Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments

Abstract

Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although non-complementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts.

Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization

“…3.1. Fab/EE rationale, cloning and biophysical properties Previously, we reported the successful engineering of both EE-specific and hexahistidine peptide-specific scFvs for potential use as crystallization chaperones (Pai et al, 2011;Kalyoncu et al, 2014). Ultimately, the anti-EE scFv proved to have higher affinity and to be preferable over its anti-hexahistidine counterpart likely owing to its chemical diversity, its insensitivity to pH especially at near-physiological conditions and its greater compatibility with a variety of peptide-insert locations (terminal and internal).…”

“…The plasmid for E. coli intimin (Fairman et al, 2012) was generously provided by Dr Susan Buchanan (NIH). The EE epitope was incorporated into an extramembraneous loop in wild-type intimin as described previously (intimin-EE1; Kalyoncu et al, 2014). Two alanines were inserted on either side of the EE tag via SDM using the QuikChange II mutagenesis kit (Agilent) and the primers listed in Supplementary Table S3 (intimin-EE2).…”

“…1d) and the CDRs are being used in the crystal contacts. Upon binding to target protein, Fab/EE would likely utilize other residues that are available for forming crystal contacts, perhaps including the crystal contact areas seen in scFv/EEs (Kalyoncu et al, 2014).…”

“…The representative -barrel membrane protein, E. coli intimin, was selected because of its high recombinant expression yield but its relative difficulty in crystallization (Fairman et al, 2012). Intimin was engineered with an internal EE peptide (residues 315-320 of the L4 loop region were mutated to EYMPME; intimin-EE1) as reported previously (Kalyoncu et al, 2014). Analogous to A 2 aR, a second construct possessing an insertion of two alanine residues on either side of the EE peptide was generated ( Supplementary Fig.…”

“…To the best of our knowledge, the only successful example of a targetindependent non-covalent chaperone involved the use of a Fab fragment that recognizes a portable small structural RNA element to crystallize a ribozyme (Koldobskaya et al, 2011;. Co-crystallization with client membrane proteins has been attempted by us using engineered scFvs (Pai et al, 2011;Kalyoncu et al, 2014) and by others using Fab fragments generated from commercially available FLAG-binding monoclonal antibodies (Roosild et al, 2006), but without documented success to date. Analysis of deposited co-crystal structures of membrane proteins using crystallization chaperones in the Protein Data Bank (PDB) reveals that the majority rely on the Fab format .…”