research papers

ISSN 2059-7983

Moving toward generalizable NZ-1 labeling for 3D
structure determination with optimized epitope-tag
insertion
Risako Tamura-Sakaguchi,a‡ Rie Aruga,a‡ Mika Hirose,b‡ Toru Ekimoto,a Takuya
Miyake,c Yohei Hizukuri,c Rika Oi,a Mika K. Kaneko,d Yukinari Kato,d,e Yoshinori
Akiyama,c Mitsunori Ikeguchi,a,f Kenji Iwasakig and Terukazu Nogia*

Received 22 December 2020
Accepted 8 March 2021
a

Edited by G. Kurisu, Osaka University, Japan‡ These authors contributed equally to thiswork.
Keywords: antibody labeling; epitope insertion;
antibody-assisted structural analysis; electron
microscopy; protein crystallography.
PDB references: NZ-1 Fab–PDZ tandem
complex, mediated by epitope inserted into the
PDZ-N domain, 7cqc; mediated by epitope
inserted into the PDZ-C domain, 7cqd
Supporting information: this article has
supporting information at journals.iucr.org/d

Graduate School of Medical Life Science, Yokohama City University, Japan, bInstitute for Protein Research, Osaka
University, Japan, cInstitute for Frontier Life and Medical Sciences, Kyoto University, Japan, dDepartment of Antibody
Drug Development, Tohoku University Graduate School of Medicine, Japan, eNew Industry Creation Hatchery Center,
Tohoku University, Japan, fRIKEN, Medical Life Sciences Innovation Hub Program, Japan, and gLife Science Center for
Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Japan. *Correspondence e-mail:nogi@yokohama-cu.ac.jp

Antibody labeling has been conducted extensively for structure determination
using both X-ray crystallography and electron microscopy (EM). However,establishing target-specific antibodies is a prerequisite for applying antibodyassisted structural analysis. To expand the applicability of this strategy, analternative method has been developed to prepare an antibody complex byinserting an exogenous epitope into the target. It has already been demonstrated
that the Fab of the NZ-1 monoclonal antibody can form a stable complex with atarget containing a PA12 tag as an inserted epitope. Nevertheless, it was also
found that complex formation through the inserted PA12 tag inevitably caused
structural changes around the insertion site on the target. Here, an attempt wasmade to improve the tag-insertion method, and it was consequently discoveredthat an alternate tag (PA14) could replace various loops on the target without
inducing large structural changes. Crystallographic analysis demonstrated thatthe inserted PA14 tag adopts a loop-like conformation with closed ends in theantigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in thebound conformation, the more optimal PA14 tag had only a minor impact on thetarget structure. In fact, the PA14 tag could also be inserted into a stericallyhindered loop for labeling. Molecular-dynamics simulations also showed a rigid
structure for the target regardless of PA14 insertion and complex formation with
the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was
performed on a bacterial site-2 protease, which enabled an approximation of thedomain arrangement based on the docking mode of the NZ-1 Fab.

1. Introduction
Antibody labeling has become a useful tool for determining
the structures of protein molecules and complexes. It has been
established that the antigen-binding fragment (Fab) and thevariable fragment (Fv) can bind to their target and serve as
chaperones to promote crystallization in X-ray crystallography, as discussed in greater depth in many previous reviews(Hino et al., 2013; Hunte & Michel, 2002; Koide, 2009).
Accordingly, a large number of crystal structures havesuccessfully been determined using antibody fragments as
crystallization chaperones. Antibody labeling has been
particularly useful for determining membrane-protein structures. The first use of antibody labeling to facilitate the crystallization of a membrane protein was in the structural analysis
of bacterial cytochrome c oxidase in complex with an Fv
fragment (Ostermeier et al., 1995). Subsequently, a highActa Cryst. (2021). D77, 645–662

https://doi.org/10.1107/S2059798321002527

645research papers
resolution crystal structure of the KcsA K+ channel was
successfully determined using a Fab as a crystallization
chaperone (Zhou et al., 2001). Utilizing Fabs has also accelerated the structure determination of G-protein-coupled
receptors such as the human 2 adrenergic receptor(Rasmussen et al., 2007; Day et al., 2007). As a consequence,
the usefulness of antibody labeling has been broadly acceptedin structural biology. Nevertheless, establishing antibodies thatstably bind to their respective targets is a prerequisite for
utilizing antibody labeling that limits its applicability.
To make antibody-assisted structural analysis more immediately applicable, we developed an alternative strategy inwhich antibody labeling is mediated through an exogenous
epitope sequence that is inserted into the target. Specifically,
we utilized the PA tag–NZ-1 antibody pair. The high-affinity
NZ-1 monoclonal antibody was established by immunizing
rats with a tetradecapeptide (EGGVAMPGAEDDVV) fromthe platelet-aggregation-stimulating domain of human podoplanin (Kato et al., 2006). It was also shown that a truncateddodecapeptide (GVAMPGAEDDVV) can bind to NZ-1 with
an affinity comparable to that of the original epitope (Fujii etal., 2014), and this has been developed as a PA tag for affinity
purification and specific labeling (in this study, for clarity, thePA tag is referred to as the PA12 tag, while the original
epitope composed of 14 residues is referred to as the PA14tag). The mode of recognition was examined by determining
the crystal structure of the NZ-1 Fab in complex with the PA14
peptide (Fujii et al., 2016). The structure shows that the PA12
portion of the PA14 peptide binds stably to NZ-1 and adopts abent loop-like conformation in the antigen-binding pocket of
NZ-1 (Supplementary Fig. S1a). These observations prompted
the prediction that the PA12 tag could bind to NZ-1 either
when fused to the termini of the target or when inserted into aloop region. In fact, our previous study demonstrated the
preparation of multiple crystallizable complexes with the
PA12 tag inserted into loop regions protruding from a globular
domain of the target (Tamura et al., 2019). Our target protein
was an integral membrane protein from the hyperthermophile
Aquifex aeolicus (Deckert et al., 1998). This membrane protein
is an orthologue of the Escherichia coli intramembraneprotease RseP that belongs to the site-2 protease family(Hizukuri et al., 2017; hereafter, E. coli RseP and theA. aeolicus orthologue are referred to as EcRseP and AaRseP,
respectively). Like EcRseP, AaRseP possesses two tandemly
arranged PSD95/Dlg/ZO-1 (PDZ) domains in the periplasmic
soluble region, referred to as the PDZ tandem (Fig. 1a). The
two PDZ domains in the tandem are each composed of six-strands and two -helices (Hizukuri et al., 2014). Both of the
PDZ domains are circular permutants of the canonical PDZ
fold (Fig. 1b). In the PDZ domains of EcRseP and AaRseP,
the new termini are formed by a chain
break between strands B and C of thecanonical fold, and the canonicaltermini on the opposite side of the PDZ
domain at strands A and F areconnected by a hairpin loop. We havedemonstrated that the PA12 tag can beinserted into the F– A loops of thetwo PDZ domains without disrupting
their structures and that the PA12inserted PDZ tandem fragment formed
a complex with the NZ-1 Fab (Tamura etal., 2019).
Our previous study also revealed that
more rigid complexes could be preparedby adjusting the insertion point to
reduce the number of residues that
undergo structural change upon binding
to the NZ-1 Fab (Tamura et al., 2019).
However, such residues could not be
eliminated completely. For instance, theinter-strand hydrogen bonds of the
F– A loops were broken around thejunctions because the C atoms of bothends of the PA12 tag were separated by
Figure 1
12–14 Å when accommodated in the
Design of PA14-insertion sites in the PDZ tandem of AaRseP. (a) 3D structure of the PDZ tandem
antigen-binding pocket of NZ-1. Thisof AaRseP. The PDZ tandem structure (PDB entry 3wkm, chain A) is shown as a ribbon model withseparation seemed to be inevitable asa transparent surface in two different views. Each PDZ domain is colored by its six -strands. The-helices conserved in the PDZ fold are colored gray. The two PA14-insertion sites are highlighted
long as we utilized the PA12 tag as thewith dotted circles. (b) Topology diagram. Both of the PDZ domains in AaRseP are circularinserted epitope. Therefore, in thispermutants of the canonical PDZ fold. The first mutant was constructed by replacing residues 182–
study we tested whether the structural
183 of the F– A loop of the PDZ-N domain with the PA14 tag. In the second mutant, PA14 waschange in the target could be reduced
inserted between residues 235 and 236 of the D– E loop of the PDZ-C domain.

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by modifying the tag sequence. Specifically, we utilized thePA14 tag, instead of PA12, which contains two additional
residues (Glu-Gly) upstream of PA12. These two residues
were flexible in the co-crystal structure of the NZ-1 Fab with
the PA14 peptide, and they may be able to change conformation to accommodate the fold of the target protein near theinsertion site. Here we again utilized the PDZ tandem of
AaRseP as the target for PA14 insertion and complex
formation with the NZ-1 Fab. In addition to structure determination by X-ray crystallography, we examined the dynamic
properties of the PA14-mediated Fab–PDZ complexes
through all-atom molecular-dynamics simulations. Finally, we
approximated the spatial arrangement of the two PDZ
domains in PA14-inserted full-length AaRseP using negativestain electron microscopy (EM) together with our improved
NZ-1 labeling technique.

2. Materials and methods
2.1. Plasmid construction for expression and in vivo cleavage
assay

The pGEX-2T-based plasmid for the PDZ tandem fragment(residues 115–292), which was constructed in our previous
study (Hizukuri et al., 2014), is hereafter termed pNO1499.
The expression plasmids for the PA14-inserted PDZ tandem
mutants were constructed by inverse PCR on pNO1499 using
primers containing the respective mutations. The PCR
products were transformed into E. coli XL-1 Blue cells afterdigestion of the pNO1499 template with DpnI. The resultant
plasmids for the PDZ tandem (181-PA24-184) and (235-PA14236) mutants are pNY1493 and pNY1468, respectively.
The DNA encoding full-length AaRseP fused with aC-terminal tag, Gly-Arg-Gly-Ser-His8 (AaRseP-His8), wasamplified by PCR using the genomic DNA of A. aeolicus
strain VF5 and primers encoding the C-terminal tag sequence.
The amplified DNA was first cloned into the NdeI/BamHI
sites of the pET-11c plasmid. Subsequently, the DNA encoding
AaRseP-His8 together with the Shine–Dalgarno sequence was
extracted and cloned into the EcoRI/BamHI sites of thepUC118 plasmid, resulting in the plasmid pNO1457. The
expression plasmids for the AaRseP mutants, pNO1461(active-site mutant E18Q), pNY1493 (181-PA14-184) andpNY1478 (235-PA14-236), were constructed by introducing
the respective mutations into pNO1457 using the inverse PCR
protocol. pTM748 (AaRseP-His8), pTM749 (active-site
mutant E18Q), pTM750 (181-PA14-184) and pTM751 (235PA14-236) were constructed by introducing a 1.5 kb fragmentof the EcoRI/BamHI-digested pUC118-based plasmid (seeabove) into the same cloning site on pTWV228. All of theplasmids used in this study are listed in Supplementary Table
S1.

thione S-transferase (GST)-fusion proteins, in which a TEV
protease recognition site was incorporated between the GST
and PDZ tandem sequences, as reported previously (Hizukuri
et al., 2014). Each PA-inserted construct was overproduced in
E. coli BL21(DE3) cells and purified from the cell lysate using
Glutathione Sepharose 4B resin (Cytiva). The mutant fragment was cleaved from the GST portion through on-column
digestion with TEV protease, and the released fragment,
which contained two additional residues (Gly-Ser) upstreamof the PDZ tandem, was further purified using cationexchange chromatography (HiTrap SP HP, Cytiva) and sizeexclusion chromatography (Superdex 200 Increase 10/300 GL,
Cytiva). In parallel, the NZ-1 Fab was prepared by cleaving
the NZ-1 antibody using papain and purifying as reported
previously (Fujii et al., 2016). NZ-1 was obtained from the
Antibody Bank (http://www.med-tohoku-antibody.com/topics/
antibody.htm) at Tohoku University, Miyagi, Japan. The
purified PDZ tandem mutant was mixed with the NZ-1 Fab in
a 2:1 molar ratio and was applied to size-exclusion chromatography to fractionate the complex. The final protein sample
was concentrated by ultrafiltration.
2.3. Crystallization and data collection

The initial crystallization conditions were screened using
the Index screening kit from Hampton Research containing 96
conditions. 0.2 ml each of protein solution and reagent weredispensed into 96-well plates using a Gryphon robotic crystallization system (Art Robbins Instruments) and equilibrated
against 60 ml reservoir solution by the sitting-drop vapordiffusion method. Diffraction-quality crystals of the PDZ
tandem (181-PA14-184) complexed with the NZ-1 Fab weregenerated from a crystallization buffer consisting of 20%(w/v)
polyethylene glycol (PEG) 3350, 0.2 M potassium sodium
tartrate. Diffraction-quality crystals of the PDZ tandem (235PA14-236) complexed with the NZ-1 Fab were obtained froma crystallization buffer consisting of 10%(w/v) PEG 3350,
0.2 M l-proline, 0.1 M HEPES–Na pH 7.5. For each crystallization condition, cryoprotectant was prepared by mixing the
crystallization buffer and ethylene glycol in a 4:1 volume ratio.
All of the crystals were quickly soaked in the cryoprotectant
and cooled in liquid nitrogen. X-ray diffraction data werecollected using a PILATUS3 S 6M photon-counting pixelarray detector (Dectris) on BL-5A and BL-17A at Photon
Factory (PF), Tsukuba, Japan. The data were processed andscaled with XDS (Kabsch, 2010) and AIMLESS (Evans &Murshudov, 2013). Diffraction intensities were converted tostructure factors using programs from CCP4 (Winn et al.,2011), where 5% of the unique reflections were randomly
selected as a test set for calculation of the free R factor. Datacollection statistics are summarized in Table 1.
2.4. Crystallographic analysis

2.2. Purification of the PA14-inserted PDZ tandem mutantsin complex with the NZ-1 FabUsing the plasmids constructed above, the PA14-inserted
PDZ tandem fragments were produced as N-terminal glutaActa Cryst. (2021). D77, 645–662

For both of the co-crystals, initial phases were determinedby the molecular-replacement method using MOLREP (Vagin& Teplyakov, 2010) in CCP4. Firstly, the Fv and constantregions of the NZ-1 Fab were assigned separately using the
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Table 1

Table 2

Data-collection statistics for Fab complexes.

Refinement statistics for Fab complexes.

Values in parentheses are for the highest resolution shell.

Values in parentheses are for the highest resolution shell.

PDZ tandem

181-PA14-184

235-PA14-236

PDZ tandem

181-PA14-184

235-PA14-236

Space groupa, b, c (Å)
, , ( )No. of complexes inasymmetric unit
X-ray source
Wavelength (Å)
Resolution limits (Å)
No. of unique reflections
Completeness (%)
MultiplicityhI/(I)i
RmergeRmeas
CC1/2

P21212152.36, 75.20, 172.53
90, 90, 90
1

P2181.36, 80.18, 168.54
90, 95.7, 90
242.97–2.50 (2.55–2.50)
0.230 (0.363)
0.258 (0.457)

38.99–3.20 (3.25–3.20)
0.265 (0.415)
0.286 (0.431)

4740

7901

BL-5A, PF
1.000045.68–2.50 (2.60–2.50)
24452 (2685)
99.9 (99.6)
6.6 (6.8)
13.1 (1.5)
0.093 (1.469)
0.102 (1.589)
0.998 (0.674)

BL-17A, PF
0.980038.99–3.20 (3.36–3.20)
35827 (4745)
99.5 (99.7)
3.4 (3.5)
8.0 (1.2)
0.115 (0.965)
0.137 (1.141)
0.994 (0.774)

Resolution limits (Å)
Rwork†
Rfree‡
No. of non-H atoms
Total
Complex 1
PDZ-N
PDZ-C
NZ-1 Fab (H)NZ-1 Fab (L)Complex 2
PDZ-N
PDZ-C
NZ-1 Fab (H)NZ-1 Fab (L)Solvent
Average B factors (Å2)
Overall
Complex 1
PDZ-N
PDZ-C
NZ-1 Fab (H)NZ-1 Fab (L)Complex 2
PDZ-N
PDZ-C
NZ-1 Fab (H)NZ-1 Fab (L)Solvent
R.m.s.d. from ideality
Bond lengths (Å)
Bond angles ( )Ramachandran plot
Favored (%)
Outliers (%)
PDB code

801
651
1597
1641

—
734
1575
1641

—
—
—
—
50

—
734
1576
1641
0

72.80

121.4461.26
122.09
63.70
68.26

—
132.18121.75121.11

—
—
—
—
55.91

—
146.29117.34109.51
—

0.002
0.54

0.003
0.85

95.17
0.33
7cqc

94.81
0.29
7cqd

atomic coordinates of the NZ-1 Fab bound to the PA14
peptide (Fujii et al., 2016; PDB entry 4yo0). Next, the PDZ-Nand PDZ-C domains were searched for separately using theatomic coordinates of the A. aeolicus PDZ tandem (Hizukuri
et al., 2014; PDB entry 3wkl) with the NZ-1 Fab models fixedin the asymmetric unit. The assigned models were manually
fitted into the electron-density map using Coot (Emsley et al.,2010). The updated models were refined with phenix.refine(Afonine et al., 2012) iteratively while monitoring the stereochemistry with MolProbity (Chen et al., 2010). As reported in
the previous study (Tamura et al., 2019), the electron densities
indicated that Asn150 side chain reacted with the main-chain
amide group of Gly151 and formed a succinimide group in the
PDZ tandem (181-PA14-184) mutant. Refinement statistics
are summarized in Table 2. The atomic coordinates of the Fabcomplexes of the PDZ tandem (181-PA14-184) and (235PA14-236) mutants have been deposited in the Protein Data
Bank with accession codes 7cqc and 7cqd, respectively.
Structural superpositions and r.m.s.d. calculations were
performed by the pairwise alignment protocol using LSQKAB(Kabsch, 1976). Figures showing protein structures wereprepared with PyMOL (version 2.3; Schrödinger).
2.5. In vivo cleavage assay of AaRseP and its mutants

The in vivo proteolytic activity of AaRseP was analyzed
using E. coli KK211 (rseA, rseP) cells (Kanehara et al.,2002) as described previously (Akiyama et al., 2015; Hizukuri
et al., 2017). E. coli KK211 (rseA, rseP) cells harboring
pYH124 [HA-MBP-RseA(LY1)148] were transformed withthe pKK11 plasmid (EcRseP-His6-Myc; Kanehara et al., 2001),
pTM748 (AaRseP-His8), pTWV228 or plasmids encoding
their derivatives. M9 medium (without CaCl2; Miller, 1972)supplemented with 20 mg ml1 of each of the 20 amino acids,
2 mg ml1 thiamine, 0.4% glucose, 1 mM isopropyl -d-1thiogalactopyranoside (IPTG) and 5 mM cAMP was inoculated with transformed E. coli KK211 cells and grown at 30 C
for 3 h. Proteins were precipitated by trichloroacetic acid
(TCA) treatment and separated by Laemmli SDS–PAGE.
Immunoblots with anti-HA, anti-His or anti-SecB antibodies

648

Tamura-Sakaguchi et al.  Antibody labeling† Rwork is the crystallographic R factor calculated for the working set consisting of 95%
of reflections used in refinement. ‡ Rfree is the crystallographic R factor calculated forthe test set consisting of 5% of reflections excluded from refinement.

were visualized using a Lumino LAS 4000 mini image analyzer(Cytiva) with ECL Prime Western Blotting Detection
Reagents (Cytiva). Rabbit polyclonal anti-HA [HA-probe(Y-11), Santa Cruz Biotechnology] and anti-SecB (Miyake etal., 2020) antibodies were used for immunoblotting. For thedetection of His-tagged proteins, anti-His antibodies from thePenta-His HRP Conjugate Kit (Qiagen) were used.

2.6. Purification of the AaRseP mutants and complex
formation with the NZ-1 Fab

For the EM analysis, the PA14-inserted AaRseP mutant was
overproduced in E. coli KK374 (rseA, rseP, degS) cells(Akiyama et al., 2004). E. coli KK374 cells transformed withthe expression plasmids (Supplementary Table 1) were grown
at 30 C to an OD600 of 0.7 in a medium containing 10 g Bacto
tryptone, 5 g yeast extract and 10 g NaCl per litre supplemented with 50 mg ml1 ampicillin, followed by the inductionof overexpression with 0.1 mM IPTG and incubation at 30 C
for an additional 4 h. The cells were harvested by centrifugation and lysed by sonication in 10 mM Tris–HCl pH 7.4,
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150 mM NaCl. The cell lysates were centrifuged at 40 000g for
45 min at 277 K. Subsequently, the supernatant was further
separated by ultracentrifugation at 200 000g for 90 min at
277 K. The membrane fraction collected as a precipitate wassuspended in 10 mM Tris–HCl pH 7.4, 150 mM NaCl and was
ultracentrifuged again under the same conditions. Finally, theprecipitated membrane fraction was suspended in 10 mM
Tris–HCl, 150 mM NaCl and the total protein was quantified
using the bicinchoninic acid (BCA) assay. The suspension of
the membrane fraction was diluted with the same buffer toadjust the protein concentration to 10 mg ml1 using bovine
serum albumin as a standard.
Membrane proteins were solubilized by adding the same
volume of a solubilization buffer consisting of 40 mM Tris–
HCl pH 8.0, 150 mM NaCl, 2% n-dodecyl-N,N-dimethylamine-N-oxide (DDAO) to the above-prepared suspension of
the membrane fraction. After incubation at 277 K for 1 h, themixture was ultracentrifuged at 210 000g for 90 min at 277 K.
The supernatant was applied onto Ni–NTA agarose resin andthe unbound fraction was washed out with a buffer consistingof 20 mM Tris–HCl pH 8.0, 300 mM NaCl, 20 mM imidazole,
0.05% DDAO. The resin was further washed with a bufferconsisting of 20 mM Tris–HCl pH 8.0, 300 mM NaCl, 50 mM
imidazole, 0.1% glyco-diosgenin (GDN) for detergent
exchange. The AaRseP mutant was eluted from the resin witha buffer consisting of 20 mM Tris–HCl pH 8.0, 300 mM NaCl,
250 mM imidazole, 0.1% GDN. The eluted mutant was thenapplied onto a Superdex 200 10/300 GL size-exclusion chromatography column (Cytiva) to isolate the monodisperse
fraction of the AaRseP mutant. Finally, the purified AaRseP
mutant was mixed with the NZ-1 Fab and was again subjected
to size-exclusion chromatography to separate the complex
fraction.

2.7. Negative-stain electron microscopy

All purified samples were diluted to 1.0 mg ml1. For
negative-stain EM, 5 ml protein solution was applied onto
glow-discharged, 600 mesh, carbon-coated grids. The grids
were negatively stained with 2% ammonium molybdate,blotted with filter paper and air-dried. A JEM2200FS (JEOL)
was operated at 200 kV to acquire EM images using a K2
camera (Gatan) in counting mode. Images were recorded at amagnification of 20 000 (1.98 Å per pixel) with a defocus of
0.5 to 2.0 mm. A movie of 50 frames was taken for each
image, and motion correction was performed using
MotionCor2 (Zheng et al., 2017). The contrast transfer function was estimated by Gctf (Zhang, 2016). All subsequent
processing was carried out using RELION3 (Zivanov et al.,2018). Particles were selected using the Relion Autopicker andthree rounds of 2D classification were performed to selectparticles for 3D reconstruction. The number of particles used
for each reconstruction of the Fab-complexed AaRseP (181PA14-184) and (235-PA14-236) mutants are indicated in Fig. 7
and Supplementary Fig. S6, respectively. Fitting of the atomic
coordinates of the Fab–PDZ complex into the 3D reconActa Cryst. (2021). D77, 645–662

struction model and figure preparation were performed withUCSF Chimera (Pettersen et al., 2004).
2.8. Molecular-dynamics (MD) simulation

Initial models were prepared by performing energy minimization using the crystal structures of the PA14-mediatedcomplexes between the NZ-1 Fab and PDZ tandems from thisstudy. For both of the two PDZ tandems, only the PDZ
domain that contains the PA14 insertion was included in theinitial model. For PDZ-N (181-PA14-184), residues 113–206,
in which Gly113 and Ser114 were derived from the expression
tag, were used in the simulation, while residues 207–292 were
included for the PDZ-C (235-PA14-236) model. The disordered loop regions in the NZ-1 Fab were built by
MODELLER (Šali & Blundell, 1993). The succinimide in
PDZ-N and the pyroglutamate at the N-terminus of the NZ-1
light chain were remodeled as aspartate and glycine and as
glutamate, respectively. For the PA12-mediated complex, thecrystal structure of PDZ-N (181-PA12-184) complexed with
the NZ-1 Fab (PDB entry 6al1; Tamura et al., 2019) was
modified to prepare the initial model for the simulationaccording to the same procedure as described above. The
model of the PDZ-N domain covered residues 113–206, while
structures for the disordered loop and terminal regions weregenerated as described above. The solvent system around each
Fab–PDZ complex was prepared by Solution Builder as
implemented in CHARMM-GUI (Jo et al., 2008; Lee et al.,2016). The protonation state of histidine was calculated by
PROPKA (Olsson et al., 2011; Søndergaard et al., 2011) as
implemented in PDB2PQR (Dolinsky et al., 2004) at pH 7.
Missing H atoms were inserted using Solution Builder, and theN- and C-termini were set to NH3+ and COO, respectively.
The MD unit cell was set to a rectangular cell with a minimumedge distance of 10 Å between the protein and the walls of thecell. The cell was filled with the TIP3P water model (Jorgensen
et al., 1983) with 150 mM NaCl and added Na+ counter-ions.
The all-atom MD simulations were carried out using the
GROMACS version 2016.3 MD program package (Abraham
et al., 2015; Pronk et al., 2013) with the CHARMM36m force
field (Huang et al., 2017; MacKerell et al., 1998, 2004) underperiodic boundary conditions. The electrostatic interactions
were handled by the smooth particle mesh Ewald method(Essmann et al., 1995), and the van der Waals interactions
were truncated by a switching function with a range of 10–
12 Å. Bond lengths involving H atoms were constrained by the
P-LINKS algorithm (Hess, 2008). According to the default
setup of CHARMM-GUI, an energy minimization and a
125 ps equilibration run as an NVT ensemble with a 1 fs time
step were executed before the production run. The production
run was performed as an NPT ensemble with a 2 fs time step.
The Nosé–Hoover scheme was used as the thermostat(Hoover, 1985, Nosé, 1984) and the Parrinello–Rahman
approach was used as the barostat (Nosé & Klein, 1983;
Parrinello & Rahman, 1981). The temperature and pressure
were set to 300 K and 1 atm, respectively. The simulation
length of the production run was 1 ms.
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R.m.s.d. and r.m.s.f. values were calculated using snapshots
extracted from each 1 ms simulation every 1 ns (1000 snapshots for each trajectory). For PDZ-N, the calculations were
performed over the C atoms of residues 123–206 because
residues 113–122 in the N-terminal region are not included in
the PDZ fold and exhibited large fluctuations during thesimulation compared with the remaining region of the PDZ-N
domain. To estimate the structural change in the PDZ
domains, r.m.s.d.s were calculated relative to the energyminimized initial models of the respective PDZ domains,which were almost identical to the atomic models in thecorresponding crystal structures, as shown in Fig. 5(a). To
estimate the degree of fluctuation in the orientation of the
PDZ domains relative to the NZ-1 Fab, r.m.s.f.s were calculated over the averaged structures of the respective complexes
after aligning the snapshot models based on the VH region(residues 20–130) in Figs. 5(b) and 5(c). In addition, r.m.s.d.swere calculated for the PDZ domains by aligning the snapshot
models within a trajectory of a Fab–PDZ complex with thecorresponding initial model based on the VH region in
Figs. 5(e) and 5( f ) and Supplementary Fig. S4. Furthermore,principal component analysis (PCA) was carried out using thesnapshot models aligned at the VH region to separate out thecharacteristic movements in the fluctuations between the
NZ-1 Fab and the respective PDZ domains. For the NZ-1 Fab–PDZ-C (235-PA14-236) complex, the two trajectories forcomplexes 1 and 2 were merged in the PCA calculation. The
structural distribution was represented as a 2D normalized
histogram of ln(Z) plotted on a principal component map
with a pixel size of 10  10 Å, where Z is the probability of agiven conformation, as shown in Supplementary Figs. S7(a)and S7(b). Hierarchical clustering was implemented using the
MMTSB tool (Feig et al., 2004) to select a representativemodel with the maximum probability for each trajectory: the605 ns snapshot for NZ-1 Fab–PDZ-N (181-PA14-184), the317 ns snapshot for complex 1 of NZ-1 Fab–PDZ-C (235PA14-236) and the 444 ns snapshot for complex 2 (Supplementary Figs. S7a and S7b).

3. Results
3.1. Reduction of the structural change in the target byinserting the PA14 tag

We first inserted the PA14 tag between F and A in the
PDZ-N domain, where we previously inserted the PA12 tag,and optimized the linkers (Tamura et al., 2019; Fig. 1). As F–
A protrudes from the globular PDZ-N domain, it is highly
possible that NZ-1 can bind to the inserted PA14 tag withoutsteric hindrance. At the same time, this must be balanced with
the need for a stiff linker to reduce conformational flexibility
in the complex with the NZ-1 Fab. Hence, we deleted two
protruding turn residues (Asn182 and Gly183) and inserted
the PA14 tag between Arg181 and Glu184 [this mutant isreferred to as PDZ tandem (181-PA14-184)]. Similar to thePA12-inserted mutants of the PDZ tandem in our previous
study, purified PDZ tandem (181-PA14-184) was stable and650

Tamura-Sakaguchi et al.  Antibody labelingmonodisperse, indicating that the insertion of PA14 did not
disrupt the folding of the PDZ tandem. The complex of PDZ
tandem (181-PA14-184) with the NZ-1 Fab was also stable andproduced crystals under several conditions. X-ray diffraction
data were collected to 2.5 Å resolution (Table 1), and weassigned a PDZ tandem and an NZ-1 Fab in the asymmetricunit by molecular replacement. The crystal packing wasmaintained by contacts in both of the molecules (Figs. 2a and2b). The electron densities were clear enough to build reliable
models for both PDZ tandem (181-PA14-184) and the NZ-1
Fab (Fig. 2c). However, the refined model of the PDZ-C
domain showed higher temperature factors, probably because
this domain made no direct contacts with the complex-forming
NZ-1 Fab in the crystal (Table 2).
As we anticipated, the folding of PDZ-N was not disrupted
by the PA14 insertion and complex formation with the NZ-1
Fab. All residues of the inserted PA14 tag were accommodated
in the antigen-binding pocket of the NZ-1 Fab, where the 14
residues assumed a loop with closed ends, in other words a
closed ring-like structure (Figs. 2d and 2e). For the 12
C-terminal residues of PA14 that constitute the PA12 tag, thestructure was almost identical to that observed in the PA12mediated complexes and in the PA14 peptide complexed with
the NZ-1 Fab (Supplementary Figs. S1b and S1c). The 80 C
atoms of PDZ-N, including Arg181 and Glu184, superposed
onto those of the wild type with an r.m.s.d. of 0.750 Å. Arg181
and Glu184 at the insertion junctions formed inter-strand
hydrogen bonds, as formed in wild-type PDZ-N (Fig. 2e). The
distance between the C atoms of Glu10 and Val140 (hereafter,
residue numbers in the PA14 tag are indicated with a prime)
at both ends of the PA14 tag was less than 7 Å, which was
remarkably shorter than the separation caused by the PA12
insertion (Fig. 2d). These observations suggest that labeling of
NZ-1 with PA14 generally has less impact on the folding of thetarget than that with PA12.
Unexpectedly, the newly introduced Glu10 residue in theinserted PA14 tag formed a salt bridge with Arg120 in the
NZ-1 heavy chain (Fig. 2f), whereas the same residue was
highly mobile in the co-crystal structure of the NZ-1 Fab withthe free PA14 peptide (Fujii et al., 2016). Besides the inserted
PA14 residues, only the junction residues in PDZ tandem(181-PA14-184) are involved in intermolecular interactions
with the NZ-1 Fab (Fig. 2f ). Arg181 on PDZ-N at the
N-terminal side formed a hydrogen bond with Tyr69 in thelight chain, and Glu184 on PDZ-N is positioned to form a salt
bridge with Arg70 in the light chain.
3.2. Insertion of the PA14 tag into a sterically hindered loop
regionWe next tested whether the PA14 tag can be successfully
inserted into multiple sites as a demonstration that PA14mediated antibody labeling can serve as a generalizablestrategy for structure determination. The insertion site for
PDZ tandem (181-PA14-184) was chosen because this loop
projects into solvent, but the context and orientation of theinsertion site will be unknown in many practical applications
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Figure 2
Crystal structure of PDZ tandem (181-PA14-184) complexed with the NZ-1 Fab. (a, b) Crystal packing in two different views. The crystallographic axes
are indicated with arrows. The complex is shown as a surface model and the neighboring complexes in the crystal lattice are shown with partial
transparency. The heavy chain and light chain of the Fab are colored dark and light orange, respectively. The PDZ-N and PDZ-C domains are coloredgreen and cyan, respectively; the inserted PA14 tag is colored magenta. (c) Overall structure of the complex. The NZ-1 Fab docks to PDZ-N throughPA14 and does not make direct contacts with PDZ-C within the complex. (d) Superposition of the C traces. The wild-type PDZ-N domain and the PA12and PA14-inserted mutants are colored as in (c) and shown in light, medium and dark colors, respectively. The structures of the wild-type and PA12inserted PDZ-N domains were extracted from previously determined crystal structures [PDB entries 3wkl (wild type) and 6al1 (PA12-inserted mutant)].
The turn residues replaced by the insertion (Asn182 and Gly183) and the inserted sequences (PA12 and PA14) are colored magenta, except for Glu10 and
Gly20 in PA14, which are colored blue. In the PA12-inserted mutant, the junction residues highlighted in yellow (Glu184–His187) alter theirconformations on PA12 insertion and complex formation. In contrast, the addition of Glu10 and Gly20 seemed to induce less structural change of thetarget PDZ-N domain, including the junction residues, in the PA14-inserted mutant. (e) Close-up view of the binding site. The NZ-1 Fab is shown as in(c). The PDZ-N domain and the inserted PA14 are shown as stick models and colored as in (d). Arg181 and Glu184 in the PDZ-N domain maintained theinter-strand hydrogen bond, as shown by dotted lines. ( f ) Residues at the binding interface. The binding interface from (e) is shown as a ribbon modeland in profile. Residues where the side chains are presumed to form salt bridges or hydrogen bonds are displayed as stick models.
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research papersof this method. To determine whether the PA14 tag could be
inserted into a more sterically hindered site, we selected theD– E loop as the insertion site (Fig. 1). The D and E
strands belong to a four-stranded -sheet, and the turn residues (Asn235 and Gly236) are also included in the flat surfaceof the -sheet. In contrast to NZ-1 labeling on a protruding
loop, steric hindrance between NZ-1 and the target PDZ
domain needs to be avoided in this case. Therefore, we
inserted the PA14 tag between the turn residues without
deletion, resulting in the mutant PDZ tandem (235-PA14-236).
The complex of PDZ tandem (235-PA14-236) with the NZ-1
Fab was also successfully crystallized and diffraction data werecollected to 3.2 Å resolution (Table 1). We assigned twocomplexes (referred to as complex 1 and 2) in the asymmetricunit by molecular replacement, but the electron densities for
the PDZ-N domains were too weak to assign reliable models
for this domain in either of the two complexes. Judging fromthe weak electron densities, the PDZ-N domains in both of thetwo complexes were exposed to the solvent and made little
contribution to lattice formation in the crystal. Hence, the two
structures of the NZ-1 Fab and the PA14-inserted PDZ-C
domain were refined and included in the final model (Figs. 3aand 3b and Table 2).
As in PDZ tandem (181-PA14-184), the PA14 tag adopts a
closed ring-like structure in both of the two complexes in theasymmetric unit. However, the conformations of the N-terminal residues were significantly different between them(Figs. 3c and 3d and Supplementary Figs. S1d and S1e). In all

Figure 3
Crystal structure of PDZ tandem (235-PA14-236) complexed with the NZ-1 Fab. (a, b) The two complexes in the asymmetric unit, complexes 1 and 2, areshown as surface models. The heavy and light chains of NZ-1 Fab in complex 1 are colored dark and light orange, respectively, while those in complex 2
are colored dark and light brown, respectively. The inserted PA14 tags are colored dark and light magenta, respectively. Due to the disorder in theelectron density, the PDZ-N domains were not included in the final model. (c, d) Close-up view of the binding site. The PDZ-C domain and the inserted
PA14 residues are shown as ribbon and stick models, respectively. Glu10 -Gly20 and Gly30 -Val40 of PA14 are colored blue and yellow, respectively. The
remaining ten C-terminal residues (Ala50 –Val140 ) are colored magenta. The residues in complexes 1 and 2 are shown in dark and light colors,
respectively.

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research papersof the crystal structures that have been determined of thePA12 and PA14 tags so far, Gly30 and Val40 in the tag werelocated in proximity to Tyr52 and Tyr113 in the NZ-1 lightchain. In complex 1, the conformations of Gly30 and Val40 wereconsistent with those in the known structures. In contrast, thetwo residues were largely separated from the NZ-1 light chain
in complex 2, although they were still located inside theantigen-binding pocket. It appeared that the conformationalchange of these two residues was related to the crystal packingof the target PDZ-C domains (Figs. 4a and 4b). In complex 2,
the PDZ-C domain closely contacted the neighboring Fab,resulting in a rigid-body reorientation of the PDZ-C domain
relative to the complex-forming NZ-1 Fab. Despite thedifferent interaction modes with the NZ-1 Fab, the main-chain
structures of the PDZ-C domains in both complexes wereconsistent with that in the structure of the wild-type PDZ
tandem without the PA14 insertion. The 81 C atoms of
PDZ-C, including the junction residues Gly235 and Asn236,
superposed onto to those of the wild type with r.m.s.d.s of
0.785 and 1.055 Å for complexes 1 and 2, respectively (Fig. 4c).
In contrast, a superposition of the Fv region between the twocomplexes showed a rotation of the PDZ-C domain byapproximately 40 around the PA14-insertion site (Fig. 4d),which was caused by the conformational change of Gly30 andVal40 . It has previously been reported that the replacement ofeither Gly30 or Val40 by alanine had no significant effect on theaffinity of the PA12 tag for NZ-1 (Fujii et al., 2014). Presumably, these two residues tolerate conformational change tosome extent as their side chains do not make a measurable
energetic contribution to the binding affinity. Overall, theseobservations indicated that the PA14 tag could be inserted
into this sterically hindered loop with less structural change inthe target PDZ-C domain.
Concerning the specific interactions between the NZ-1 Faband PDZ-C, Glu10 formed salt bridges with Arg70 in the NZ-1
light chain as well as with Arg120 in the heavy chain in bothcomplexes 1 and 2 (Figs. 4e and 4f ). In addition to the PA14
residues, Lys255 in PDZ-C also formed intermolecular
contacts with the NZ-1 Fab, although Lys255 made different
contacts in the two complexes. In complex 1, the amino groupof Lys255 formed hydrogen bonds to the carbonyl group of
Arg70 in the light chain as well as the side-chain carboxylgroup of Asp72 in the light chain. In complex 2, the sameamino group formed a hydrogen bond with the hydroxyl groupof Tyr52 in the light chain. Thus, the interaction mode of
Lys255 is also affected by the reorientation of PDZ-C relative
to the NZ-1 Fab.
3.3. Molecular-dynamics simulations to analyze
conformational variation in PA14-mediated Fab–PDZ
complexes

For the broadest application of this method, a fixed
conformation of the Fab is desirable. Because crystallization
may artificially fix the conformation of the complex subunits,
we turned to molecular-dynamics (MD) simulations to characterize the complex stability and intermolecular motions in
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the NZ-1 Fab–PDZ complexes. For all complex structures,
only the PDZ domain that contains the PA14 insertion was
included in the complex model. For the 235-PA14-236 mutant,the structures of both complexes 1 and 2 were simulated inindividual MD trajectories. During all simulations, the inserted
PA14 tag maintained the closed ring-like conformation inside
the antigen-binding pocket, and the PDZ domains neverdissociated from the NZ-1 Fab (Supplementary Fig. S2). In allcases, the structures of the PA14-inserted PDZ domains wererelatively rigid over 1 ms of simulation, and the r.m.s.d.s relative to the initial models were 2 Å, excluding the inserted
PA14 residues (Fig. 5a). The results of the simulations
suggested that the impact of the PA14 insertion on the foldingof the target PDZ domain was relatively small. Focusing on
the inserted PA14 tag, the four N-terminal residues Glu10 –Val40 showed relatively high root-mean-square fluctuations(r.m.s.f.s; Fig. 5b), which coincides with the observation thatthe conformation of Gly30 and Val40 was dependent on the
crystal packing in PDZ tandem (235-PA14-236) with the NZ-1
Fab (Figs. 3c and 3d). As a comparison, we also performed an
MD simulation on our previously reported structure of thePA12-inserted PDZ-N mutant [PDZ-N (181-PA12-184)]
complexed with the NZ-1 Fab (Tamura et al., 2019). During
the simulation, the inserted PA12 residues and junction residues showed high r.m.s.f.s, while the rest of the target PDZ-N
domain was stable (Supplementary Fig. S3a). In particular, thefour C-terminal residues of PA12, Asp110 –Val140 , fluctuated toa larger extent (Supplementary Figs. S3b–S3d). The conformation of the junction residues also seemed to be unstable in
the PA12-inserted mutant, where the distance between
Arg181 and Glu184 in the heavy chain fluctuated greatly
during the simulation (Supplementary Fig. S3e). Theseobservations suggested that in the context of a loop insertion,
the PA14 tag binds to the NZ-1 Fab more stably than the PA12
tag.
With respect to the subunit arrangement in the complex,
PDZ-N (181-PA14-184) with the NZ-1 Fab was more flexible
even though there was only one conformation in the asymmetric unit of the crystal structure. PDZ-N (181-PA14-184)
showed higher r.m.s.d. and r.m.s.f. values than PDZ-C (235PA14-236) in the simulations where the structures of thecomplex were aligned based on the variable region of theheavy chain (VH region) (Figs. 5c, 5e, 5f and Supplementary
Fig. S4). At 358 ns in the 1 ms simulation, PDZ-N (181-PA14184) showed the highest r.m.s.d. value (19 Å) relative to theinitial model (Supplementary Figs. S2a and S4). In fact, almost
no hydrogen-bonding pairs were observed between the NZ-1
Fab and the PDZ-N domain during the simulation, except for
those formed by the inserted PA14 tag. Additionally, Glu10 in
PA14 became separated from the NZ-1 Fab, whereas thisresidue was placed close to Arg120 in the heavy chain in the
crystal structure (Fig. 5d). For PDZ-C (235-PA14-236)
complexed with the NZ-1 Fab, the preferential conformations
were more similar to complex 1 than to complex 2 (Figs. 5e and5f ). In the simulation initialized from complex 2, the model
immediately sampled conformations that were more similar tocomplex 1, although conformations similar to complex 2 also
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Figure 4
Comparison of the two conformations of PDZ tandem (235-PA14-236) complexed with the NZ-1 Fab. (a, b) Comparison of crystal contacts in complexes
1 and 2 with PA14-inserted PDZ-C domains. The structures are shown as in Figs. 3(a) and 3(b). In both complexes 1 and 2, the PDZ-C domains were in
direct contact with two neighboring complexes, labeled neighbor-1 and neighbor-2. The PDZ-C domain of complex 1 had a relatively small contact areawith neighbor-2 where solvent-accessible space was present between them. The relative arrangement of the NZ-1 Fab and PDZ-C in complex 1 did notappear to be affected by the crystal packing. In contrast, the PDZ-C domain of complex 2 intimately interacted with neighbor-2, indicating that thepositioning of the PDZ-C domain with respect to the NZ-1 Fab was restricted by the crystal packing. (c) Superposition of the C traces. The wild-type
PDZ-C domain (PDB entry 3wkl) and the PA14-inserted mutants in complexes 1 and 2 are shown in dark, medium and light colors, respectively. The
PDZ-C domain is shown in cyan, while the insertion site (Asn235 and Gly236) is colored orange. The inserted PA14 tags are colored as in Figs. 3(c) and
3(d). (d) Superposition of the two complexes based on the NZ-1 Fab. The NZ-1 Fab in complex 1 is shown as a surface model. The heavy chain and lightchains are colored gray and white, respectively. The interaction modes of Gly30 and Val40 of PA14 shown in yellow were different between the twocomplexes. As a result, the superposition showed a rigid-body rotational movement of the PDZ-C domain around the PA14-insertion site. (e, f ) Residues
at the binding interfaces of complexes 1 (e) and 2 ( f ). Residues presumed to form salt bridges or hydrogen bonds are displayed as stick models in the
ribbon models.

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Figure 5
MD simulation of NZ-1 Fab–PDZ complexes. (a) Trajectories for the target PDZ structures. R.m.s.d.s were calculated for the snapshot models relative tothe initial model to estimate the structural changes in the PDZ domains during the simulation trajectories. The inserted PA14 residues and the flexible
N-terminal region upstream of the PDZ-N domain were excluded from the calculation. R.m.s.d.s are plotted for trajectories of the PDZ-N domain(green) and for the PDZ-C domain in both complex 1 (cyan) and complex 2 (blue). (b) Structural fluctuations in the residues within the PA14 tag. In the
r.m.s.f. calculations, the complex models within each trajectory were aligned with the initial model based on the VH region of the NZ-1 Fab, and thestructural fluctuation from the averaged structure was calculated for each residue in PA14. (c) Histogram of PDZ-domain residues binned by r.m.s.f.
values calculated relative to the NZ-1 Fab. Similar to the process for calculating r.m.s.f. values in (b), the snapshot models within a trajectory were alignedwith the averaged structure at the respective VH regions for each of the three complexes. Subsequently, the fluctuation in the atomic coordinates wascalculated for each residue. (d) The representative snapshot of PDZ-N (181-PA14-184) complexed with NZ-1 Fab from the trajectory. The snapshot at
605 ns, representing the most frequently observed conformation, is shown as a ribbon model. The PDZ-N domain is colored green. PA14 residues arecolored light magenta, except for Glu10 and Gly20 at the N-terminus, which are colored light blue. The heavy and light chains in NZ-1 are colored dark
and light orange, respectively. Glu10 in PA14 was separated both from Arg120 in the NZ-1 heavy chain and from Arg70 in the NZ-1 light chain. Arg181
was also separated from Tyr69 in the light chain. Glu184 in PDZ-N interacted with Tyr51 and Ser119 in the NZ-1 heavy chain as opposed to theinteraction with Arg70 in the NZ-1 light chain in the crystal structure (see Fig. 2f ). (e, f ) Reorientation of the PDZ-C domains relative to the NZ-1 Fab in
the MD simulations initialized from complexes 1 (e) and 2 ( f ). For both of the calculations, the snapshot models were aligned with the initial models ofcomplexes 1 and 2 based on the VH region, and the r.m.s.d.s for the PDZ-C domains were calculated relative to the initial models. The r.m.s.d.s calculatedusing complexes 1 and 2 as references are shown in cyan and blue, respectively. The conformations of the 347 ns snapshot in (e) and the 689 ns snapshotin ( f ) are similar to that of complex 2, as analyzed in Supplementary Fig. S7(c).
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appeared as a minor population (Fig. 5f). Comparing thesnapshot structures, we found that Lys255 in PDZ-C formedhydrogen bonds to residues in the NZ-1 light chain such as
Asp72 in conformations similar to complex 1 (Supplementary
Fig. S5a). In contrast, Lys255 was separated from Asp72 inconformations similar to complex 2 (Supplementary Fig. S5b).
Furthermore, the contribution of Glu10 in PA14 to complex
formation depended on the PA-insertion sites. Glu10 in PDZ-C(235-PA14-236) formed a hydrogen bond to Arg70 in the
NZ-1 heavy chain in 72.6% of the snapshots on the trajectory,
whereas Glu10 in PDZ-N (181-PA14-184) reoriented into thesolvent and away from the interaction with Arg120 in the
NZ-1 heavy chain in the MD simulation, as mentioned above.
3.4. Preparation of PA14-inserted full-length AaRseP for
NZ-1 labeling

Using our improved NZ-1 labeling technique, we next
attempted to determine the location and orientation of thetwo PDZ domains in the context of the full-length AaRseP
using antibody-assisted negative-stain EM. Although no highresolution 3D structural data have been generated to date forany full-length RseP orthologue, we have previously estimatedthe spatial arrangement of the PDZ domains using biochemical methods (Hizukuri et al., 2014). Specifically, we performed
chemical modification analysis to estimate the solvent accessibility of the surface residues of EcRseP based on structural
data for the PDZ tandem fragment from X-ray crystallography and small-angle scattering analysis. These previous
results suggested that the two PDZ domains adopted a clamlike structure and that the putative ligand-binding grooves in
the PDZ domains would be inaccessible in the context of the
full-length protein on the cell membrane. Based on this estimation, the two PA14-insertion sites tested in the present
study were presumed to project into solvent because they werelocated on the back side of the putative ligand-binding
grooves. Therefore, it was expected that PA14 insertion would
not destabilize the structure of the full-length protein in bothmutants.
We therefore performed negative-stain EM structural
analysis on full-length AaRseP constructs with PA14 tag
insertions. For these full-length constructs, we used the same
insertion sites as used for the PDZ tandem fragments andproduced AaRseP (181-PA14-184) and AaRseP (235-PA14236) for PDZ-N and PDZ-C, respectively. After confirmingthat both mutants accumulated in the E. coli membrane, we
attempted to examine whether or not the PA14 insertion
affected proteolytic activity in AaRseP. However, no physiological substrates have yet been identified for AaRseP. It is
known that EcRseP cleaves the anti-sigma factor RseA (Albaet al., 2002; Kanehara et al., 2002), but no orthologue of RseA
has been identified in the A. aeolicus genome. Looking for analternative, we discovered that AaRseP is able to cleave amodel substrate for EcRseP derived from RseA (Fig. 6a). In
this model substrate, the periplasmic domain of RseA is fusedto the first transmembrane region of lactose permease (LY1)
with a recombinant cytoplasmic domain containing hemag-

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Tamura-Sakaguchi et al.  Antibody labelingglutinin (HA)-tagged maltose-binding protein (MBP).
Furthermore, the C-terminal periplasmic region of the model
substrate was truncated at Val148 because EcRseP performs
the intramembrane proteolysis only after the periplasmic
region of RseA has first been cleaved by DegS (Alba et al.,
2002; Kanehara et al., 2002). Hence, the model substrate
mimics the DegS-cleaved product and can be detected by antiHA antibody labeling. This model substrate is referred to as
HA-MBP-RseA(LY1)148 (Hizukuri & Akiyama, 2012). Whenco-expressed with either wild-type AaRseP or EcRseP, theband for HA-MBP-RseA(LY1)148 shifts compared with thevector control containing neither AaRseP nor EcRseP. We
also observed that the band for HA-MBP-RseA(LY1)148
does not shift when the catalytically important glutamate
residue of the HEXXH sequence that is conserved among thesite-2 protease family of intramembrane proteases was
mutated to a glutamine in either EcRseP or AaRseP, which isconsistent with intramembrane proteolysis of HA-MBPRseA(LY1)148 by the respective RsePs. These observations
confirm for the first time that AaRseP is functionally orthologous to EcRseP and belongs to the site-2 protease family.
Finally, both AaRseP (181-PA14-184) and AaRseP (235-PA14236) were also able to cleave HA-MBP-RseA(LY1)148 atcomparable levels to wild-type AaRseP (Fig. 6b), indicatingthat the PA14 insertion did not abrogate proteolytic activity.
3.5. Antibody-assisted negative-stain EM of full-length
AaRsePWe next overproduced the PA14-inserted AaRseP mutantsin E. coli and purified them using immobilized metal-affinity
and size-exclusion chromatography. We fractionated monodisperse mutants solubilized with glyco-diosgenin (GDN)
detergent and mixed them with the NZ-1 Fab. We thensubjected the mixture to size-exclusion chromatography again
to fractionate the stable complex. Subsequently, the purifiedcomplex was negatively stained with ammonium molybdate
for EM single-particle analysis. For AaRseP (181-PA14-184)
complexed with the NZ-1 Fab, four different classes of 2D
averages were reconstructed into 3D models (Fig. 7a). In eachmodel, the putative Fab fragment was identified by a characteristic ellipsoidal shape with a hole at the center. The
remaining part is constituted of a sphere and two protrusions,and presumably corresponds to the detergent-solubilized TM
domain and the two PDZ domains of AaRseP. Structuralalignment of the four 3D models with the putative AaRseP
molecule showed that the relative orientation of the NZ-1 Fabamong the models hinges around a fixed point (Fig. 7b). Thishinge-like variation indicates that the contacting points,
namely the locations of the PA14 insertion sites, are consistentamong the four classes despite the variable orientation of theNZ-1 Fab. It was therefore highly possible that the arrangement of the PDZ-N domain was also consistent among thefour classes. In fact, the abovementioned MD simulation also
demonstrated that the orientation of the NZ-1 Fab relative to
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lation time. For AaRseP (235-PA14-236) complexed with the
NZ-1 Fab, the 3D reconstruction also produced four different
classes. In each of these four classes, however, the densities

presumed to be the NZ-1 Fab were located in a common
location relative to the spherical profile, although the resolutions of the EM maps were relatively low for three classes(Supplementary Fig. S6). This observation is also consistent with the result
from the MD simulation, where theorientation of the NZ-1 Fab relative to
PDZ-C (235-PA14-236) was more fixedcompared with PDZ-N (181-PA14-184).
In the most well averaged class thedistinctive shape of the NZ-1 Fab wasrecognized, while two protrusions wereagain identified on the spherical part(Fig. 7c). Taken together, we could
successfully obtain 3D structural data
for the full-length RseP orthologue forthe first time, which could be utilized toanalyze the spatial arrangement of thetwo PDZ domains.
3.6. Approximation of the domain
arrangement in full-length AaRseP

Figure 6
In vivo cleavage of a model substrate by wild-type AaRseP and PA-inserted mutants. (a) Design ofthe model substrate. The wild-type RseA from E. coli is a type II membrane protein composed of
216 amino-acid residues. The full-length RseA binds  E and RseB in the cytoplasmic andperiplasmic domains, respectively. DegS cleaves RseA at the C-terminal side of Val148 and liberates
RseB. The model substrate, HA-MBP-RseA(LY1)148, contains the first transmembrane region of
lactose permease (LacY TM1; LY1) with a recombinant cytoplasmic domain composed of HAtagged MBP. The C-terminal periplasmic domain was truncated at Val148 to mimic the DegScleaved product. (b) Immunoblotting with anti-HA antibody detected the model substrate. The fulllength model substrate appeared at the ‘Uncleaved’ position. ‘Cleaved’ indicates the model
substrate that was cleaved by RseP within the membrane, as illustrated in (a). Immunoblotting with
anti-His antibody detected His-tagged EcRseP, AaRseP and their derivatives. Immunoblotting of
the cytoplasmic protein SecB serves as a loading control. Molecular-size marker positions are shownin kDa on the left.
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We then attempted to superpose therepresentative models of the Fab–PDZ
complexes obtained from MD simulation onto the 3D reconstruction models
of full-length AaRseP with the NZ-1
Fab from EM analysis. To select theconformation that appeared with highprobability as a representative conformation, we performed principal
component analysis (PCA) for thesnapshots within the MD trajectories.
For the 181-PA14-184 mutant, therepresentative model obtained from
PCA (605 ns snapshot in Fig. 5d andSupplementary Fig. S7a) fitted class 1 of
the 3D reconstruction model best.
Aligning the representative model andthe 3D reconstruction at the Fv region
resulted in good overlap between the
PDZ-N model and one of the
protruding lobes of the EM map(Fig. 8a).
For the 235-PA14-236 mutant, we firstattempted to perform a structuralalignment onto the 3D reconstruction
using the representative models of the
PDZ-C domain complexed with the
NZ-1 Fab obtained from PCA (Supplementary Figs. S5a and S7b). However,neither the representative models
calculated from complex 1 nor complex
2 fitted the 3D reconstruction well.
Superposition of the NZ-1 Fab region
positioned the A– B loop of the

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PDZ-C domain outside the EM map in both models(Supplementary Figs. S8a and S8c). These two models adopted
conformations similar to that of complex 1 from the crystal
structure. In fact, the atomic model of complex 1 also fitted the
3D reconstruction poorly (Supplementary Figs. S8a and S8c).
The PDZ-C domain in complex 1 was located closer to the
NZ-1 Fab than that in complex 2. In most trajectory snapshots,
the PDZ-C domains were also located relatively close to the
NZ-1 Fab, as observed in complex 1 (Supplementary Figs. S2band S2c). In contrast, the PDZ-C domain in complex 2 wasrelatively separated from the NZ-1 Fab, and structural alignment of complex 2 onto the 3D reconstruction resulted in abetter overlap of the PDZ-C domain with the protruding lobe
in the EM map (Supplementary Figs. S8b and S8d). Within the
trajectories initialized from both complexes 1 and 2, there
were minor populations of snapshots with conformations
similar to that in complex 2 (Supplementary Fig. S7b). In thesesnapshots, the PDZ-C domains were relatively separated fromthe NZ-1 Fab. In addition, comparison of the snapshot models

indicated that Lys255 in PDZ-C and Asp72 in the NZ-1 lightchain were not close enough to form a hydrogen bond in theconformations similar to that in complex 2. In fact, the r.m.s.d.
of the snapshot model relative to complex 2 showed a negative
correlation with the distance between Lys255 and Asp72(Supplementary Fig. S7c). To analyze the structural features ofthe MD models that fit the 3D reconstruction, we selected one
snapshot that had both a low r.m.s.d. relative to complex 2 anda large distance between Lys255 and Asp72 for each of the
trajectories initialized from complexes 1 and 2 (Supplementary Figs. S5b and S7c). According to the PC surface, theselected snapshots were intermediate conformations between
complexes 1 and 2 (Supplementary Fig. S7b). As a result, bothof the two selected MD models (the 347 ns snapshot fromcomplex 1 and the 689 ns snapshot from complex 2) seemed to
fit the density similarly to complex 2 (Fig. 8b and Supplementary Figs. S8b and S8d). From these observations, weconcluded that the hydrogen bond between Lys255 in PDZ-C
and Asp72 in the NZ-1 light chain is broken in the 3D
reconstruction model of the complex.
Finally, combination of the two EM reconstructions enabled
us to approximate the spatial arrangement of the PDZ tandem
in the context of the full-length protein (Fig. 8c). Firstly, thetwo EM maps were aligned based on the position of thespherical part and two protrusions. Next, structural alignmentsof the MD models onto the EM maps were performed for the
Fab–PDZ-N complex (the 605 ns snapshot) and the Fab–PDZC complex (the 347 ns snapshot from complex 1), respectively.
Subsequently, the crystal structure of the wild-type PDZ-Nand PDZ-C domains were aligned onto the MD models andmerged to build the entire PDZ tandem model. In the resultant model, the putative ligand-binding grooves of both of the
PDZ domains pointed towards the TM domain containing theactive center. In addition, both of the 3D reconstruction
models commonly indicated that a gap was present between
the PDZ-C domain and the TM domain, whereas it seemedthat the PDZ-N domain directly contacts the sphericaldetergent micelle covering the TM domain.

4. Discussion

Figure 7
Antibody-assisted negative-staining EM analysis of PA14-inserted fulllength AaRseP. (a) 3D reconstruction models of AaRseP (181-PA14-184)
complexed with the NZ-1 Fab. Four different 3D reconstructions from the
2D class average images are shown in different colors. The numbers ofparticles used for the reconstruction are indicated below each model. (b)Superposition of the 3D reconstructions. The four models in (a) werealigned based on the putative AaRseP region. The NZ-1 Fabs appear tocontact AaRseP at almost the same point in each model. (c) 3D
reconstruction model of AaRseP (235-PA14-236) complexed with the
NZ-1 Fab. The most well averaged class is drawn as a representative.

658

Tamura-Sakaguchi et al.  Antibody labeling

Antibody labeling has been widely used in the structural
analysis of protein molecules and complexes. The use of
inserted exogenous epitopes has the potential to expand theapplicability of antibody labeling, even to cases where no
antibodies have been established for the target protein. In
fact, antibody-labeling methods for membrane proteins
mediated by fusion with a thermostabilized variant of
apocytochrome b562 have been reported very recently (Miyagi
et al., 2020; Mukherjee et al., 2020). Our method provides a
new option for antibody labeling through exogenous epitopes.
However, the structural integrity of the target protein must be
maintained for the resulting structural model to be relevant inany of the methods. In our previous study of antibody-assisted
structural analysis with NZ-1 labeling, the inserted PA12 tag
caused a large structural change around the insertion site in
the target protein. In the present study, we attempted to
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Figure 8
Estimation of the arrangement of the two PDZ domains in full-length AaRseP. (a, b) Structuralalignment of the Fab–PDZ models onto the 3D reconstruction. (a) The 605 ns snapshot in the MD
trajectory of the NZ-1 Fab–PDZ-N (181-PA14-184) pair, whose conformation was most frequently
sampled during the MD simulation, was fitted into the 3D reconstruction model of class 1 shown in
Fig. 7(a) based on the position of the Fv region. (b) The 347 ns snapshot, which showed a similar
conformation to that of the complex 2 structure, in the MD trajectory calculated from the complex 1
structure of the NZ-1 Fab–PDZ-C (235-PA14-236) pair was fitted into the 3D reconstruction model
shown in Fig. 7(c) based on the position of the Fv region. (c) Structural model of size exclusion. The
approximate arrangement of the two PDZ domains from AaRseP is shown together with the modelof RseA from E. coli to help understand the size-exclusion mechanism. The crystal structure of theperiplasmic domain of RseA in complex with RseB (PDB entry 3m4w) is drawn in the RseA model.
RseB is shown as a blue ribbon model with a partial transparent surface. The RseA periplasmic
domain is shown as magenta ribbon model, and the side chain of Val148 is highlighted with anorange sphere model. The atomic model of the PDZ tandem from AaRseP was built based on the
structural alignments in (a) and (b). Firstly, the two 3D reconstruction models were aligned basedon the AaRseP molecule composed of a sphere and two protrusions. Next, the position of the PA14inserted PDZ-N and PDZ-C domains were aligned onto the respective 3D models as described in(a) and (b). Finally, the crystal structures of wild-type PDZ-N and PDZ-C domains wereindependently aligned onto the models of the PA14-inserted mutants. The aligned models of
PDZ-N and PDZ-C were merged to build the entire PDZ tandem model. The PA14-insertion sites
and the carboxylate-binding loops within the ligand-binding grooves are highlighted with magenta
and red sphere models, respectively. Glu158 and Asp162, shown as blue sphere models, weremodeled to be close to the transmembrane region based on the structural alignment. The
corresponding residues in EcRseP were estimated to be membrane-proximal residues based onprotection from chemical modification. In both 3D reconstruction models the PDZ-C domain was
separated from the TM domain by a gap, which might suppress the entry of the RseB-bound fulllength RseA to the active center as the size-exclusion filter.
Acta Cryst. (2021). D77, 645–662

circumvent the deformation that PA12
caused by utilizing the PA14 tag thatpossesses a Glu–Gly pair of residues
upstream of PA12. We anticipated thatthe two additional residues would act asa buffer region to reduce the structuralchange in the target. We observed thattwo different PA14-inserted PDZ
tandem mutants both produced cocrystals with the NZ-1 Fab, which indicated that stable antibody–target
complexes were successfully formed
through the inserted PA14 tag. In thesecrystal structures, the loop-like conformations of the inserted PA14 tags were
almost the same as those observed in
the co-crystals of PA12-inserted PDZ
tandems complexed with the NZ-1 Fab
as well as the PA14-free peptide
complexed with the NZ-1 Fab (Supplementary Fig. S1). Besides, the additionof Glu–Gly residues at the N-terminus
resulted in a closed ring-like conformation for the entire 14-residue epitope
when recognized by the NZ-1 Fab inboth cases. In these conformations, theends of the PA14 tag were located lessthan 7 Å apart based on the C –C
distance of Glu10 and Val140 . Due to thisstructural property of the inserted PA14
tag, no significant structural changes
were observed in the target PDZ
domains in comparison to the wild-type
structure without the PA14 insertion. In
the previous study on inserting thePA12 tag, the insertion point needed to
be optimized to produce a rigid complex
with the NZ-1 Fab. In particular, eliminating residues that undergo structuralchange upon complex formation is quite
important. However, prior structural
data for the target are necessary forsuch an optimization. In contrast, we
expect that the PA14 tag, adopting aclosed ring-like structure, can beinserted into a target with fewer optimization steps. In addition, the MD
simulations suggested that an inserted
PA14 tag exhibits less structural fluctuation inside the NZ-1 antigen-binding
pocket than an inserted PA12 tag. Taken
together, we conclude that the PA14 tag
is more suitable both for insertion into
the target and for complex formation
with NZ-1 compared with thepreviously used PA12 tag. This result
opens the possibility that the PA14 tag

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can be inserted into other proteins with varying structuralfeatures.
In the present study, we also demonstrated that NZ-1
labeling via an inserted PA14 tag can be applied to negativestain EM for structural analyses, as demonstrated using the
PDZ tandem in AaRseP. As the PDZ tandem is thought toplay a pivotal role in substrate discrimination in RseP, structural data on the spatial arrangement of the PDZ domains
should help to delineate the regulatory mechanism of intramembrane proteolysis. Our previous chemical modification
analysis has suggested that the putative ligand-binding
grooves of the two PDZ domains form a pocket-like space
sitting just above the active center of RseP that is sequestrated
within the membrane (Hizukuri et al., 2014). The spatial
arrangement approximated by EM analysis is fully consistent
with the chemical modification analysis, wherein the putative
ligand-binding grooves indeed point towards the TM region.
In the chemical modification of EcRseP, Asp162 and Leu167
in the B helix were assigned as membrane-proximal residues.
In the PDZ tandem model of AaRseP, the corresponding
residues, Glu158 and Asp162, were located at the interface
with the TM domain (Fig. 8c). As structural changes in the
PDZ domains were not induced by the use of a PA14 insertion,
we conclude that the arrangement of the PDZ tandem reflects
the native state. The biochemical analysis confirmed thatwithin the cell membrane the PA14-inserted mutants at least
have a proteolytic activity similar to that of the wild type. In
our previous study, we proposed that the pocket-forming PDZ
tandem serves as a size-exclusion filter that only accommodates the RseA substrate for cleavage after pre-cleavage by
DegS (Hizukuri et al., 2014). Based on the 3D reconstruction
model, the PDZ-C domain was separated from the TM
domain. The degree of separation may control substrate
discrimination (Fig. 8c). The structural data from our present
hybrid analysis will advance biochemical analyses in theinvestigation of the ‘size-exclusion model’.
The present study has raised a new issue to be solved when
designing PA14-insertion sites for EM structural analysis
assisted by NZ-1 labeling. While antibody labeling has applications in negative-stain EM (Boisset et al., 1993, 1995), theimpact of this method has been particularly pronounced in 3D
structure determination by cryo-EM. Although cryo-EM
techniques have been drastically improved through the‘resolution revolution’ of recent years (Kühlbrandt, 2014), it isstill difficult to determine high-resolution structures of small
proteins due to the weak signal from individual particles in the
EM images (Rubinstein, 2007). Antibody labeling is expected
to resolve this size-limitation problem both by increasing theparticle size and by facilitating accurate image alignment (Wuet al., 2012), as has already been exemplified by recent cryoEM structure determinations (Kim et al., 2019; Coleman et al.,2019). Similarly to as in X-ray crystallography, more rigid
complexes with Fabs are however desirable in order todetermine high-resolution structures by cryo-EM. Whilelattice contacts can stabilize the arrangement of proteins in acomplex to some extent for X-ray crystallography, individualcomplex particles in EM images may display more structural

660

Tamura-Sakaguchi et al.  Antibody labelingheterogeneity. In fact, our negative-stain EM analysis indicated that the orientation of the bound NZ-1 Fab relative tothe target PDZ domains was variable. In particular, AaRseP(181-PA14-184) complexed with the NZ-1 Fab showed a highdegree of conformational flexibility between the two molecules. Crystallographic analysis showed that only the junction
residues of the target PDZ-N domain made direct contacts
with the NZ-1 Fab in the 181-PA14-184 mutant. In addition,
MD simulations indicated that the intermolecular interaction
between Glu10 in PA14 and NZ-1 can readily be broken in thismutant. For the 181-PA14-184 mutant, we attempted to optimize the insertion site by deleting the turn residues to eliminate the intermolecular space between the target PDZ-N
domain and the bound NZ-1 Fab, as this insertion site
protruded from the globular domain. Nevertheless, it seems
that some conformational flexibility still exists (Fig. 7b) due tothe absence of specific interactions between the NZ-1 Fab andthe target PDZ-N domain other than through the inserted
PA14 tag. In contrast, the orientation of the NZ-1 Fab seemed
to be relatively fixed in the complex with AaRseP (235-PA14236). Lys255 in PDZ-C interacted with Asp72 in the NZ-1 lightchain in complex 1 of the crystal structure of the PDZ tandem(235-PA14-236) with the NZ-1 Fab, and this interaction was
reproduced in the MD simulation. We therefore hypothesizedthat it could stabilize the subunit arrangements in thecomplex. However, the structural alignment suggested that
only the PA14 residues were involved in intermolecularinteractions with the NZ-1 Fab because the interaction
mediated by Lys255 was broken in the atomic models that fit
the 3D reconstruction from the EM analysis (SupplementaryFigs. S5b, S8b and S8d). As the PA14 tag was inserted into aloop that packs against a flat surface on this insertion mutant,the orientation of the NZ-1 Fab in the EM model might belocked in position by steric hindrance from the flat surface of
the PDZ domain. These observations indicated that creating
an NZ-1-labeled complex with reduced flexibility will require
further optimization of the insertion strategy by consideringboth specific interactions and steric hindrance between thetarget and the Fab.
In conclusion, we have optimized the insertion method of
the NZ-1 epitope to reduce structural changes in the target
protein. Using a target with known structure, we showed that
inserting a PA14 tag is more suitable for NZ-1 labelingcompared with PA12 insertion. The advantage of the PA14 tagis that it can reduce structural changes in the target protein
during labeling and structure determination even when it isinserted into sterically hindered sites such as loops that packagainst the protein. If these advantages are general properties
of PA14, the use of PA14 as an inserted epitope tag will
expand the range of application of the NZ-1 labeling technique. To further advance our generalizable method of antibody-assisted structural analysis for application in EM studies,
future work will optimize the insertion site to fix the orientation of the NZ-1 Fab relative to the target and minimize
conformational variability. Other optimization challenges,
such as Fab dissociation upon vitrification, will also need to beaddressed. This endeavor may require mutations of both theActa Cryst. (2021). D77, 645–662

research papers
epitope and the paratope. Computational approaches, such as
the modeling of PA14-inserted mutants and/or NZ-1 Fabmutants followed by MD simulation to assess complex rigidity,
will aid these future optimizations. Ultimately, a generalizable
method for antibody labeling that produces stable and rigid
antibody–target complexes will be an important new tool forhigh-resolution structural analysis by both X-ray crystallography and cryo-EM.

Acknowledgements
We are grateful to the beamline staff of the Photon Factory,
Tsukuba, Japan for providing data-collection facilities andsupport. We thank Samuel Thompson for editing the manuscript and Professor Junichi Takagi for useful discussion. This
work was performed in part under the Cooperative Research
Program (Joint Usage/Research Center Program) of theInstitute for Frontier Life and Medical Sciences, Kyoto
University, under the Collaborative Research Program of theInstitute for Protein Research, Osaka University (CR-19-05)
and under the Cooperative Research Project Program of Life
Science Center for Survival Dynamics, Tsukuba Advanced
Research Alliance (TARA Center), University of Tsukuba.
Author contributions are as follows. TN conceived the project.
RT-S and TN designed the research. RT-S and RA prepared
the target proteins and performed the crystallographic
analysis. MKK and YK produced the antibody. RO prepared
the antibody fragment for structural analysis. KI supervised
the EM work and MH obtained and analyzed the EM images.
TM, YH and YA examined the enzymatic activity of themutants. TE and MI performed the MD simulations. RT-S, RA
and TN analyzed the data from structural and functional
analysis. All of the authors contributed to paper preparation
and TN compiled the paper. The corresponding author
declares no financial or nonfinancial competing interests on
the behalf of all authors.

Funding information
This research was partially supported by the Japan Society for
the Promotion of Science (JSPS) KAKENHI under Grant
Nos. JP26291016, JP17K19206 and 19H03170 (to TN),JP19K06562 (to YH) and JP18H02404 (to YA), by a Grant-inAid for Scientific Research on Innovative Areas from the
Ministry of Education, Culture, Sports, Science and Technology (MEXT; Grant No. 18H05426) (to MI), by the Platform Project for Supporting Drug Discovery and Life Science
Research (Platform for Drug Discovery, Informatics andStructural Life Science) from the Japan Agency for Medical
Research and Development (AMED) under Grant No.
JP16am0101020 (to TN), the Platform Project for Supporting
Drug Discovery and Life Science Research (Basis for
Supporting Innovative Drug Discovery and Life Science
Research; BINDS) from AMED under Grant Nos.
19am0101072j0003 (support number 0418) (to MH and KI),
JP20am0101009 (support number 2633) (to MI),
JP20am0101078 (support number 1948) (to YK), the Programfor Promoting Research on the Supercomputer Fugaku (MDActa Cryst. (2021). D77, 645–662

Driven Precision Medicine) under Project ID hp200129, by
the RIKEN Dynamic Structural Biology Project (to MI)
and by AMED under Grant Nos. JP20am0401013 andJP20ae0101028 (to YK).

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