Biomolecular NMR Assignments最新文献

UEV domains are catalytically dead variants of the E2 enzymes which play an intermediate role in ubiquitin signaling. UEV domain containing proteins, like the ESCRT-I factor Tsg101 often play critical roles in trafficking of ubiquitylated cargos or in modulating ubiquitin processivity, or in determining the type of signal that is transferred to a target protein. Ubiquitin-conjugating enzyme E2 variant (UEV) and lactate/malate dehydrogenase (UEVLD), also known as UEV3, is a human paralogue of Tsg101 with apparent associations to cancer, innate immunity, NF-κB signaling, and autophagy. It contains an N-terminal UEV domain with 56% identity to that of Tsg101 and a C-terminal lactate dehydrogenase domain. Here, we show the backbone assignments of the UEV domain from UEVLD and find that its Cα shifts are consistent with a UEV domain composition. Further experiments suggest that it may have regions corresponding to the known binding pockets of Tsg101, but further structural and functional work will be required to uncover critical determinants of UEV domain function, and the role of these domains in Ubiquitin signaling as a whole.

Drebrin (developmentally regulated brain protein) is a vital component of the Postsynaptic Density (PSD). It performs important biological roles as it interacts with the postsynaptic protein Homer and anchors the complete protein network to the cellular skeleton through actin filaments. Drebrin contains unique structural elements including an evolutionarily unconventional actin-depolymerizing factor homology (ADFH) domain that has lost its strong actin-binding ability, and a Single Alpha-Helix (SAH) motif harbored by long flexible regions. In vivo studies have suggested that a disordered segment in Drebrin plays a key role in binding filamentous actin, yet the atomic-level characterization of the binding interface between these proteins has not been reported. To bridge this gap, we designed the intrinsically disordered construct D233 and employed 3D (HN)CO(CO)NH NMR spectroscopy to accomplish a near-complete backbone resonance assignment. This work serves as an essential step toward a detailed structural and functional investigation of the interaction between Drebrin and F-Actin.

EmrE is a bacterial membrane-embedded multidrug transporter that functions as an asymmetric homodimer. EmrE is implicated in antibiotic resistance but is now known to confer either resistance or susceptibility depending on the identity of the small molecule substrate. Here, we report both solution- and solid-state NMR assignments of S64V-EmrE at pH 5.8, below the pKa of critical residues E14 and H110. This includes ¹H, ¹⁵N, and ¹³C resonance assignments of the backbone, methyl groups (isoleucine, leucine, valine, threonine and alanine) from solution NMR experiments in bicelles, and backbone and side-chain assignments from solid-state NMR ¹³C-detected experiments in liposomes.

NRAS^Q61R is a frequent mutation in melanoma. Hydrolysis of GTP by NRAS^Q61R is reported to be much slower than other KRAS and NRAS mutants. Recent structural biology efforts for KRAS and NRAS proteins have been limited to X-ray crystallography and therefore lack insight into the structure and dynamics of these proteins in solution. Here we report the ¹H^N, ¹⁵N, and ¹³C backbone and sidechain resonance assignments of the G-domain of oncogenic NRAS^Q61R-GTP (MW 19.3 kDa; aa 1–169) using heteronuclear, multidimensional NMR spectroscopy. NRAS^Q61R-GTP is a conformationally stable protein in solution. The ¹H–¹⁵N correlation cross-peaks in a 2D ¹H–¹⁵N HSQC spectrum collected after 48 h at 298 K remained intact and only minimal signs of peak-broadening were noted for select residues. High resolution NMR allowed unambiguous assignments of the ¹H–¹⁵N correlation cross-peaks for all aa residues, except Y40, in addition to a significantly large number of aliphatic and aromatic sidechain resonances. NRAS^Q61R-GTP exhibits canonical secondary structural elements in the 5 (five) α-helices, 6 (six) β-strands, and associated loop regions as predicted in TALOS-N and CSI. Order parameter (RCI-S²) values predicted by TALOS-N indicate that the NRAS^Q61R-GTP switch (SW) regions and overall backbone are less flexible than observed in KRAS4b-GTP. The SW region rigidification was validated in heteronuclear NOE measurements. ³¹P NMR experiments indicate that the G-domain of NRAS^Q61R-GTP is in a predominant state 2 (active) conformation.

KKT23 is a kinetoplastid-specific kinetochore protein that has a C-terminal GCN5-related histone acetyltransferase domain that acetylates the C-terminal tail of histone H2A. Here, we present the ¹H, ¹³C and ¹⁵N resonance assignments for the C-terminal region of KKT23 (KKT23^125–348) from Trypanosoma brucei in complex with known cofactors for acetyltransferases, acetyl coenzyme A and coenzyme A. These assignments provide the starting point for detailed investigation of the structure, dynamics and interactions of KKT23 in solution.

Human La-related protein 6 (HsLARP6) participates in the post-transcriptional regulation of type I collagen biosynthesis and is involved in the onset and progression of fibroproliferative disease. The RNA-binding protein HsLARP6 recognizes a hairpin structure known as the 5’ stem-loop (5’SL) located at the junction of 5’ untranslated and coding regions of type I collagen mRNA. Despite extensive biochemical and functional studies of the interaction between HsLARP6 and the 5’SL motif, the lack of high-resolution molecular data significantly hampers our understanding of the binding mechanism. Here, we introduced a shorter 5’SL model, named A2M5, reducing the molecular size of the protein-RNA complex as well as spectral overlap in RNA-based spectra. Furthermore, we reported the near-complete backbone and side chain resonance assignment of the La domain of HsLARP6 in a 1:1 complex with the A2M5 model RNA. These results will provide a significant platform for future NMR spectroscopic studies of 5’SL binding to the La domain of HsLARP6.

The Interferon Regulatory Factor (IRF) family of transcription factors is well known for its anti-viral activity in vertebrates. The IRF family comprises nine members (IRF1-9) which have the ability to induce the Interferon beta (IFNβ) promotor. The IRF3 and IRF7 are the key family members involved in the production of type I and type III IFN. IRF3 and IRF7 both comprise of a DNA binding domain (DBD) which binds to its cognate interferon responsive element (IRE) on its target gene promoters. Here, we report near complete backbone and partial side-chain resonance assignments of the DBD domain of the IRF3 subunit of the IRF family. The predicted secondary structure using the backbone chemical shifts largely conforms with that obtained from the crystal structure, with the TALOS-N predicted secondary structures showing slightly elongated β-strands.

Non-ribosomal peptide synthetases (NRPSs) are macromolecular enzymatic complexes responsible for the biosynthesis of an array of microbial natural products, many of which have important applications for human health. The nature of the NRPS machinery, which has been likened to an assembly line, should be amenable to bio-engineering efforts directed towards efficient synthesis of novel and tailored molecules. However, the success of such endeavours depends on a detailed understanding of the mechanistic principles governing the various steps in the peptide assembly-line. Here, we report the near-complete assignment of the Ile, Met, Leu and Val methyl-groups of the 59-kDa adaptor-condensation di-domain (BN-BC) from the Tomaymycin NRPS. These assignments will provide the foundation for future NMR studies of the complex dynamic behaviour of the condensation domain both in isolation and in the context of the enzymatic cycle, which will themselves form the basis for developing a complete mechanistic description of the central condensation reaction in this prototypical NRPS.

Protein Tyrosine Phosphatase Non-receptor type 22 (PTPN22) is a tyrosine-phosphatase that plays a major role in inhibiting T-cell activation in immune cells. Recent studies have revealed that downregulation of PTPN22 triggers T-cell activation and enhances antitumor immune response, thereby identifying PTPN22 as a potential pharmacological target in cancer-immunology.

Here we report the ¹H, ¹⁵N and ¹³C backbone resonance assignment for the 35.6 kDa catalytic domain of human PTPN22. This assignment will provide an essential experimental tool to identify and characterize potential PTPN22 inhibitors.