Cytosolic Ric-8A has guanine nucleotide exchange factor (GEF) activity and is a chaperone for several classes of heterotrimeric G protein α subunits in vertebrates. Using Hydrogen-Deuterium ...Exchange-Mass Spectrometry (HDX-MS) we show that Ric-8A disrupts the secondary structure of the Gα Ras-like domain that girds the guanine nucleotide-binding site, and destabilizes the interface between the Gαi1 Ras and helical domains, allowing domain separation and nucleotide release. These changes are largely reversed upon binding GTP and dissociation of Ric-8A. HDX-MS identifies a potential Gα interaction site in Ric-8A. Alanine scanning reveals residues crucial for GEF activity within that sequence. HDX confirms that, like G protein-coupled receptors (GPCRs), Ric-8A binds the C-terminus of Gα. In contrast to GPCRs, Ric-8A interacts with Switches I and II of Gα and possibly at the Gα domain interface. These extensive interactions provide both allosteric and direct catalysis of GDP unbinding and release and GTP binding.
The application of sulfur single‐wavelength anomalous dispersion (S‐SAD) to determine the crystal structures of macromolecules can be challenging if the asymmetric unit is large, the crystals are ...small, the size of the anomalously scattering sulfur structure is large and the resolution at which the anomalous signals can be accurately measured is modest. Here, as a study of such a case, approaches to the SAD phasing of orthorhombic Ric‐8A crystals are described. The structure of Ric‐8A was published with only a brief description of the phasing process Zeng et al. (2019), Structure, 27, 1137–1141. Here, alternative approaches to determining the 40‐atom sulfur substructure of the 103 kDa Ric‐8A dimer that composes the asymmetric unit are explored. At the data‐collection wavelength of 1.77 Å measured at the Frontier micro‐focusing Macromolecular Crystallography (FMX) beamline at National Synchrotron Light Source II, the sulfur anomalous signal strength, |Δano|/σΔano (d′′/sig), approaches 1.4 at 3.4 Å resolution. The highly redundant, 11 000 000‐reflection data set measured from 18 crystals was segmented into isomorphous clusters using BLEND in the CCP4 program suite. Data sets within clusters or sets of clusters were scaled and merged using AIMLESS from CCP4 or, alternatively, the phenix.scale_and_merge tool from the Phenix suite. The latter proved to be the more effective in extracting anomalous signals. The HySS tool in Phenix, SHELXC/D and PRASA as implemented in the CRANK2 program suite were each employed to determine the sulfur substructure. All of these approaches were effective, although HySS, as a component of the phenix.autosol tool, required data from all crystals to find the positions of the sulfur atoms. Critical contributors in this case study to successful phase determination by SAD included (i) the high‐flux FMX beamline, featuring helical‐mode data collection and a helium‐filled beam path, (ii) as recognized by many authors, a very highly redundant, multiple‐crystal data set and (iii) the inclusion within that data set of data from crystals that were scanned over large ω ranges, yielding highly isomorphous and highly redundant intensity measurements.
Multiple approaches to data merging and scaling and anomalous substructure determination were used to solve the structure of a large asymmetric unit from weakly diffracting crystals by sulfur SAD.
The structure of the first ubiquitin-associated domain from HHR23A, UBA(1), was determined by X-ray crystallography at a 1.60 Å resolution, and its stability, folding kinetics, and residual structure ...under denaturing conditions have been investigated. The concentration dependence of thermal denaturation and size-exclusion chromatography indicate that UBA(1) is monomeric. Guanidine hydrochloride (GdnHCl) denaturation experiments reveal that the unfolding free energy, Δ
°'(H
O), of UBA(1) is 2.4 kcal mol
. Stopped-flow folding kinetics indicates sub-millisecond folding with only proline isomerization phases detectable at 25 °C. The full folding kinetics are observable at 4 °C, yielding a folding rate constant,
, in the absence of a denaturant of 13,000 s
and a Tanford β-value of 0.80, consistent with a compact transition state. Evaluation of the secondary structure via circular dichroism shows that the residual helical structure in the denatured state is replaced by polyproline II structure as the GdnHCl concentration increases. Analysis of NMR secondary chemical shifts for backbone
NH,
CO, and
Cα atoms between 4 and 7 M GdnHCl shows three islands of residual helical secondary structure that align in sequence with the three native-state helices. Extrapolation of the NMR data to 0 M GdnHCl demonstrates that helical structure would populate to 17-33% in the denatured state under folding conditions. Comparison with NMR data for a peptide corresponding to helix 1 indicates that this helix is stabilized by transient tertiary interactions in the denatured state of UBA(1). The high helical content in the denatured state, which is enhanced by transient tertiary interactions, suggests a diffusion-collision folding mechanism.
H chain-only Igs are naturally produced in camelids and sharks. Because these Abs lack the L chain, the Ag-binding domain is half the size of a traditional Ab, allowing this type of Ig to bind to ...targets in novel ways. Consequently, the H chain-only single-domain Ab (sdAb) structure has the potential to increase the repertoire and functional range of an active humoral immune system. The majority of vertebrates use the standard heterodimeric (both H and L chains) structure and do not produce sdAb format Igs. To investigate if other animals are able to support sdAb development and function, transgenic chickens (Gallus gallus) were designed to produce H chain-only Abs by omitting the L chain V region and maintaining only the LC region to serve as a chaperone for Ab secretion from the cell. These birds produced 30-50% normal B cell populations within PBMCs and readily expressed chicken sequence sdAbs. Interestingly, the H chains contained a spontaneous CH1 deletion. Although no isotype switching to IgY or IgA occurred, the IgM repertoire was diverse, and immunization with a variety of protein immunogens rapidly produced high and specific serum titers. mAbs of high affinity were efficiently recovered by single B cell screening. In in vitro functional assays, the sdAbs produced by birds immunized against SARS-CoV-2 were also able to strongly neutralize and prevent viral replication. These data suggest that the truncated L chain design successfully supported sdAb development and expression in chickens.
Ric-8A is a 530-amino acid cytoplasmic molecular chaperone and guanine nucleotide exchange factor (GEF) for i, q, and 12/13 classes of heterortrimeric G protein alpha subunits (Gα). We report the ...2.2-Å crystal structure of the Ric-8A Gα-binding domain with GEF activity, residues 1–452, and is phosphorylated at Ser435 and Thr440. Residues 1–429 adopt a superhelical fold comprised of Armadillo (ARM) and HEAT repeats, and the C terminus is disordered. One of the phosphorylated residues potentially binds to a basic cluster in an ARM motif. Amino acid sequence conservation and published hydrogen-deuterium exchange data indicate repeats 3 through 6 to be a putative Gα-binding surface. Normal mode modeling of small-angle X-ray scattering data indicates that phosphorylation induces relative rotation between repeats 1–4, 5–6, and 7–9. 2D 1H-15N-TROSY spectra of 2H,15N-labeled Gαi1 in the presence of R452 reveals chemical shift perturbations of the C terminus and Gαi1 residues involved in nucleotide binding.
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•The G protein α subunit binding domain of Ric-8A is an Armadillo/HEAT repeat fold•Hydrogen-deuterium exchange data suggest a Gα binding surface on Ric-8A•Ric-8A binds elements of Gα also recognized by G protein-coupled receptors•The Ric-8A fold harbors a putative binding site for its C-terminal phosphoserine
Zeng et al. use X-ray crystallography and small-angle scattering, in conjunction with NMR spectroscopy, to reveal the structure of and dynamics of the G protein chaperone and activator Ric-8A and probe its interaction with the G protein alpha subunit i1.
Heterotrimeric G-proteins (Gαβγ) regulate many cellular processes in the G-protein signaling pathways. The α-subunit (Gα) in the heterotrimer is activated by G-protein-coupled receptor (GPCR) as the ...guanine-nucleotide exchange factor (GEF), which catalyzes the GDP-release and GTP-binding reactions at G? nucleotide-binding site, at the cell membrane. Intracellular GEFs for G? subunits have been identified; among them, the mammalian isoform A of resistance to inhibitors of cholinesterase-8 (Ric-8A) catalyzes nucleotide exchange and functions as a folding chaperone for inhibitory Gα (Gαi1). In a nucleotide-free complex with Gαi1, Ric-8A likely assumes the GEF and chaperone roles by inducing a molten globule-like state. Tall et al. recently discovered that Casein Kinase II phosphorylates Ric-8A at two conserved sites (S435 and T440), which upon phosphorylation, elevate both the GEF and chaperone activities. To understand the molecular mechanism under which Ric-8A interacts with G?i1, we conducted hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) and identified a putative protein-protein interactive site (residues 454-470) on Ric-8A. Site-directed mutagenesis generated single alanine mutants of Ric-8A along the putative Gαi1-binding sequence and tryptophan fluorescence GEF assays identified five residues (V455, T456, R458, P466, and G469) as binding "hotspot". We also solved a 2.2Å resolution, X-ray crystal structure of a 452-residue long fragment (R452) of the full-length Ric-8A. The crystal structure depicts a phosphorylated Ric-8A 1-452 molecule (pR452). Mapping sequence conservation scores and HDX protection profile on the pR452 crystal structure provides insights about the Ric-8A, Gαi1 interaction. Low-resolution, solution structures of both R452 and pR452 were also determined using Small Angle X-ray Scattering (SAXS). Phosphorylation of R452 at S435 and T440 likely induces subtle conformational changes on the molecule. Steady-State GTPase assay results indicated that not only does R452 retain measurable GEF activity towards Gαi1, phosphorylation of R452 also elevates the GEF activity at high Ric-8A concentrations. With information from the biochemical assessments and Ric-8A protein structures, we conclude that (A) Gαi1 likely binds to Ric-8A residues 454 to 470 and other under-characterized sites on Ric-8A because (B) R452 retains important structural elements for the GEF activity towards Gαi1 and (C) phosphorylation of Ric-8A induces elevated Ric-8A GEF activity which is accompanied by conformational changes.
Ric-8A is a 530-amino acid cytoplasmic molecular chaperone and guanine nucleotide exchange factor (GEF) for i, q, and 12/13 classes of heterortrimeric G protein alpha subunits (Gα). In this work, we ...report the 2.2-Å crystal structure of the Ric-8A Gα-binding domain with GEF activity, residues 1–452, and is phosphorylated at Ser435 and Thr440. Residues 1–429 adopt a superhelical fold comprised of Armadillo (ARM) and HEAT repeats, and the C terminus is disordered. One of the phosphorylated residues potentially binds to a basic cluster in an ARM motif. Amino acid sequence conservation and published hydrogen-deuterium exchange data indicate repeats 3 through 6 to be a putative Gα-binding surface. Lastly, normal mode modeling of small-angle X-ray scattering data indicates that phosphorylation induces relative rotation between repeats 1–4, 5–6, and 7–9. 2D 1H-15N-TROSY spectra of 2H,15N-labeled Gαi1 in the presence of R452 reveals chemical shift perturbations of the C terminus and Gαi1 residues involved in nucleotide binding.
Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This “knob” domain is analogous to natural ...cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC
50
potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.
Background/Aims: Emerging studies on brain-derived neurotrophic factor (BDNF) have shown that might be novel biomarkers and therapeutic targets for cancer. We explore the role of BDNF in the ...tumorigenesis of bladder cancer and the underlying molecular mechanism. Methods: 368 patients with diagnosed bladder cancer and 352 healthy controls were enrolled to evaluate the association of BDNF and the miR-146b. Bioinformatics algorithm analysis and luciferase assay were performed to identify the target genes of miR-146b. Real-time PCR and western-blot were carried out to validate the relationship between miR-146b and CRK. MTT assay and FACS were used to evaluated the proliferation and apoptosis of cancer cells. MVs were isolated and transfect into the culture cells to confirm the above observation. Results: The clinical study shows that BDNF Met/Met was significantly associated with the risk of bladder cancer. In addition, comparing with Val/Val and Val/Met, Met/Met has lower miR-146b level. Luciferase assay shows that BDNF Val/Val is apparently enhanced miR-146b promoter-luciferase, but not BDNF Met/Met. Based on luciferase assay, CRK is a direct target gene of miR-146b. MiR-146b mimics significantly inhibited the expression of CRK and activation of AKT level. The expression of CRK and the activation of AKT (p-AKT) were significantly inhibited by MV-BDNF Val/Val-miR-146b or MV-BDNF Val/Met-miR-146b, but not MV-BDNF Met/Met-miR-146b. MV-BDNF Val/Val-miR-146b or Val/Met-miR-146b obviously inhibited cell proliferation, which eliminated by CRK. Meanwhile, with MV-BDNF Met/Met-miR-146b or Met/Met-miR-146b+CRK did not affect the proliferation. MV-BDNF Val/Val-miR-146b or Val/Met-miR-146b enhanced cell apoptosis, which could be eliminated by CRK. Meanwhile, MV-BDNF Met/Met-miR-146b or Met/Met-miR-146b+CRK did not promote apoptosis. Conclusion: BDNF VAL66MET polymorphism is associated with miR-146b and its target gene CRK. MiR-146b and CRK mediated BDNF VAL66MET polymorphism associated proliferation and apoptosis via activation of AKT. Thus, BDNF Val66Met is associated with the risk of bladder cancer, and the BDNF variant could be used a biomarker for the diagnosis of bladder cancer.
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