Side Chain NMR Research Articles

Backbone and sidechain NMR assignments of residues 1-81 from yeast Sis1 in complex with an Hsp70 C-terminal EEVD peptide.

Molecular chaperones aid proteins to fold and assemble without modifying their final structure, requiring, in several folding processes, the interplay between members of the Hsp70 and Hsp40 families. Here, we report the NMR chemical shift assignments for 1H, 15N, and 13C nuclei of the backbone and side chains of the J-domain of the class B Hsp40 from Saccharomyces cerevisiae, Sis1, complexed with the C-terminal EEVD motif of Hsp70. The data revealed information on the structure and backbone dynamics that add significantly to the understanding of the J-domain-Hsp70-EEVD mechanism of interaction.

Backbone and side chain NMR assignments and secondary structure calculation of the pheromone binding protein3 of Ostrinia nubilalis, an agricultural pest.

Ostrinia nubilalis, also known as European Corn Borer (ECB), is a serious pest in Europe and North America, as well as in Central Asia and Northern Africa. It damages a variety of agricultural crops such as corn, oats, buckwheat, millet, and soybeans. causing annually at least one billion dollars in loss. The Ostrinia nubilalis pheromone-binding protein3 (OnubPBP3), preferentially expressed in the male moth antenna, has been implicated in the detection of the female-secreted pheromone blend during the mating process. Understanding the structure of and function of OnubPBP3, including the mechanism of pheromone binding and its release at the dendritic olfactory neuron (ORN), is essential if integrated pest management through sensory inhibition is to be achieved. We report here the backbone and side-chain resonance assignments of OnubPBP3 at pH 6.5 using various triple resonance NMR experiments on a 13C, 15N-labeled protein sample. The secondary structure of OnubPBP3 consists of six α-helices and an unstructured C-terminus based on backbone chemical shifts.

Fitting Force Field Parameters to NMR Relaxation Data.

We present an approach to optimize force field parameters using time-dependent data from NMR relaxation experiments. To do so, we scan parameters in the dihedral angle potential energy terms describing the rotation of the methyl groups in proteins and compare NMR relaxation rates calculated from molecular dynamics simulations with the modified force fields to deuterium relaxation measurements of T4 lysozyme. We find that a small modification of Cγ methyl groups improves the agreement with experiments both for the protein used to optimize the force field and when validating using simulations of CI2 and ubiquitin. We also show that these improvements enable a more effective a posteriori reweighting of the MD trajectories. The resulting force field thus enables more direct comparison between simulations and side-chain NMR relaxation data and makes it possible to construct ensembles that better represent the dynamics of proteins in solution.

Backbone and ILV side-chain methyl NMR resonance assignments of human Rev7/Rev3-RBM1 and Rev7/Rev3-RBM2 complexes.

Rev7 is a versatile HORMA (Hop1, Rev7, Mad2) family adaptor protein with multiple roles in mitotic regulation and DNA damage response, and an essential accessory subunit of the translesion synthesis (TLS) DNA polymerase Polζ employed in replication of damaged DNA. Within Polζ, the two copies of Rev7 interact with the two Rev7-bonding motifs (RBM1 and RBM2) of the catalytic subunit Rev3 by a mechanism characteristic of HORMA proteins whereby the "safety-belt" loop of Rev7 closes on the top of the ligand. Here we report the nearly complete backbone and Ile, Val, Leu side-chain methyl NMR resonance assignments of the 27kDa human Rev7/Rev3-RBM1 and Rev7/Rev3-RBM2 complexes (BMRB deposition numbers 51651 and 51652) that will facilitate future NMR studies of Rev7 dynamics and interactions.

Backbone and side chain NMR assignments for the ribosome maturation factor P (RimP) from Staphylococcus aureus.

The ribosomal maturation factor (RimP) is a 17.7kDa protein and is the assembly factor of the 30S subunit. RimP is essential for efficient processing of 16S rRNA and maturation (assembly) of the 30S ribosome. It was suggested that RimP takes part in stabilization of the central pseudoknot at the early stages of the 30S subunit maturation, and this process may occur before the head domain assembly and later stages of the 30S assembly, but the mechanism of this interaction is still not fully understood. Here we report the assignment of the 1H, 13C and 15N chemical shift in the backbone and side chains of RimP from Staphylococcus aureus. Analysis of chemical shifts of the main chain using TALOS + suggests that the RimP contains eight β-strands and three α-helices with the topology α1-β1-β2-α2- β3- α3- β4- β5- β6- β7- β8. Structural studies of RimP and its complex with the ribosome by integrated structural biology approaches (NMR spectroscopy, X-ray diffraction analysis and cryoelectron microscopy) will allow further screening of highly selective inhibitors of the translation of S. aureus.

Backbone and side chain NMR assignment of the heme-nitric oxide/oxygen binding (H-NOX) domain from Nostoc punctiforme

Soluble guanylate cyclase (sGC) is considered as the primary NO receptor across several known eukaryotes. The main interest regarding the biological role and its function, focuses on the H-NOX domain of the β1 subunit. This domain in its active form bears a ferrous b type heme as prosthetic group, which facilitates the binding of NO and other diatomic gases. The key point that still needs to be answered is how the protein selectively binds the NO and how the redox state of heme and coordination determines H-NOX active state upon binding of diatomic gases. H-NOX domain is present in the genomes of both prokaryotes and eukaryotes, either as a stand-alone protein domain or as a partner of a larger polypeptide. The biological functions of these signaling modules for a wide range of genomes, diverge considerably along with their ligand binding properties. In this direction, we examine the prokaryotic H-NOX protein domain from Nostoc punctiforme (Npun H-NOX). Herein, we first report the almost complete NMR backbone and side-chain resonance assignment (1H, 13C, 15 N) of Npun H-NOX domain together with the NMR chemical shift-based prediction of the domain’s secondary structure elements.

ILV methyl NMR resonance assignments of the 81kDa E. coli β-clamp.

The ring-shaped E. coli β-clamp protein is an 81kDa head-to-tail homodimer, which serves as a processivity factor anchoring the replicative polymerase to DNA, thereby increasing replication processivity and speed. In addition, it facilitates numerous protein transactions that take place on DNA during replication, repair, and damage response. We used a structure-based approach to obtain nearly complete Ile, Leu and Val side-chain methyl NMR resonance assignments of the wild-type β-clamp and its stabilized T45R/S107R variant based on site directed mutagenesis and the analysis of methyl-methyl NOESY data. The obtained assignments will facilitate future studies of the β-clamp interactions and dynamics.

1H, 13C, 15 N backbone and side-chain NMR assignments for three MAX effectors from Magnaporthe oryzae

Effectors are small and very diverse proteins secreted by fungi and translocated in plant cells during infection. Among them, MAX effectors (for Magnaporthe Avrs and ToxB) were identified as a family of effectors that share an identical fold topology despite having highly divergent sequences. They are mostly secreted by ascomycetes from the Magnaporthe genus, a fungus that causes the rice blast, a plant disease leading to huge crop losses. As rice is the first source of calories in many countries, especially in Asia and Africa, this constitutes a threat for world food security. Hence, a better understanding of these effectors, including structural and functional characterization, constitutes a strategic milestone in the fight against phytopathogen fungi and may give clues for the development of resistant varieties of rice. We report here the near complete 1H, 15N and 13C NMR resonance assignment of three new putative MAX effectors (MAX47, MAX60 and MAX67). Secondary structure determination using TALOS-N and CSI.3 demonstrates a high content of β-strands in all the three proteins, in agreement with the canonic ß-sandwich structure of MAX effectors. This preliminary study provides foundations for further structural characterization, that will help in turn to improve sequence predictions of other MAX effectors through data mining.

Backbone and ILV side-chain NMR resonance assignments of the catalytic domain of human deubiquitinating enzyme USP7.

Ubiquitin specific protease 7 (USP7) is a deubiquitinating enzyme, which removes ubiquitin tag from numerous protein substrates involved in diverse cellular processes such as cell cycle regulation, apoptosis and DNA damage response. USP7 affects stability, interaction network and cellular localization of its cellular and viral substrates by controlling their ubiquitination status. The large 41kDa catalytic domain of USP7 harbors the active site of the enzyme. Here we present a nearly complete (93%) NMR resonance assignment of isoleucine, leucine and valine (ILV) side-chains of the USP7 catalytic domain along with a refined nearly complete (93%) assignment of its backbone resonances. The reported ILV methyl group assignment will facilitate further NMR investigations of structure, interactions and conformational dynamics of the USP7 enzyme.

1H, 15N and 13C chemical shift assignments of the N-terminal domain of the two isoforms of the human apolipoprotein E.

Apolipoprotein E (ApoE) is one of the major lipid transporters in humans. It is also implicated in pathological conditions like Alzheimer's and cardiovascular diseases. The N-terminal domain of ApoE binds low-density lipoprotein receptors (LDLR) while the C-terminal domain binds to the lipid. I report the backbone and aliphatic side-chain NMR chemical shifts of the N-terminal domain of two isoforms of ApoE, namely ApoE3 NTD (BMRB No. 51,122) and ApoE4 NTD (BMRB No. 51,123) at pH 3.5 (20°C).

Fitting Side-Chain NMR Relaxation Data Using Molecular Simulations.

Proteins display a wealth of dynamical motions that can be probed using both experiments and simulations. We present an approach to integrate side-chain NMR relaxation measurements with molecular dynamics simulations to study the structure and dynamics of these motions. The approach, which we term ABSURDer (average block selection using relaxation data with entropy restraints), can be used to find a set of trajectories that are in agreement with relaxation measurements. We apply the method to deuterium relaxation measurements in T4 lysozyme and show how it can be used to integrate the accuracy of the NMR measurements with the molecular models of protein dynamics afforded by the simulations. We show how fitting of dynamic quantities leads to improved agreement with static properties and highlight areas needed for further improvements of the approach.

1H, 13C, 15N backbone and side-chain chemical shift assignments of the polyketide cyclase from Mycobacterium tuberculosis.

Polyketide cyclase from Mycobacterium tuberculosis (MtPC) is related to the formation of sterol derivatives, which may play a role in immune escape in the initial stage of macrophage infection by Mycobacterium tuberculosis. However, the structure and specific functions of MtPC are still unknown. Here we report the backbone and side-chain NMR resonance assignments for the MtPC. Most resonances were assigned and the secondary structure was predicted according to the assigned backbone resonances by TALOS-N and PECAN. These NMR assignments represent a first step towards researching the structure and function of MtPC.

NMR resonance assignments for the nucleotide binding domains of the E. coli clamp loader complex γ subunit.

The E. coli γ clamp loader is a pentameric complex of δ, δ' and three γ subunits that opens and loads β-clamp proteins onto DNA in an ATP-dependent process essential for efficient DNA replication. ATP binding to the γ subunits promotes conformational changes that enable the clamp loader to bind and open the ring-shaped β-clamp homodimer. Here we report the nearly complete backbone and side-chain 1H, 13C and 15N NMR resonance assignments of the 242-residue truncated γ subunit of the clamp loader complex, which includes the N-terminal mini (domain I) and lid (domain II) domains. This construct represents the nucleotide binding module in the clamp loader complex and provides a model system for studies of conformational rearrangements of the clamp loader induced by nucleotide binding.

Backbone and sidechain NMR assignments for the ribosome maturation factor RbfA from Escherichia coli.

RbfA (ribosome binding factor A; 15.2kDa) is a protein involved in ribosome biogenesis and has been shown to be important for growth at low temperatures and to act as a suppressor for a cold-sensitive mutation (C23U) in the ribosomal RNA of the small 30S ribosomal subunit. The 3D structure of isolated RbfA has been determined from several organisms showing that RbfA has type-II KH-domain fold topology similar to the KH domain of another assembly factor, Era, whose overexpression can compensate for the deletion of rbfA, suppressing both the cold sensitivity and abnormal accumulation of 17S rRNA in rbfA knockout stains. Interestingly, a RbfAΔ25 variant used in previous NMR studies, truncated at the C-terminal domain to remove 25 unstructured residues causing aggregation at room temperature, was biologically active in the sense that it could complement a knock-out of wildtype RbfA, although it did not act as a suppressor for a 16S cold-sensitive mutation (C23U), nor did it interact stably with the 30S subunit. To complement this work, we report the 1H, 13C, and 15N backbone and sidechain NMR resonance assignments of full length RbfA from Escherichia coli measured under physiological conditions (pH 7.6). This construct contains seven additional C-terminal residues from the cloning (i.e. one alanine and six residues from the HRV 3C cleavage site) and no aggregation issues were observed over a 1-week period at 293K. The assignment data has been deposited in the BMRB data bank under Accession No. 27857.

Backbone and sidechain NMR assignments for the ribosome maturation factor RimP from Escherichia coli.

Ribosome biogenesis is an energetically expensive and complex cellular process that involves the coordinated folding of the ribosomal RNA and dozens of ribosomal proteins. It proceeds along multiple parallel pathways and is guided by trans-acting factors called ribosome assembly factors. Although this process has been studied for decades, there are still many open questions regarding the role of the ribosome assembly factors in directing the folding of ribosome biogenesis intermediates. RimP is one of the early acting factors and guides the assembly of the small 30S ribosomal subunit by facilitating the binding of ribosomal proteins uS5 and uS12. Here we report the virtually complete 1H, 15N, and 13C chemical shift assignment of RimP from Escherichia coli. The NMR chemical shift data, deposited in the BMRB data bank under Accession No. 28014, indicates a widely folded protein composed of three alpha helices and eight beta strands.

15N, 13C and 1H resonance assignments of FKBP12 proteins from the pathogenic fungi Mucor circinelloides and Aspergillus fumigatus

Invasive fungal infections are a leading cause of death in immunocompromised patients and remain difficult to treat since fungal pathogens, like mammals, are eukaryotes and share many orthologous proteins. As a result, current antifungal drugs have limited clinical value, are sometimes toxic, can adversely affect human reaction pathways and are increasingly ineffective due to emerging resistance. One potential antifungal drug, FK506, establishes a ternary complex between the phosphatase, calcineurin, and the 12-kDa peptidyl-prolyl isomerase FK506-binding protein, FKBP12. It has been well established that calcineurin, highly conserved from yeast to mammals, is necessary for invasive fungal disease and is inhibited when in complex with FK506/FKBP12. Unfortunately, FK506 is also immunosuppressive in humans, precluding its usage as an antifungal drug, especially in immunocompromised patients. Whereas the homology between human and fungal calcineurin proteins is > 80%, the human and fungal FKBP12s share 48–58% sequence identity, making them more amenable candidates for drug targeting efforts. Here we report the backbone and sidechain NMR assignments of recombinant FKBP12 proteins from the pathogenic fungi Mucor circinelloides and Aspergillus fumigatus in the apo form and compare these to the backbone assignments of the FK506 bound form. In addition, we report the backbone assignments of the apo and FK506 bound forms of the Homo sapiens FKBP12 protein for evaluation against the fungal forms. These data are the first steps towards defining, at a residue specific level, the impacts of FK506 binding to fungal and mammalian FKBP12 proteins. Our data highlight differences between the human and fungal FKBP12s that could lead to the design of more selective anti-fungal drugs.

Resonance assignments for the tandem PWWP-ARID domains of human RBBP1.

Retinoblastoma-binding protein 1 (RBBP1), also known as AT-rich interaction domain 4A (ARID4A), is a tumour suppressor involved in the regulation of the epigenetic programming in leukemia and Prader-Willi/Angelman syndromes. The ARID domain of RBBP1 binds to DNA non-specifically and has gene suppression activity. However, no structural data has been obtained for the human RBBP1 ARID domain so far. Here we report the near-complete 1H, 13C, 15N backbone and side-chain NMR assignment of a 27kDa tandem PWWP-ARID domain construct that spans residues 171-414 with the removal of a short disordered region between the two domains. The predicted secondary structure based on the assigned chemical shifts is consistent with the structures of the isolated PWWP domain of human RBBP1 previously solved and the homologous ARID domains of other proteins.

Backbone and side-chain chemical shift assignments of full-length, apo, human Pin1, a phosphoprotein regulator with interdomain allostery.

Pin1 is a human peptidyl-prolyl cis-trans isomerase important for the regulation of phosphoproteins that are implicated in many diseases including cancer and Alzheimer's. Further biophysical study of Pin1 will elucidate the importance of the two-domain system to regulate its own activity. Here, we report near-complete backbone and side-chain 1H, 13C and 15N NMR chemical shift assignments of full-length, apo Pin1 for the purpose of studying interdomain allostery and dynamics.

Backbone and side chain NMR assignments for the ribosome binding factor A (RbfA) from Staphylococcus aureus.

Ribosome binding factor A (RbfA) is a 14.9kDa adaptive protein of cold shock, which is important for bacterial growth at low temperatures. RbfA can bind to the free 30S ribosomal subunit and interacts with the 5'-terminal helix (helix I) of 16S rRNA. RbfA is important for the efficient processing of 16S rRNA and for the maturation (assembly) of 30S ribosomal subunits. Here we report backbone and side chains 1H, 13C and 15N chemical shift assignments of RbfA from Staphylococcus aureus. Analysis of the backbone chemical shifts by TALOS+ suggests that RbfA contains four α-helixes and three β-strands with α1-β1-β2-α2-α3-β3-α4 topology. Secondary structure of RbfA have KH-domain fold topology with βααβ subunit which is characterized by a helix-kink-helix motif in which the GxxG sequence is replaced by a conserved AxG sequence, where an Ala residue at position 70 forming an interhelical kink. The solution of the structure of this protein factor and its complex with the ribosome by NMR spectroscopy, X-ray diffraction analysis and cryo-electron microscopy will allow further development of highly selective substances for slowing or completely stopping the translation of the pathogenic bacterium S. aureus, which will interfere with the synthesis and isolation of its pathogenicity factors.

Direct amide 15N to 13C transfers for solid-state assignment experiments in deuterated proteins

The assignment of protein backbone and side-chain NMR chemical shifts is the first step towards the characterization of protein structure. The recent introduction of proton detection in combination with fast MAS has opened up novel opportunities for assignment experiments. However, typical 3D sequential-assignment experiments using proton detection under fast MAS lead to signal intensities much smaller than the theoretically expected ones due to the low transfer efficiency of some of the steps. Here, we present a selective 3D experiment for deuterated and (amide) proton back-exchanged proteins where polarization is directly transferred from backbone nitrogen to selected backbone or sidechain carbons. The proposed pulse sequence uses only 1H-15N cross-polarization (CP) transfers, which are, for deuterated proteins, about 30% more efficient than 1H-13C CP transfers, and employs a dipolar version of the INEPT experiment for N-C transfer. By avoiding HN-C (HN stands for amide protons) and C-C CP transfers, we could achieve higher selectivity and increased signal intensities compared to other pulse sequences containing long-range CP transfers. The REDOR transfer is designed with an additional selective π pulse, which enables the selective transfer of the polarization to the desired 13C spins.