satellite cell-independent source of muscle tissue in vivo. Summary StatementHoxa11 expression marks a novel population of muscle interstitial cells capable of extensive, satellite cell-independent contribution to skeletal muscle fibers during adult homeostasis.

developmental biology↗

bioRxiv2022

GTP-bound E. coli FtsZ filaments are composed of Tense monomers: A DNP NMR study using interface detection.

FtsZ filaments are the major structural component of the bacterial Z-ring and are drivers of bacterial division. While crystal structures for FtsZ from some gram positive bacteria in the presence of GTP-analog like compounds suggest the possibility of a high energy "Tense" conformation, to date it remains an important question to elucidate whether this Tense form is the dominant form in filaments. Using dynamic nuclear polarization (DNP) solid-state NMR and differential isotopic labelling, we directly detect residues located at the inter-monomer interface of GTP-bound WT Escherichia coli FtsZ filaments. We combine chemical shift prediction, homology modelling, and heteronuclear dipolar recoupling techniques to characterize the E. coli FtsZ filament interface and demonstrate that the monomers in active filaments assume a Tense conformation.

microbiology↗

bioRxiv2020

Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel

As the first potassium channel with a X-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related in particular to the allosteric coupling between its gates remain open. The many currently available X-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state NMR (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible X-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs. fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.

biophysics↗

bioRxiv2020

Scaled Recoupling of Chemical Shift Anisotropies at High Magnetic Fields under MAS with Interspersed C-elements

The power of chemical shift anisotropy (CSA) measurements for probing structure and dynamics of molecules has been long recognized. NMR pulse sequences that allow measurement of CSA values in an indirect dimension of a protein correlation spectrum have been employed for aliphatic groups, but for practical reasons carbonyl functional groups have been little studied, despite the fact that carbonyls are expected to give particularly varied and informative CSA values. Specifically, the wide spectral widths of carbonyl tensors make their measurements difficult with typically attainable spectrometer settings. We present here an extended family of experiments that enable the recovery of static CSA lineshapes in a indirect dimension of magic angle spinning (MAS) solid-state NMR experiments, except for various real valued scaling factors. The experiment is suitable for uniformly labeled material, at moderate MAS rates (10-30 kHz), and at higher magnetic fields (ν0H > 600 MHz). Specifically, the experiments are based on pulse sequence elements from a previous commonly used pulse sequence for CSA measurement, ROCSA, while modification of scaling factors is achieved by interspersing different blocks of C-elements of the same cycle. Using experimental conditions similar to the parent ROCSA sequence, a CSA scaling factor between 0 and 0.272 can be obtained, thus allowing a useful practical range of possibilities in experimental conditions for measurement of larger CSA values. Using these blocks it is also possible to make a constant-time CSA recoupling sequence. The utility and effectiveness of this approach, fROCSA, is shown on model compounds 1-13C-Gly, U-13C,15N-l-His, and microcrystalline U-13C,15N-Ubiquitin.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Phe-Gly Motifs Drive Fibrillization of TDP-43's Prion-Like Domain Droplets

TDP-43 assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g. muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct endpoint species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs, and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions, and illustrate structural features that distinguishes between aggregate forms.

biophysics↗

bioRxiv2019

Stability of Nitroxide Biradical TOTAPOL in Biological Samples

We characterize chemical reduction of a nitroxide biradical, TOTAPOL, used in dynamic nuclear polarization (DNP) experiments, specifically probing the stability in whole-cell pellets and lysates, and present a few strategies to stabilize the biradicals for DNP studies. DNP solid-state NMR experiments use paramagnetic species such as nitroxide biradicals to dramatically increase NMR signals. Although there is considerable excitement about using nitroxide-based DNP for detecting the NMR spectra of proteins in whole cells, nitroxide radicals are reduced in minutes in bacterial cell pellets, which we confirm and quantify here. We show that addition of the covalent cysteine blocker N-ethylmaleimide to whole cells significantly slows the rate of reduction, suggesting that cysteine thiol radicals are important to in vivo radical reduction. The use of cell lysates rather than whole cells also slows TOTAPOL reduction, which suggests a possible role for the periplasm and oxidative phosphorylation metabolites in radical degradation. Reduced TOTAPOL in lysates can also be efficiently reoxidized with potassium ferricyanide. These results point to a practical and robust set of strategies for DNP of cellular preparations.

biophysics↗

bioRxiv2019

Allosteric studies of an open pH gate KcsA mutant in its activated and inactivated states

Transmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the mean open time of the prototypical potassium channel KcsA in the context of C-type inactivation. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. We tested this mechanism for inactivation using a KcsA mutant (H25R/E118A) that has the pH gate open across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. The potassium binding affinity in the selectivity filter of this mutant, 81 mM, is about 4 orders of magnitude weaker than that of wild type KcsA at neutral pH and is comparable to the value for wild type KcsA at low pH (pH [~] 3.5). This result strongly supports our assertion that the open pH gate allosterically effects the potassium binding affinity of the selectivity filter. In this mutant the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery.\n\nSignificance statementInactivation of potassium channels controls mean open times and provides exquisite control over biological processes. In the highly conserved C-type inactivation process, opening of the activation gate causes subsequent inactivation. We test whether the open state of the channel simply has a poor ability to bind the K+ ion. Previously, activated and inactivated states were stabilized using truncations or a significant pH drop. Here, we use the H25R/E118A constitutively open mutant of KcsA and also observe a large drop in potassium binding affinity. This provides strong evidence that channel opening causes an allosteric loss of ion affinity, and that the central feature of this universal channel inactivation process is loss of ion affinity at the selectivity filter.

biophysics↗