Structural basis for the assembly and translocation of the Vip1-Vip2 insecticidal toxin from Bacillus thuringiensis

Domain: biology
Published: 2026-03-30
Editorial outcome: major_revision

Abstract
Insecticidal toxins from Bacillus thuringiensis (Bt) have been extensively and successfully used in genetically
engineered crops for decades but continue to face challenges from the adaptive resistance in the insect population.
The Bt binary toxin Vip1Ad1(Vip1) and Vip2Ag1(Vip2), a promising next-generation candidate gene
combination for transgenic crops, have demonstrated high efficacy against the destructive coleopteran pest white
grubs, however, their mode of action remains largely elusive. In this study, we report cryo-EM structures of the
heptameric Vip1-pore and Vip2-bound Vip1-pore complex, capturing a series of putative assembly-related
intermediates that suggest a binary toxin assembly and translocation pathway. Together with structure-guided
mutagenesis, these data provide insights into a sequential assembly of binary complex and a sequence-
independent translocation mechanism. Proof-of-principle experiments showed successful delivery of a desired
protein cargo into host cells based on the mini-Vip2-Vip1 pore system, paving the way for developing much
needed extracellular pesticidal protein delivery platforms. These findings not only clarify the assembly and
translocation mechanism of the binary insecticidal toxin pair but also offer an excellent alternative model to
investigate human-pathogenic pore-forming toxin because of its similarity and biosafety.

Introduction
Bacillus thuringiensis (Bt) is an entomopathogenic bacterium whose secreted toxins have been used as
biopesticides for pest control over several decades 1, such as the crystal proteins (Cry) family and the vegetative
insecticidal proteins (Vip) family 2-5. Most commercially deployed Bt toxins belong to the Cry family. However,
the increasing incidence of insect resistance to traditional Cry toxins has created an urgent need for alternative
pest control strategies 6. The Vip toxin family has been classified into four different subfamilies, namely Vip1,
Vip2, Vip3, and Vip4, based on their protein sequence similarity 7,8. Unlike the widely used single-component
Cry toxins, Vip1 and Vip2 act as binary toxins, with Vip2 exhibiting ADP-ribosyltransferase activity 9. The
nomenclature of Bt proteins generally consists of two components. The first denotes the protein’s type and
functional characteristics, while the second distinguishes different hierarchical ranks within that protein family.
These ranks follow a four-tier system-Arabic numerals, uppercase letters, lowercase letters, and a final set of
Arabic numerals-corresponding to sequence similarities of 45%, 78%, and 95% respectively 10. Vip1Ad1(Vip1)
and Vip2Ag1(Vip2) from Bt strain HBF-18 are members of the Vip1-Vip2 binary toxin translocation system,
and they can be effective against the notorious coleoptera insects Holotrichia parallela larvae that severely
damage many crops 11,12.

Binary toxins typically contain two components that are responsible for forming transmembrane pores and
inducing cytotoxicity, respectively. The pore-forming component can specifically recognize receptors on the
surface of host cells and forms a transmembrane pore that facilitate the translocation of toxin proteins into the
host cytosol 13,14. Binary toxin systems have been broadly used for infection by different human pathogens, such
as Clostridioides difficile CDT toxin (CDTa and CDTb) 15, Clostridium botulinum C2 toxin (C2I and C2II) 16,
Clostridium perfringens iota toxin (Ia and Ib) 17, Clostridium spiroforme CST toxin (CSTa and CSTb) 18, and
Bacillus anthracis anthrax toxin (PA, EF and LF) 19. In contrast to these human pathogens, Bt is widely used for
plant protection, providing significant benefits to agriculture worldwide.

As two of the few effective toxins, the Vip1-Vip2 binary toxins have shown significant potential in
controlling corn rootworm (e.g., Diabrotica virgifera) 6 and white grubs (e.g., Holotrichia parallela) 6, which
are major insect pests worldwide. These toxins are considered as strong candidates for second-generation
transgenic crops. However, two key unknowns have hindered their application. First, the molecular mechanism
underlying the functional assembly of the Vip1-Vip2 complex and its subsequent insecticidal actions on target
cells remains largely unclear, despite the elucidation of the crystal structures of Vip1 (PDB: 6SMS
[https://doi.org/10.2210/pdb6sms/pdb]) and Vip2 9. Second, while Vip2 prevents actin polymerization, which
makes its expression in planta lethal, some Vip2 mutants with reduced ADP-ribosyltransferase activity has been
engineered 20. Although previous studies have provided important insights, the lack of understanding of Vip2
translocation remains a major limitation of understanding and engineering Vip1-Vip2 system. Therefore,
elucidating the functional assembly mechanism of Vip1-Vip2 and the translocation mechanism of Vip2 is
essential for leveraging these toxins in the development of effective and targeted strategies for future
biotechnological applications.

Here, we determined the cryo-EM structures of the functional heptameric Vip1-pore and its complex with
Vip2, revealing that the heptameric β-barrel channel is highly hydrophilic which is crucial for sequence
independent Vip1 translocation. Additionally, we captured critical putative assembly-related intermediates
during the assembly of both the heptameric pore and the Vip2-bound Vip1-pore complexes, uncovering a putative
stepwise assembly mechanism. Moreover, through domain swapping of Vip2, we provided proof-of-concept
evidence for using N-terminal domain of Vip2 as a mini vector for protein translocation, which has high potential
for the translocation of other biopesticides. Together, these findings indicate the putative stepwise assembly and
sequence-independent translocation mechanism of the binary toxins and offer strategies for developing next-
generation biopesticides.

Results
Overall structure of Vip1 heptameric pore
It is widely accepted that Bt Vip1 protoxin is digested by proteases in the midgut of targeting pest. The protease
digestion of Vip1 protoxin is indispensable for the pore formation that was assumed to deliver the enzymatic
Vip2 component into target cells2. We monitored the pore formation systematically by digesting Vip1 using
trypsin and obtained a stable oligomerization condition suitable for cryo-EM structure studies (Fig. 1a,
Supplementary Fig. 1a and 1c-f). In principle, the Vip1 protoxin was digested into a stable fragment within 2
hours under tested conditions, which further assembled into an SDS-resistant and heat-stable homogeneous
oligomer (Fig. 1b). We then determined the Vip1 oligomer at a resolution of 3.31 Å (Supplementary Table 1),
including data processing and representative density (Supplementary Fig. 2 and 3b). The structure of Vip1
oligomer adopted a stable heptamer manner (Fig. 1c-d), and the protomer of which is composed by four distinct
domains: D1 (D223-P305), D2 (S306-K512), D3 (T513-S622), and D4 (S623-F878) (Fig. 1e). It is worth noting
that the long stem of the pore structure, spanning residues K320 to F381, is encased by the D4 domain, which
comprises two sub-domains, D4I (S623-I756) and D4II (I769-F878), connected by a linker (S757-E768). The
D4 domain was reported as receptor-binding domain (RBD) in the B component of other binary toxins, such as
Ib and CDTb 21-23.

The Vip1 heptamer resembles a funnel shaped structure, termed Vip1-pore hereafter, with the upper part of
the open region containing four conserved motifs, which were involved in the recognition of enzyme components
in other binary toxin 22,24, and the lower part consisting of a cylindrical stem structure. In detail, the upper half
of the open region consists of four checkpoints. The first checkpoint, with an inner diameter of 36.2 Å, includes
seven di-calcium binding sites and was defined as Ca2+-edge. The second checkpoint, which is similar to the
NSS-loop in CDTb, is defined as the DTH-loop (residues D491-H493) in Vip1 (Fig. 1f-h). The narrowest
checkpoint formed by seven Phe residues (F455) from the heptamer, with an inner diameter of 6.0 Å, was defined
as ϕ-clamp 25. The fourth checkpoint locating at the top of the stem region has an inner diameter of 20.0 Å (Fig.
1h).

In the cylindrical stem, the pore forming region (K320-F381) of each protomer adopts an antiparallel β-
sheet structure and twists together to form the β-barrel stem, with a height of about 80.0 Å (Fig. 1d). A structural
comparison between the protomer and the predicted protoxin revealed drastic secondary structural differences
in the long stem region (Supplementary Fig. 4a-b). Strikingly, the secondary structure of the pore forming region
flipped nearly 180°, switching from a flexible loop to a rigid antiparallel β-sheet (Supplementary Fig. 4c). This
may have been triggered by the release of restriction by the N-terminal domain (D32-I222) of Vip1, which
provides the structural basis for the initiation of β-barrel pore formation.

Structure of Vip2-bound Vip1-pore heptamer complex
To explore the recognition and translocation mechanism of Vip2-Vip1 binary toxin, we established a pull-down
assay to monitor the interaction between Vip2 and Vip1 heptamer, which was also used for Vip2 and Vip1
heptamer complex assembly for cryo-EM studies (Fig. 2a-b and Supplementary Fig. 1b). We determined the
structure of Vip2 in complex with the Vip1 heptamer at a high resolution of 2.40 Å (Supplementary Table 1,
Supplementary Fig. 3c, 5 and 6), with Vip2 positioned within the wide opening of funnel-like Vip1 heptamer via
asymmetric interactions with four of the seven protomers (Fig. 2c-d). The total interaction area of Vip2-Vip1 is
about 1661.1 Å2 (Supplementary Table 2). In the complex, the overall structure of Vip1 heptamer is almost
identical to the Vip1-pore alone, with an RMSD of 0.862 Å. In short, Vip2 is a mixed α/β protein and could be
divided into two domains, an N-terminal Vip1 binding domain (residues A73-S265) and a C-terminal ADP-
ribosyltransferase domain (residues L266-T461), which is essential for actin modification (Fig. 2e) 9. The N-
terminal domain (NTD) of Vip2 is recognized by the upper part of the Vip1 heptamer funnel via extensive
interactions with the Ca2+-edge and DTH loop, whereas the C-terminal domain (CTD) remains largely
disengaged from the heptamer (Fig. 2f).

Vip2 is specifically recognized by the Vip1 heptamer mainly through subunit B, C, D, E via hydrogen bonds,
salt bridges, as well as hydrophobic interaction (Fig. 2g-k, and Supplementary Tables 2). In detail, K250 of Vip2
forms a hydrogen bond with T492 of the subunit B, while K251 interacts with E282, D287, and K490 of the
same subunit (Fig. 2h, and Supplementary Table 2). Right beside this, S232, I248, and Y281 of the subunit B
interact with the main-chain of the K251-F253 (KGF) loop of Vip2, which helps K251 of Vip2 to root in the
groove of the subunit B (Fig. 2g-h). Moreover, N163 of Vip2 interacts with S232 of the subunit B, which
stabilizes the conformation of the G162-T164 loop of Vip2 , further enhancing the interactions between Vip2 and
Vip1 (Fig. 2h). Meanwhile, the β-sheet of K244-E254 and the helix of I165-L178 of Vip2 forms extensive
interaction with the subunits C of Vip1 heptamer (Fig. 2i, and Supplementary Table 2). In detail, the K244,
K247 and V249 of Vip2 interact with the backbone of Ca2+-edge of the subunit C, and K174 of Vip2 interacts with
D225 in the Ca2+-edge of the subunit C (Fig. 2i).

Subunit D plays a crucial role in the recognition and translocation of Vip2. The N-terminal helix 1 (residues
A73-E83), N137-S139 loop, and S211-T216 loop of Vip2 participate in the interaction with the subunit D (Fig.
2j, and Supplementary Table 2). Additionally, Vip2 interacts with the DTH loop of subunit E via its T87-E90
loop (Fig. 2k). The N-terminal helix of Vip2 is nearly parallel to the main-chain of the subunit D from T498 to
E501 involving van der Waals interactions (Fig. 2l). Furthermore, W76 of Vip2 forms an interaction with the
DTH loop of the subunit D (Fig. 2l), and the on-going unfolded N-terminus of Vip2 is 17.7 Å away from the ϕ-
clamp checkpoint (Fig. 2l). We also observed an additional asymmetric density in the narrow checkpoint
surrounded by the ϕ-clamp in the complex structure of Vip2-Vip1 heptamer (Supplementary Fig. 7a-b). This
density should be part of the distal N-terminal region (residues N34-K72) of Vip2, indicating that the currently
obtained heptameric state of Vip1-Vip2 represents a putative translocation intermediate. Previous studies
revealed that deletion of the same region (α1-α3 helices, residues E27-Y59) of CDTa did not affect the binding
of toxin proteins, but the translocation efficiency decreased significantly, further supporting that the N-terminal
helix is essential for toxin protein translocation 23.

Compared with the structure of the other six subunits within the Vip1-Vip2 complex, the side-chain of H493
in subunit D undergoes a conformational shift upon complex formation and directly participates in Vip2 binding
(Supplementary Fig. 7c-e). This suggests that H493, located within the DTH loop of Vip1, may act as a switch
for the recognition of Vip2. The extensive interactions between Vip2 and Vip1 heptamer reveals structural basis
for the recognition of Vip2 by Vip1 heptamer.

Stepwise assembly of the Vip2-bound heptameric pore complex
The Vip1-Vip2 complex, as a binary toxin, has to be fully functional in the insect's midgut, where the toxins have
to be exposed to numerous proteases and endure fluctuating pH levels, particularly highly alkaline environment
26 , raising concerns about the stability of the complex. We challenged the stability of the pre-assembled Vip2-
bound Vip1 heptamer complex under a pH gradient and verified its integrity using a two-way rebinding assay,
which employs two distinct affinity tags on Vip2 or Vip1, respectively (Supplementary Fig. 8).

Using the Twin-strep tagged Vip2 for rebinding, we found that the binding ratios between Vip1 and Vip2
are comparable at pH ranging from 4.2 to 9.5 (Supplementary Fig. 8a). The clear off-ratio of the complex began
to emerge at pH 10.0, indicating either some artifacts or pH-induced disassembly of the complex. To further
explore these possibilities, we performed analytical ultracentrifugation (AUC) at pH 10.4. In the absence of Vip2,
Vip1 alone exhibited multiple oligomeric species, consistent with partial pH-induced disassembly of the pore.
Notably, upon addition of Vip2, the sedimentation coefficients of Vip1 tetramers, pentamers, and heptamers
increased by 1.19%, 4.38%, and 11.83%, respectively, whereas no change was observed for Vip1 monomers
(Supplementary Fig. 9). These results indicate that Vip2 can associate with oligomeric forms of Vip1 and that
this interaction scales with oligomer size.

The appearance of off-ratio binding at high pH prompted us to investigate whether trace amounts of the
complex could survive at high pH, potentially adopting a conformation distinct from that observed at regular pH.
Motivated by the observed off-ratio results, in which an excess of Twin-Strep-tagged Vip2 was captured, we
intentionally reversed the affinity configuration to selectively isolate the trace amount of complex present at high
pH while eliminating the overwhelming excess of Vip1. Hence, we reversed the rebinding approach using Vip1
heptamer to bind to Ni resin under screened pH from 9.0 to 10.6 and found that the presence of Vip1-Vip2
complex at these harsh pH conditions (Supplementary Fig. 8b). This suggested the possible co-existence of the
Vip1-Vip2 complex and previously uncharacterized species at extremely alkaline environment 26, after
comparing the two-way rebinding assay.

To further characterize these species, we collected a full cryo-EM dataset about 8,961 movies at this pH
10.4. During 2D classification, we identified numerous putative assembly-related intermediates, corresponding
to Vip2-bound Vip1 heptamer complex, such as the two-subunit partial pore, the incomplete 1:3 (Vip2:Vip1)
complex, and the 1:4 to 1:7 complex (Fig. 3a). Because these species were observed under highly alkaline
conditions, we considered whether they might reflect pH-induced artifacts or disassembly products rather than
biologically relevant complexes. To address this concern, we performed chemical cross-linking experiments
under pH 7.5. As a control, pre-oligomerized Vip1 was crosslinked with comparable efficiency to the input,
showing no apparent accumulation of intermediate species (Supplementary Fig. 10a). In contrast, distinct pre-
assembly intermediate states were clearly captured by the crosslinking assay in the experimental group, in which
the crosslinker and trypsin were added simultaneously to the Vip1 protoxin (Supplementary Fig. 10b).
Furthermore, when Vip2 was present, combined cross-linking and Western blot analyses revealed Vip2-
associated complexes corresponding to several Vip1 binding intermediate states (Supplementary Fig. 11b).
Together, these observations support the existence of Vip1-Vip2 complexes involving distinct oligomeric forms
of Vip1 and suggest that the species identified by cryo-EM represent putative assembly-related intermediates,
rather than exclusively pH-induced disassembly products.

We then successfully reconstructed a low-resolution 3D map for an incomplete 1:4 (Vip2:Vip1) complex,
which matched the complete 1:7 (Vip2:Vip1) complex well, indicating that this 1:4 complex may represent a
putative intermediate state during complex assembly (Fig. 3b, Supplementary Fig. 12 and 13). Meanwhile, we
also solved a partial heptameric complex structure of 1:5 (Vip2:Vip1) complex at a resolution of 3.05 Å
(Supplementary Table 1, Supplementary Fig. 3d and 13), which includes the Vip2 in complex with five Vip1
subunits, while missing two subunits for a complete heptamer (Fig. 3c-d).

The overall structure of Vip1 in the 1:5 complex closely resembles that of the Vip2 bound Vip1 heptamer
complex (RMSD = 0.569 Å) (Fig. 3e). Although most regions of Vip2 are highly identical between the two
complexes, the distal N-terminal helix 1 of Vip2 exhibits a pronounced conformational difference. Specifically,
when helix 2 of Vip2 is aligned between the 1:7 and 1:5 complexes, helix 1 shows an approximately 50° shift
(Fig. 3f-g). Furthermore, relative to the key ϕ-clamp residue F455 in Vip1, Vip2 appears to penetrate more deeply
into the heptamer in the 1:7 complex (Fig. 3e), which also differs from the configuration observed in CDTa and
Ia (Supplementary Fig. 14b-e). To our knowledge, this is the most in-depth intermediate complex structure of
homologous binary toxin in the translocation process (Supplementary Fig. 14b-e). In addition, the short loop
(residues K62-K70) and a 13 amino acids α-helix 1 (E71-E83) of Vip2 in the 1:5 complex structure shifted to a
disordered loop and an even shorter α-helix in the Vip2-bound heptamer complex structure, indicating that the
Vip2 conformations we captured here may represent an initial binding state as well as a pre-translocation state
(Fig. 3f-g). Notably, the overall structure of Vip2 in the 1:5 complex is highly consistent with that of the crystal
structure of Vip2 monomer (RMSD = 0.627 Å) (Supplementary Fig. 14a) 9, further supporting that this Vip2
conformation may represent an initial binding state for complex assembly.

In the 1:5 complex, the interface between Vip2 and Vip1 is strikingly different from that in the heptameric
structure, as only protomers B and C are involved in binding to Vip2 (Supplementary Fig. 14f). This indirectly
implies that subunits B and C of Vip1 are sufficient for initiating Vip2 recognition, while subunits D and E might
be essential for further translocation. Further 3D variability analysis (3DVA) of the 1:5 complex showed a
putative stepwise assembly process for the Vip2-bound Vip1 oligomers from the 1:5 complex to 1:6 complex
(Supplementary movie S1).

Dynamic translocation of Vip2 by the heptameric pore
As mentioned above, the distal N-terminal helix (A73-E83) of Vip2 in the heptameric complex has undergone
drastic secondary structure shift and significant conformational change (Fig. 3f-g). Remarkably, the K62-A73
region at the N-terminus of Vip2 in the Vip2-bound Vip1 pentamer complex is displayed as a loop connected to
the first helix, with E64 and E68 of Vip2 interact with the DTH loop of subunit C and B, respectively
(Supplementary Fig. 14g). In contrast, the same region is almost invisible when binding to the Vip1-pore in the
heptamer, indicating that the two structures may represent the initial binding and pre-translocation stages, with
unfolding of Vip2 occurring upon binding to the pore. Although there are significant differences in the
conformation of Vip2 in the two complex structures, the mode of interaction between Vip1 and KGF loop of
Vip2 is highly consistent (Fig. 3e), suggesting that the KGF loop is crucial for both recognition and translocation
of Vip2.

We were also able to reconstruct a 1:7 complex using the same dataset, which is highly similar to the
aforementioned Vip2-bound Vip1 heptamer complex under regular pH. The 3D variability analysis (3DVA) of
the 1:7 complex displayed the dynamic translocation of Vip2 by the heptamer (Supplementary movie S2). Firstly,
Vip2 binds to the pore of the Vip1 heptamer via specific interactions between the KGF loop of Vip2 and the
Ca2+-edge and DTH loop of B subunit of the Vip1 heptamer (Fig. 3h). Then, the N-terminal helix of Vip2 binds
to the DTH loop, preparing for the translocation of Vip2 (Fig. 3i). Subsequently, Vip2 rotates inward toward the
pore of the Vip1 heptamer, anchoring via the KGF loop (Fig. 3e). Concurrently, as Vip2 rotates and the DTH
loop is extruded, its N-terminal helical structure unfolds and begins to penetrate the pore in the form of a linear
peptide chain. Once the Vip2 peptide chain passes through the constriction formed by F455, it is further
translocated into the β-barrel channel (Fig. 3j). Together with the 3DVA, the successful capture of the putative
assembly-related intermediates provides a delicate scenario for the assembly and dynamic translocation of the
binary toxin (Fig. 3h-j).

Translocation of the binary toxin is hydrophilicity dependent and sequence independent
Vip1 adopts a classical heptameric β-barrel structure, which is similar to those of CDTb, Ib and anthrax toxin
PA 24,27. Surprisingly, 96% of residues in the interior of Vip1-pore are hydrophilic and 4% of them are
hydrophobic (Fig. 4a). However, in the predicted protoxin structure, the stem region of Vip1 is generally
composed of an amphipathic flexible loop, containing 69.4% of hydrophilic and 30.6% of hydrophobic residues.
These hydrophilic residues converged at the inner pore portion, providing a highly hydrophilic pore (Fig. 4a).

The highly hydrophilic pore is supported by other binary toxins, as revealed by structural comparison combined
with sequence analysis of the stem region. In addition, 72.4% of the residues lining in the inner tunnel of Vip1
heptamer are small side-chain and hydrophilic amino acids, such as Ser, Thr and Gly (Fig. 4b). 13.8% of the
residues on the inner surface of the Vip1-pore are charged residues, including E327, H329, E370, and E360 (Fig.
4a), and only one hydrophobic residue A368 could be observed. The highly hydrophilic residue preference of
the pore intrigues us to ask whether it is essential for the translocation of unfolded Vip2 peptide across the pore,
as all the enzymatic proteins in binary toxins have to be unfolded for translocation 28.

To explore the influence of hydrophilic residues on the translocation of Vip2, structure-based mutagenesis
on key residues inside the pore of Vip1 were generated and further evaluated by insecticidal assay. We first
generated a hydrophilic mutant AT368S, which retained similar insecticidal activity to the WT, suggesting that
the hydrophobic residue is not essential for translocation (Fig. 4c). Next, three charged residues on the stem were
mutated to serine, generating E327S/H329S double mutant and E370S single mutant. E327S/H329S and E370S
did not display significant effects on insecticidal activity, indicating that charged residues might be dispensable
and hydrophilic serine replacement could be tolerated by Vip2 for translocation. Similarly, the reverse charge
mutant H329E showed minor effects on insecticidal activity, which might be caused by its large side-chain that
delayed the translocation efficiency (Fig. 4c). Reversely, two hydrophobic variants E327A/H329A as well as
E370V demonstrated minor decrease in insecticidal activity. We then combined the two variants and generated
an E327A/H329A/E370V triple mutant, which showed significant decrease in insecticidal activity. In contrast,
an E327S/H329S/E370S triple mutant still maintained high insecticidal activity, supporting that the sequence-
independent hydrophilic environment in the pore is critical for translocation (Fig. 4c). The folding of all mutants
was comparable with that of the wild type as shown in the thermal shift assays as well as the trypsin digestion
induced oligomerization assay (Supplementary Fig. 15). Consistently, hydrophobic variants caused dramatic
effects on ion selectivity, and changes in single-channel conductance were observed in Clostridium perfringens
iota toxin 29.

In order to verify the physiological relevance of the hydrophilic residues in the Vip1-pore regarding Vip2
translocation, histopathological staining on the larvae of H.parallela were analyzed. The histopathological
staining demonstrated that Vip1 combined with Vip2 toxins were able to cause substantial damage on the gut of
the larvae, including destruction of tissue structure and shedding of cellular content into the gut lumen (Fig. 4d).
In contrast, Vip1-E327A/H329A/E370V variant and Vip2 caused only slight damage to the gut, characterized by
loss of microvilli and intracellular vacuolation, while the tissue structure remained largely intact (Fig. 4e). As a
negative control, the gut treated with saline alone showed clear microvilli and intact gut cells (Fig. 4f). The
destruction of these tissues was further assessed, revealing that the damage caused by Vip1 and Vip2 toxins are
significantly higher than that in the saline-treated control group. In contrast, the Vip1-E327A/H329A/E370V
variant was less severe than the damage induced by Vip1 and Vip2 toxins (Fig. 4g). The insecticidal activity of
the triple hydrophobic variant significantly decreased, supporting the hydrophilic environment within the pore
is critical for the translocation and efficacy of Vip1-Vip2 binary toxin (Fig. 4c). Taken together, structural
analysis combined with mutagenesis data revealed that translocation of Vip2 is highly dependent on
hydrophilicity and sequence independent.

The hydrophilic pore of Vip1 has the potential for desired protein delivery
The sequence-independent and hydrophilicity-dependent translocation mechanism adopted by the Vip1-Vip2
complex prompted us to investigate whether it could be exploited for non-specific cargo delivery. To explore
this possibility, we established an assay to test the delivery capacity of the complex. We constructed a fusion
protein by attaching green fluorescent protein (GFP) to the C-terminus of full length Vip2, herein referred to as
Vip2-GFP, to assess whether it could be translocated into target cells (Fig. 5a). As shown in Figure 5b, after 3h
of incubation, successful translocation of Vip2-GFP into H.parallela HP9-2 cells could be observed by
subcellular localization assay. In contrast, the Vip1 loss-of-function triple mutant (E327A/H329A/E370V) failed
to translocate Vip2-GFP into cells, which is similar to the control group (Fig. 5b-c). This result supports the
notion that the Vip1-mediated translocation is sequence-independent, as otherwise the GFP domain might have
impeded translocation. Moreover, given that Vip2 could be divided into two functional domains, NTD which
mediates binding to Vip1, and CTD which exerts toxic activity, we next asked whether the NTD alone is
sufficient to drive translocation. To address this, we generated a second fusing protein Vip2NTD-GFP by replacing
the whole CTD of Vip2 with a GFP tag (Fig. 5a). Remarkably, the mini Vip2NTD-GFP was translocated with even
better efficiency into the H.parallela HP9-2 cell, as revealed by the subcellular localization assay (Fig. 5d-e).
These results further support the idea that translocation through the Vip1-pore is independent of the specific
sequence of the cargo.

Taken together, our findings provide comprehensive insights into the putative assembly and sequence-
independent translocation mechanism of binary complex, which may serve as a promising platform for non-
specific delivery of other target proteins (Fig. 6). In brief, Vip1 is proteolytically activated by host proteases to
form a pore structure (Vip1-pore). The fusion of a target protein with mini Vip2NTD, which acts as a guidance
module, enables specifical interaction with the Vip1-pore, thereby facilitating translocation across the cell
membrane. Upon entry into the cytosol, the fusion protein is likely assisted by molecular chaperones such as
Hsp70 and Hsp90 30-32, which promote proper refolding and functional recovery.

The successful delivery of a non-specific sequence not only supports our vision that Vip2 translocation is
sequence-independent, but also provides a molecular tool for future insecticidal drug development and delivery.
Vip2NTD-GFP displayed higher efficiency in the subcellular localization assay demonstrating that the NTD of
Vip2 could serve as a promising transport carrier for other protein biopesticides, which has high potential for the
next-generation drug development against rigorous drug resistance.

Methods
Cloning, expression and purification of recombinant proteins
The full-length Vip1 (GenBank ID: AGC08395.1) and Vip2 (GenBank ID: AGC08396.1) proteins were natively
expressed in Bt and purified via multi-step chromatographic purification 33. Briefly, Bt Vip1 and Vip2 were
cultured in LB at 30℃ for 24 h. The natively expressed and secreted Vip1 and Vip2 were purified using ion
exchange chromatography. Prior to loading, each sample was diluted 10-fold by buffer A (25 mM Tris-HCl, pH
8.0, 40 mM NaCl). Vip1 was purified using a HiTrap Q HP column, while Vip2 was purified on a HiTrap SP HP
column. Both columns were pre-equilibrated with buffer C and eluted with a linear gradient of buffer B (25 mM
Tris-HCl, pH 8.0, 1 M NaCl), increasing from 0% to 100 % Buffer B over 75 minutes. Then, the sample was
loaded onto a Superdex 200 Increase column (Cytiva, Marlborough, USA) and eluted with a buffer C (25 mM
Tris, pH 8.0, 100 mM NaCl). The purity of the eluted fractions was subsequently assessed by SDS-PAGE.

The Vip1 protein with C-terminal His6-tag was cloned into pET-28a vector (Supplementary Table 3) and
further overexpressed in E. coli BL21 (DE3) for pull-down assay and insecticidal bioassay. All mutants were
generated by overlapping PCR and further verified by DNA sequencing, with primers listed in Supplementary
Table 4. Vip2 (E41-K462) with C-terminal His-tag and N-terminal Twin-Strep tag was cloned into pET-28a and
overexpressed in E. coli BL21 (DE3). Bacteria were cultured in LB medium containing 100 μg/mL kanamycin.
Expression was induced with 1 mM IPTG (isopropyl 1-thio-β-d-galactopyranoside) when OD reached 0.6-
0.8, followed by culturing at 18°C for 18 h and harvested by centrifugation.

The harvested cells were lysed by sonication in lysis buffer D (50 mM Tris, pH 8.5, 400 mM NaCl),
followed by centrifugation at 30,624×g for 30 min. The supernatant containing soluble Vip1 or Vip2 protein was
loaded onto a Ni-NTA column pre-equilibrated with lysis buffer D. Vip1 or Vip2 protein was eluted using buffer
E (50 mM Tris, pH 8.5, 400 mM NaCl, 300 mM imidazole). Further purification of the recombinant Vip1 and
Vip2 proteins was performed similarly to that of the native tagless Vip1 and Vip2, respectively 33 .

Assembly of Vip1 heptameric pore and Vip1-Vip2 complex
To establish conditions that promote Vip1 oligomerization, the effects of trypsin concentration and digestion
duration were systematically evaluated. A series of controlled proteolytic digestions was performed at 37℃ using
varying trypsin concentrations (Trypsin:Vip1 mass ratios ranging from 1:10 to 1:100), digestion times (0.5-60
h), ethanol concentrations (0-20%), and different oligomerization times (0-36 h). The results show that a mass
ratio of 1:100 (Trypsin:Vip1) at 37°C for 2 h is sufficient to achieve homogeneous activation of Vip1. The
digestion reactions were subsequently quenched by the addition of 2 mM PMSF to inhibit further proteolysis
and allow Vip1 self-assembly to proceed.

The tagless Vip2 was mixed with Vip1 oligomer at a molar ratio of 3:1 (Vip2:Vip1 oligomer) and incubated
at 8 °C for 12 h. The Vip1-Vip2 complex was further purified by Ni-NTA column, as we serendipitously found
that the oligomeric pore could bind to Ni-NTA resin, which is probably due to multiple adjacent histidine residues.
The complex was then buffer-exchanged into a solution containing 25 mM Tris-HCl (pH 8.0) and 100 mM NaCl
for cryo-EM sample preparation.

Cryo-EM sample preparation and data collection
The trypsin-activated Vip1 heptamer was applied to glow-discharged carbon-coated copper grids (300 mesh,
QUANTIFOIL® R 1.2/1.3) at a final concentration of 1.5 mg/mL. The grids were blotted and plunge-frozen in
liquid ethane using an FEI Vitrobot-MarkI (Thermo Fisher Scientific, Rockford, USA). The frozen grids were
screened and then transferred to a Glacios 200kV TEM (Thermo Fisher Scientific, Rockford, USA) equipped
with a Falcon 4 direct electron detector for data acquisition. Cryo-EM images were automatically collected using
a defocus range of -1 to -2 μm, a nominal magnification of 150,000×, a calibrated pixel size of 0.94 Å/pixel. An
exposure time of 5.52 s was employed at an electron dose rate of 9.05 e⁻/Å²/s.

The two Vip1-Vip2 complexes were applied to the glow-discharged Quantifoil R1.2/1.3 grids at a final
concentration of 1.5 mg/mL and plunge-frozen in liquid ethane cooled by liquid nitrogen using the same Vitrobot.
The screened frozen grids were then transferred to a 300kV cryo-electron microscope (Titan Krios G4, Thermo
Fisher Scientific, Rockford, USA) equipped with a Gatan K3 electron detector and a GIF BioQuantum energy
filter for data acquisition. Images were collected on a Titan Krios microscope using a defocus range of -1 to -
2 μm, a nominal magnification of 105,000×, and a binned pixel size of 0.84 Å/pixel. Energy filtering was
performed with a slit width of 20 eV. An exposure time of 2.16 s was employed at an electron dose rate of
23.148 e⁻/Å²/s. Micrographs were recorded as movie stacks consisting of 40 frames, with a total electron dose
of 50 e⁻/Å² and an electron fluence of 1.25 e⁻/Å² per frame. All data collection was automated using EPU
software.

Cryo-EM data processing
Patch motion correction and contrast transfer function (CTF) estimation were performed for all acquired using
the patch CTF estimation module in cryoSPARC v4.0. For the Vip1-pore, a total of about 0.2 million particles
were automatically picked using Blob picker. Among these, 74,122 particles corresponding to heptamers with
high-resolution features were selected for further ab-initio reconstruction and heterogeneous refinement. A
predominant class containing 59,851 particles were selected for further refinement, resulting in a heptamer map
at an overall resolution of 3.31 Å with C7 symmetry.

Data processing of the Vip2-bound Vip1-pore complex followed the same procedure as above. Similarly,
particles were automatically picked using Blob picker, and around 3.2 million particles were selected for 2D
classification. A class representing the heptamer complex, containing 265,183 particles, was selected for
homogeneous refinement and NU-refinement with C1 symmetry, yielding a final map of 2.40 Å resolution.

Processing of the 1:4 and 1:5 (Vip2:Vip1) complex followed a similar workflow using the dataset collected
under pH 10.4. Approximately 4.5 million particles automatically picked using Blob picker and subjected to 2D
classification. The well-defined class representing for each complex were selected for ab-initio reconstruction,
followed by homogeneous refinement and NU-refinement with C1 symmetry.

Three-dimensional variability analysis (3DVA) was performed in cryoSPARC. The analysis was conducted
using the final particle stack obtained from the consensus refinement. The soft mask generated during the
consensus refinement was directly used for the 3DVA calculation. Three variability modes were calculated to
visualize conformational heterogeneity. No particle subset selection, clustering, or further refinement was
performed following the 3DVA analysis. The resulting volumes were exported to generate the two supplementary
movies, respectively.

Model building and refinement
Model building of the four structures was accomplished in an iterative process involving SWISS-MODEL 51,
Coot 52, Chimera 53, and PHENIX 54. Briefly, for the structure determination of the 3.31 Å resolution heptameric
pore, the transmembrane antiparallel β-sheet of CDTb (PDB: 7YVQ [https://doi.org/10.2210/pdb7yvq/pdb]) and
a model generated by SWISS-MODEL server were modified and used as an initial model for manually fitting to
the heptameric density map. Together with iterative manual building in Coot and real space refinement in Phenix,
the final heptameric structure was generated by applying the C7 symmetry and further refined in PHENIX.
Notably, the D4-II domain (S763-F878) was excluded in the Vip1-pore due to weak map density.

Using the structures of Vip1-pore and Vip2 (PDB: 1QS1 [https://doi.org/10.2210/pdb1qs1/pdb]) as input
models, we were able to generate an initial model for the Vip2-bound Vip1 heptamer complex using Chimera.
This initial structure model was used for iterative manual building in Coot and real space refinement in Phenix.
The N-terminal region (N34-K72 residues) of Vip2 was omitted due to weak density which might indicate high
flexibility. The model of partial D4-II domain (P783-F878) was excluded because of weak map density. The
model of the Vip2-bound Vip1 pentamer complex was built similarly as above. Briefly, the partial Vip1-pore
structure and Vip2 monomer were applied to fitting into the density map using Chimera.

Protein melting assay
The thermal stability of Vip1 variants was assessed using a fluorescence-based thermal shift assay performed on
a Real-Time PCR system. Each protein, at a concentration of approximately 0.5 mg/mL, was mixed with the
fluorescent dye SYPRO Orange (Sigma-Aldrich, St. Louis, USA) and subjected to heating from 25°C to 95°C
with a linear temperature ramp. The melting midpoint (Tm) of each protein was determined using the analysis
software provided by the instrument manufacturer. The data from three independent experiments were averaged
to generate the bar graph.

Insect and cell line
The larva of Holotrichia parallela were obtained from the field of peanut supplied by Cangzhou Academy of
Agriculture and Forestry Sciences. HP9-2, an established cell line derived from embryonic cell of H.parallela
was obtained as a seed culture from Qingdao Agricultural University, and grown at Institute of Plant Protection
in Trichoplusia ni Medium-Formulation Hink (TNM-FH) insect cell culture medium (Pricella, Wuhan, China)
55 supplemented with 10% fetal bovine serum (FBS) (VivaCell Biosciences, Shanghai, China). The cells were
grown at 28°C in 25 cm2 tissue culture flasks and transferred to fresh medium every 5-7 days.

Insecticidal bioassay
Second instar larvae of H.parallela were starved for 12 h. Vip1 or its mutant proteins was mixed with Vip2 or
its mutant proteins with molar mass ratio 1:1 in 20 μL buffer (25 mM Tris-HCl containing 100 mM NaCl).
Twenty microgram mixed proteins were injected into larvae through their mouthparts. The buffer without protein
was treated as a negative control. Twelve larvae were injected for each treatment. Three repeats were performed.
The larvae were fed in soil mixed with shredded carrot at 26°C in artificial climate box. The mortality of larvae
was checked after 7 days. Corrected mortality was calculated by subtracting the mortality of the negative control
group from the mortality of each treatment group. Each assay was repeated at least three times 56.

Histopathological analyses
Six second-instar larvae of H. parallela were starved for 6 h for each treatment. Twenty microgram Vip1 or its
mutants and Vip2 proteins were injected into larvae through their mouthparts. The buffer without protein was
treated as a negative control. The larvae were fed in soil mixed with shredded carrot for 48 h at 26°C in artificial
climate box. The midgut tissues were dissected from these larvae and fixed in 4% paraformaldehyde (Solarbio
Life Sciences, Beijing, China) at 4°C overnight. The tissues were embedded in paraffin and made 5 µm thick
sections. The sections were stained with hematoxylin solution for 10 min and eosin solution for 30 s by using
Hematoxylin-Eosin (HE) Stain Kit according to manufacturer’s instructions (Solarbio Life Sciences, Beijing,
China), and sealed with neutral gum. The pathological changes in midgut tissues were observed under
Pannoramic 250 Flash digital microscopes (3DHISTECH, Budapest, Hungary). To further examine the damage
to tissues, we carried out histological analysis with paraffin embedded midgut tissue sections. These tissues were
scored according to four histological criteria including disruption of the epithelial microvilli, disorder of cell
arrangement, cell shedding, and cytoplasmic vacuolation on a scale of 0 to 3 (normal, mild, moderate, or severe).

Glutaraldehyde cross-linking
Purified Vip1 and Vip2 were buffer-exchanged into 20 mM HEPES, pH 7.5, 100 mM NaCl using a Superdex
200 Increase column (Cytiva, Marlborough, USA). To assess Vip1 oligomerization, in the first group, purified
Vip1 was proteolytically activated by trypsin digestion and then pre-incubated for 12 hours to allow oligomer
formation. In the second group, trypsin-activated Vip1 was used without extended incubation, and
glutaraldehyde was added immediately after proteolytic activation. Both sets of samples were treated with
increasing concentrations of glutaraldehyde at 37°C for 2 h, followed by quenching with 1 M Tris-HCl (pH 8.0).
The reactions were resolved by SDS-PAGE and visualized by Coomassie staining. In addition, eluted Vip1 was
mixed with Vip2 at molar ratios of 3:0, 3:1 or 3:3, followed by addition of trypsin and 2.5 mM CaCl₂.
Glutaraldehyde was then added to 0.0075% (w/v), and the mixture was incubated at 37°C for 2 h. All reactions
were quenched with Tris (pH 8.0, final 0.1 M) for 20 min. Samples were resolved by SDS-PAGE and visualized
by Coomassie staining.

Western blotting
For western blotting analysis, samples were separated by Young PAGE™ 4-20% Bis-Tris (GenScript, Piscataway,
USA). Proteins were transferred onto 0.45 μm PVDF membrane (Beyotime, Shanghai, China) in Tris-Glycine
transfer buffer (25mM Tris, 200mM Glycine). Membranes were blocked in 5% milk in TBS-T for 1 h prior to
incubation with StrepII Tag mouse monoclonal antibody (AF2924, 1:1,500 dilution, Beyotime, Shanghai, China)
overnight. The following day, membranes were incubated with HRP-labeled Goat Anti-Mouse IgG (H+L)
(A0216, 1:1,500 dilution, Beyotime, Shanghai, China) in TBS-T and visualized using ECL Prime reagent
(Beyotime, Shanghai, China).

Analytical ultracentrifugation (AUC)
Sedimentation velocity experiments were carried out at 20℃ and 90,000×g on a Beckman Coulter Optima AUC
following standard protocols 57. Samples containing either 1 mg/mL Vip1 alone or 1 mg/mL Vip1 pre-mixed
with Vip2 at a 1:3 mole ratio were prepared in buffer (50 mM CAPS, 100 mM NaCl, 2.5 mM CaCl₂, pH 10.4).
The mixtures were then treated with trypsin at a Vip1:trypsin mass ratio of 100:1 and incubated at 37°C for 2 h
before being loaded into 12-mm Epon centerpieces for analytical ultracentrifugation. Absorbance (280 nm) and
interference scans were collected at 60 s intervals. Data analysis was performed with SEDFIT (Version 18p1)
using a Continuous c(s) Distribution model as described by Schuck et al. 58.

Observation of subcellular localization
H.parallela HP9-2 cells were suspended at 1×10⁷ cells/mL in TNM-FH insect cell culture medium supplemented
with 10% FBS, and 500 μL aliquots were transferred to glass bottom cell culture dishes (801001, NEST, Wuxi,
China). The dishes were incubated at 28°C overnight. Four hundred microliters TNM-FH culture medium were
eliminated. One hundred microliters of 2000 nM Vip1, mixed with Vip2-GFP or Vip2NTD-GFP proteins were
added to the cells and the dishes incubated for 2 h at 28°C. Vip2-GFP or Vip2NTD-GFP protein with same amount
was added solely to the cells as negative control. One hundred microliters of 5000 nM Vip1, mixed with Vip2-
GFP or Vip2NTD-GFP proteins were added to the cells and the dishes incubated for 1.5 h or 3.5 h at 28°C. Cells
were washed twice with pH 7.4 PBS (Phosphate Buffer Saline) and fixed in 200 μL 4% paraformaldehyde for
30 min at room temperature. After washed three times with PBS, they were carefully permeabilized with 2%
Triton X-100 in PBS for 5 min at room temperature. After washing with PBS for 3 times, the cells were labeled
with 1×Phalloidin-iFluor 594 (Abcam, Cambridge, UK) for 1 h and 1×Hoechst33342 (Thermo Fisher Scientific,
Rockford, USA) for 10 min at room temperature. After washing with PBS for 3 times, Prolong Diamond
Antifade (Invitrogen, Oregon, USA) was added into the dishes. Images were obtained using confocal LSM 980
microscopes (Zeiss, Germany) 59.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "1. What are the noteworthy results?\n>I think the finding that the N-terminus of Vip2 can be fused to non-Vip proteins to alter the mode of action of the toxin\ncomponent is very important. Obtaining the cryo-EM structures of Vip2 bound to the Vip1 heptomer is also noteworthy,\nespecially defining the sub-domains and AA residues important in contact and Vip2 translocation etc. The data relating to\nthe in-depth intermediate complex structures is also quite unique and very interesting as are the findings relating to the\nstructures of homologous binary toxins in the translocation process\n2. Will the work be of significance to the field and related fields? How does it compare to the established literature? If the\nwork is not original, please provide relevant references.\n>I believe this work is of significance to the field, especially in the light of the emergence of Bt cry toxin resistant pests,\ndriving the need for the next generation of pest control options.\n3. Does the work support the conclusions and claims, or is additional evidence needed?\n>While structural studies of this nature are not exactly my expertise, I do believe the data presented very clearly supports the\nclaims and conclusions.\n4. Are there any flaws in the data analysis, interpretation and conclusions? Do these prohibit publication or require revision?\n>I do not recognise and obvious flaws or omissions in this study, in the data analysis, interpretation or conclusions.\n5. Is the methodology sound? Does the work meet the expected standards in your field?\n>I believe this methodology is sound.\n6. Is there enough detail provided in the methods for the work to be reproduced?\n>I believe so. The methods are detailed, comprehensive and clearly described.", "reviewer_2": "Groups of Jie Zhang and Peng Chen present a structural and functional study of the Vip1-Vip2 toxin complex from Bacillus\nthuringiensis. In the manuscript, I see the following three major messages:\nI) The study reports a structure of the Vip1 heptamer and a structure of the Vip1-Vip2 complex, showing their similarity to\nother binary proteins.\nII) Following alkaline pH treatment, the researchers show the presence of Vip1-Vip2 complexes with different stoichiometry,\nsuggesting that they represent \"critical intermediates\".\nIII) The researchers demonstrate that Vip1-Vip2 complex can be used for translocation of the desired protein cargos.\nAs I have major concerns to all three messages, either in terms of methodological approaches, or in terms of novelty, I do not\nrecommend this paper for publishing.\nMajor points:\nMessage I.\n1. In line 87, the authors write that 'it is widely accepted that Bt Vip1 prototoxin is digested...'. I find such statement without a\nproper citation non appropriate for scientific literature. Please provide a reference.\n2. In line 89 the authors report that they 'systematically monitored pore formation' with a reference to Fig 1a. However, I don't\nsee any systematic analysis there. What was it? How did the authors perform it? In Fig 1b the authors show an SDS-PAGE\nsuggesting that a high molecular weight band is the pore. It is quite an overstatement.\n3. In line 105 the authors describe a region of Vip1 that is essential for the Vip2 translocation. How can it be deduced only\nfrom the structure of Vip1?\n4. Line 107. The authors built many metal ion binding sites. Indeed, they are of a good quality in cryo-EM maps, however,\nwhat is the basis for being sure that it is calcium, and not, for example, magnesium?\n5. The authors applied C7 symmetry for the Vip1 structure. What was the rationale for doing this? Could it be that the protein\nhas a pseudosymmetry that was lost when applying C7? Later, the authors compare Vip1 alone (refined with C7) and when\nit complex with Vip2 (C1), is such a comparison justified?\nI inspected the final cryo-EM maps and the structures and found them of a good fit. I appreciate that the authors voluntarily\nprovided them with the submission. However, after reading supplementary information, PDB reports and materials and\nmethods, I am raising the following concerns:\n6. The cryo-EM reconstructions are of good resolution, however clashscores of the deposited structures are surprisingly bad.\nFor 9VEK is it 9 and for 9VEG is it 12. Considering this and the following points, I believe that authors need to revisit the\nmodel building.\n7. 9VEK contains 6 side chain outliers. It is fine having them, however the structure has 7 chains built into C7-symmetry\ncryo-EM density. How is it possible to have 6 outliers?\n8. All three structures contain bong angle outliers. Are they supported by the density?\n9. Supplementary table 1 reports 'model resolution'. What is it? How is it different to 'map resolution' the authors provide\nabove other than it is given at different FSC threshold?\n10. Please provide data on what residues have been built into the density, as the structures do not cover complete protein\nsequences.\n11. Supplementary figure 1, 3 and 5 are missing exemplary cryo-EM micrographs and 2D class averages.\n12. Finally, it looks to me that the obtained structures are very similar to other binary toxins, like C2, Iota and others. What is\nthe new information gained from the Vip1-Vip2 complex?\nMessage 2.\n13. In line 173 the authors mention 'two-way rebinding assay' but do not describe it. Could it be that at extreme pHs the\nproteins do not bind to strep sepharose? I think this experiment misses a control.\n14. While I find it interesting that at high pH the authors found the Vip1-Vip2 complex at different stoichiometry, I do not think\nthat based on that, one can claim that these are 'intermediate states' and deduce an assembly process (line 213). It all might\nbe artifacts of high pH or rather 'disassembly process'. I believe that functional assays are required to show that 5:1 Vip1-\nVip2 complex is a true intermediate and can be assembled further.\n15. Line 223. The same as above. It is possible that high pH created nonphysiological artifacts that the authors interpreted\nas intermediate states.\n16. Fig 3a misses scale bars. I have my doubts that the 2D class on the very left represents a single Vip1. The class is of low\nquality and can just contain some debris. I suggest reextracting these particles with lower box size and redoing 2D\nclassification to get some high-resolution features to prove that it is really a single Vip1.\nMessage 3.\n17. While I find this interesting and a well-done set of experiments, I am questioning the novelty of them. For example,\nBacillus anthracis PA is used for decades for similar purpose. A few recent articles describing it:\nhttps://pubmed.ncbi.nlm.nih.gov/40758882/ https://pubmed.ncbi.nlm.nih.gov/25250705/\nMinor points:\n1. Abstract line 31. I would not use 'irrelevant' protein cargo but maybe non-specific, engineered or desired?\n2. Introduction has several strange sentences that I could not follow. For example, first you describe 4 families of Vip proteins\n(line 47) and, right after it, on line 50, talk about the Vip1-Vip2 family. Why?\n3. What means Ad1 in Vip1Ad1 in line 49?\n4. What is a pore-forming protein in line 54?\n5. Anthrax toxin has two payloads, EF and LF. Why only to mention the second?\n6. Line 71-72: the authors state that the lack of the Vip2 translocation mechanism is a major limitation... of what? I couldn't\nfollow. Of engineering of Vip2 to be nontoxic to plants?\n7. Line 87-88. 'which is indispensable for the pore formation'. Is it the pest, which is indispensable?\n8. Line 101, what homologous protein you have in mind?\n9. Line 192 cites Supplementary Fig 5a-e, while it is cryo-EM supplementary figure.\n10. Line 397. Please describe the method of purification of Vip1 in detail.", "reviewer_3": "The manuscript presents novel cryo-EM structures formed by insecticidal toxins from Bacillus thuringiensis.\n1. The high resolution structures of Vip1 heptameric pore and Vip2-bound Vip1-pore heptamer complex are well described\nand analysed and provide a valuable new structural information that supports a sequence-independent translocation\nmechanism of the binary toxins. The findings will be a helpful addition to the field. To evaluate the quality of these structures\nthe reviewer requests access to cryo-EM maps including non-symmetrised maps, maps without masking and masks applied\nprior to CTF estimation. We remind the authors that EMDB now encourages and accepts deposition of these together with\n‘main’ structure. In addition, we will appreciate up-to-date PDB validation reports for all structures. Representative cryo-EM\nimages and averages of common views of Vip1 pore and Vip2/Vip1- complex should be also added to supplementary\ninformation to aid understanding and evaluation of data presented.\nWhile data analysis and image processing steps are well described in Methods section, some important information is\nmissing: please add which detector was used for data collection on Glacios instrument (Vip1 data), correct Vitrobot model\nand replace ‘flash frozen’ ‘plunge frozen’ for cryo-Em grids. In addition, information about electron flux (e/A2/s), exposure\ntime (s) and electron fluence per frame (e/A2) should be added for each solved structure in Methods and/or in Table 1.\nTo put new structural information in context and show the significance to the field, Vip1 and Vip2/Vip1 structures should be\naccompanied by analysis of sequence homology with other bacterial toxins: α-hemolysin, anthrax protective antigen pore,\nClostridium perfringens iota toxin and Vibrio cholerae cytolysin and comparison with known structures of membrane-inserted\npores of binary toxins.\nWhile discussing translocation mechanism, we also suggest including speculations on driving forces for substrate protein\ntranslocation: differences of proton concentration or electrical potential across the ϕ-clamp. The former requires protonation-\ndependent conformational changes of two or more alternating gates and protonation-dependent conformational change in ϕ-\nclamp region. Alternatively, translocation could follow charge-state dependent Brownian ratchet model similar to the one\ndescribed for anthrax protective antigen pore. Electrophysiological studies – if possible – would be a valuable addition.\n2. Authors also described multiple incomplete assemblies of Vip2-bound Vip-pore heptamer complex after treatment with\nhigh pH (pH 10.4): two-subunit partial pore,1:3 (Vip2:Vip1) complex, 1:4 and 1:7 complex. Low-resolution cryo-EM 3D\nreconstructions of an incomplete 1:4 (Vip2:Vip1) complex and complete 1:7 (Vip2:Vip1) complex were obtained from this\npreparation and discussed as intermediate states of complex assembly. This interpretation is only supported by fluctuating\npH levels in insect’s midgut, including highly alkaline environment. The authors did express some ‘concerns about\nphysiological relevance’ of these findings in Discussion section but nonetheless propose a step-wise assembly pathway\nbased on these ‘intermediate’ structures. No high pH treatment step was performed for Vip2/Vip1 preparation used for high\nresolution structures discussed earlier, neither for subcellular localization experiments with insect cells reported in this\nmanuscript. To enable mechanistic understanding of Vip1/Vip1 complex formation and action, further in vitro experiments\nare required using model liposomes and Vip2/Vip1 (or Vip2-GFP/Vip1): Vip2/Vip1 assemblies on liposomes could be\ntreated with high pH and then used for cryo-EM sample preparation. Image processing of these cryo-EM data would follow\nalready established single particle workflow using cryoSPARC and could yield direct visualisation of intermediate\nstructures. These experiments could also shed some light on nature of Vip1 pore receptor. It seems too early to consider\npotential use of Vip1/Vip2 system for development therapeutic or delivering proteins to specific cellular targets without\nfurther knowledge about receptor binding and pore formation mechanism.\n3. And lastly, it is important to complete the nonspecific GFP delivery experiments with negative control when cells are\ntreated with Vip2-GFP or Vip2NTD-GFP only. Surprising translocation of relatively large folded protein (GFP) through pore\nwith an inner diameter of 6.0 Å seems inconsistent with Vip1 structure presented in tne same manuscript and requires further\nchecks and validation."}, "author_response": "Manuscript number: NCOMMS-25-64397\nWe thank the editor and the reviewers for their careful reading of the manuscript and their\nconstructive suggestions that have guided our revision. We have revised the manuscript\nto address reviewers’ concerns as outlined below.\nReviewer #1 (Remarks to the Author):\n1. What are the noteworthy results?\n>I think the finding that the N-terminus of Vip2 can be fused to non-Vip proteins to alter the\nmode of action of the toxin component is very important. Obtaining the cryo-EM structures\nof Vip2 bound to the Vip1 heptomer is also noteworthy, especially defining the sub-domains\nand AA residues important in contact and Vip2 translocation etc. The data relating to the\nin-depth intermediate complex structures is also quite unique and very interesting as are\nthe findings relating to the structures of homologous binary toxins in the translocation\nprocess\n2. Will the work be of significance to the field and related fields? How does it compare to\nthe established literature? If the work is not original, please provide relevant references.\n>I believe this work is of significance to the field, especially in the light of the emergence\nof Bt cry toxin resistant pests, driving the need for the next generation of pest control\noptions.\n3. Does the work support the conclusions and claims, or is additional evidence needed?\n>While structural studies of this nature are not exactly my expertise, I do believe the data\npresented very clearly supports the claims and conclusions.\n4. Are there any flaws in the data analysis, interpretation and conclusions? Do these\nprohibit publication or require revision?\n>I do not recognise and obvious flaws or omissions in this study, in the data analysis,\ninterpretation or conclusions.\n5. Is the methodology sound? Does the work meet the expected standards in your field?\n>I believe this methodology is sound.\n6. Is there enough detail provided in the methods for the work to be reproduced?\n>I believe so. The methods are detailed, comprehensive and clearly described.\nResponse: We thank the reviewer for the positive comments.\nReviewer #2 (Remarks to the Author):\nGroups of Jie Zhang and Peng Chen present a structural and functional study of the Vip1-\nVip2 toxin complex from Bacillus thuringiensis. In the manuscript, I see the following three\nmajor messages:\nI) The study reports a structure of the Vip1 heptamer and a structure of the Vip1-Vip2\ncomplex, showing their similarity to other binary proteins.\nII) Following alkaline pH treatment, the researchers show the presence of Vip1-Vip2\ncomplexes with different stoichiometry, suggesting that they represent \"critical\nintermediates\".\nIII) The researchers demonstrate that Vip1-Vip2 complex can be used for translocation of\nthe desired protein cargos.\nAs I have major concerns to all three messages, either in terms of methodological\napproaches, or in terms of novelty, I do not recommend this paper for publishing.\nMajor points:\nMessage I.\n1. In line 87, the authors write that 'it is widely accepted that Bt Vip1 prototoxin is digested...'.\nI find such statement without a proper citation non appropriate for scientific literature.\nPlease provide a reference.\nResponse: Thanks for your suggestion. We have now added the reference #2 \"Bacterial\nVegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria\", (line 104) which\nstates: \"The molecular mechanism of the insecticidal activity of the Vip1/Vip2 toxin is not\ntotally understood. The multistep process begins with the ingestion of the toxin by the larva,\nfollowed by proteolytic activation in the midgut by trypsin-like proteases.\"\n2. In line 89 the authors report that they 'systematically monitored pore formation' with a\nreference to Fig 1a. However, I don't see any systematic analysis there. What was it? How\ndid the authors perform it? In Fig 1b the authors show an SDS-PAGE suggesting that a\nhigh molecular weight band is the pore. It is quite an overstatement.\nResponse: Thanks for your correction. Initially, we adapted the published CDTb activation\nconditions as a foundation for optimizing Vip1 oligomerization. To establish conditions that\npromote oligomerization, we evaluated the effects of trypsin concentration, digestion time\nand oligomerization time, and hydrophobicity environments. We have added these data to\nthe Supplementary Fig. 1 and expanded the Methods section to provide additional details\non the optimization procedure.\nWe have added the sentences as follows: “To establish conditions that promote Vip1\noligomerization, the effects of trypsin concentration and digestion duration were\nsystematically evaluated. A series of controlled proteolytic digestions was performed at\n37°C using varying trypsin concentrations (Trypsin:Vip1 mass ratios ranging from 1:10 to\n1:100), digestion times (0.5-60 h), ethanol concentrations (0-20%), and different\noligomerization times (0-36 h). The results show that a mass ratio of 1:100 (Trypsin:Vip1)\nat 37°C for 2 h is sufficient to achieve homogeneous activation of Vip1. The digestion\nreactions were subsequently quenched by the addition of 2 mM PMSF to inhibit further\nproteolysis and allow Vip1 self-assembly to proceed.” (line 558-565)\nThanks again for your valuable suggestion, as such details may benefit researchers\nworking on the activation of enzyme-activated toxins. We agree it is too early for claiming\nthe high molecular weight band as a pore at Fig 1b, and revised it accordingly.\nSupplementary Fig. 1 Purification of Vip1 and Vip2 proteins and Optimization and\ncharacterization of Vip1 oligomerization conditions. a-b The quality of the Vip1 and Vip2 used\nfor cryo-EM studies was characterized by SDS-PAGE, a representative result from 3 similar results\nwas reported. Inset: SDS-PAGE analysis of the purified Vip1 and Vip2 proteins, with molecular\nweight markers (M) shown on the left. The gels are representative of three independent experiments.\nc SDS-PAGE analysis of Vip1 samples digested with trypsin at varying enzyme-to-substrate mass\nratios (1:10 to 1:100) for 2-10 h at 37°C. d Time course of Vip1 digestion under fixed enzyme-to-\nsubstrate ratio (Trypsin:Vip1=1:100, w/w). Samples were digested for 0-60 h at 37°C and analyzed\nby SDS-PAGE. e Effect of ethanol on Vip1 oligomerization. Samples were treated with increasing\nethanol concentrations (0-20%, v/v) during activation and analyzed by SDS-PAGE/Coomassie\nstaining. f Influence of digestion time (0.5-2 h) and subsequent oligomerization duration (0-36 h)\non Vip1 assembly, as assessed by SDS-PAGE. Molecular weight markers (kDa) are indicated on the\nleft of each panel. All data shown are representative of at least three independent replicates.\n3. In line 105 the authors describe a region of Vip1 that is essential for the Vip2\ntranslocation. How can it be deduced only from the structure of Vip1?\nResponse: We agree that it is premature to designate this region as essential for Vip2\ntranslocation prior to reporting the complex structure. To clarify this point, we have revised\nthe statement accordingly and a similar statement is already included on line 164.\n4. Line 107. The authors built many metal ion binding sites. Indeed, they are of a good\nquality in cryo-EM maps, however, what is the basis for being sure that it is calcium, and\nnot, for example, magnesium?\nResponse: Thanks for your suggestion. Previous studies have shown that other binary\ntoxins, such as CDTb, PA, and Ib, contain calcium-binding motifs. Sequence alignment\nrevealed that Vip1 also harbors conserved calcium-binding sites (Supplementary Fig. 17).\nAccordingly, 2.5 mM calcium ions were included during sample preparation and calcium\nions were modeled at the corresponding conserved sites in the cryo-EM structures.\nSupplementary Fig. 17: Sequence alignment of Vip1, PA-6UZB, Ib-6KLW, and CDTb-7VNN.\nResidues are color-coded based on their conservation: identical residues are highlighted in red.\nThe secondary structure elements are indicated above the sequences, with α-helices shown as coils and\nβ-strands as arrows. The pink bar denotes the Ca²⁺-edge motif, which includes conserved secondary\nstructure elements. Green stars mark residues directly involved in Ca²⁺ coordination.\n5. The authors applied C7 symmetry for the Vip1 structure. What was the rationale for\ndoing this? Could it be that the protein has a pseudosymmetry that was lost when applying\nC7? Later, the authors compare Vip1 alone (refined with C7) and when it complex with\nVip2 (C1), is such a comparison justified?\nResponse: Thank you for raising this important point. Applying C7 symmetry to Vip1 was\nguided by both structural precedent and our own experimental data. Nearly all heptameric\npore-forming components of binary toxins solved by cryo-EM-such as PDB entries 3J9C,\n6KLX, 6O2N, 9UE9, 6RB9, and 5JZT-have been refined using the corresponding cyclic\nsymmetry (see attached table), reflecting the well-established sevenfold architecture of this\nprotein family. Consistently, our survey of pore-forming toxins in the PDB shows that single-\ncomponent heptameric pores are almost universally reconstructed with symmetry imposed,\nas this improves resolution without obscuring biologically relevant features.\nIn our own processing pipeline, we initially refined the Vip1 heptamer in C1, which\nyielded a map at ~3.8 Å resolution. When C7 symmetry was applied, the resolution\nimproved to ~3.3 Å. Importantly, we did not observe evidence of pseudosymmetry or\nasymmetry that would be lost upon symmetry imposition. The use of C7 was therefore\nmotivated primarily by the substantial gain in resolution and the apparent structural\nhomogeneity of Vip1 alone.\nWe agree with the reviewer that a direct comparison between Vip1 alone refined\nwith C7 symmetry and the Vip1-Vip2 complex refined in C1 is not strictly justified, as\nsymmetry imposition may mask or average out subtle asymmetric features. Accordingly,\nwe have revised the manuscript to avoid such comparisons.\nInstead, we now focus on an internal comparison among the seven Vip1 protomers\nwithin the asymmetric Vip1-Vip2 complex. As shown in Supplementary Fig. 7c-e, six of the\nseven Vip1 subunits adopt highly similar conformations, whereas H493 in chain D displays\na distinct conformation relative to the other subunit. This observation supports our original\nconclusion that Vip2 binding induces localized conformational differences in Vip1, while\navoiding cross-symmetry comparisons.\nPDB Symmetry Reconstruction Protein References\ncode method information\n3J9C C7 Cryo-EM Anthrax PA 10.1038/nature14247\npore\n6KLX C7 Cryo-EM Heptameric Ib- 10.1038/s41594-020-0388-6\npore\n6O2N C7 Cryo-EM CDTb\n10.1038/s41564-019-0601-8\nPrepore/Pore\n6OKT C7 Cryo-EM CDTb\nHeptamer\nLong Form\n9UE9 C7 Cryo-EM Vibrio cholerae\n10.1016/j.str.2025.10.013\ncytolysin\n6RB9 C7 Cryo-EM Epsilon toxin 10.1038/s41467-019-10645-8\n5JZT C7 Cryo-EM Aerolysin 10.1038/ncomms12062\n7T4E C8 Cryo-EM Epx1 pore 10.1016/j.cell.2022.02.002\n5GAQ C9 Cryo-EM Lysenin Pore 10.1038/ncomms11293\nI inspected the final cryo-EM maps and the structures and found them of a good fit. I\nappreciate that the authors voluntarily provided them with the submission. However, after\nreading supplementary information, PDB reports and materials and methods, I am raising\nthe following concerns:\n6. The cryo-EM reconstructions are of good resolution, however clashscores of the\ndeposited structures are surprisingly bad. For 9VEK is it 9 and for 9VEG is it 12.\nConsidering this and the following points, I believe that authors need to revisit the\nmodel building.\nResponse: Thanks for your insightful comment. We have carefully refined and re-\nevaluated our structural models. The updated clashscores for 9VEK, 9VEJ, and 9VEG are\nnow 5, 5, and 8, respectively.\n7. 9VEK contains 6 side chain outliers. It is fine having them, however the structure has 7\nchains built into C7-symmetry cryo-EM density. How is it possible to have 6 outliers?\nResponse: Thanks for your careful observation. Following your comment, we performed\nadditional real-space refinement in Phenix, which allowed us to correct these outliers. We\nhave updated the Methods section and PDB accordingly.\n8. All three structures contain bong angle outliers. Are they supported by the density?\nResponse: Thanks for your valuable suggestion. We have re-visited our structural models\nand identified a small number of bond angle outliers: five in 9VEK, two in 9VEJ, and one\nin 9VEG. Notably, none of these outliers are located at the Vip1-Vip1-Vip2 interaction interface;\nrather, they are confined to peripheral or flexible regions and therefore do not affect the\ncore structural interpretations or functional conclusions of the study. However, we\nrecognize that the fit of these outlier residues to the cryo-EM density was suboptimal. We\nhave since refined the models to address these issues and re-uploaded the corrected\nstructures to the PDB. The updated models do not have bond angle outliers now, and the\nrevised validation reports are also provided.\n9. Supplementary table 1 reports 'model resolution'. What is it? How is it different to 'map\nresolution' the authors provide above other than it is given at different FSC threshold?\nResponse: Thanks for your correction. The term \"model resolution\" can be ambiguous\nand misleading. To avoid confusion, we have removed this descriptor from supplementary\ntable 1.\n10. Please provide data on what residues have been built into the density, as the structures\ndo not cover complete protein sequences.\nResponse: Thanks for your suggestion. We have added a new panel in Supplementary\nFigure 3 that explicitly illustrates the modeled residue ranges for each chain in all reported\nstructures (9VEK, 9VEJ, and 9VEG). We hope this addition enhances transparency and\naids readers in interpreting the structural data.\nSupplementary Fig. 3: Schematic representation of residue coverage for Vip1 and Vip2 in\nthe three structural models. a Full-length Vip1 contains an N-terminal region (residues 1-223)\nthat is proteolytically cleaved upon activation. b In the Vip1 heptameric pore structure, Vip1\nshows a fully resolved central region (residues 223-762) and an unresolved C-terminal segment\n(762-878, shown with shadows). c In the 1:7 (Vip2:Vip1) complex structure, Vip1 has a slightly\nshorter unresolved C-terminal region (782-878, shadows); Vip2 contains an unresolved N-terminal\nregion (1-73, shadows). d In the 1:5 (Vip2:Vip1) complex structure, Vip1 displays two unresolved\nregions: an internal loop (323-381, shadows) and a C-terminal segment (782-878, shadows); Vip2\nhas an unresolved N-terminal region (1-62, shadows). Colored bars represent solved regions: dark\nblue for Vip1, yellow for Vip2.\n11. Supplementary figure 1, 3 and 5 are missing exemplary cryo-EM micrographs and 2D\nclass averages.\nResponse: Thanks for your suggestion. We have now included representative exemplary\ncryo-EM micrographs and 2D class averages in Supplementary Figures 2, 5, and 11.\n12. Finally, it looks to me that the obtained structures are very similar to other binary toxins,\nlike C2, Iota and others. What is the new information gained from the Vip1-Vip2 complex?\nResponse: Thanks for your insightful comment. We agree that many binary toxins share\nsimilar overall architectures, which is why they are classified within the same toxin family,\nmuch like many GPCRs which can be structurally conserved yet functionally diverse.\nHowever, sequence alignment analyses reveal that Vip1/Vip2 shares ~30% sequence\nidentity with other well-characterized binary toxins (such as PA/EF/LF, CDTa/b, and Ia/b),\nindicating substantial divergence at the primary sequence level.\nImportantly, the Vip1/Vip2 binary toxin exhibits behavior markedly differently from\npreviously characterized binary toxins. While most known binary toxins target humans or\nmammals, Vip1/Vip2 is insect-specific, raising a fundamental question: how do binary\ntoxins with similar overall folds but low sequence identity give rise to such divergent\nbiological specificities and disease outcomes?\nOur structural analysis directly addresses this question. Notably, the regions critical\nfor Vip2 translocation, including the DTH loop and β-barrel stem, exhibit substantial\nsequence divergence (Supplementary Fig.17). As a result, the precise functional roles of\nthese elements could not be reliably inferred from homology models and required direct\nstructural determination of the Vip1-Vip2 complex.\nIndeed, solving the Vip1-Vip2 complex structure has yielded several novel\nmechanistic insights. First, we captured the highest-resolution structural view to date of a\ntranslocation intermediate state among binary toxins, providing an unprecedented\nstructural snapshot of N-terminal conformational rearrangements during translocation (Fig\n3j). Second, we identified the KGF loop as a key structural determinant for specific\nrecognition and translocation, a role not previously recognized in homologous systems.\nThird, analysis of the hydrophobicity profile within the lumen of the β-barrel stem provides\ndirect evidence supporting a translocation mechanism governed by a hydrophilic pore\nenvironment rather than strict sequence specificity. Fourth, we captured and solved the\nstructure of the 1:5 (Vip2:Vip1) intermediate complex, revealing an interface between Vip2\nand Vip1 that differs from that observed in the heptameric assembly, thereby providing\ndirect structural evidence for a putative assembly process of Vip2-bound Vip1 oligomers.\nFinally, functional assays examining the subcellular localization of the Vip2NTD-GFP fusion\nprotein provide evidence for the translocation mechanism that is sequence-independent\nbut lumen hydrophilicity dependent (Fig 5d-e), and highlighting the potential of this system\nfor protein delivery.\nTogether, these findings substantially advance our understanding of binary toxin\nfunction and highlight the necessity of obtaining the Vip1-Vip2 complex structure to uncover\nsystem-specific mechanistic features that cannot be inferred from existing binary toxin\nstructures alone.\nMessage 2.\n13. In line 173 the authors mention 'two-way rebinding assay' but do not describe it. Could\nit be that at extreme pHs the proteins do not bind to strep sepharose? I think this\nexperiment misses a control.\nResponse: Thanks for your comment. We previously did not clearly explain the rationale\nfor performing the two-way rebinding assay. This experiment was motivated by the off-ratio\nresults that show disproportionate capture of excess Twin-Strep-tagged Vip2\n(Supplementary Fig. 8a). To address this issue, we then intentionally reversed the affinity\nbinding strategy to selectively enrich the low-abundance Vip1-Vip2 complex at high pH and\nminimize contamination from excess Vip1.\nTo enhance clarity, we added the following sentence to the main text: “Motivated\nby the observed off-ratio results, in which an excess of Twin-Strep-tagged Vip2 was\ncaptured, we intentionally reversed the affinity configuration to selectively isolate the trace\namount of complex present at high pH while eliminating the overwhelming excess of\nVip1.”(line 227-230) We also detailed the two rebinding assays in the methods and figure\nlegends of Supplementary Fig. 8a-b.\nTo address this point, we performed additional control experiments demonstrating\nthat Twin-Strep-tagged Vip2 retains robust binding to Twin-Strep Sepharose over a wide\npH range (3.8-10.4) (Supplementary Fig. 8d). In contrast, negative controls showed no\ndetectable non-specific interaction of Vip1 oligomers with the resin (Supplementary Fig.\n8c).\nSupplementary Fig. 8 SDS-PAGE Analysis of two-way rebinding assay for Vip1 Oligomer and\nVip2. a SDS-PAGE/Coomassie staining profile of the Twin-Strep tagged Vip1 rebinding assay using\nthe pre-assembled Vip2-bound Vip1 heptamer complex, subjected to a pH gradient from 3.8 to 10.4.\nNotably, precipitation was observed at pH 3.8, however, the ratio of the trace amount of complex\nappeared comparable. Lane M represents molecular weight markers. These gels represent results\nobtained from three independent trials. b SDS-PAGE/Coomassie staining profile of Ni resin\nrebinding assay of the pre-assembled Vip2-bound Vip1 heptamer complex treated by a pH gradient\nfrom 9.0 to 10.6. Notably, neither Vip2 nor Vip1 contains any His-tag. We accidentally found that\nthe heptameric Vip1-pore could bind to Ni-NTA resin, which is presumably due to the seven\nadjacent histidine residues surrounding the stem of the pore. The pH-treated Vip2-bound Vip1\nheptamer complex might be a mixture of different conformation at high pH, however, only a full\nheptamer could be captured by Ni-NTA resin. Results from three independent experiments are\nshown in these gels. c SDS-PAGE/Coomassie staining profile of the Twin-Strep tagged Vip1\nrebinding assay using the pre-assembled Vip1 heptamer, subjected to a pH gradient from 3.8 to 10.4.\nNotably, binding of Vip1 oligomers to the Twin-Strep tag resin was specific, with no evidence of\nnon-specific interactions. Lane M represents molecular weight markers. These gels represent results\nobtained from three independent trials. d SDS-PAGE/Coomassie staining profile of the Twin-Strep\ntagged Vip2 rebinding assay using the Vip2, subjected to a pH gradient from 3.8 to 10.4. Lane M\nrepresents molecular weight markers. These gels represent results obtained from three independent\ntrials.\n14. While I find it interesting that at high pH the authors found the Vip1-Vip2 complex at\ndifferent stoichiometry, I do not think that based on that, one can claim that these are\n'intermediate states' and deduce an assembly process (line 213). It all might be artifacts of\nhigh pH or rather 'disassembly process'. I believe that functional assays are required to\nshow that 5:1 Vip1-Vip2 complex is a true intermediate and can be assembled further.\nResponse: Thank you for raising this important concern regarding the interpretation of the\ndifferent Vip1-Vip2 stoichiometries. We agree that the heterogeneity observed under high-\npH conditions could, in principle, reflect partial disassembly or experimental artifacts rather\nthan bona fide assembly intermediates.\nTo address this, we expanded our analysis using complementary approaches\nunder distinct conditions. While analytical ultracentrifugation and cryo-EM were carried out\nunder alkaline conditions, our chemical cross-linking experiments were performed\nindependently at pH 7.5 (Supplementary Fig. 9-11). As a control, pre-oligomerized Vip1\nwas crosslinked with comparable efficiency to the input, showing no apparent accumulation\nof intermediate species (Supplementary Fig. 10a). In contrast, distinct pre-assembly\nintermediate states were clearly captured by the crosslinking assay in the experimental\ngroup, in which the crosslinker and trypsin were added simultaneously to the Vip1 protoxin\n(Supplementary Fig. 10b). Furthermore, when Vip2 was present, combined cross-linking\nand Western blot analyses revealed Vip2-associated complexes corresponding to several\nVip1 binding intermediate states (Supplementary Fig. 11). Together, these observations\nsupport the existence of Vip1-Vip2 complexes involving distinct oligomeric forms of Vip1\nand suggest that the species identified by cryo-EM represent putative assembly-related\nintermediates, rather than exclusively pH-induced disassembly products.\nSecond, using a two-way rebinding strategy, we found that trace amounts of Vip1-\nVip2 complexes persist at high pH and can be selectively enriched, demonstrating that\nthese complexes retain stable interactions even under alkaline conditions.\nFinally, we have revised the manuscript to avoid definitive claims of “intermediate\nstates” or a specific assembly pathway. We now describe these species more cautiously\nas putative assembly-related intermediates (revised lines 236 and 265).\n15. Line 223. The same as above. It is possible that high pH created nonphysiological\nartifacts that the authors interpreted as intermediate states.\nResponse: Thanks for your comment. The midgut environment of PBL (Plutella xylostella\nlarvae) is highly alkaline, with a pH of approximately 10.6 (DOI:10.1186/s40168-022-\n01291-2). Based on our rebinding assay (Supplementary Fig. 8b), we finally selected pH\n10.4 to mimic condition. Under pH 10.4, we also confirmed the existence of Vip1-Vip2\ncomplexes with varying stoichiometries through ultracentrifugation density gradient\nexperiments, providing further experimental evidence for the formation of Vip1-Vip2\ncomplexes with variable stoichiometries (Supplementary Fig. 9).\nSimilarly as we mentioned above, we were also able to capture variable\nstoichiometries through chemical cross-linking experiments at pH (7.5) (Supplementary Fig.\n10 and Supplementary Fig. 11). We believe these experiments support two key points: (1)\nVip1 undergoes a stepwise oligomerization process, and (2) the existence of Vip1-Vip2\ncomplexes with different stoichiometries reflects a biologically relevant and reproducible\nstate during toxin assembly. This data reinforces the mechanistic model presented in our\nstudy and helps clarify the dynamic nature of binary toxin complex formation.\nSupplementary Fig. 9: Ultracentrifugation analysis of the oligomerization of Vip1 and Vip2.\nThe continuous size distribution C(s) as a function of sedimentation coefficient S shows the\ncharacteristics of samples under two conditions: Vip1 (red curve) and Vip1+Vip2 (black curve). The\naddition of Vip2 results in a shift of the sedimentation profile to higher coefficients, consistent with\nthe assembly of higher-order oligomeric states.\nSupplementary Fig. 10. Cross-linking assay reveals distinct pre-assembly intermediate states\nof Vip1. a Pre-oligomerized Vip1 was crosslinked under increasing concentrations of\nglutaraldehyde (0-0.05% w/v) and analyzed by Coomassie staining. b Distinct pre-assembly\nintermediate states were captured in the experimental group, in which the crosslinker and trypsin\nwere added simultaneously to the Vip1 protoxin (0-0.05% w/v). Distinct bands corresponding to\ndimer (~140 kDa), trimer (~210 kDa), and high-molecular-weight oligomers are indicated on the\nright.\nSupplementary Fig. 11: SDS-PAGE and Western blot analysis of Vip1 and Vip2 following\nglutaraldehyde cross-linking assay. a Coomassie-stained SDS-PAGE analysis of glutaraldehyde-\nmediated cross-linking between Vip1 protoxin and His-Vip2-Strep (His-tag at the N-terminus,\nStrep-tag at the C-terminus) under varying conditions. Lanes 1-3: Equal amounts of Vip1 were\nincubated with decreasing concentrations of His-Vip2-Strep and cross-linked with glutaraldehyde,\nVip2 was absent in lane 3; Lanes 4-5: control reactions without glutaraldehyde or Vip1; lanes 6-10:\ninput proteins before cross-linking. Lane 8 represents molecular weight markers. b The high-\nmolecular-weight complexes observed in panel a were verified to contain Vip2 through Western blot\nanalysis using anti-Strep-tag antibodies. All samples in panel b were loaded in the same order. Major\ncross-linked bands are labeled as: 1 (Vip1 prototoxin), 1′ (Vip1 oligomers), 2 (Vip2 monomer), 2′\n(Vip2 dimer), α (Vip2 bound to Vip1), β (Vip2 bound to Vip1 oligomers with varying\nstoichiometric ratios). The representative blot from three independent assay was shown here.\n16. Fig 3a misses scale bars. I have my doubts that the 2D class on the very left represents\na single Vip1. The class is of low quality and can just contain some debris. I suggest\nreextracting these particles with lower box size and redoing 2D classification to get some\nhigh-resolution features to prove that it is really a single Vip1.\nResponse: Thanks for your valuable suggestion. We have now added scale bars to the\nrelevant figures for clarity. Additionally, due to the small size of the monomer, it was difficult\nto obtain high-quality 2D class averages. Therefore, the figure showing the 2D class\naverages for the “0:1” condition has been removed. We appreciate your careful attention\nto this detail.\nMessage 3.\n17. While I find this interesting and a well-done set of experiments, I am questioning the\nnovelty of them. For example, Bacillus anthracis PA is used for decades for similar purpose.\nA few recent articles describing it: https://pubmed.ncbi.nlm.nih.gov/40758882/\nhttps://pubmed.ncbi.nlm.nih.gov/25250705/\nResponse: We thank the reviewer for this thoughtful comment and agree that Bacillus\nanthracis protective antigen (PA) represents an important and well-established precedent\nfor binary toxin–mediated protein delivery. Our study does not aim to claim novelty for the\ngeneral concept of toxin-based delivery, but rather to define the distinct biological context\nand mechanistic features of the Vip1/Vip2 system.\nUnlike PA-mediated delivery, the Vip1/Vip2 toxin functions in the highly alkaline\ninsect midgut, an environment characterized by markedly different pH, protease\ncomposition, and receptor usage. Our work specifically addresses how the Vip1-Vip2\ncomplex assembles, remains stable, and mediates protein translocation under these\nalkaline conditions, which have not been examined in PA-based systems.\nAt the molecular level, Vip1/Vip2 differs substantially from PA/LF. Vip2 shares only\n~17% sequence identity with LF and lacks recognizable PA-binding motifs, making direct\nmechanistic extrapolation from PA unreliable. Consistent with this, we show that Vip1-\nmediated translocation is hydrophilicity-dependent but sequence-independent, supported\nby structural analysis of a predominantly hydrophilic transmembrane pore and by\nmutational disruption of pore polarity that abolishes insecticidal activity. We further\ndemonstrate efficient delivery of both full-length Vip2-GFP and minimal N-terminal fusion\nconstructs, indicating a level of cargo tolerance specific to this system.\nFinally, while PA has been extensively developed as a mammalian delivery\nplatform, our objective is to establish Vip1/Vip2 as an insect-adapted protein delivery\nsystem with potential applications in precision pest control. We therefore believe that the\nnovelty of this work lies in revealing how a distinct binary toxin system achieves protein\ndelivery under insect-specific physiological constraints, rather than in the general principle\nof binary toxin-mediated translocation.\nMinor points:\n1. Abstract line 31. I would not use 'irrelevant' protein cargo but maybe non-specific,\nengineered or desired?\nResponse: Thanks for your advice. “desired” cargo is a perfect word for this (line 31). We\nhave revised the statement accordingly, thanks again.\n2. Introduction has several strange sentences that I could not follow. For example, first you\ndescribe 4 families of Vip proteins (line 47) and, right after it, on line 50, talk about the Vip1-\nVip2 family. Why?\nResponse: Thanks for your helpful suggestion. On original line 47, the term “family” refers\nto the broad Vip toxin protein family, which has been classified into four distinct subfamilies.\nWe agree that the original use of “family” on line 50 was kind of misleading. To improve\nclarity, we have revised them accordingly (line 59).\n3. What means Ad1 in Vip1Ad1 in line 49?\nResponse: We added an explanation of classification and nomenclature method for Bt\nproteins, line 53-58: “The nomenclature of Bt proteins generally consists of two\ncomponents. The first denotes the protein’s type and functional characteristics, while the\nsecond distinguishes different hierarchical ranks within that protein family. These ranks\nfollow a four-tier system-Arabic numerals, uppercase letters, lowercase letters, and a final\nset of Arabic numerals-corresponding to sequence similarities of 45%, 78%, and 95%\nrespectively 10.”\n4. What is a pore-forming protein in line 54?\nResponse: Thanks for your suggestion. We revised the term “pore-forming protein” to\n“pore-forming component” (line 62), as our intention was to refer specifically to one of the\ntwo components of binary toxin.\n5. Anthrax toxin has two payloads, EF and LF. Why only to mention the second?\nResponse: Thanks for your careful reading and correction. The absence of EF in the initial\ndescription (line 68) was an oversight, although EF was mentioned later in the manuscript\n(line 450) and in Supplementary Fig. 18. We have now corrected line 68 to read: “Bacillus\nanthracis anthrax toxin (PA, EF and LF).”\n6. Line 71-72: the authors state that the lack of the Vip2 translocation mechanism is a major\nlimitation... of what? I couldn't follow. Of engineering of Vip2 to be nontoxic to plants?\nResponse: Thanks for your insightful suggestion. Determining the structure of the Vip1–\nVip2 complex not only clarifies the molecular mechanisms governing Vip2 recognition and\ntranslocation but also supports the rational engineering of Vip2 to enhance its insecticidal\nefficacy. We have revised it accordingly. (line 80)\n7. Line 87-88. 'which is indispensable for the pore formation'. Is it the pest, which is\nindispensable?\nResponse: Thanks for your helpful suggestion. We intentionally mean the protease\ndigestion process. To improve clarity, we have revised the sentence as follows: “It is widely\naccepted that Bt Vip1 protoxin is digested by proteases in the midgut of targeting pest. The\nprotease digestion of Vip1 protoxin is indispensable for the pore formation that was\nassumed to deliver the enzymatic Vip2 component into target cells 2.” (line 102-104)\n8. Line 101, what homologous protein you have in mind?\nResponse: Thanks for your correction. We have revised the sentence to: “The D4 domain\nwas reported as receptor-binding domain (RBD) in the B component of other binary toxins,\nsuch as Ib and CDTb 21-23.” (line 115-116)\n9. Line 192 cites Supplementary Fig 5a-e, while it is cryo-EM supplementary figure.\nResponse: Thanks for your suggestion. We have revised the original line 192 (now line\n273) properly.\n10. Line 397. Please describe the method of purification of Vip1 in detail.\nResponse: Thanks for your suggestion. We have now added detailed information on the\npurification of Vip1 (and Vip2) to the Methods section.\nThe updated text reads as follows: “Briefly, Bt Vip1 and Vip2 were cultured in LB\nat 30°C for 24 h. The natively expressed and secreted Vip1 and Vip2 were purified using\nion exchange chromatography. Prior to loading, each sample was diluted 10-fold by buffer\nA (25 mM Tris-HCl, pH 8.0, 40 mM NaCl). Vip1 was purified using a HiTrap Q HP column,\nwhile Vip2 was purified on a HiTrap SP HP column. Both columns were pre-equilibrated\nwith buffer C and eluted with a linear gradient of buffer B (25 mM Tris-HCl, pH 8.0, 1 M\nNaCl), increasing from 0% to 100 % Buffer B over 75 minutes. Then, the sample was\nloaded onto a Superdex 200 Increase column (Cytiva, Marlborough, USA) and eluted with\na buffer C (25 mM Tris, pH 8.0, 100 mM NaCl). The purity of the eluted fractions was\nsubsequently assessed by SDS-PAGE.” (line 527-536)\nReviewer #3 (Remarks to the Author):\nThe manuscript presents novel cryo-EM structures formed by insecticidal toxins from\nBacillus thuringiensis.\n1. The high resolution structures of Vip1 heptameric pore and Vip2-bound Vip1-pore\nheptamer complex are well described and analysed and provide a valuable new structural\ninformation that supports a sequence-independent translocation mechanism of the binary\ntoxins. The findings will be a helpful addition to the field. To evaluate the quality of these\nstructures the reviewer requests access to cryo-EM maps including non-symmetrised\nmaps, maps without masking and masks applied prior to CTF estimation. We remind the\nauthors that EMDB now encourages and accepts deposition of these together with ‘main’\nstructure. In addition, we will appreciate up-to-date PDB validation reports for all structures.\nRepresentative cryo-EM images and averages of common views of Vip1 pore and\nVip2/Vip1-complex should be also added to supplementary information to aid\nunderstanding and evaluation of data presented.\nResponse: Thanks for this valuable suggestion. In response, we have added\nrepresentative cryo-EM micrographs and 2D class averages of the Vip1 pore and Vip2/Vip1\ncomplex to the Supplementary Information (Supplementary Fig. 2, 5, and 12.) to facilitate\ndata evaluation.\nNon-symmetrised maps and maps without masking were uploaded to Figshare server. No\nuser-defined masks were applied prior to CTF estimation for this study. We also provided\nthe up-to-date PDB validation reports.\nWhile data analysis and image processing steps are well described in Methods section,\nsome important information is missing: please add which detector was used for data\ncollection on Glacios instrument (Vip1 data), correct Vitrobot model and replace ‘flash\nfrozen’ ‘plunge frozen’ for cryo-Em grids. In addition, information about electron flux\n(e/A2/s), exposure time (s) and electron fluence per frame (e/A2) should be added for each\nsolved structure in Methods and/or in Table 1.\nResponse: Thanks for your careful reading and helpful suggestions. We have now\nupdated the Methods section to specify that a Gatan K3 electron detector was used for\nVip1 data collection on the Glacios microscope, corrected the Vitrobot model to Vitrobot\nMark I, and replaced “flash frozen” with “plunge-frozen”. Additionally, we have added the\nelectron flux, total exposure time, and electron fluence per frame for each structure in the\nMethods section (line 574-589).\nThe updated text reads as follows: “The frozen grids were screened and then\ntransferred to a Glacios 200kV TEM (Thermo Fisher Scientific, Rockford, USA) equipped\nwith a Falcon 4 direct electron detector for data acquisition. Cryo-EM images were\nautomatically collected using a defocus range of -1 to -2 μm, a nominal magnification of\n150,000×, a calibrated pixel size of 0.94 Å/pixel. An exposure time of 5.52 s was employed\nat an electron dose rate of 9.05 e⁻/Å²/s.” “Images were collected on a Titan Krios\nmicroscope using a defocus range of -1 to -2 μm, a nominal magnification of 105,000×,\nand a binned pixel size of 0.84 Å/pixel. An exposure time of 2.16 s was employed at an\nelectron dose rate of 23.148 e⁻/Å²/s. Micrographs were recorded as movie stacks\nconsisting of 40 frames, with a total electron dose of 50 e⁻/Å² and an electron fluence of\n1.25 e⁻/Å² per frame. All data collection was automated using EPU software.”\nTo put new structural information in context and show the significance to the field, Vip1 and\nVip2/Vip1 structures should be accompanied by analysis of sequence homology with other\nbacterial toxins: α-hemolysin, anthrax protective antigen pore, Clostridium perfringens iota\ntoxin and Vibrio cholerae cytolysin and comparison with known structures of membrane-\ninserted pores of binary toxins.\nResponse: Thanks for this insightful suggestion. To better contextualize our findings, we\nhave now included an analysis of sequence homology between Vip1/Vip2 and\nrepresentative bacterial pore-forming toxins-including α-hemolysin, anthrax protective\nantigen (PA), Clostridium perfringens iota toxin (Ib), and Vibrio cholerae cytolysin (VCC)-\nas well as structural comparisons with known membrane-inserted pores of toxins.\nThese analyses are presented in line 440 as follows: “As a member of the pore-\nforming toxins, Vip1 shares approximately 30% sequence identity with the binary toxins\nPA, Ib, and CDTb (Supplementary Fig. 16a-b). Despite this low sequence identity, the\narchitecture of Vip1 pore is more closely resembles that of the binary toxins PA, Ib, and\nCDTb (Supplementary Fig. 16c). Notably, the regions critical for Vip2 translocation,\nincluding the DTH loop and β-barrel stem, exhibit substantial sequence divergence\n(Supplementary Fig.16). In contrast, Vip1 exhibits lower sequence identity with the single-\ncomponent toxins α-hemolysin (α-HL) and Vibrio cholerae cytolysin (VCC), at 20.28% and\n15.53%, respectively (Supplementary Fig. 16a-b).”\nSupplementary Fig. 16: Sequence identity and structural similarity between Vip1 and other\npore-forming toxins. a Sequence identity matrix showing the percentage identity between Vip1\nand other pore-formation proteins (PA, Ib, CDTb, α-HL, VCC). b Phylogenetic tree of Vip1 and\nthese pore-forming toxins. c The heptameric pore structures of Vip1 and these pore-forming toxins.\nColors denote different proteins: Vip1 (PDB: 9VEK), PA (PDB: 3J9C), Ib (PDB: 6KLX), CDTb\n(PDB: 6OKU), α-HL (PDB: 7AHL), VCC (PDB: 3O44).\nWhile discussing translocation mechanism, we also suggest including speculations on\ndriving forces for substrate protein translocation: differences of proton concentration or\nelectrical potential across the ϕ-clamp. The former requires protonation-dependent\nconformational changes of two or more alternating gates and protonation-dependent\nconformational change in ϕ-clamp region. Alternatively, translocation could follow charge-\nstate dependent Brownian ratchet model similar to the one described for anthrax protective\nantigen pore. Electrophysiological studies-if possible-would be a valuable addition.\nResponse: We greatly appreciate your insightful suggestion regarding the translocation\nmechanism. In the revised manuscript, we have expanded the discussion to include\nspeculative models for the driving forces of Vip2 translocation, as follows: “In the PA-LF/EF\nsystem, the translocation of LF or EF by the PA-pore is driven by the proton motive force\n(PMF), which consists of a proton gradient (ΔpH) and membrane potential (ΔΨ) 22,36-39. A\nΔpH-driven Brownian ratchet mechanism has been proposed, wherein protonation and\ndeprotonation of acidic residues on the translocating polypeptide facilitate directional\nmovement 22,39,40. The translocation of Ia and CDTa was also thought to rely on an acidic\npH-dependent and ΔpH-driven Brownian ratchet mechanism 22,24. Similarly, the Vip1-Vip2\nsystem features a conserved ϕ-clamp gate, suggesting it may employ a ϕ-clamp-mediated,\nΔpH-driven charge-state Brownian ratchet mechanism as well. However, unlike other\nsystems where the extracellular environment is acidic, in the Vip1-Vip2 system, the\nextracellular pH corresponds to the alkaline conditions of insect midgut. This extracellular\npH is higher than the neutral pH inside the target cell-potentially reversing the directionality\nor operational details of the proton gradient-driven translocation. Notably, Michael et al.\npreviously demonstrated that the Vip1Ac pore exhibits an asymmetric voltage response in\nartificial lipid bilayer membranes 41. The Vip1Ac channel shows slightly higher permeability\nto anions 41, suggesting that the transmembrane voltage gradient may serve as the driving\nforce for Vip2 translocation, similar to proton gradient-driven translocation in other binary\ntoxin systems.” (line 466-481)\nWhile electrophysiological experiments would indeed provide valuable functional\nvalidation, they exceed our current experimental conditions; nonetheless, we now explicitly\nacknowledge this as an important direction for future work.\n2. Authors also described multiple incomplete assemblies of Vip2-bound Vip-pore\nheptamer complex after treatment with high pH (pH 10.4): two-subunit partial pore,1:3\n(Vip2:Vip1) complex, 1:4 and 1:7 complex. Low-resolution cryo-EM 3D reconstructions of\nan incomplete 1:4 (Vip2:Vip1) complex and complete 1:7 (Vip2:Vip1) complex were\nobtained from this preparation and discussed as intermediate states of complex assembly.\nThis interpretation is only supported by fluctuating pH levels in insect’s midgut, including\nhighly alkaline environment. The authors did express some ‘concerns about physiological\nrelevance’ of these findings in Discussion section but nonetheless propose a step-wise\nassembly pathway based on these ‘intermediate’ structures. No high pH treatment step\nwas performed for Vip2/Vip1 preparation used for high resolution structures discussed\nearlier, neither for subcellular localization experiments with insect cells reported in this\nmanuscript. To enable mechanistic understanding of Vip1/Vip1 complex formation and\naction, further in vitro experiments are required using model liposomes and Vip2/Vip1 (or\nVip2-GFP/Vip1): Vip2/Vip1 assemblies on liposomes could be treated with high pH and\nthen used for cryo-EM sample preparation. Image processing of these cryo-EM data would\nfollow already established single particle workflow using cryoSPARC and could yield direct\nvisualisation of intermediate structures. These experiments could also shed some light on\nnature of Vip1 pore receptor. It seems too early to consider potential use of Vip1/Vip2\nsystem for development therapeutic or delivering proteins to specific cellular targets without\nfurther knowledge about receptor binding and pore formation mechanism.\nResponse: Thanks for your insightful comment. We agree that the current evidence-\nparticularly the data obtained under high-pH conditions-is insufficient to establish a\nstepwise assembly pathway or to support immediate therapeutic applications. In the\nrevised manuscript, we have explicitly clarified that these assemblies likely represent in\nvitro snapshots whose physiological relevance remains uncertain.\nRegarding your suggestion to reconstitute Vip1-Vip2 complexes on model\nliposomes and visualize intermediates by cryo-EM, we fully recognize the value of such\nexperiments. However, performing liposome-based reconstitution at pH 10.4 is technically\nprohibitive owing to rapid phospholipid hydrolysis, membrane destabilization, and the\nresulting incompatibility with vitrification-based cryo-EM sample preparation. As a result,\nwe were unable to generate stable liposome samples at these extreme pH conditions.\nTo address the underlying mechanistic question using feasible and complementary\napproaches, we expanded our experimental analysis using complementary approaches\nperformed under distinct conditions. Analytical ultracentrifugation (AUC) detected distinct\nVip1 oligomeric states at pH 10.4 (Supplementary Fig. 9), while the adding of Vip2 increase\nthe sedimentation coefficients of distinct oligomeric states, supporting the intrinsic ability of\nVip1 to populate multiple assemblies with/without Vip2. Independent chemical cross-linking\nat pH 7.5 further revealed Vip2 association with several Vip1 oligomeric species\n(Supplementary Fig. 10 and Supplementary Fig. 11), consistent with the presence of\ntransient intermediates under activation conditions. Together, these data indicate\nstoichiometric heterogeneity that may reflect progressive stages of assembly, while not\nallowing us to specify the exact sequence of events.\nTo avoid overstating these findings, we have tempered our interpretation\nthroughout the manuscript. The revised text now states that the observed assemblies\n“represent possible intermediate states” rather than establishing a stepwise pathway. While\nthe identity of the native receptor for Vip1 remains unknown, our data provide a foundation\nfor future mechanistic studies, including receptor identification and engineering Vip2 for a\nmore potent bioinsecticide.\nSupplementary Fig. 9: Ultracentrifugation analysis of the oligomerization of Vip1 and Vip2.\nThe continuous size distribution C(s) as a function of sedimentation coefficient S shows the\ncharacteristics of samples under two conditions: Vip1 (red curve) and Vip1+Vip2 (black curve). The\naddition of Vip2 results in a shift of the sedimentation profile to higher coefficients, consistent with\nthe assembly of higher-order oligomeric states.\nSupplementary Fig. 10. Cross-linking assay reveals distinct pre-assembly intermediate states\nof Vip1. a Pre-oligomerized Vip1 was crosslinked under increasing concentrations of\nglutaraldehyde (0-0.05% w/v) and analyzed by Coomassie staining. b Distinct pre-assembly\nintermediate states were captured in the experimental group, in which the crosslinker and trypsin\nwere added simultaneously to the Vip1 protoxin (0-0.05% w/v). Distinct bands corresponding to\ndimer (~140 kDa), trimer (~210 kDa), and high-molecular-weight oligomers are indicated on the\nright.\nSupplementary Fig. 11: SDS-PAGE and Western blot analysis of Vip1 and Vip2 following\nglutaraldehyde cross-linking assay. a Coomassie-stained SDS-PAGE analysis of glutaraldehyde-\nmediated cross-linking between Vip1 protoxin and His-Vip2-Strep (His-tag at the N-terminus,\nStrep-tag at the C-terminus) under varying conditions. Lanes 1-3: Equal amounts of Vip1 were\nincubated with decreasing concentrations of His-Vip2-Strep and cross-linked with glutaraldehyde,\nVip2 was absent in lane 3; Lanes 4-5: control reactions without glutaraldehyde or Vip1; lanes 6-10:\ninput proteins before cross-linking. Lane 8 represents molecular weight markers. b The high-\nmolecular-weight complexes observed in panel a were verified to contain Vip2 through Western\nblot analysis using anti-Strep-tag antibodies. All samples in panel b were loaded in the same order.\nMajor cross-linked bands are labeled as: 1 (Vip1 prototoxin), 1′ (Vip1 oligomers), 2 (Vip2\nmonomer), 2′ (Vip2 dimer), α (Vip2 bound to Vip1), β (Vip2 bound to Vip1 oligomers with\nvarying stoichiometric ratios). The representative blot from three independent assay was shown here.\n3. And lastly, it is important to complete the nonspecific GFP delivery experiments with\nnegative control when cells are treated with Vip2-GFP or Vip2NTD-GFP only. Surprising\ntranslocation of relatively large folded protein (GFP) through pore with an inner diameter\nof 6.0 Å seems inconsistent with Vip1 structure presented in tne same manuscript and\nrequires further checks and validation.\nResponse: Guided by your valuable advices, the negative control and a loss-of-function\ntriple mutant (E327A/H329A/E370V) were included for the nonspecific GFP delivery\nexperiments, which further support the nonspecific delivery.\nWe agree that the reported translocation of folded GFP pore with an inner diameter\nof 6.0 Å seems inconsistent with Vip1 structure. To clarify, similar to Vip2, GFP is not\nexpected to cross the pore in its native folded state. Instead, based on the dynamic\ntranslocation events captured in our study that GFP translocation does not occur in a fully\nfolded state. Instead, based on the dynamic translocation process we captured (Fig 5,\nmovie 2), Vip2 undergoes a stepwise partially unfold prior to translocation and passes\nthrough the ϕ-clamp as an extended linear polypeptide chain, which could be then refolded\nin the cytosol with assistance from as assisted by chaperon such as Hsp70/90 (Fig 6, DOI:\n10.1098/rsob.200089, DOI: 10.1038/nrm.2017.20, DOI: 10.1155/2012/745962).\nFurthermore, recent work by Vera et al. has demonstrated the feasibility of a protein-based\nanti-CRISPR delivery platform (LF -Acr/PA; DOI: 10.1073/pnas.2426960122), illustrating\nthat binary toxin systems are capable of delivering fusion proteins, consistent with our\nobservations. Nonetheless, we agree that further validation will be valuable for\nunderstanding the precise determinants governing GFP translocation.\nREVIEWERS' COMMENTS\nReviewer #2 (Remarks to the Author):\nThe authors addressed my concerns, and I recommend the article for publication.\nPlease add scale bars to 2D classes in supplementary figures.\nResponse: Thanks, scale bars have been included for the 2D classes in the\nsupplementary figures.\nReviewer #3 (Remarks to the Author):\nThe revised manuscript addresses many questions and concerns raised by the reviewers\nand presents a more comprehensive study with a better balanced and justified\nconclusions. I appreciate that this revision must have required considerable time and\neffort.\nPlease find below few additional comments and suggestions:\nInterpretation of different stoichiometry Vip1-Vip2 complexes found in high pH as\nintermediate states remains a concern. In the revised manuscript these are now\ncollectively called ‘putative assembly-related intermediates’. New cross-linking\nexperiments (Supplementary Fig. 11) conducted at pH 7.5 included in the revised\nmanuscripts provide indirect support for the proposed Vip1/Vip2 assembly pathway.\nHowever, experimental validation of the step-wise mechanism of Vip1 pore formation\nand Vip2 translocation requires membrane- and receptor- binding. Hence, I suggest to\nedit the manuscript lines 28, 86-88, 97, 463 and 967 accordingly, replacing verbs such\nas ‘define’ and ‘establish’ with ‘suggest’, ‘indicate’, etc.\nResponse: Thank you for highlighting the need for nuanced language concerning the\nVip1-Vip2 complexes observed at high pH. We agree that characterizing these as\n'putative assembly-related intermediates' is the most appropriate approach given the\ncurrent data. Accordingly, we have carefully edited the manuscript to replace strong\nassertions with more measured phrasing. Verbs such as 'define' and 'establish' have been\nsubstituted with 'suggest' and 'indicate' across lines 28, 97, and 417. Furthermore, we\nhave integrated the descriptor 'putative' into lines 86-88, 463, 269, 968 and 967 to\nensure the tentative status of the step-wise mechanism is clearly communicated. These\nedits ensure that our description of the Vip1/Vip2 assembly pathway accurately mirrors\nthe indirect evidence provided by our new cross-linking data, avoiding\noverinterpretation while clearly outlining our proposed model. All relevant instances\nhave been carefully reviewed and corrected to ensure consistency.\nSupplementary Figures 2a and 5a: please check scale bars included in representative\ncryo-EM images as particle size appears to be too big\nResponse: Thank you for highlighting this issue. We have verified the particle sizes\nand corrected the scale bars in Supplementary Figures 2a and 5a.\nSupplementary Figures 2b, 5b, 12b and 12c are missing scale bars\nResponse: Thank you for highlighting this issue. We have verified the particle sizes\nand corrected the scale bars in Supplementary Figures 2b, 5b, 12b and 12c. The revised\nfigures now display accurate scaling consistent with the reported cryo-EM data.\nDetails of 3DVA are missing from the Methods sections: please specify if/which masks\nwere used and if the analysis was followed up by subsets selection/clustering and\nrefinement? If structures presented in Suppl. Movies 1 and 2 are direct outputs of 3DVA\njobs with no refinement, this should be stated in the accompanying text.\nResponse: Thank you for this detailed comment. We have clarified in the revised\nMethods: “Three-dimensional variability analysis (3DVA) was performed in\ncryoSPARC. The analysis was conducted using the final particle stack obtained from\nthe consensus refinement. The soft mask generated during the consensus refinement\nwas directly used for the 3DVA calculation. Three variability modes were calculated to\nvisualize conformational heterogeneity. No particle subset selection, clustering, or\nfurther refinement was performed following the 3DVA analysis. The resulting volumes\nwere exported to generate the two supplementary movies, respectively.”\nPlease specify GIF slit width used during Vip1/Vip2 data collection\nResponse: Thank you for pointing out this omission. The GIF slit width employed\nduring data collection was 20 eV. We have revised the manuscript to include this\nspecific detail in the data collection parameters."}]

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