Complete NMR chemical shift assignments of odorant binding protein 22 from the yellow fever mosquito, Aedes aegypti , bound to arachidonic acid The article listed above was initially published with incorrect copyright information. Upon publication of this Correction, the copyright of the article is changed to “The Author(s)”. The original article has been corrected.

1 H N, 13 C, and 15 N resonance assignments of human calmodulin bound to a peptide derived from the STRA6 vitamin A transporter (CaMBP2) Abstract Vitamin A is a necessary nutrient for all mammals, and it is required for the transcription of many genes and vital for vision. While fasting, the vitamin A alcohol form (Retinol) from storage in the liver is mobilized and transported through the bloodstream while bound to retinol binding protein (RBP). Details of how exactly vitamin A is released from RBP and taken into the cells are still unclear. As part of the effort to elucidate the specifics of this process, single-particle cryo-electron microscopy structural studies of STRA6 (the RBP receptor 75-kDa transmembrane receptor protein) were recently reported by Chen et al. (Science, https://doi.org/10.1126/science.aad8266, 2016). Interestingly, STRA6 from zebrafish was shown to be a stable dimer and bound to calmodulin (CaM), forming a 180-kDa complex. The topology of the STRA6 complex includes 18 transmembrane helices (nine per protomer) and two long horizontal intramembrane helices interacting at the dimer core (Chen et al., in Science, https://doi.org/10.1126/science.aad8266, 2016). CaM was shown to interact with three regions of STRA6, termed CaMBP1, CaMBP2, and CaMBP3, with the most extensive interactions involving CaMBP2. To further our understanding of Ca2+-dependence of CaM-STRA6 complex formation, studies of the structure and dynamic properties of the CaMBP2–CaM complex were initiated. For this, the 1HN, 13C, and 15N backbone resonance assignments of the 148 amino acid Ca2+-bound calmodulin protein bound to the 27-residue CaMBP2 peptide derived from STRA6 were completed here using heteronuclear multidimensional NMR spectroscopy.

Solution NMR backbone assignment reveals interaction-free tumbling of human lineage-specific Olduvai protein domains Abstract Olduvai protein domains, encoded primarily by NBPF genes, have been linked to both human brain evolution and cognitive diseases such as autism and schizophrenia. There are six primary domains that comprise the Olduvai family: three conserved domains (CON1-3) and three human lineage-specific domains (HLS1-3), which typically occur as a triplet (HLS1, HLS2 and HLS3). Herein, we present the solution NMR assignment of the backbone chemical shifts of the separate HLS1, 2 and 3 domains of NBPF15. Our data suggest that there is no change in the structure of the separate domains when compared to the full-length triplet (HLS1–HLS2–HLS3). We also demonstrate that there is no direct interaction between the three domains.

1 H, 13 C, 15 N backbone and side chain resonance assignment of the HNH nuclease from Streptococcus pyogenes CRISPR-Cas9 Abstract HNH is one of two endonuclease domains of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9 that perform site-specific cleavage of double-stranded DNA. We engineered a novel construct of this critical nuclease from Streptococcus pyogenes Cas9 that not only maintains the wild-type amino acid sequence and fold, but displays enhanced thermostability when compared to the full-length Cas9 enzyme. Here, we report backbone and side chain assignments of the HNH nuclease as a foundational step toward the characterization of protein dynamics and allostery in CRISPR-Cas9.

Backbone resonance assignments and secondary structure of Ebola nucleoprotein 600–739 construct Abstract Ebola viral infections have resulted in several deadly epidemics in recent years in West and Central Africa. Because only one of the seven proteins encoded by the viral genome possesses enzymatic activity, disruption of protein–protein interactions is a promising route for antiviral drug development. We carried out a screening campaign to identify small, drug-like compounds that bind to the C-terminal region of the multifunctional Ebola nucleoprotein (eNP) with the objective of discovering ones that disrupt its binding to other Ebola proteins or to the single-stranded RNA genome. In the course of this effort we assigned the backbone 1H, 15N, and 13C resonances of residues 600‒739, the region that contains the critical eVP30 binding region 600‒615 targeted by host factors, and used the assigned chemical shifts to predict secondary structural features and peptide dynamics. This work supports and extends the previous X-ray crystal structures and NMR studies of residues 641‒739. We found that the 600‒739 domain consists of separate regions that are largely disordered and ordered.

Backbone and side chain resonance assignments of the C-terminal domain of human TGIF1 Abstract TGIF1 is an essential regulator of cell differentiation in various biological processes, and is associated with holoprosencephaly and many cancers. The C-terminal domain of TGIF1 that was originally defined as repressive domain 2 can interact with a variety of proteins, such as transcription factor Smad2 and co-repressor Sin3A, to mediate the regulative roles of TGIF1 in diverse cell signaling pathways. However, the recognition mechanism of TGIF1 C-terminal domain for different interacting proteins remains unknown. Here, we report the nearly complete 1H, 13C, and 15N backbone and side chain resonance assignments of TGIF1 C-terminal domain (residues 256–375), laying a foundation for further research on the structure–function relationship of TGIF1.

1 H N , 13 C, and 15 N backbone resonance assignments of the human DNA ligase 3 DNA-binding domain (residues 257-477) Abstract In mammalian cells, the process of DNA ligation is necessary during DNA replication to create an intact “lagging” strand from a series of smaller Okazaki fragments and to repair DNA strand breaks that arise either due to the direct action of a DNA damaging agent or as a consequence of DNA damage excision during DNA repair. In humans, there are three genes that encode for members of the DNA ligase family (LIG1, LIG3 and LIG4) (Ellenberger and Tomkinson in Ann Rev Biochem 77:313–338. 2008). Although these genes code for polypeptides with overlapping functions in the nucleus, the only mitochondrial DNA ligase (DNA ligase IIIα), which is essential for mitochondrial genome maintenance, is encoded by the LIG3 gene (Lakshmipathy and Campbell in Mol Cell Biol 19:3869–3876, 1999; Zong et al. in Mol Cell 61:667–676, 2016) Because mitochondria play a central and multifunctional role in malignant tumor progression, there is emerging interest in targeting key mitochondrial proteins. Notably, there is evidence in pre-clinical models that inhibitors of DNA ligase IIIα, which is frequently up-regulated in cancer, preferentially target cancer cells via their effect on mitochondria (Zong et al. 2016). Since NMR spectroscopy provides unique capabilities for identifying small molecules that bind specifically to DNA ligase IIIα versus the other DNA ligases), the backbone 1HN, 13C, and 15N NMR resonance assignments were completed for a 222 amino acid DNA-binding domain of human DNA ligase III. These NMR assignments represent a vital first step towards developing DNA ligase III-selective inhibitors.

NMR chemical shift backbone assignment of the viral protein P1 encoded by the African Rice Yellow Mottle Virus Abstract RNA silencing describes a pan-eukaryotic pathway of gene regulation where doubled stranded RNA are processed by the RNAse III enzyme Dicer or homologs. In particular, plants use it as a way to defend themselves against pathogen invasions. In turn, to evade the plant immune response, viruses have developed anti-RNA silencing mechanisms. They may indeed code for proteins called “viral suppressor of RNA silencing” which block the degrading of viral genomic or messenger RNA by the plant. The Rice Mottle Virus is an African virus of the sobemovirus family, which attacks the most productive rice varieties cultivated on this continent. It encodes P1, a cysteine-rich protein described as a potential RNA silencing suppressor. P1 is a 157 amino-acid long protein, characterized by a high propensity to aggregate concomitant with a limited stability with time in the conditions used in structural studies. To overcome this problem, shorter fragments were also studied. This strategy enabled the assignment of more than 90% backbone resonances of P1. This assignment should set the base of future NMR investigation of the protein structure and of its interactions with rice cellular partners.

NMR resonance assignments for the GSPII-C domain of the PilF ATPase from Thermus thermophilus in complex with c-di-GMP Abstract The natural transformation system of the thermophilic bacterium Thermus thermophilus is one of the most efficient DNA transport systems in terms of DNA uptake rate and promiscuity. The DNA transporter of T. thermophilus plays an important role in interdomain DNA transfer in hot environments. PilF is the traffic ATPase that provides the energy for the assembly of the DNA translocation machinery and the functionally linked type IV pilus system in T. thermophilus. In contrast to other known traffic ATPases, the N-terminal region of PilF harbors three consecutive domains with homology to general secretory pathway II (GSPII) domains. These GSPII-like domains influence pilus assembly, twitching motility and transformation efficiency. A structural homolog of the PilF GSPII-like domains, the N-terminal domain of the traffic ATPase MshE from Vibrio cholerae, was recently crystallized in complex with the bacterial second messenger c-di-GMP. In order to study the consequences of c-di-GMP binding on the three-dimensional architecture of PilF, we initiated structural studies on the PilF GSPII-like domains. Here, we present the 1H, 13C and 15N chemical shift assignments for the isolated PilF GSPII-C domain from T. thermophilus in complex with c-di-GMP. In addition, the structural dynamics of the complex was investigated in an {1H},15N-hetNOE experiment.

NMR resonance assignments for the GTP-binding RNA aptamer 9-12 in complex with GTP Abstract Ligand binding RNAs such as artificially created RNA-aptamers are structurally highly diverse. Therefore, they represent important model systems for investigating RNA-folding, RNA-dynamics and the molecular recognition of chemically very different ligands, ranging from small molecules to whole cells. High-resolution structures of RNA-aptamers in complex with their cognate ligands often reveal unexpected tertiary structure elements. Recent studies on different classes of aptamers binding the nucleotide triphosphate GTP as a ligand showed that these systems not only differ widely in binding affinity but also in their ligand binding modes and structural complexity. We initiated the NMR-based structure determination of the high-affinity binding GTP-aptamer 9-12 in order to gain further insights into the diversity of ligand binding modes and structural variability of those aptamers. Here, we report 1H, 13C and 15N resonance assignments for the GTP 9-12-aptamer bound to GTP as the prerequisite for the structure determination by solution NMR.