Antigen-binding fragments with improved crystal lattice packing and enhanced conformational flexibility at the elbow region as crystallization chaperones

Abstract

It has been shown previously that a set of three modifications-termed S1, Crystal Kappa, and elbow-act synergistically to improve the crystallizability of an antigen-binding fragment (Fab) framework. Here, we prepared a phage-displayed library and performed crystallization screenings to identify additional substitutions-located near the heavy-chain elbow region-which cooperate with the S1, Crystal Kappa, and elbow modifications to increase expression and improve crystallizability of the Fab framework even further. One substitution (K141Q) supports the signature Crystal Kappa-mediated Fab:Fab crystal lattice packing interaction. Another substitution (E172G) improves the compatibility of the elbow modification with the Fab framework by alleviating some of the strain incurred by the shortened and bulkier elbow linker region. A third substitution (F170W) generates a split-Fab conformation, resulting in a powerful crystal lattice packing interaction comprising the biological interaction interface between the variable heavy and light chain domains. In sum, we have used K141Q, E172G, and F170W substitutions-which complement the S1, Crystal Kappa, and elbow modifications-to generate a set of highly crystallizable Fab frameworks that can be used as chaperones to enable facile elucidation of Fab:antigen complex structures by x-ray crystallography.

Keywords: crystal lattice packing interactions; phage display technology; protein domain swapping; surface entropy reduction; x‐ray crystallography.

FIGURE 1

Fab library design and results. (a) Fab^S1CE‐F1 (PDB entry 8T7I shown as a representative structure) (Bruce et al., 2023) with the heavy chain and light chain colored light blue or gray, respectively. Constant and variable domains of the light chain (CL/VL) and heavy chain (CH/VH) are indicated. In addition, residue positions in key regions shown as spheres are colored as follows: S1 (dark blue), Crystal Kappa (green), elbow (magenta), KGP site (cyan), FPE site (orange), GLY site (yellow). (b) The principal Fab molecule heavy chain is colored light blue, while the packing Fab molecule heavy chain and light chain are colored dark blue or green, respectively. The principle Fab light chain is omitted. (Left panel) In the crystal lattice, the Crystal Kappa mediated β‐sheet stacking interaction (Lieu et al., 2020) occurs between the CH domain (from the principal Fab in this representation) and CL′ domain of the packing Fab molecule (indicated by prime symbol). (Middle panel) Due to their proximity, the Crystal Kappa substitution (green) in the packing Fab, and the elbow substitution (magenta) in the principle Fab, form a contiguous crystal lattice packing interface. Residues Phe^136H, Asn^137H, Gln^138H, and Ile^139H of the elbow region form weak intermolecular interactions with residues Thr^155H′, Ser^156H′, and Gly^157H′ (light blue) in the CH domain of the packing Fab molecule. The KGP site, which directly follows the elbow region, is colored cyan. (Right panel) Rotating the packing Fab molecules in the crystal lattice by 180° reveals a sub‐optimal crystal lattice packing site; residues Lys^141H, Gly^142H, and Pro^143H of the KGP site do not participate in the Fab:Fab packing interaction at the Crystal Kappa:elbow junction. The flexible side chain of Lys^141H projects away from the crystal lattice packing site toward the solvent. (c) Sequences of Fab variants selected from the phage‐displayed library, The WT sequence (S1CE) is shown at the top and variant sequences (S1CE1–7) are shown below with dashes indicating identities. The number of times each variant was observed amongst 94 sequenced clones (N) is shown to the right of each sequence, followed by the protein yield from transient transfection of mammalian cell cultures, and the melting temperature (T _m ) determined by differential scanning fluorimetry.

FIGURE 2

Crystal screening results for Fabs and Fab:antigen complexes. Crystal hits for Fab^S1CE‐EPR‐1 with substitution E172G or F170W, with or without K141Q, are plotted for (a) the apo Fab‐EPR‐1 and (b) the Fab‐EPR‐1 in complex with EPOR‐ECD. The numbers of crystal hits (left y‐axis) or % of crystal hits (right y‐axis) obtained for each Fab variant (x‐axis) are shown from screening a total of 192 conditions (96 conditions each from JCSG+Eco and PACT). Putative apo Fab crystal hits (gray) in the Fab:antigen complex crystallization screening were evaluated by comparing crystal morphology for each complex drop to the corresponding apo Fab drop (see Excel document in Data S1).

FIGURE 3

Substitutions K141Q, F170W, and E172G alter Fab^S1CE‐EPR‐1 structural conformation and crystal lattice packing. (a) Crystal lattice packing arrangement of Fab‐EPR‐1 with the following frameworks: (i) Fab^S1CE (orthorhombic crystal system, space group P2₁2₁2₁), (ii) Fab^S1CE1 (trigonal crystal system, space group P3₁21), (iii) Fab^S1CE2 (trigonal crystal system, space group P3₂), (iv) Fab^S1CE3 (trigonal crystal system, space group P3₁21), (v) Fab^S1CE3 (orthorhombic crystal system, space group P2₁2₁2₁), and (vi) Fab^S1CE4 (triclinic crystal system, space group P1). The lower panel displays the asymmetric unit (ASU). Fab light and heavy chains are colored light blue or gray, respectively. The upper panel shows the ASU, in addition to the packing Fab molecules of selected symmetry mates in the crystal lattices with those light and heavy chains colored green or dark blue, respectively. Substitutions specific to each Fab variant are underscored. (b) In the Fab^S1CE1, Fab^S1CE3, and Fab^S1CE4 crystal lattice structures, the canonical VH:VL domain interaction occurs between packing Fab molecules in the crystal lattice as opposed to within the Fab molecule itself. The light and heavy chains of one Fab molecule are colored gray or light blue, respectively, while the light and heavy chains of selected packing Fabs are colored green or dark blue, respectively. Packing Fab molecule domains outside of the principal ASU are indicated with a prime symbol. Packing Fab molecule domains within the principal ASU are underscored. (i) In the Fab^S1CE1‐EPR‐1 or Fab^S1CE3‐EPR‐1 crystal structures (both P3₁21 space group), the Fab in the principal ASU interacts with a Fab in another ASU through VH:VL association in the crystal lattice to form a closed dimer arrangement (see 3aii and 3aiv). (ii) In the Fab^S1CE3‐EPR‐1 structure (P2₁2₁2₁ space group), an open‐ended crystal lattice structural arrangement forms in which the principal ASU Fab molecule interacts with two different packing Fab molecules through VH:VL association, priming the formation of an interconnected lattice array (see 3av). (iii) In the Fab^S1CE4‐EPR‐1 structure (see 3avi), a single ASU contains two Fab molecules (colored gray/light blue or green/dark blue) that associate through their respective pairings of VH:VL domains to form a closed dimer formation, distinct from that found in the other split‐Fab structures.

FIGURE 4

Molecular details of the incorporation of substitution F170W into the Fab^S1CE framework. Residues in the elbow site (WT sequence or elbow substitution sequence) are colored magenta. Residues in the FPE site and “molecular socket” site (Stanfield et al., 2006) are colored orange or blue, respectively. The principle Fab heavy and light chains are colored light blue or gray, respectively, whilst the packing Fab light chain is colored green. Fab domains from a packing ASU (in Fab^S1CE3 structures) are indicated with a prime symbol. Packing Fab domains within the principal ASU (in Fab^S1CE4 structure) are underscored. (a) In Fab structures with WT elbow sequence, residue Ser^136H, along with residues Leu^12H and Thr^134H of the VH domain, mediate weak intramolecular interactions with residues Phe^170H and Pro^171H of the FPE site in the CH domain (Fab^C‐F1 structure used for representation (Bruce et al., 2023) (PDB entry 8T7G)). (b) The elbow substitution reduces Fab conformational flexibility. (Left panel) Superposition of the Fab Fv (VH/VL) region from two representative structures that contain the WT elbow sequence (PDB entries 1BBD and 1PLG, in dark red or pink, respectively, and with elbow angles of 127.0° or 189.5°, respectively). (Right panel) Superposition of the Fv regions from two different Fab ASU molecules from a structure containing the elbow substitution (PDB entry 6AZ2, with elbow angles of 166.6° or 188.0°, respectively, colored dark blue or light blue, respectively). (c) In the Fab^S1CE structure, the side chains of Phe^170H and Pro^171H form Van der Waals interactions with the residues in the elbow region and the VH domain (Leu^12H and Thr^134H). (d–f) Incorporating the F170W substitution results in destabilization at the CH:VH interface; the bulky Trp^170H side chain cannot be accommodated in the limited space, resulting in a split Fab conformation for structures: (d) Fab^S1CE3 (space group P3₁21), (e) Fab^S1CE3 (space group P2₁2₁2₁), and (f) Fab^S1CE4 (space group P1).

FIGURE 5

Molecular details of the impact of the K141Q substitution on crystal lattice packing at the Crystal Kappa:elbow junction. Residues in the KGP site and elbow region are colored cyan or magenta, respectively. The ASU Fab heavy chain constant (CH) and variable (VH) domains are colored light blue, whilst the packing Fab heavy chain constant domain (CH′) is colored dark blue. (a) (i) In the Fab^S1CE‐EPR‐1 structure (used as a representative), the side chain of residue Lys^141H projects away from the crystal lattice packing site, toward the solvent. (ii) In the Fab^S1CE2‐EPR‐1 structure, Gln^141H and Gln^138H form hydrogen bonds and Van der Waals interactions with residues Thr^155H′, Ser^156H′, Gly^157H′, Gly^158H′, and Ser^210H′ in the packing Fab CH domain. (b) (i) In the Fab^S1CE3‐EPR‐1 structure (P2₁2₁2₁ space group structure used as a representative), the side chain of residue Lys^141H is forced unfavorably away from residues in the packing Fab CH domain. Meanwhile, a small section of the packing Fab CH domain (Ser^152H′ and Lys^153H′) remains unresolved from the electron density (dotted line). (ii) In the Fab^S1CE4‐EPR‐1 structure, the side chain of Gln^141H participates in hydrogen bonds and Van der Waals interactions with Gly^157H′ in the packing Fab CH domain, which in turn transforms the Crystal Kappa:elbow junction into a more favorable crystal lattice packing site with an extensive interaction network, supported by interactions between residues in the packing Fab CH domain and residues in the principle Fab CH and VH domains, and elbow region (see main text for more details). (c) (i) In the Fab^S1CE1‐EPR‐1:EPOR‐ECD complex structure, the side chain of Lys^141H is forced away from the crystal lattice packing site, and minimal intermolecular interactions form between residues in the principle Fab elbow region and packing Fab CH domain. (ii) In the Fab^S1CE2‐EPR‐1:EPOR‐ECD complex structure, the side chain of Gln^141H forms hydrogen bond interactions with the side chain of Ser^156H′ and peptide backbone amide group of Gly^158H′ in the packing Fab CH domain, while elbow residue Gln^138H forms interactions with Ser^156H′ in the packing CH domain.

FIGURE 6

Molecular details of the incorporation of substitution E172G into the Fab^S1CE framework. Residues in the elbow region, FPE/FPG site in the CH domain, and “molecular socket” site (Stanfield et al., 2006) in the VH domain, are colored magenta, orange, or blue, respectively. Residues Tyr^169H, Ala^192H, and Tyr^200H (in the CH domain) are colored blue. (a) In the Fab^S1CE structure, the side chain of residue Glu^172H forms various hydrogen bonds and Van der Waals interactions with residues Tyr^169H, Ala^192H, and Tyr^200H in the CH domain, which supports the position and conformation of the FPE loop region. Phe^170H and Pro^171H of the FPE site form weak intramolecular interactions with elbow residues, and Leu^12H and Thr^134H of the VH domain. (b,c) In the (b) Fab^S1CE1 and (c) Fab^S1CE2 structures, Gly^172H cannot participate in the network of intramolecular interactions in an equivalent way to Glu^172H. In addition, the φ and ψ dihedral angles in the peptide backbone are altered at positions 171 and 172 (see Figure S7). While a split‐Fab conformation was captured in the Fab^S1CE1 crystal structure, no changes to the Fab quaternary or tertiary structure occur in the Fab^S1CE2 crystal structure (see Figure 3a, ii and iii). Due to flexibility, the side chain of Phe^136H was unresolved from the electron density in the Fab^S1CE1‐EPR‐1 structure.

FIGURE 7

Substitution E172G facilitate an alternative crystal lattice packing arrangement for the Fab‐EPR‐1:EPOR‐ECD complex. Crystal lattice packing arrangement (upper panel) with symmetry mates, and asymmetric unit (lower panel) of the Fab‐EPR‐1:EPOR‐ECD complex. The ASU Fab light and heavy chains are colored light blue or gray, respectively, while packing Fab light and heavy chains are colored green or dark blue, respectively. The antigen EPOR‐ECD is colored magenta. (a) In the Fab^S1CE:EPOR‐ECD complex structure (orthorhombic crystal system, space group P2₁2₁2₁), one Fab:antigen complex is present in the ASU. (b) In the Fab^S1CE1:EPOR‐ECD complex structure (orthorhombic crystal system, space group P2₁2₁2₁), two Fab molecules and one antigen molecule are present in the ASU. (c) In the Fab^S1CE2:EPOR‐ECD complex structure (orthorhombic crystal system, space group P2₁2₁2₁), two Fab molecules and one antigen molecule are present in the ASU. For the Fab^S1CE1 and Fab^S1CE2 complex structures, the secondary Fab molecule (i.e., unbound to the antigen) domains are underscored. (d,e) The Fv:EPOR‐ECD region of the Fab^S1CE‐EPR‐1:EPOR‐ECD complex structure is superposed with the corresponding region in (d) the Fab^S1CE1‐EPR‐1:EPOR‐ECD complex structure or (e) the Fab^S1CE2‐EPR‐1:EPOR‐ECD complex structure. The Fab^S1CE1‐EPR‐1:EPOR‐ECD and Fab^S1CE2:EPR‐1:EPOR‐ECD complex structures are colored cyan.