Evolution of Thyroglobulin Loop Kinetics in EpCAM

Chen, Serena H.; Bell, David R.

doi:10.3390/life11090915

Cited by 4 publications

(5 citation statements)

References 38 publications

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“…The CHARMM36 protein force field was used for all protein parameters 51 . MD simulations were performed using NAMD2.13, similar to previous work 52 : 20,000 steps of solvent minimization followed by 20,000 steps of protein + solvent minimization, then 500,000 steps of equilibration with a 0.5 fs timestep. Production MD simulations were run for 500 ns with a 2 fs timestep.…”

Section: Methodsmentioning

confidence: 99%

Variants in the WDR44 WD40-repeat domain cause a spectrum of ciliopathy by impairing ciliogenesis initiation

Accogli,

Shakya,

Yang

et al. 2024

Nat Commun

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WDR44 prevents ciliogenesis initiation by regulating RAB11-dependent vesicle trafficking. Here, we describe male patients with missense and nonsense variants within the WD40 repeats (WDR) of WDR44, an X-linked gene product, who display ciliopathy-related developmental phenotypes that we can model in zebrafish. The patient phenotypic spectrum includes developmental delay/intellectual disability, hypotonia, distinct craniofacial features and variable presence of brain, renal, cardiac and musculoskeletal abnormalities. We demonstrate that WDR44 variants associated with more severe disease impair ciliogenesis initiation and ciliary signaling. Because WDR44 negatively regulates ciliogenesis, it was surprising that pathogenic missense variants showed reduced abundance, which we link to misfolding of WDR autonomous repeats and degradation by the proteasome. We discover that disease severity correlates with increased RAB11 binding, which we propose drives ciliogenesis initiation dysregulation. Finally, we discover interdomain interactions between the WDR and NH2-terminal region that contains the RAB11 binding domain (RBD) and show patient variants disrupt this association. This study provides new insights into WDR44 WDR structure and characterizes a new syndrome that could result from impaired ciliogenesis.

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Section: Methodsmentioning

confidence: 99%

Variants in the WDR44 WD40-repeat domain cause a spectrum of ciliopathy by impairing ciliogenesis initiation

Accogli,

Shakya,

Yang

et al. 2024

Nat Commun

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“…The canonical VL-VL configuration structure was selected as the top-ranked AlphaFold2 model. MD simulations were conducted using NAMD2 following a similar protocol used previously [25]. Briefly, both systems were solvated in TIP3P water boxes with 100 mM of NaCl concentration.…”

Section: Molecular Dynamics Simulationsmentioning

confidence: 99%

Crystal Structure of a Chimeric Antigen Receptor (CAR) scFv Domain Rearrangement Forming a VL-VL Dimer

et al. 2023

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Chimeric Antigen Receptor T-cell (CAR-T) immunotherapies are dependent upon designed transmembrane proteins to bind target antigens and stimulate an immune response. The success or failure of these CARs is only partially predictable, yet recent work has highlighted the importance of antigen binding scFvs driving distinct oligomerization states with varied CAR-T efficacy. Here, we sought to determine the extracellular structure of the anti-CD19 CAR 47G4-CD828Z. Unexpectedly, the resolved crystal structure revealed an IgVL homodimer bound along an inverted VL|VL interface. We found that the VL-VH linker, designed to be cleavage resistant, was cleaved, and the VH and CAR hinge domains were absent from the crystal structure lattice. Molecular Dynamics simulations revealed that the inverted VL|VL interface was more stable than the canonical VL|VL configuration. Our work substantiates the need to interrogate the scFv structure and CAR oligomerization state for optimal CAR-T design.

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“…Prevalent across the three domains of life, intrinsically disordered proteins (IDPs) are important for a wide range of biological functions from molecular recognition to regulation of enzyme activity (Chen & Bell, 2021; Dunker et al, 2000; Dunker et al, 2002; Huber & Bennett Jr., 1983). As the function of a protein depends on its structural dynamics, it is inevitable to study the structural ensembles of IDPs to uncover their physiological roles.…”

Section: Introductionmentioning

confidence: 99%

Structural characterization of an intrinsically disordered protein complex using integrated small‐angle neutron scattering and computing

Chen,

Weiss,

Stanley

et al. 2023

Protein Science

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Characterizing structural ensembles of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) of proteins is essential for studying structure–function relationships. Due to the different neutron scattering lengths of hydrogen and deuterium, selective labeling and contrast matching in small‐angle neutron scattering (SANS) becomes an effective tool to study dynamic structures of disordered systems. However, experimental timescales typically capture measurements averaged over multiple conformations, leaving complex SANS data for disentanglement. We hereby demonstrate an integrated method to elucidate the structural ensemble of a complex formed by two IDRs. We use data from both full contrast and contrast matching with residue‐specific deuterium labeling SANS experiments, microsecond all‐atom molecular dynamics (MD) simulations with four molecular mechanics force fields, and an autoencoder‐based deep learning (DL) algorithm. From our combined approach, we show that selective deuteration provides additional information that helps characterize structural ensembles. We find that among the four force fields, a99SB‐disp and CHARMM36m show the strongest agreement with SANS and NMR experiments. In addition, our DL algorithm not only complements conventional structural analysis methods but also successfully differentiates NMR and MD structures which are indistinguishable on the free energy surface. Lastly, we present an ensemble that describes experimental SANS and NMR data better than MD ensembles generated by one single force field and reveal three clusters of distinct conformations. Our results demonstrate a new integrated approach for characterizing structural ensembles of IDPs.

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Evolution of Thyroglobulin Loop Kinetics in EpCAM

Cited by 4 publications

References 38 publications

Variants in the WDR44 WD40-repeat domain cause a spectrum of ciliopathy by impairing ciliogenesis initiation

Variants in the WDR44 WD40-repeat domain cause a spectrum of ciliopathy by impairing ciliogenesis initiation

Crystal Structure of a Chimeric Antigen Receptor (CAR) scFv Domain Rearrangement Forming a VL-VL Dimer

Structural characterization of an intrinsically disordered protein complex using integrated small‐angle neutron scattering and computing

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