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Biomedical subjects

Yun-Xing Wang

Publications and source records attributed to Yun-Xing Wang.

14 recordsLinked to original sources
PubMed2025

Structural investigation of an RNA device that regulates PD-1 expression in mammalian cells.

Synthetic RNA devices are engineered to control gene expression and offer great potential in both biotechnology and clinical applications. Here, we present multidisciplinary structural and biochemical data for a tetracycline (Tc)-responsive RNA device (D43) in both ligand-free and bound states, providing a structure-dynamical basis for signal transmission. Activation of self-cleavage is achieved via ligand-induced conformational and dynamical changes that stabilize the elongated bridging helix harboring the communication module, which drives proper coordination of the catalytic residues. We then show the utility of CRISPR-integrated D43 in EL4 lymphocytes to regulate programmed cell death protein 1 (PD-1), a key receptor of immune checkpoints. Treatment of these cells with Tc showed a dose-dependent reduction in PD-1 by immunostaining and a decrease in messenger RNA levels by quantitative PCR as compared with wild type. PD-1 expression was recoverable upon removal of Tc. These results provide mechanistic insight into RNA devices with potential for cancer immunotherapy or other applications.

Programmed Cell Death 1 Receptor↗
PubMed2006

RAP uses a histidine switch to regulate its interaction with LRP in the ER and Golgi.

The receptor associated protein (RAP) is an antagonist and molecular chaperone that binds tightly to low-density lipoprotein receptor family members in the endoplasmic reticulum (ER). After escorting these receptors to the Golgi, RAP dissociates from the receptors. The molecular mechanism of the dissociation has been unknown until now. The solution structure of RAP-D3 domain presented here reveals a striking increase in positively charged residues on the surface of this RAP domain due to protonation of solvent-exposed histidine sidechains as the pH is reduced from a near neutral pH of the ER to the acidic pH of the Golgi. Structure-based mutagenesis studies in vitro and in cells confirm that the protonation of histidine residues as a consequence of the pH changes modulate the binding/release of RAP from LRP. This histidine switch may serve as a general mechanism for regulating cell trafficking events.

Alanine↗
PubMed2005

Periodicity, planarity, residual dipolar coupling, and structures.

The periodic behavior of residual dipolar couplings (RDCs) arising from nucleic acid and protein secondary structures is shown to be more complex and information-rich than previously believed. We have developed a theoretical framework which allows the bond vector orientation of nucleic acids and the peptide plane orientations of protein secondary structures to be extracted from their Dipolar waves. In this article, we focus on utilizing "Dipolar waves" of peptides to extract structure information, and describe in more detail the fundamental principles of the relationship between the periodicities in structure and RDCs, the practical procedure to extract peptide plane orientation information from RDC data, and assessment of errors using Monte-Carlo simulations. We demonstrate the utility of our method for two model alpha-helices, one kinked and one curved, and as well as an irregular beta-strand.

Monte Carlo Method↗
PubMed2005

Determining a helical protein structure using peptide pixels.

The residual dipolar coupling-periodicity planarity correlation makes it possible to determine peptide plane orientations in regular periodic protein secondary structure elements. Each peptide plane orientation represents a "pixel" of protein structure, and is expressed in terms of three angles referred to as tilt, phase, and pitch angles. In this report, we present the novel "3P" (periodicity, planarity, and pixels) method that allows one to determine secondary and tertiary structure of alpha-helical proteins. We demonstrate the 3P method by determining the structure of domain 1 of the receptor-associated protein (RAP) to a backbone accuracy of 1.0 Angstrom using RDCs measured in a single alignment medium, together with a minimal number of NOE distance restraints, using a new Xplor-NIH module.

Nuclear Magnetic Resonance, Biomolecular↗
PubMed2004

Periodicity in residual dipolar couplings and nucleic acid structures.

The periodicity in nucleic acid duplex structures is shown to be correlated to the periodicity in residual dipolar couplings (RDCs) in the form of an "RDC wave". This "RDC wave" is characteristic of the alignment of the duplex in the magnetic field, and hence fitting of the data allows the duplex global orientation (, Phi) to be extracted. Further, because the "RDC wave" is fit as a data set of a corresponding secondary structure element, the degeneracy problem is greatly reduced. Consequently, with the global orientation (, Phi) determined, local bond vector conformations are defined. The fit is demonstrated in the examples of the imino RDCs of the negative regulator of splicing RNA fragment (NRS23) and for the C1'H1' RDCs of the Dickerson dodecamer.

Models, Molecular↗
PubMed2004

NMR structural studies of domain 1 of receptor-associated protein.

The 39 kDa receptor-associated protein (RAP) is an endoplasmic reticulum resident protein that binds tightly to the low-density lipoprotein receptor-related protein (LRP) as well as to other members of the low-density lipoprotein receptor superfamily. The association of RAP with LRP prevents this receptor from interacting with ligands. RAP is a three-domain protein that contains two independent LRP binding sites; one located within domains 1 and 2, and one located within domain 3. As the first step toward defining the structure of the full-length protein and understanding the interaction between RAP and this family of receptors, we have determined the 3D structure of domain 1 using constraints derived from heteronuclear multi-dimensional NMR spectra, including NOEs, dihedral angles, J-couplings and chemical shifts, as well as two sets of non-correlated residual dipolar couplings measured from the protein solutions in anisotropic media of Pf1 and 6% polyacrylamide gel. The backbone C(alpha) rmsd between the current structure and a homo-nuclear NOE-based structure is about 2 A. The large rmsd mainly reflects the significant differences in helical orientation and in the structural details of the long helix (helix 2) between the two structures.

Acrylic Resins↗
PubMed2004

Solution structure of the pseudo-5' splice site of a retroviral splicing suppressor.

Control of Rous sarcoma virus RNA splicing depends in part on the interaction of U1 and U11 snRNPs with an intronic RNA element called the negative regulator of splicing (NRS). A 23mer RNA hairpin (NRS23) of the NRS directly binds U1 and U11 snRNPs. Mutations that disrupt base-pairing between the loop of NRS23 and U1 snRNA abolish its negative control of splicing. We have determined the solution structure of NRS23 using NOEs, torsion angles, and residual dipolar couplings that were extracted from multidimensional heteronuclear NMR spectra. Our structure showed that the 6-bp stem of NRS23 adopts a nearly A-form duplex conformation. The loop, which consists of 11 residues according to secondary structure probing, was in a closed conformation. U913, the first residue in the loop, was bulged out or dynamic, and loop residues G914-C923, G915-U922, and U916-A921 were base-paired. The remaining UUGU tetraloop sequence did not adopt a stable structure and appears flexible in solution. This tetraloop differs from the well-known classes of tetraloops (GNRA, CUYG, UNCG) in terms of its stability, structure, and function. Deletion of the bulged U913, which is not complementary to U1 snRNA, increased the melting temperature of the RNA hairpin. This hyperstable hairpin exhibited a significant decrease in binding to U1 snRNP. Thus, the structure of the NRS RNA, as well as its sequence, is important for interaction with U1 snRNP and for splicing suppression.

Avian Sarcoma Viruses↗
PubMed2004

Codon-optimized cloning, expression and characertization of the C-terminal region of human apoptotic protein GADD34 in Escherichia coli.

The human GADD34 (Growth Arrest and DNA Damage-inducible 34) is the product of an apoptosis- and DNA-damage-inducible gene. The C-terminus domain of GADD34 is highly homologous to HSV-1 gamma-1 34.5, HSV-2 and the African swine fever virus virulence-associated factor NL-S. Among these viral proteins, HSV-1 gamma 34.5 protein is known to prevent apoptosis of viral-infected cells. Because of the difficulty in expressing GADD34 protein or any of its fragments, including the C-terminus (amino acids 533-632) in E. coli, partially due to sub-optimal expression of eukaryotic codons in prokaryotic E. coli, we used a codon-optimized cloning scheme to construct the eukaryotic gene that codes for GADD34(533-632). We derived a novel PCR protocol to assemble 20 oligonucleotides into the synthetic GADD34(533-632) gene. The clear advantage of using this protocol is that the assembled gene is without the mutation and deletion that are usually of a major problem in constructing synthetic genes. The synthetic GADD34(533-632) gene was cloned, expressed, and purified in large quantity. We obtained approximately 50 mg of GADD34(533-632) protein per liter minimum-medium culture. To our knowledge, this is the first report of a large-scale production of the C-terminus of GADD34. The production and purification of GADD34(533-632) in large quantity are essential for structure determination as well as for understanding its interactions with other proteins such as phosphatase 1-alpha using NMR spectroscopy and other biophysical methods.

Amino Acid Motifs↗
PubMed2003

1H, 13C and 15N resonance assignments of domain 1 of receptor associated protein.

The 39 kDa receptor associated protein (RAP) is a modular protein consisting of multiple domains. There has been no x-ray crystal structure of RAP available and the full-length protein does not behave well in a NMR tube. To elucidate the 3D structure of the RAP, we undertook structure determination of individual domains of the RAP. As the first step, here we report the nearly complete assignments of the (1)H, (13)C and (15)N chemical shift signals of domain 1 of the RAP.

Amino Acid Sequence↗
PubMed2002

Rapid structural fluctuations of the free HIV protease flaps in solution: relationship to crystal structures and comparison with predictions of dynamics calculations.

Crystal structures have shown that the HIV-1 protease flaps, domains that control access to the active site, are closed when the active site is occupied by a ligand. Although flap structures ranging from closed to semi-open are observed in the free protease, crystal structures reveal that even the semi-open flaps block access to the active site, indicating that the flaps are mobile in solution. The goals of this paper are to characterize the secondary structure and fast (sub-ns) dynamics of the flaps of the free protease in solution, to relate these results to X-ray structures and to compare them with predictions of dynamics calculations. To this end we have obtained nearly complete backbone and many sidechain signal assignments of a fully active free-protease construct that is stabilized against autoproteolysis by three point mutations. The secondary structure of this protein was characterized using the chemical shift index, measurements of (3h)J(NC') couplings across hydrogen bonds, and NOESY connectivities. Analysis of these measurements indicates that the protease secondary structure becomes irregular near the flap tips, residues 49-53. Model-free analysis of (15)N relaxation parameters, T(1), T(2) (T(1rho)) and (15)N-[(1)H] NOE, shows that residues in the flap tips are flexible on the sub-ns time scale, in contrast with previous observations on the inhibitor-bound protease. These results are compared with theoretical predictions of flap dynamics and the possible biological significance of the sub-ns time scale dynamics of the flap tips is discussed.

Amino Acid Sequence↗