Order and disorder bound together in SARS-CoV-2 Nsp1 suppress host translation
Structure, Feb 1, 2023
In this issue of Structure, Wang et al. investigate the interplay between folded and disordered r... more In this issue of Structure, Wang et al. investigate the interplay between folded and disordered regions of the SARS-CoV-2 non-structural protein 1 (Nsp1) that promotes the suppression of host protein translation. Their investigation will lead to novel avenues to therapeutically target critical viral functions necessary for host immune-response suppression.
Proceedings of the National Academy of Sciences, 2019
The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic doma... more The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic domain (htt NT ), a polyglutamine (Q n ) tract, and a proline-rich sequence. Polyglutamine expansion results in an aggregation-prone protein responsible for Huntington’s disease. Here, we study the earliest events involved in oligomerization of a minimalistic construct, htt NT Q 7 , which remains largely monomeric over a sufficiently long period of time to permit detailed quantitative NMR analysis of the kinetics and structure of sparsely populated ( ≲ 2% ) oligomeric states, yet still eventually forms fibrils. Global fitting of concentration-dependent relaxation dispersion, transverse relaxation in the rotating frame, and exchange-induced chemical shift data reveals a bifurcated assembly mechanism in which the NMR observable monomeric species either self-associates to form a productive dimer (τ ex ∼ 30 μs, K diss ∼ 0.1 M) that goes on to form a tetramer ( τ ex ≲ 25 μs; K diss ∼ 22 μM), or e...
Solution structure of an immunodominant epitope of myelin basic protein (MBP) -Conformational dependence on environment of an intrinsically unstructuredprotein
Plasmids encoding the wild type (SH3 WT ) and triple A39V/N53P/V55L mutant (SH3 Mut ) SH3 domains... more Plasmids encoding the wild type (SH3 WT ) and triple A39V/N53P/V55L mutant (SH3 Mut ) SH3 domains (residues 85-142, numbered 2-59 in the current work) of the Gallus gallus tyrosine kinase Fyn 1 were synthesized by Genscript. The gene sequences were codon optimized for expression in E. coli and included an N-terminal His 6 -tag and a tobacco etch virus (TEV) protease cleavage site 2 as previously described. 3 The codon encoding Ala55 or Pro56 was replaced by a stop codon using the QuickChange mutagenesis kit to generate the Δ56 and Δ57 variants respectively, for both SH3 WT and SH3 Mut . The resulting plasmids were transformed into E. coli BL21 Star (DE3) competent cells (Life Technologies). Expression and purification of the truncation mutants followed the procedure described previously for full length SH3 Mut . 3 Briefly, E. coli containing the appropriate plasmid was grown at 37°C in M9 minimal medium, supplemented with either 15 NH 4 Cl and/or 13 C 6 -glucose as the sole nitrogen and carbon sources, respectively, to an OD 600 ~ 0.6, induced with 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) and grown for a further 4 h. Cells were harvested via centrifugation, resuspended in a denaturing buffer, lysed by two passes through a high-pressure homogenizer (Avestin), and initially fractionated by passing the cleared lysate over a 5 mL HisTrap column (GE Healthcare). Fractions containing the SH3 protein were pooled, refolded in a step dialysis, and had the His 6 -tag removed by TEV protease. A second pass over a HisTrap column removed the His 6 -tag remnant, and the flow-through, containing the SH3 domain, was concentrated and applied to a Superdex 75 HiLoad size exclusion column as a final purification step. All purified SH3 domains were subjected to liquid chromatography/mass spectrometry to confirm correct molecular weight, mutations and isotopic incorporation. GroEL and ribulose-1,5-bisphosphate carboxylase (Rubisco) were expressed in an E. coli recombinant system and purified as previously described. [3][4] For the quantitative binding studies described in this work, endogenous E. coli proteins remaining in the cavity of GroEL after initial purification were removed by subjecting purified GroEL to two rounds of acetone (45% v/v) precipitation. 3,4b The removal of contaminating proteins was monitored by tryptophan fluorescence, a sensitive diagnostic of extraneous protein contamination since GroEL does not contain any native tryptophans. Purification of Rubisco followed a previously published protocol. 4a Purified Rubisco was buffer exchanged into 2 mM Tris pH
The Classic Basic Protein of Myelin–Conserved Structural Motifs and the Dynamic Molecular Barcode Involved in Membrane Adhesion, Protein-Protein Interactions, and Pathogenesis in Multiple Sclerosis
In embryonal rhabdomyosarcoma (ERMS) and generally in sarcomas, the role of wild-type and loss or... more In embryonal rhabdomyosarcoma (ERMS) and generally in sarcomas, the role of wild-type and loss or gain of function TP53 mutations remains largely undefined. Eliminating mutant or restoring wild-type p53 is challenging; nevertheless, understanding TP53 effects on tumorigenesis remains central to realizing better treatment outcomes. In ERMS, >70% of patients retain wild-type TP53, yet TP53 mutations when present in tumors are associated with poor prognosis. Employing a kRASG12D-driven ERMS tumor model and newly generated tp53 null (tp53-/-) zebrafish, we define both wild-type and patient-specific TP53 mutant effects on tumorigenesis. We demonstrate that tp53 is a major suppressor of tumor initiation, where tp53 loss expands tumors initiation from <35% to >97% of animals. Next, characterizing three patient-specific mutants finds that TP53C176F partially retains wild-type p53 apoptotic activity that can be exploited, while the TP53P153Δ and TP53Y220C mutants define two structur...
The journal of physical chemistry letters, Jan 21, 2018
The chaperonin GroEL is a 800 kDa nanomachine comprising two heptameric rings, each of which encl... more The chaperonin GroEL is a 800 kDa nanomachine comprising two heptameric rings, each of which encloses a large cavity or folding chamber. The GroEL cycle involves ATP-dependent capping of the cavity by the cochaperone GroES to create a nanocage in which a single protein molecule can fold. We investigate how protein substrates sample the cavity prior to encapsulation by GroES using paramagnetic relaxation enhancement to detect transient, sparsely populated interactions between apo GroEL, paramagnetically labeled at several sites within the cavity, and three variants of an SH3 protein domain (the fully native wild type, a triple mutant that exchanges between a folded state and an excited folding intermediate, and a stable folding intermediate mimetic). We show that the substrate not only interacts with the hydrophobic inner rim of GroEL at the mouth of the cavity but also penetrates deep within the cavity, transiently contacting the disordered C-terminal tail, and, in the case of the f...
Structural Characterization of the RNA-Binding Protein SERBP1 Reveals Intrinsic Disorder and Atypical RNA Binding Modes
Frontiers in Molecular Biosciences
RNA binding proteins (RBPs) are essential for critical biological processes such as translation r... more RNA binding proteins (RBPs) are essential for critical biological processes such as translation regulation and mRNA processing, and misfunctions of these proteins are associated with diseases such as cancer and neurodegeneration. SERBP1 (SERPINE1 mRNA Binding Protein 1) is an RBP that comprises two RG/RGG repeat regions yet lacks other recognizable RNA-binding motifs. It is involved in mRNA maturation, and translational regulation. It was initially identified as a hyaluronic acid binding protein, but recent studies have identified central roles for SERBP1 in brain function and development, especially neurogenesis and synaptogenesis. SERBP1 regulates One-carbon metabolism and epigenetic modification of histones, and increased SERBP1 expression in cancers such as leukemia, ovarian, prostate, liver and glioblastoma is correlated with poor patient outcomes. Despite these important regulatory roles for SERBP1, little is known about its structural and dynamic properties, nor about the mol...
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Papers by David S Libich