Affinity maturation of a portable Fab–RNA module for chaperone-assisted RNA crystallography

Abstract

Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3–6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5′-GNGACCC-3′ consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab–RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography.

RNA Structure and Cellular Applications of Fluorescent Light‐Up Aptamers

“…Their structure resolves the loop in its rigidified conformation ( Figure 4D). [66] Acloser look at the actual DFHBI-binding region reveals as tack of two G-quadruplex tiers (1 &2 )t owards the basal stem. [66] Acloser look at the actual DFHBI-binding region reveals as tack of two G-quadruplex tiers (1 &2 )t owards the basal stem.…”

“…[59,60] Initially in 2013, aR NA aptamer was selected for the sulforhodamine moiety of the sulforhodamine-dinitroaniline (SR-DN) conjugate ( Figure 2C). [66] Nevertheless three-dimensional (3D) structures of RNA molecules with atomic resolution are one of the main ways to gain in-depth insight into these highly flexible molecules. This effect might come from the chosen experimental settings in vitro,h owever,t his FLAP was also successfully utilized in vivo for imaging in E. coli.…”

“…Crystallization of RNAm olecules is known within the structural community to be challenging for several reasons. [66] Nevertheless three-dimensional (3D) structures of RNA molecules with atomic resolution are one of the main ways to gain in-depth insight into these highly flexible molecules. ForFLAPs,obtaining crystal structures has had ahuge impact because it has resolved the exact coordination of their fluorogens as well as their overall conformation.…”

“…[69] Interestingly,Fab BL3-6 builds up to 99 %ofthe buried surface area within the crystal lattice,r esulting in aRNA aptamer structure that is less influenced by the crystal packing itself.The same research group was able to show that aimproved Fab3-6 protein binding resulted in an even better crystal structure with the so far highest resolution of 1.64 ( Table 2). [66] Warner et al independently solved aS pinach structure using no assisting protein chaperone. [62] Here,t he initial published Spinach sequence was split into two halves at aflexible loop (L3-loop), resulting in two hybridizing Spinach RNAs trands ( Figure 4C).…”

“…[64] Affinity maturation of the Fab3-6 crystallization chaperon resulted in the highest resolution structure up to now (1.64 ,P DB ID: 6b14). [66] Acloser look at the actual DFHBI-binding region reveals as tack of two G-quadruplex tiers (1 &2 )t owards the basal stem. Each is built up in aclassical manner by four guanosines complexing apotassium ion ( Figure 5A,C).…”