Engineering 2’O-mRNA methyltransferases for industrial biocatalysis

Eukaryotic messenger RNA (mRNA) are universally modified at their 5’ end into a cap 0 structure consisting of an N7-methylguanosine and an inverted 5’-5’ triphosphate bridge linking the penultimate nucleoside. Multicellular eukaryotes possess the capacity to further modify this cap by 2’O-methylating the ribose of the penultimate nucleotide producing a so-called cap 1 structure1. This methylation seems to be a molecular signature for the discrimination between self and non-self mRNA2. In order to escape the innate immune system of the infected cell, some viruses have also evolved the ability to methylate their cap structures1. By analogy, therapeutic mRNAs must be non-immunogenic in order to restore or supplement the function of altered genes by mRNA-based therapy3. In this context, we propose to exploit the capacity of Vaccinia virus to produce non-immunogenic mRNAs. More specifically, VP39 is a 39 kDa-enzyme directly involved in the mRNAs’ post-transcriptional modifications. It catalyses the 2’O-methylation in the 5’ cap structure producing the cap 1 mRNA and acts by heterodimerisation as a processivity factor with the poly(A) RNA polymerase4. However, the low expression level of VP39 in Escherichia coli (E. coli) as well as its low in vitro catalytic efficiency have so far limited its use for industrial biocatalysis. Here, the two above-mentioned limitations are tackled by complementary approaches: i) we use a Split-GFP5 strategy coupled with ultrahigh throughput screening to select for higher soluble expression in E. coli and ii) we design smart libraries seeking to directly improve the catalytic turnover of the enzyme. 1. Leung, D. W. & Amarasinghe, G. K. When your cap matters: structural insights into self vs non-self recognition of 5’ RNA by immunomodulatory host proteins. Curr. Opin. Struct. Biol. 36, 133–141 (2016). 2. Zust, R. et al. Ribose 2’-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5. Nat Immunol 12, 137–143 (2011). 3. Sahin, U., Kariko, K. & Tureci, O. mRNA-based therapeutics - developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014). 4. Hodel, A. E., Gershon, P. D., Shi, X. & Quiocho, F. A. The 1.85 A structure of vaccinia protein VP39: A bifunctional enzyme that participates in the modification of both mRNA ends. Cell 85, 247–256 (1996). 5. Cabantous, S. & Waldo, G. S. In vivo and in vitro protein solubility assays using split GFP. Nat. Methods 3, 845–854 (2006)

RNA kink turns to the left and to the right

A helix-loop-helix within the group I intron has most of the canonical sequence elements of a kink turn (K-turn), yet it bends in the opposite direction. The reverse K-turn kinks toward the major rather than the minor grooves of the flanking helices. This suggests that there are two distinct subclasses of tertiary structures that a K-turn secondary structure can adopt. The final structure may be specified by external factors, such as protein binding or the tertiary structural context, rather than the intrinsic conformation of the RNA

Crystal structure of a group I intron splicing intermediate

A recently reported crystal structure of an intact bacterial group I self-splicing intron in complex with both its exons provided the first molecular view into the mechanism of RNA splicing. This intron structure, which was trapped in the state prior to the exon ligation reaction, also reveals the architecture of a complex RNA fold. The majority of the intron is contained within three internally stacked, but sequence discontinuous, helical domains. Here the tertiary hydrogen bonding and stacking interactions between the domains, and the single-stranded joiner segments that bridge between them, are fully described. Features of the structure include: (1) A pseudoknot belt that circumscribes the molecule at its longitudinal midpoint; (2) two tetraloop-tetraloop receptor motifs at the peripheral edges of the structure; (3) an extensive minor groove triplex between the paired and joiner segments, P6-J6/6a and P3-J3/4, which provides the major interaction interface between the intron’s two primary domains (P4-P6 and P3-P9.0); (4) a six-nucleotide J8/7 single stranded element that adopts a μ-shaped structure and twists through the active site, making critical contacts to all three helical domains; and (5) an extensive base stacking architecture that realizes 90% of all possible stacking interactions. The intron structure was validated by hydroxyl radical footprinting, where strong correlation was observed between experimental and predicted solvent accessibility. Models of the pre-first and pre-second steps of intron splicing are proposed with full-sized tRNA exons. They suggest that the tRNA undergoes substantial angular motion relative to the intron between the two steps of splicing

Enzymatic Excision of Uracil Residues in Nucleosomes Depends on the Local DNA Structure and Dynamics

The excision of uracil bases from DNA is accomplished by the enzyme uracil DNA glycosylase (UNG). Recognition of uracil bases in free DNA is facilitated by uracil base pair dynamics, but it is not known whether this same mechanistic feature is relevant for detection and excision of uracil residues embedded in nucleosomes. Here we investigate this question using nucleosome core particles (NCPs) generated from <i>Xenopus laevis</i> histones and the high-affinity “Widom 601” positioning sequence. The reactivity of uracil residues in NCPs under steady-state multiple-turnover conditions was generally decreased compared to that of free 601 DNA, mostly because of anticipated steric effects of histones. However, some sites in NCPs had equal or even greater reactivity than free DNA, and the observed reactivities were not readily explained by simple steric considerations or by global DNA unwrapping models for nucleosome invasion. In particular, some reactive uracils were found in occluded positions, while some unreactive uracils were found in exposed positions. One feature of many exposed reactive sites is a wide DNA minor groove, which allows penetration of a key active site loop of the enzyme. In single-turnover kinetic measurements, multiphasic reaction kinetics were observed for several uracil sites, where each kinetic transient was independent of the UNG concentration. These kinetic measurements, and supporting structural analyses, support a mechanism in which some uracils are transiently exposed to UNG by local, rate-limiting nucleosome conformational dynamics, followed by rapid trapping of the exposed state by the enzyme. We present structural models and plausible reaction mechanisms for the reaction of UNG at three distinct uracil sites in the NCP

High Apparent Dielectric Constant Inside a Protein Reflects Structural Reorganization Coupled to the Ionization of an Internal Asp

The dielectric properties of proteins are poorly understood and difficult to describe quantitatively. This limits the accuracy of methods for structure-based calculation of electrostatic energies and pK(a) values. The pK(a) values of many internal groups report apparent protein dielectric constants of 10 or higher. These values are substantially higher than the dielectric constants of 2–4 measured experimentally with dry proteins. The structural origins of these high apparent dielectric constants are not well understood. Here we report on structural and equilibrium thermodynamic studies of the effects of pH on the V66D variant of staphylococcal nuclease. In a crystal structure of this protein the neutral side chain of Asp-66 is buried in the hydrophobic core of the protein and hydrated by internal water molecules. Asp-66 titrates with a pK(a) value near 9. A decrease in the far UV-CD signal was observed, concomitant with ionization of this aspartic acid, and consistent with the loss of 1.5 turns of α-helix. These data suggest that the protein dielectric constant needed to reproduce the pK(a) value of Asp-66 with continuum electrostatics calculations is high because the dielectric constant has to capture, implicitly, the energetic consequences of the structural reorganization that are not treated explicitly in continuum calculations with static structures