Structural characterization of B. subtilis m1A22 tRNA methyltransferase TrmK: insights into tRNA recognition.

1-Methyladenosine (m1A) is a modified nucleoside found at positions 9, 14, 22 and 58 of tRNAs, which arises from the transfer of a methyl group onto the N1-atom of adenosine. The yqfN gene of Bacillus subtilis encodes the methyltransferase TrmK (BsTrmK) responsible for the formation of m1A22 in tRNA. Here, we show that BsTrmK displays a broad substrate specificity, and methylates seven out of eight tRNA isoacceptor families of B. subtilis bearing an A22. In addition to a non-Watson-Crick base-pair between the target A22 and a purine at position 13, the formation of m1A22 by BsTrmK requires a full-length tRNA with intact tRNA elbow and anticodon stem. We solved the crystal structure of BsTrmK showing an N-terminal catalytic domain harbouring the typical Rossmann-like fold of Class-I methyltransferases and a C-terminal coiled-coil domain. We used NMR chemical shift mapping to drive the docking of BstRNASer to BsTrmK in complex with its methyl-donor cofactor S-adenosyl-L-methionine (SAM). In this model, validated by methyltransferase activity assays on BsTrmK mutants, both domains of BsTrmK participate in tRNA binding. BsTrmK recognises tRNA with very few structural changes in both partner, the non-Watson-Crick R13-A22 base-pair positioning the A22 N1-atom close to the SAM methyl group.

Modulation of the HIV nucleocapsid dynamics finely tunes its RNA-binding properties during virion genesis.

During HIV-1 assembly and budding, Gag protein, in particular the C-terminal domain containing the nucleocapsid domain (NCd), p1 and p6, is the site of numerous interactions with viral and cellular factors. Most in vitro studies of Gag have used constructs lacking p1 and p6. Here, using NMR spectroscopy, we show that the p1-p6 region of Gag (NCp15) is largely disordered, but interacts transiently with the NCd. These interactions modify the dynamic properties of the NCd. Indeed, using isothermal titration calorimetry (ITC), we have measured a higher entropic penalty to RNA-binding for the NCd precursor, NCp15, than for the mature form, NCp7, which lacks p1 and p6. We propose that during assembly and budding of virions, concomitant with Gag oligomerization, transient interactions between NCd and p1-p6 become salient and responsible for (i) a higher level of structuration of p6, which favours recruitment of budding partners; and (ii) a higher entropic penalty to RNA-binding at specific sites that favours non-specific binding of NCd at multiple sites on the genomic RNA (gRNA). The contributions of p6 and p1 are sequentially removed via proteolysis during Gag maturation such that the RNA-binding specificity of the mature protein is governed by the properties of NCd.

Bisubstrate analogues as structural tools to investigate m6A methyltransferase active sites.

RNA methyltransferases (MTases) catalyse the transfer of a methyl group to their RNA substrates using most-often S-adenosyl-L-methionine (SAM) as cofactor. Only few RNA-bound MTases structures are currently available due to the difficulties in crystallising RNA:protein complexes. The lack of complex structures results in poorly understood RNA recognition patterns and methylation reaction mechanisms. On the contrary, many cofactor-bound MTase structures are available, resulting in well-understood protein:cofactor recognition, that can guide the design of bisubstrate analogues that mimic the state at which both the substrate and the cofactor is bound. Such bisubstrate analogues were recently synthesized for proteins monomethylating the N6-atom of adenine (m6A). These proteins include, amongst others, RlmJ in E. coli and METLL3:METT14 and METTL16 in human. As a proof-of-concept, we here test the ability of the bisubstrate analogues to mimic the substrate:cofactor bound state during catalysis by studying their binding to RlmJ using differential scanning fluorimetry, isothermal titration calorimetry and X-ray crystallography. We find that the methylated adenine base binds in the correct pocket, and thus these analogues could potentially be used broadly to study the RNA recognition and catalytic mechanism of m6A MTases. Two bisubstrate analogues bind RlmJ with micro-molar affinity, and could serve as starting scaffolds for inhibitor design against m6A RNA MTases. The same analogues cause changes in the melting temperature of the m1A RNA MTase, TrmK, indicating non-selective protein:compound complex formation. Thus, optimization of these molecular scaffolds for m6A RNA MTase inhibition should aim to increase selectivity, as well as affinity.

Vascular Endothelial Growth Factor Peptide Ligands Explored by Competition Assay and Isothermal Titration Calorimetry.

The v114* cyclic peptide has been identified as a tight vascular endothelial growth factor (VEGF) ligand. Here we report on the use of isothermal titration calorimetry (ITC), 96-well plate competition assay, and circular dichroism (CD) to explore the binding determinants of a new set of related peptides. Anti-VEGF antibodies are currently used in the clinic for regulating angiogenesis in cancer and age-related macular degeneration treatment. In this context, our aim is to develop smaller molecular entities with high affinity for the growth factor by a structure activity relationship approach. The cyclic disulfide peptide v114* was modified in several ways, including truncation, substitution, and variation of the size and nature of the cycle. The results indicated that truncation or substitution of the four N-terminal amino acids did not cause severe loss in affinity, allowing potential peptide labeling. Increase of the cycle size or substitution of the disulfide bridge with a thioether linkage drastically decreased the affinity, due to an enthalpy penalty. The leucine C-terminal residue positively contributed to affinity. Cysteine N-terminal acetylation induced favorable ΔΔG° and ΔΔH° of binding, which correlated with free peptide CD spectra changes. We also propose a biochemical model to extrapolate Ki from IC50 values measured in the displacement assay. These calculated Ki correlate well with the Kd values determined by extensive direct and reverse ITC measurements.

Weak protein-cationic co-ion interactions addressed by X-ray crystallography and mass spectrometry.

The adsorption of Rb(+), Cs(+), Mn(2+), Co(2+) and Yb(3+) onto the positively charged hen egg-white lysozyme (HEWL) has been investigated by solving 13 X-ray structures of HEWL crystallized with their chlorides and by applying electrospray ionization mass spectrometry (ESI-MS) first to dissolved protein crystals and then to the protein in buffered salt solutions. The number of bound cations follows the order Cs(+) < Mn(2+) ≃ Co(2+) < Yb(3+) at 293 K. HEWL binds less Rb(+) (qtot = 0.7) than Cs(+) (qtot = 3.9) at 100 K. Crystal flash-cooling drastically increases the binding of Cs(+), but poorly affects that of Yb(3+), suggesting different interactions. The addition of glycerol increases the number of bound Yb(3+) cations, but only slightly increases that of Rb(+). HEWL titrations with the same chlorides, followed by ESI-MS analysis, show that only about 10% of HEWL binds Cs(+) and about 40% binds 1-2 Yb(3+) cations, while the highest binding reaches 60-70% for protein binding 1-3 Mn(2+) or Co(2+) cations. The binding sites identified by X-ray crystallography show that the monovalent Rb(+) and Cs(+) preferentially bind to carbonyl groups, whereas the multivalent Mn(2+), Co(2+) and Yb(3+) interact with carboxylic groups. This work elucidates the basis of the effect of the Hofmeister cation series on protein solubility.

Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55(Gag), reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNA(Lys) 3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity.

Use of a redox probe for an electrochemical RNA-ligand binding assay in microliter droplets.

In this work, we report an affordable, sensitive, fast and user-friendly electroanalytical method for monitoring the binding between unlabeled RNA and small compounds in microliter-size droplets using a redox-probe and disposable miniaturized screen-printed electrochemical cells.

In Vitro/In Vivo Production of tRNA for X-Ray Studies.

tRNAs occupy a central role in the cellular life, and they are involved in a broad range of biological processes that relies on their interaction with proteins and RNA. Crystallization and structure resolution of tRNA or/and tRNA/partner complexes can yield in valuable information on structural organizations of key elements of cellular machinery. However, crystallization of RNA, is often challenging. Here we review two methods to produce and purify tRNA in quantity and quality to perform X-ray studies.

The Interaction between tRNA(Lys) 3 and the primer activation signal deciphered by NMR spectroscopy.

The initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) requires the opening of the three-dimensional structure of the primer tRNA(Lys) 3 for its annealing to the viral RNA at the primer binding site (PBS). Despite the fact that the result of this rearrangement is thermodynamically more stable, there is a high-energy barrier that requires the chaperoning activity of the viral nucleocapsid protein. In addition to the nucleotide complementarity to the PBS, several regions of tRNA(Lys) 3 have been described as interacting with the viral genomic RNA. Among these sequences, a sequence of the viral genome called PAS for "primer activation signal" was proposed to interact with the T-arm of tRNA(Lys) 3, this interaction stimulating the initiation of reverse transcription. In this report, we investigate the formation of this additional interaction with NMR spectroscopy, using a simple system composed of the primer tRNA(Lys) 3, the 18 nucleotides of the PBS, the PAS (8 nucleotides) encompassed or not in a hairpin structure, and the nucleocapsid protein. Our NMR study provides molecular evidence of the existence of this interaction and highlights the role of the nucleocapsid protein in promoting this additional RNA-RNA annealing. This study presents the first direct observation at a single base-pair resolution of the PAS/anti-PAS association, which has been proposed to be involved in the chronological regulation of the reverse transcription.