Improvement of the crystallizability and expression of an RNA crystallization chaperone

Abstract

Crystallizing RNA has been an imperative and challenging task in the world of RNA research. Assistive methods such as chaperone-assisted RNA crystallography (CARC), employing monoclonal antibody fragments (Fabs) as crystallization chaperones have enabled us to obtain RNA crystal structures by forming crystal contacts and providing initial phasing information. Despite the early successes, the crystallization of large RNAFab complex remains a challenge in practice. The possible reason for this difficulty is that the Fab scaffold has not been optimized for crystallization in complex with RNA. Here, we have used the surface entropy reduction (SER) technique for the optimization of "C209 P4P6/Fab2 model system. Protruding lysine and glutamate residues were mutated to a set of alanines or serines to construct Fab2SMA or Fab2SMS. Expression with the shake flask approach was optimized to allow large scale production for crystallization. Crystal screening shows that significantly higher crystal-forming ratio was observed for the mutant complexes. As the chosen SER residues are far away from the CDR regions of the Fab, the same set of mutations can now be directly applied to other Fabs binding to a variety of ribozymes and riboswitches to improve the crystallizability of FabRNA complex.Keywords: chaperone assisted RNA crystallography/ Fab/protein engineering/shake flask expression/ surface entropy reduction.With every new discovery of a functional RNA, there is a sprint to obtain its crystal structure and elucidate the structurefunction relationship. The dynamic RNA research is mired by the difficulty in obtaining RNA crystals due to its structural property. The negatively charged phosphate backbone of RNA disfavours good crystal forming lattices. Compared to proteins, RNAs have fewer functional groups and have weaker tertiary interactions. The flexible tertiary structure adopting elongated shapes and inter-domain movements within RNA makes crystal packing even more difficult (1). Assistive methods for crystallization are constantly pursued to alleviate these complications (2, 3). Chaperone-assisted RNA crystallography (CARC) is one such assistive method which employs a monoclonal antibody fragment to bind RNAs of interest and aid in crystallization. Synthetic antigenbinding fragments (Fabs) selected from a phage display library can bind to large functional RNAs recognizing tertiary structures. These Fabs have large surface area, participate extensively in crystal contacts and provide good initial phasing information (4, 5).YSGX synthetic library is a reduced genetic codon library that generates randomized CDRs using codons enriched in tyrosines, serines and glycines (6). This was used to obtain the first RNAFab complex crystal structure (1.95 Å ) of ÁC209 P4P6 RNA binding to Fab2 (1, 4). ÁC209 P4P6 RNA is a single site mutant form of the P4P6 RNA of the Tetrahymena group I intron which catalyses its own excision from precursor RNA. The single site mutation increases the tertiary stability by reducing the conformational f...

An energetically beneficial leader–linker interaction abolishes ligand-binding cooperativity in glycine riboswitches

“…S6-S8); (2) the longer VCLD runs slightly faster than VCIII in the absence of glycine on native gel, while running comparably or slightly slower than VCIII in the presence of glycine ( Supplemental Fig. S9); (3) two antibody fragments (Fabs) selected against VCIII (Ravindran et al 2011) bind VCLD with similar affinities, but higher percentage binding, at saturating Fab concentrations, presumably because VCLD is less flexible and retained better during the washing step in the filter-binding assay (data not shown). The more compact and organized structure of full-length glycine riboswitches may have advantages in glycine sensing under physiological conditions in vivo if fast on rate is desired, similar to other riboswitches under kinetic control (Wickiser et al 2005;Smith and Strobel 2011;Trausch et al 2011).…”

A historical perspective on protein crystallization from 1840 to the present day

“…This applies to all types of proteins, including membrane proteins, as well as RNAs. In that case, the chaperone can be a general RNA module [318,319] or a protein [320][321][322]. Interestingly, this allowed the opportunity to crystallize biologically significant RNA:protein complexes [320,[322][323][324].…”

Detecting and Comparing Non-Coding RNAs in the High-Throughput Era

“…The accumulation of crystals is prevented by the high RNA flexibility. RNAs have flexible structures adopting inter-domain movements and with respect to proteins have weaker tertiary interactions [67]. The polyanionic charge of the phosphate backbone makes the nucleotide sequence move much more than in proteins and this makes the packaging of crystals much harder to achieve.…”