Structurally constrained phosphonate internucleotide linkage impacts oligonucleotide-enzyme interaction,and modulates siRNA activity and allele specificity

Oligonucleotides is an emerging class of chemically-distinct therapeutic modalities, where extensive chemical modifications are fundamental for their clinical applications. Inter-nucleotide backbones are critical to the behaviour of therapeutic oligonucleotides, but clinically explored backbone analogues are, effectively, limited to phosphorothioates. Here, we describe the synthesis and bio-functional characterization of an internucleotide (E)-vinylphosphonate (ⁱE-VP) backbone, where bridging oxygen is substituted with carbon in a locked stereo-conformation. After optimizing synthetic pathways for ⁱE-VP-linked dimer phosphoramidites in different sugar contexts, we systematically evaluated the impact of the ⁱE-VP backbone on oligonucleotide interactions with a variety of cellular proteins. Furthermore, we systematically evaluated the impact of ⁱE-VP on RNA-Induced Silencing Complex (RISC) activity, where backbone stereo-constraining has profound position-specific effects. Using Huntingtin (HTT) gene causative of Huntington''s disease as an example, ⁱE-VP at position 6 significantly enhanced the single mismatch discrimination ability of the RISC without negative impact on silencing of targeting wild type htt gene. These findings suggest that the ⁱE-VP backbone can be used to modulate the activity and specificity of RISC. Our study provides (i) a new chemical tool to alter oligonucleotide-enzyme interactions and metabolic stability, (ii) insight into RISC dynamics and (iii) a new strategy for highly selective SNP-discriminating siRNAs.     

Redox-controlled backbone dynamics of human cytochrome c revealed by N NMR relaxation measurements

Redox-controlled backbone dynamics in cytochrome c (Cyt c) were revealed by 2D ¹⁵N NMR relaxation experiments. ¹⁵N T₁ and T₂ values and ¹H-¹⁵N NOEs of uniformly ¹⁵N-labeled reduced and oxidized Cyt c were measured, and the generalized order parameters (S²), the effective correlation time for internal motion (τ_e), the ¹⁵N exchange broadening contributions (R_ex) for each residue, and the overall correlation time (τ_m) were estimated by model-free dynamics formalism. These dynamic parameters clearly showed that the backbone dynamics of Cyt c are highly restricted due to the covalently bound heme that functions as the stable hydrophobic core. Upon oxidation of the heme iron in Cyt c, the average S² value was increased from 0.88 ± 0.01 to 0.92 ± 0.01, demonstrating that the mobility of the backbone is further restricted in the oxidized form. Such increases in the S² values were more prominent in the loop regions, including amino acid residues near the thioether bonds to the heme moiety and positively charged region around Lys87. Both of the regions are supposed to form the interaction site for cytochrome c oxidase (CcO) and the electron pathway from Cyt c to CcO. The redox-dependent mobility of the backbone in the interaction site for the electron transfer to CcO suggests an electron transfer mechanism regulated by the backbone dynamics in the Cyt c-CcO system.     

The interaction of cholesterol with bilayers of phosphatidylethanolamine

The interaction of cholesterol with the glycerol backbone segments of phospholipids was studied in bilayers of phosphatidylethanolamine containing equimolar amounts of cholesterol. Glycerol selectively deuterated at various positions was supplied to the growth medium of Escherichia coli strain 131 GP which is defective in endogeneous glycerol synthesis. The procedure enables the stereospecific labeling of the three glycerol backbone segments of the membrane phospholipids. Phosphatidylethanolamine with wild-type fatty acid composition was purified from E. coli cells and deuterium magnetic resonance spectra were obtained either from dispersions of pure phosphatidylethanolamine or from equimolar mixtures of phosphatidylethanolamine with cholesterol. For comparative purposes 1,2-di[9,10-²H₂]elaidoyl-sn-glycero-3-phosphoethanolamine and [3-α-²H]cholesterol were synthesized in order to monitor the behavior of the fatty acyl chains and of the cholesterol molecule itself. For all deuterated segments the deuterium quadrupole splittings as well as the deuterium spin-lattice (T₁) relaxation times were measured as a function of temperature. The glycerol backbone was found to be a remarkably stable structural element of the phospholipid molecule. The quadrupole splittings of the backbone segments changed only by at most 2 kHz upon incorporation of 50 mol % cholesterol. This was in contrast to the fatty acyl chains where the same amount of cholesterol increased the quadrupole splitting by more than 20 kHz. The glycerol segments exhibited the shortest T₁ relaxation times of all CH₂ segments indicating that the glycerol backbone is the slowest motional moiety of the lipid molecule. Addition of cholesterol has no effect on the backbone motion but the fast reorientation rate of the trans-double bonds in 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine is increased dramatically.     

1H, 15N and 13C assignments of a putative peptidyl prolyl cis–trans isomerase FKBP12 from Trypanosoma brucei

TbFKBP12 is a putative peptidyl prolyl cis–trans isomerase from Trypanosoma brucei, causative agent of the African trypanosomiasis or sleeping sickness. It interacts with the immunosuppressive drug rapamycin inhibiting the formation of TORC2 complex leading to parasite death by inhibiting cell proliferation through cytokinesis blockade. Moreover, RNAi silencing of TbFKBP12 revealed essential function in both procyclic and bloodstream forms. Both facts make TbFKBP12 an attractive target for ligand development and thus structural data is desirable. In this work we report the NMR resonance assignments for ¹H, ¹⁵N and ¹³C nuclei in the backbone and side chains of the TbFKBP12 as basis for further studies of structure, backbone dynamics, interaction mapping and drug screening.     

Structural dynamics of protein backbone φ angles: extended molecular dynamics simulations versus experimental 3 J scalar couplings

³ J scalar couplings report on the conformational averaging of backbone φ angles in peptides and proteins, and therefore represent a potentially powerful tool for studying the details of both structure and dynamics in solution. We have compared an extensive experimental dataset with J-couplings predicted from unrestrained molecular dynamics simulation using enhanced sampling available from accelerated molecular dynamics or using long timescale trajectories (200 ns). The dynamic fluctuations predicted to be present along the backbone, in agreement with residual dipolar coupling analysis, are compatible with the experimental ³ J scalar couplings providing a slightly better reproduction of these experimental parameters than a high-resolution static structure.     

Reduced dimensionality (4,3)D-hnCOCANH experiment: an efficient backbone assignment tool for NMR studies of proteins

Sequence specific resonance assignment of proteins forms the basis for variety of structural and functional proteomics studies by NMR. In this context, an efficient standalone method for rapid assignment of backbone (¹H, ¹⁵N, ¹³C^α and ¹³C′) resonances of proteins has been presented here. Compared to currently available strategies used for the purpose, the method employs only a single reduced dimensionality experiment—(4,3)D-hnCOCANH and exploits the linear combinations of backbone (¹³C^α and ¹³C′) chemical shifts to achieve a dispersion relatively better compared to those of individual chemical shifts (see the text). The resulted increased dispersion of peaks—which is different in sum (CA + CO) and difference (CA ? CO) frequency regions—greatly facilitates the analysis of the spectrum by resolving the problems (associated with routine assignment strategies) arising because of degenerate amide ¹⁵N and backbone ¹³C chemical shifts. Further, the spectrum provides direct distinction between intra- and inter-residue correlations because of their opposite peak signs. The other beneficial feature of the spectrum is that it provides: (a) multiple unidirectional sequential (i→i + 1) ¹⁵N and ¹³C correlations and (b) facile identification of certain specific triplet sequences which serve as check points for mapping the stretches of sequentially connected HSQC cross peaks on to the primary sequence for assigning the resonances sequence specifically. On top of all this, the F ₂–F ₃ planes of the spectrum corresponding to sum (CA + CO) and difference (CA ? CO) chemical shifts enable rapid and unambiguous identification of sequential HSQC peaks through matching their coordinates in these two planes (see the text). Overall, the experiment presented here will serve as an important backbone assignment tool for variety of structural and functional proteomics and drug discovery research programs by NMR involving well behaved small folded proteins (MW < 15 kDa) or a range of intrinsically disordered proteins.      

Secondary structure and 1H, 13C and 15N resonance assignments of BamE, a component of the outer membrane protein assembly machinery in Escherichia coli

We report the ¹H, ¹³C and ¹⁵N backbone and side chain chemical shift assignments and secondary structure of the Escherichia coli protein BamE, a subunit of the BAM (Omp85) complex, the β-barrel assembly machinery present in all Gram-negative bacteria, mitochondria and chloroplasts and is essential for viability.     

1H, 13C and 15N backbone and side-chain resonance assignments of reduced CcmG from Escherichia coli

CcmG is a periplasmic, membrane-anchored protein widely distributed in a variety of species. In Escherichia coli, the CcmG protein always acts as a weak reductant in the electron transport chain during cytochrome c maturation (Ccm). Here we report ¹H, ¹⁵N and ¹³C backbone and side-chain resonance assignments of the reduced CcmG protein (residues 19–185, renumbered as 1–167) from E. coli. This work lays the essential basis for the further structural and functional analysis of reduced CcmG.     

Protein chemical shift assignments of the unbound and RNA-bound forms of the alternative splicing factor SUP-12 from C. elegans

The splicing factor SUP-12 from Caenorhabditis elegans binds to regulatory RNA elements in pre-mRNA in order to generate tissue-specific alternative splicing for genes such as the fibroblast growth factor receptor egl-15. In nematode muscle cells, SUP-12 promotes the use of a mutually exclusive exon to impart variant binding specificity to the EGL-15 extracellular protein domain. Here we report the side chain and backbone ¹H, ¹³C and ¹⁵N chemical shift assignments for the bacterially expressed RNA recognition motif domain from SUP-12, both in isolation as well as bound to a short RNA derived from the intron sequence between exon 4 and exon 5B of egl-15. Comparison of protein chemical shift values for both the backbone and side chain nuclei, coupled with secondary chemical shift analysis, reveal initial details of the RNA recognition.     

Backbone and side-chain assignments of an effector membrane localization domain from Vibrio vulnificus MARTX toxin

¹H, ¹³C, and ¹⁵N chemical shift assignments are presented for the isolated four-helical bundle membrane localization domain from the domain of unknown function 5 (DUF5) effector (MLD_VvDUF5) of the MARTX toxin from Vibrio vulnificus in its solution state. We have assigned 97 % of all backbone and side-chain carbon atoms, including 96 % of all backbone residues. Secondary chemical shift analysis using TALOS+ demonstrates four helices that align with those predicted by structure homology modeling using the MLDs of Pasteurella multocida toxin (PMT) and the clostridial TcdB and TcsL toxins as templates. Future studies will be towards solving the structure and determining the dynamics in the solution state.     

Backbone and side-chain resonance assignments of the membrane localization domain from Pasteurella multocida toxin

¹H, ¹³C, and ¹⁵N chemical shift assignments are presented for the isolated four-helical bundle membrane localization domain (MLD) from Pasteurella multocida toxin (PMT) in its solution state. We have assigned 99 % of all backbone and side-chain carbon atoms, including 99 % of all backbone residues excluding proline amide nitrogens. Secondary chemical shift analysis using TALOS+ demonstrates four helices, which align with those observed within the MLD in the crystal structure of the C-terminus of PMT (PDB 2EBF) and confirm the use of the available crystal structures as templates for the isolated MLDs.     

Backbone and side-chain resonance assignment of the A147T polymorph of mouse TSPO in complex with a high-affinity radioligand

The integral polytopic membrane protein TSPO is the target for numerous endogenous and synthetic ligands. However, the affinity of many ligands is influenced by a common polymorphism in TSPO, in which an alanine at position 147 is replaced by threonine, thereby complicating the use of several radioligands for clinical diagnosis. In contrast, the best-characterized TSPO ligand (R)-PK11195 binds with similar affinity to both variants of mitochondrial TSPO (wild-type and A147T variant). Here we report the ¹H, ¹³C, ¹⁵N backbone and side-chain resonance assignment of the A147T polymorph of TSPO from Mus Musculus in complex with (R)-PK11195 in DPC detergent micelles. More than 90 % of all resonances were sequence-specifically assigned, demonstrating the ability to obtain high-quality spectral data for both the backbone and the side-chains of medically relevant integral membrane proteins.     

The initiation of GTP hydrolysis by the G-domain of FeoB: insights from a transition-state complex structure

The polytopic membrane protein FeoB is a ferrous iron transporter in prokaryotes. The protein contains a potassium-activated GTPase domain that is essential in regulating the import of iron and conferring virulence to many disease-causing bacteria. However, the mechanism by which the G-domain of FeoB hydrolyzes GTP is not well understood. In particular, it is not yet known how the pivotal step in GTP hydrolysis is achieved: alignment of a catalytic water molecule. In the current study, the crystal structure of the soluble domains from Streptococcus thermophilus FeoB (NFeoB^St) in complex with the activating potassium ion and a transition-state analogue, GDP⋅AlF₄ ⁻, reveals a novel mode of water alignment involving contacts with the protein backbone only. In parallel to the structural studies, a series of seven mutant proteins were constructed that targeted conserved residues at the active site of NFeoB^St, and the nucleotide binding and hydrolysis properties of these were measured and compared to the wild-type protein. The results show that mutations in Thr35 abolish GTPase activity of the protein, while other conserved residues (Tyr58, Ser64, Glu66 and Glu67) are not required for water alignment by NFeoB^St. Together with the crystal structure, the findings suggest a new mechanism for hydrolysis initiation in small G-proteins, in which the attacking water molecule is aligned by contacts with the protein backbone only.     

Reversible sheet-turn conformational change of a cell-penetrating peptide in lipid bilayers studied by solid-state NMR

The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The ¹³C chemical shifts of ¹³C, ¹⁵N-labeled residues in the peptide indicate a reversible conformational change from β-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C^α and C^β sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.     

Characterization of the N-methyltransferase CalM involved in calcimycin biosynthesis by Streptomyces chartreusis NRRL 3882

Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the calcimycin biosynthesis gene cluster is required for calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl calcimycin to calcimycin in vitro. The enzyme was determined to have a kinetic parameter of K_m 276 μM, k_cat 1.26 min⁻¹ and k_cat/K_m 76.2 M⁻¹ s⁻¹. These results proved that CalM is a N-methyltransferase that is required for calcimycin biosynthesis, and they set the stage for generating much desired novel calcimycin derivatives by rational genetic and chemical engineering.     

NMR Backbone Dynamics of VEK-30 Bound to the Human Plasminogen Kringle 2 Domain

To gain insights into the mechanisms for the tight and highly specific interaction of the kringle 2 domain of human plasminogen (K2_Pg) with a 30-residue internal peptide (VEK-30) from a group A streptococcal M-like protein, the dynamic properties of free and bound K2_Pg and VEK-30 were investigated using backbone amide ¹⁵N-NMR relaxation measurements. Dynamic parameters, namely the generalized order parameter, S², the local correlation time, τ_e, and the conformational exchange contribution, R_ex, were obtained for this complex by Lipari-Szabo model-free analysis. The results show that VEK-30 displays distinctly different dynamic behavior as a consequence of binding to K2_Pg, manifest by decreased backbone flexibility, particularly at the binding region of the peptide. In contrast, the backbone dynamics parameters of K2_Pg displayed similar patterns in the free and bound forms, but, nonetheless, showed interesting differences. Based on our previous structure-function studies of this interaction, we also made comparisons of the VEK-30/K2_Pg dynamics results from different kringle modules complexed with small lysine analogs. The differences in dynamics observed for kringles with different ligands provide what we believe to be new insights into the interactions responsible for protein-ligand recognition and a better understanding of the differences in binding affinity and binding specificity of kringle domains with various ligands.     

A maximum entropy approach to the study of residue-specific backbone angle distributions in α-synuclein,an intrinsically disordered protein

α-Synuclein is an intrinsically disordered protein of 140 residues that switches to an α-helical conformation upon binding phospholipid membranes. We characterize its residue-specific backbone structure in free solution with a novel maximum entropy procedure that integrates an extensive set of NMR data. These data include intraresidue and sequential H^N–H^α and H^N–H^N NOEs, values for ³J_HNHα, ¹J_HαCα, ²J_CαN, and ¹J_CαN, as well as chemical shifts of ¹⁵N, ¹³C^α, and ¹³C′ nuclei, which are sensitive to backbone torsion angles. Distributions of these torsion angles were identified that yield best agreement to the experimental data, while using an entropy term to minimize the deviation from statistical distributions seen in a large protein coil library. Results indicate that although at the individual residue level considerable deviations from the coil library distribution are seen, on average the fitted distributions agree fairly well with this library, yielding a moderate population (20–30%) of the PP_II region and a somewhat higher population of the potentially aggregation-prone β region (20–40%) than seen in the database. A generally lower population of the α_R region (10–20%) is found. Analysis of ¹H–¹H NOE data required consideration of the considerable backbone diffusion anisotropy of a disordered protein.