As a target for PA‐insertion, we selected the periplasmic soluble fragment from a Site‐2 protease (S2P) homolog from the hyper‐thermophilic bacterium Aquifex ( A .) aeolicus . 14 This soluble fragment contains two tandemly arranged PSD95/Dlg/ZO‐1 (PDZ) domains (hereafter, the soluble fragment is referred to as the PDZ tandem). In our previous work, we obtained crystals of the PDZ tandem fragment, which diffracted X‐rays up to 2.8 Å resolution. 15 We found that the PDZ tandem was loosely packed in the crystal with a relatively high solvent content (74%), which is probably due to the abundance of flexible lysine residues on the molecular surface. In that study, we generated a monoclonal antibody against the PDZ tandem to utilize the Fab fragment as the crystallization chaperone. As a result, we improved the crystallographic resolution to 2.2 Å and determined the crystal structure more precisely. 15 The successful previous application of the standard approach for chaperone‐assisted crystallography makes the PDZ tandem a good system to test and evaluate a novel approach.

Antibody fragments such as antigen‐binding fragments (Fab) and variable fragments (Fv) can serve as crystallization chaperones in protein crystallography. 1 - 4 Antibody fragments increase the probability of forming inter‐molecular contacts required for lattice formation. This strategy is particularly important for difficult‐to‐crystallize proteins such as membrane proteins covered with fluidic detergent micelles and glycoproteins containing flexible sugar moieties. Although various immunization and screening techniques have been developed, 5 - 7 the establishment of antibodies that promote co‐crystallization is still a difficult task. We, therefore, attempted to explore an alternative strategy to promote crystallization by inserting the epitope sequence of an established antibody into target proteins and subsequently preparing complexes with antibody fragments. Specifically, we utilized a monoclonal antibody NZ‐1 and its epitope tag. NZ‐1 was originally generated by immunizing rats with the platelet‐aggregation‐stimulating domain of human podoplainin and the epitope was identified as the tetradecapeptide PA14 (EGGVAMPGAEDDVV). 8 Although the central portion, PA10 (GVAMPGAEDD), is indispensable and sufficient for specific binding with NZ‐1, 9 PA14 and PA12 (GVAMPGAEDDVV) both show exceptionally high affinity and slow dissociation from NZ‐1. 10 Due to these properties, the PA12 epitope has been developed as the PA tag for protein purification. In addition, a crystallographic analysis of the NZ‐1 Fab–PA14 peptide complex has shown that the epitope binds to NZ‐1 in a bent conformation with a Type II β‐turn between Pro‐5 and Gly‐6, making it possible to insert a PA tag into the loop regions of target proteins. 11 In fact, a PA‐insertion has already been used as a probe to detect conformational states of a modular cell‐surface receptor by comparing the accessibilities of the PA tag to NZ‐1. 11 NZ‐1 labeling of an inserted PA tag is also useful for locating subunits or domains within macromolecular complexes by electron microscopy (EM) imaging. 12 , 13 In this study, we further examined the applicability of the inserted PA tag‐NZ‐1 Fab pair in generating well‐diffracting crystals of target proteins.

Comparison between the Fab‐free and Fab‐bound forms indicated that the structure of target PDZ‐C domain remarkably changed in Met‐267 at the PA tag junction as a result of Fab‐binding. The βA–βB loop of PDZ‐C also showed a larger conformational change with a 6 Å shift at the Cα atom of Lys‐279 in the β‐turn, which is presumably affected by lattice contacts. However, the main chain conformation was maintained between the Fab‐free and Fab‐bound forms with an RMSD of 0.823 Å for the 71 Cα atoms excluding the PA tag and the βA–βB loop region. These observations indicate that the inserted PA tag can adopt a conformation suitable for the interaction with NZ‐1 without affecting the folding of the target PDZ‐C domain [Fig. 7 (D)].

Crystal structure of PDZ tandem (263‐PA12‐267) in the Fab‐free form. (A) Ribbon model of the entire PDZ tandem. The inserted PA tag and Met‐267 are colored in magenta and yellow, respectively. The N‐ and C‐terminal residues of the PA tag are incorporated into the β‐strands of the target PDZ‐C domain. (B) Stick model of the inserted PA tag. Inter‐strand hydrogen bonds are indicated with dotted lines. (C) Lattice contacts for PDZ tandem (263‐PA12‐267). PDZ tandem (263‐PA12‐267) is shown in surface model with the same coloring as in (A). The inserted PA tag makes direct contacts with four neighboring molecules (#1–#4) in the crystal lattice. (D) Conformational change of the inserted PA tag upon the Fab‐binding. The Cα trace model in bright colors represents the conformation in the Fab‐bound form while the pale‐colored model represents the Fab‐free form. The NZ‐1 Fab in the complex is shown as a surface model. Met‐267 alone has undergone a conformational change in the target PDZ‐C domain. In contrast, Pro‐5 and Gly‐6 maintain a β‐turn conformation after Fab‐binding.

In contrast to the pre‐modified mutant, PDZ tandem (263‐PA12‐267) produced crystals in the Fab‐free form as well, which resulted in a structure determined to 1.9 Å resolution [Fig. 7 (A)]. The electron density of the inserted PA tag sequence was also sufficiently clear to build the model. Of note, the PA tag was partly incorporated into the β‐hairpin structure of PDZ‐C. Gly‐1 and Val‐2 participated in the βF strand, and formed hydrogen bonds with Asp‐10, Val‐11, and Val‐12 included in the βA strand [Fig. 7 (B)]. In addition, the Type II β‐turn was constituted by Met‐4, Pro‐5, Gly‐6, and Ala‐7 in the typical conformation whereas the inter‐strand hydrogen bonds were disrupted at Ala‐3, Glu‐8, and Asp‐9. The extended β‐hairpin protrudes out of PDZ‐C to a length of about 16 Å, and it makes contacts with four neighboring molecules in the crystal [Fig. 7 (C)]. It appears that the lattice contacts with the β‐hairpin generate a rigidly packed crystal with a solvent content of 44.5%, which is much lower than that of the Fab‐free wild type crystal.

Although the removal of the junction residues noticeably changed the orientation of the NZ‐1 Fab with respect to the PDZ tandem, the NZ‐1 Fab again made no direct contacts with the PDZ tandem other than at the inserted PA tag in both complexes (Figs. 2 and 3 ). It is, therefore, presumed that some flexibility still remained in the relative arrangement between the NZ‐1 Fab and the PDZ tandem. Again, the PA‐insertion and Fab‐binding in these new mutants has little effect on the fold of the target PDZ domains. The 76 Cα atoms of PDZ‐N in PDZ tandem (181‐PA12‐186) were superposed on their counterparts from the wild‐type structure with an RMSD of 0.630 Å. The PDZ‐C in PDZ tandem (263‐PA12‐267) again showed a slightly larger structural change from that of the wild type, but the RMSD was reduced to 0.722 Å for 71 Cα atoms excluding the βA–βB loop region. In contrast, the relative arrangement of PDZ‐N and ‐C domains is flexible and varies between the individual models.

The two modified mutants were stable and monodisperse after purification, as shown by the elution profiles of the size‐exclusion chromatography [Supporting Information Fig. S1(E, F) ]. We successfully obtained co‐crystals with the NZ‐1 Fab for both of them. As hypothesized, the resolutions of the X‐ray diffraction were improved remarkably as compared to those of the respective pre‐modified mutants. The co‐crystal structures with PDZ tandem (181‐PA12‐186) and (263‐PA12‐267) were finally refined to resolutions of 2.0 Å and 2.6 Å, respectively [Figs. 2 (D–F) and 3 (D–F)]. In the complex with PDZ tandem (181‐PA12‐186), the 12.4 Å separation of the Cα atoms in Gly‐1 and Val‐12 caused a flip‐out of Leu‐186 at the junction into the solvent, but the inter‐strand hydrogen bonds with the βF strand were maintained in the downstream of this residue [Figs. 4 (D) and 5 (D), and Supporting Information Fig. S2(C) ]. In the complex with the PDZ tandem (263‐PA12‐267), it appeared as if removing Lys‐266 had no noticeable effect on the main chain conformation of the flanking residues and the inter‐strand hydrogen bonds [Fig. 6 (A, D)]. The recognition mode of the PA tag as well as the distance between Gly‐1 and Val‐12 were essentially consistent with those from other complexes [Figs. 4 (E) and Supporting Information Fig. S2(D) ].

The above structural analysis does not support the conclusion that fold destabilization from insertion of the PA tag caused the reduced resolution for the co‐crystal structure. Apparently, the inserted PA tag did not disrupt the fold topology of the PDZ domains in either mutant. Another possibility was that the flexibility between the PA‐inserted PDZ domain and NZ‐1 Fab increased the thermal vibration of the entire complex in the crystal lattice and decreased the resolutions of the co‐crystals. In both cases, the NZ‐1 Fab was tethered to the PDZ tandem mutants only through the inserted PA tag, without direct contacts with the remainder of the target PDZ tandem [Figs. 2 (C) and 3 (C)], while it formed lattice contacts with both the NZ‐1 Fab and the PDZ tandem from the symmetry neighbors. Furthermore, some residues located at the junction with the PA tag appeared to be highly flexible in both mutants, as mentioned above. These observations suggested that the junction residues permit some extent of a swinging motion of the NZ‐1 Fab with respect to the PDZ domain. Hence, we constructed two new mutants, PDZ tandem (181‐PA12‐186) and (263‐PA12‐267), to make more rigid complexes by deleting the respective solvent‐exposed residues at the junction sites.

Compared with the PDZ‐N domain of PDZ tandem (181‐PA12‐184), the structure of the PA‐inserted PDZ‐C domain deviated from that of the wild type to a greater extent. The 77 Cα atoms of PDZ‐C excluding the PA tag were superposed on those of the wild type with an RMSD of 0.980 Å. Unexpectedly, the largest deviation was due to a conformational change of the βA‐βB loop, rather than one near the PA‐insertion site. It had been found that the βA–βB loop was relatively flexible in the crystallographic analysis of the wild‐type PDZ tandem. 15 The conformation of this loop region is easily affected by lattice contacts because it largely projects away from the domain. Accordingly, the RMSD decreased to 0.696 Å when the six residues belonging to the βA–βB loop were excluded from the superposition.

Structural change of the PDZ‐C domain around the PA insertion site. (A) Superposition of the Cα traces. The wild‐type PDZ‐N domain and the two PA‐inserted mutants are displayed as varying shades of the same color scheme. The rigidly folded part of the PDZ‐C domain is shown in cyan where the residues undergoing significant structural changes due to the PA insertion (Lys‐266 and Met‐267) are highlighted in yellow. The two residues (Asn‐264 and Gly‐265) deleted to construct the PDZ tandem (263‐PA12‐266) are shown in dark magenta. The PA12 residues of PDZ tandem (263‐PA12‐266) and (263‐PA12‐267) are shown in bright magenta and light magenta, respectively. Close‐up view of the PA insertion site of the wild‐type PDZ‐C domain (B), PDZ tandem (263‐PA12‐266) (C), and PDZ tandem (263‐PA12‐267) (D). Inter‐strand hydrogen bonds are shown as orange dotted lines.

The recognition mode of the PA tag was also conserved in the NZ‐1 Fab‐PDZ tandem (263‐PA12‐266) complex where the Cα atoms of Gly‐1 and Val‐12 were separated by 12.1 Å [Fig. 4 (C) and Supporting Information Fig. S2(B) ]. However, a slight difference was found in the side chain conformation of Arg‐120 on the NZ‐1 heavy chain. It had been shown in the NZ‐1 Fab‐PA14 complex that this residue penetrated into the loop formed by the PA tag and seemed to fix the loop in the antigen‐binding pocket where the guanidinium group of Arg‐120 interacted with the main chain carbonyl groups of Glu‐8 and Asp‐10 of PA14 [Fig. 4 (A)]. In contrast, Arg‐120 pointed to Arg‐263 of PDZ‐C and Gly‐1 of PA12 in the NZ‐1 Fab‐PDZ tandem (263‐PA12‐266) complex [Fig. 4 (C)]. As in the above case of PDZ tandem (181‐PA12‐184), some junction residues of the βA strand, such as Lys‐266 and Met‐267, were displaced by the PA‐insertion [Fig. 6 (A–C)]. In particular, Lys‐266 made no direct contacts with the NZ‐1 Fab nor the remainder of PDZ‐C [Figs. 3 (C) and Supporting Information Fig. S2(B) ]. Both Lys‐266 and Met‐267 appeared to be highly mobile although they are located relatively close to one of the symmetry neighbors in the crystal packing.

Structural change of the PDZ‐N domain around the PA insertion site. (A) Superposition of the Cα traces. The wild‐type PDZ‐N domain and the two PA‐inserted mutants are displayed as varying shades of the same color scheme. The rigidly‐folded part of the PDZ‐N domain is shown in green where the residues undergoing significant structural changes due to the PA insertion (Glu‐184 to His‐187) are highlighted in yellow. The two residues (Asn‐182 and Gly‐183) deleted to construct the PDZ tandem (181‐PA12‐184) are shown in dark magenta. The PA12 residues of PDZ tandem (181‐PA12‐184) and (181‐PA12‐186) are shown in bright magenta and light magenta, respectively. Close‐up view of the PA insertion site of the wild type PDZ‐N domain (B), PDZ tandem (181‐PA12‐184) (C), and PDZ tandem (181‐PA12‐186) (D). Inter‐strand hydrogen bonds are shown as orange dotted lines.

As the NZ‐1 Fab rigidifies the main chain conformation of the PA tag when it is accommodated into the antigen‐binding pocket, the Cα atoms of Gly‐1 and Val‐12 were separated by 14.1 Å, which is comparable to the 12.4 Å distance in the NZ‐1 Fab‐PA14 co‐crystal [Fig. 4 (A,B)]. The separation of these two residues caused significant conformational changes in the target PDZ‐N domain at the junction with the PA tag. For instance, Glu‐184 and Val‐185 that originally belonged to the βA strand were separated from their hydrogen‐bonding partners of the βF strand. In addition, the inter‐strand hydrogen bonds were also broken at Leu‐186 and His‐187 due to a main chain distortion [Fig. 5 (A–C)]. These conformational changes at the junction are expected to increase the flexibility of the relative positioning between the PDZ tandem and the NZ‐1 Fab. Glu‐184 and His‐187 were also co‐ordinated to a metal ion with a fixed conformation in the co‐crystal, but these side chains will likely be solvent exposed and disordered under metal‐free conditions. In contrast, we observed that the overall fold of the target PDZ‐N domain was hardly affected by PA‐insertion and Fab‐binding. The 76 Cα atoms of PDZ‐N excluding the four junction residues, Glu‐184 to His‐187, could be superposed onto to those of the wild type with a root‐mean‐square deviation (RMSD) of 0.697 Å.

Conformation of the PA tag in the antigen‐binding pocket. (A) The PA14 peptide in complex with the NZ‐1 Fab (PDB code: 4YO0 ). The PA peptide is shown in magenta while the heavy and light chains of the NZ‐1 Fab are colored with light orange and light pink, respectively. Lys‐76 and Arg‐120 of the heavy chain are highlighted in blue where the hydrogen bonds formed by the side chain of Arg‐120 are indicated with dotted lines. Asp‐75 of the heavy chain is also highlighted in red. (B) PDZ tandem (181‐PA12‐184) in complex with the NZ‐1 Fab. The PDZ‐N domain is shown in green where the residues showing significant structural changes compared with the wild type are highlighted in yellow as shown in Figure 5 . Metal ions presumed to be cadmium ions are shown with sphere models. (C) PDZ tandem (263‐PA12‐266) in complex with the NZ‐1 Fab. The PDZ‐C domain is shown in cyan where the residues showing significant structural changes compared to the wild type are highlighted in yellow as shown in Figure 6 . (D) PDZ tandem (181‐PA12‐186) in complex with the NZ‐1 Fab. (E) PDZ tandem (263‐PA12‐267) in complex with the NZ‐1 Fab. The models are colored as in previous figures but the PDZ‐N and ‐C domains are shown in pale colors. The distances between the Cα atoms of Gly‐1 and Val‐12 are indicated under the respective models.

In the NZ‐1 Fab‐PDZ tandem (181‐PA12‐184) complex, the interaction mode of the complementarity determining regions with the inserted PA tag was essentially consistent with that observed in the NZ‐1 Fab‐PA14 co‐crystal, excepting the involvement of a metal ion [Fig. 4 (A, B)]. The electron density indicated that a metal ion is co‐ordinated by Glu‐8 and Asp‐9 from the PA tag together with Asp‐75 from the NZ‐1 heavy chain, although these two PA residues were recognized by Lys‐76 of the heavy chain in the NZ‐1 Fab‐PA14 complex [Supporting Information Fig. S2(A) ]. The metal ion is presumed to be a cadmium ion present in the crystallization buffer. However, we could not determine the identities of the residual electron densities due to the absence of anomalous data near the absorption edge. Likewise, coordination geometry, such as monodentate or bidentate co‐ordination by carboxyl groups, could not be unambiguously assigned due to the poor quality of electron density map.

Complex formation of the PDZ tandem with the NZ‐1 Fab through a PA‐inserted PDZ‐C domain. (A, B) Surface model of PDZ tandem (263‐PA12‐266) in complex with the NZ‐1 Fab in two different views. The inserted PA tag is shown in magenta. The residues undergoing significant structural changes compared with the wild type, as shown in Figure 6 , are highlighted in yellow. (C) Close‐up view of the binding site. The PDZ‐C domain and the inserted PA tag are shown in stick models with a transparent surface. The solvent‐accessible space between the rigidly‐folded part of the PDZ‐C domain and the NZ‐1 Fab is indicated with a double‐headed arrow. (D, E) Surface model of PDZ tandem (263‐PA12‐267) in complex with NZ‐1 Fab in two different views. (F) Close‐up view of the binding site. The models are colored as in (A, B, and C) but the PDZ domains are shown in pale colors.

Complex formation of the PDZ tandem with the NZ‐1 Fab through a PA‐inserted PDZ‐N domain. (A, B) Surface model of PDZ tandem (181‐PA12‐184) in complex with the NZ‐1 Fab in two different views. The inserted PA tag is shown in magenta. The residues undergoing significant structural changes compared with the wild type, as shown in Figure 5 , are highlighted in yellow. (C) Close‐up view of the binding site. The PDZ‐N domain and the inserted PA tag are shown as stick models with a transparent surface. The solvent‐accessible space between the rigidly folded part of the PDZ‐N domain and the NZ‐1 Fab is indicated with a double‐headed arrow. (D, E) Surface model of PDZ tandem (181‐PA12‐186) in complex with the NZ‐1 Fab in two different views. (F) Close‐up view of the binding site. The models are colored as in (A, B, and C) while the PDZ domains are shown in pale colors.

Although the mutants seemed to become less crystallizable, we discovered several crystallization conditions for both of the two mutants in complex with the NZ‐1 Fab. Many poorly diffracting co‐crystals were screened before we selected crystals that allowed us to determine crystal structures to resolutions of 3.2 Å and 4.0 Å for PDZ tandems (181‐PA12‐184) and (263‐PA12‐266) in complex with the NZ‐1 Fab, respectively (Figs. 2 (A–C) and 3 (A–C)). Inspection of the crystal packing indicated that not only the NZ‐1 Fab but also the PDZ tandem substantially contributed to the lattice formation in both cases. In particular, the moderate resolution of the co‐crystal with PDZ tandem (181‐PA12‐184) was probably due to loose lattice contacts. In the course of model building, we observed weak and disordered electron densities for the PDZ‐C portion. PDZ‐C is connected with the PA‐inserted PDZ‐N domain through a short linker, and the PDZ tandem, therefore, showed conformational flexibility. The weak electron densities suggested that PDZ‐C was involved in the lattice contacts to some extent, but we could not assign a reliable model for this portion in the end [Fig. 2 (A)].

The two PDZ tandem mutants were produced as glutathione‐S‐transferase (GST)‐fusion proteins. The mutant fragments with the PA‐insertion were monodisperse after removal of the GST portion. In size‐exclusion chromatography, both of the mutants eluted at elution volumes comparable to that of the wild type (Supporting Information Fig. S1(A–C) ), indicating that the PA insertion did not affect the folding of the PDZ domains. As a control, we introduced an L259R mutation to PDZ tandem (263‐PA12‐266). Leu‐259 participates in the hydrophobic core and contributes to the structural integrity of the PDZ‐C domain. The mutation was therefore expected to cause partial unfolding of the PDZ tandem fragment. As expected, we observed a significant peak shift to a smaller elution volume than that of wild type in the size‐exclusion chromatography (Supporting Information Fig. S1(D) ). The peak shifts observed in the PDZ tandem (181‐PA12‐184) and (263‐PA12‐266) were negligible compared with that observed in the L259R mutant, which is supportive of the correct folding for both of the PDZ tandem (181‐PA12‐184) and (263‐PA12‐266). Subsequently, we prepared complexes with the NZ‐1 Fab. Both mutants bound stably to the NZ‐1 Fab, and almost no dissociation was observed during fractionation by size‐exclusion chromatography. After purification, we attempted to crystallize the mutants alone and in complex with the NZ‐1 Fab. However, we obtained no crystals of the solitary mutants at least within the search space of our screening. As mentioned above, the wild‐type PDZ tandem tends to crystallize through loose lattice contacts. Both of the PA‐insertion sites were involved in lattice contacts for the wild‐type crystal, so there is a possibility that the insertions disrupt that form.

3D structure of the PDZ tandem from the A. aeolicus Site‐2 protease homolog. (A) Ribbon model of PDZ tandem. The six β‐strands of the respective PDZ domains are colored differently where PDZ‐N and PDZ‐C are shown in bright and pale colors, respectively. The deleted loop residues for the PA‐insertion in the β‐hairpins are indicated in magenta with dotted circles. (B) Topology diagram of the circular‐permutant PDZ domain. The loop connecting the βF and βA strands is colored in magenta and indicated with a dotted circle. In the present work, the PA tag was inserted here.

The canonical PDZ domain consists of six β‐strands, βA‐F, and two α‐helices, αA–B. Both of the two PDZ domains (PDZ‐N and ‐C) forming the PDZ tandem are circular permutants of the canonical PDZ‐fold (Fig. 1 ). As a result, the non‐canonical PDZ domains of the PDZ tandem possess the N‐terminus before βC and the C‐terminus after βB while βF and βA are connected by a loop. We selected these two βF‐βA loops of PDZ‐N and ‐C as the PA‐insertion sites (Fig. 1 (A)) because both of the two loops protrude outside of the PDZ tandem fragment. In the present work, we specify the respective PDZ tandem mutants based on the residues flanking the PA tag sequence and the length of the insertion. For instance, the mutant with the PA12 sequence inserted between Arg‐181 and Glu‐184 in the PDZ‐N is termed as PDZ tandem (181‐PA12‐184). Similarly, the PDZ‐C mutant with the PA‐insertion is termed as PDZ tandem (263‐PA12‐266).

Discussion

Application of antibody fragments has brought great success for structural biology. In particular, crystallographic analyses of membrane proteins have been accelerated dramatically by utilizing antibody fragments as crystallization chaperones.1, 2, 4 One benefit for crystallization from antibody fragments is expanded solvent‐exposed area in targets such as membrane proteins. Antibody fragments are also useful for crystallizing other difficult‐to‐crystallize proteins such as glycoproteins with conformational flexibilities.16 Most of the antibody fragment‐assisted crystallizations have been performed using an antibody that specifically recognizes the tertiary structure of the target protein. Our study further raised the possibility that an anti‐tag antibody can be applied to the same kind of crystallization strategy under the condition that the tag sequence can be inserted into the target protein without disrupting its tertiary structure. In the present work, we selected the β‐hairpins in the PDZ tandem fragment of a bacterial S2P homolog as the insertion sites because the Fab‐bound PA tag is known to adopt a loop‐like structure with a β‐turn between Pro‐5 and Gly‐6. Although the size of the PA tag is not negligible compared with the total size of the individual 80‐residue PDZ domains, the PA‐insertion did not disrupt the fold topology of the target PDZ domains for any of the mutants constructed in the present work (Supporting Information Fig. S1). Furthermore, our results have shown that the inserted PA tag can serve as a scaffold to generate a ready‐to‐crystallize complex between the NZ‐1 Fab and the target PDZ tandem. In fact, we obtained co‐crystals with the NZ‐1 Fab for all of the PA‐inserted mutants, even in cases where crystallization was not successful for the Fab‐free form.

In addition, our results strongly indicated that optimization of the insertion site should be an important process for obtaining a co‐crystal with a higher diffraction quality. We attempted to insert the PA tag into the two different sites and initially obtained co‐crystals with moderate diffraction qualities for both cases. Based on our inspection of the co‐crystal structures, we hypothesized that the conformational flexibilities between the NZ‐1 Fab and the target PDZ domains lowered the diffraction qualities. The NZ‐1 Fab did not form any specific interactions with the target PDZ domains, but was tethered to them only through the inserted PA tag within the complex [Figs. 2(C, F) and 3(C, F)]. Hence, the solvent‐accessible space present between the NZ‐1 Fab and the rigidly folded part of the PDZ domain is thought to increase thermal vibration throughout the entire complex. To test the hypothesis, we modified both of the two mutants by eliminating the flexible residues present at the insertion sites. Ultimately, the two modified mutants produced the co‐crystals with higher diffraction qualities than those of the respective pre‐modified mutants. The averaged temperature factors in the co‐crystals significantly decreased after the modification of the insertion sites (Table 2), although it is not a direct measurement of decreased conformational flexibilities in the complexes. Remarkably, the diffraction qualities were also higher than that of the Fab‐free wild‐type crystal. These results support the case that the NZ‐1 Fab plays a beneficial role in the crystallization and improved diffraction quality of the PDZ tandem mediated through the inserted PA tag.

The structure determination of PDZ tandem (263‐PA12‐267) has demonstrated that the inserted PA has potential to form a β‐hairpin structure. The formation of the β‐hairpin structure of the PA tag in the Fab‐free PDZ tandem (263‐PA12‐267) crystal is probably not only affected by the lattice contact but also dependent on the sequence context. Nevertheless, it is highly possible that β‐hairpins are generally competent as PA‐insertion sites. As the Pro‐Gly sequence generally has a higher β‐turn propensity,17, 18 it is presumed that Pro‐5 and Gly‐6 at the middle of the PA tag sequence are predisposed to form a turn at the insertion site. When incorporated as a β‐hairpin, the PA tag is expected to dock to the NZ‐1 Fab with an intact β‐turn conformation between Pro‐5 and Gly‐6. Furthermore, it is noteworthy that other than for Met‐267 the main chain conformations of the residues flanking the PA tag are almost identical in both the Fab‐free and Fab‐bound PDZ tandem (263‐PA12‐267) [Fig. 7(D)]. It seems that the inter‐strand hydrogen bonds maintained the folding of the target PDZ domain during the Fab‐binding, which also suggests that the β‐hairpins are suitable sites for PA insertion.

Although we here inserted the PA tag into a protein for which the 3D structure was initially available, it is presumed that the same co‐crystallization strategy can be applied to proteins whose 3D structures have not yet been determined experimentally. Accumulation of structural data in Protein Data Bank (PDB) and development of structure prediction algorithms19, 20 should enable us to predict the positions of loop regions that become candidates for PA‐insertion sites in proteins without an initial mid‐ to low‐resolution structure model. For instance, glycoproteins secreted to extracellular space or ectodomain fragments of membrane proteins should be ideal targets for the PA insertion as they are abundant in the β‐hairpin structures. Although multi‐pass membrane proteins are generally α‐helical in the transmembrane regions, the loop regions connecting the α‐helices can be candidates for the insertion sites. Based on our structural analysis, an additional criterion can be proposed to select candidate insertion sites. It has been shown in the complex structures that docking to the NZ‐1 Fab separates Gly‐1 and Val‐12 of the PA tag by at least 12 Å (Fig. 4). Therefore, PA tag insertion sites that conform to this distance constraint can be selected from a predicted structural model. In fact, some residues belonging to the βA strand were deleted along with the loop residues for PA insertion in the two modified mutants of the PDZ tandem. The additional deletions extended the end‐to‐end distance of the PA tag in the host proteins and thereby minimized the structural distortion upon the Fab‐binding in both two cases. We expect some difficulty in finding an optimal insertion site in the case of de novo structure determination. Nevertheless, once initial co‐crystal structures can be determined it should be possible in principle to improve the diffraction qualities of co‐crystals from low‐resolution starting points by optimizing the insertion sites, as we demonstrated in this work.

In addition to the merit for protein crystallography, it is also highly probable that complex formation with an antibody fragment through an inserted epitope will be advantageous for structural determination via cryo‐EM. In recent years, cryo‐EM analysis has undergone dramatic development and has produced a growing number of atomic models for difficult‐to‐crystallize targets.21-23 However, it is still difficult to resolve high‐resolution cryo‐EM structures for targets with smaller particle sizes and lower symmetries. Several characteristics of our approach should have benefit toward these technical challenges in cryo‐EM analysis. An antibody fragment bound to the target should increase the size of the particle and serve to identify the orientation. While the conformational flexibility between the target and the bound antibody is expected to disturb the averaging procedure, it could be addressed by optimizing the insertion site based on the preliminary structural analysis as discussed above.