Biomolecular NMR Assignments

Streptococcus pneumoniae is a Gram-positive human pathogen that causes millions of infections worldwide with an increasing occurrence of antibiotic resistance. Iron acquisition is essential for its survival and virulence, especially under host-imposed nutritional immunity. S. pneumoniae expresses several ATP-binding cassette (ABC) transporters to facilitate acquisition under iron limitation, including PitABCD, PiaABCD, and PiuBCDA. The substrate specificity of PiuBCDA is not fully established. Herein, we report the backbone 1H, 13C and 15N resonance assignments of the 31 kDa soluble, extracellular domain of the substrate binding protein PiuA in the apo form and in complex with Ga(III) and the catechol siderophore-mimic 4-LICAM. These studies provide valuable information for further functional studies of interactions with other proteins, metals, and small molecules.

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.

We report the 94 % assignment of DVU2108, a protein belonging to the Orange Protein family, that in Desulfovibrio vulgaris Hildenborough forms a protein complex named the Orange Protein complex. This complex has been shown to be implicated in the cell division of this organism. DVU2108 is a conserved protein in anaerobic microorganisms and in Desulfovibrio gigas the homologous protein was isolated with a novel Mo–Cu cluster non-covalently attached to the polypeptide chain. However, the heterologously produced DVU2108 did not contain any bound metal. These assignments provide the means to characterize the interaction of DVU2108 with the proteins that form the Orange Protein complex using NMR methods.

Cytoplasmic dynein is a motor protein that walks toward the minus end of microtubules (MTs) by utilizing the energy of ATP hydrolysis. The heavy chain of cytoplasmic dynein contains the microtubule-binding domain (MTBD). Switching of MTBD between high and low affinity states for MTs is crucial for processive movement of cytoplasmic dynein. Previous biochemical studies demonstrated that the affinity of MTBD is regulated by the AAA+ family ATPase domain, which is separated by 15 nm long coiled-coil helix. In order to elucidate the structural basis of the affinity switching mechanism of MTBD, we designed two MTBD constructs, termed MTBD-High and MTBD-Low, which are locked in high and low affinity state for MTs, respectively, by introducing a disulfide bond between the coiled-coil helix. Here, we established the backbone and side-chain assignments of MTBD-High and MTBD-Low for further structural analyses.

Leishmania donovani cofilin displays low sequence similarity to other mammalian cofilins and also possesses characteristic activity of its own. Determination of its solution structure would facilitate understanding of the molecular mechanism of actin dynamics regulation in this disease causing pathogen.

The non-structural gene V protein (pV, gVp) from fd virus is a non-specific single-stranded DNA binding protein. The role of gVp is to sequester the single-stranded DNA thus reducing the generation of the replicative DNA form and leading to the formation of progeny phage. In this study, we assigned the 13C and 15N resonances of the crystalline unbound protein by magic-angle spinning solid-state NMR. The secondary structure predicted by the NMR shifts is in excellent agreement with the X-ray structure of the same 87-residue protein.

LmrA from Lactococcus lactis is a multidrug transporter and a member of the ATP binding cassette (ABC) transporter family. ABC transporters consist of a transmembrane domain (TMD) and a nucleotide binding domain (NBD). The NBD contains the highly conserved signature motifs of this transporter superfamily. In the case of LmrA, the TMD and the NBD are expressed as a single polypeptide. LmrA catalyzes the extrusion of hydrophobic compounds including antibiotics from the cell membrane at the expense of ATP hydrolysis. ATP binds to the NBD, where binding and hydrolysis induce conformational changes that lead to the extrusion of the substrate via the TMD. Here, we report the 1H, 13C and 15N backbone chemical shift assignments of the isolated 263 amino acid containing NBD of LmrA in its ADP bound state.