Wolbachia pipientis is an intracellular bacterium infecting ~40% of all terrestrial arthropod species and certain filarial nematodes1,2,3. They are primarily inherited maternally4. Their remarkable success in spreading in arthropod populations is in part driven by the ability of Wolbachia to increase the number of infected females5, often by acting as reproductive parasites that distort host sex ratios or reproductive outcomes6. The most common type of reproductive parasitism is cytoplasmic incompatibility (CI)6, which is a type of post-zygotic male sterility characterized by improper paternal chromatin condensation and separation in the first mitosis cycle7,8,9. CI causes Wolbachia-infected male insects to be sterile in matings with uninfected females but not similarly infected females (called ‘rescue’), providing a selective advantage for infected females and thus driving Wolbachia into the host population6,10. Insect species infected by two or more different Wolbachia strains can be bidirectional incompatible11,12,13, which causes reproductive isolation of populations infected with different strains11,14.

Based on the outcomes of crosses between Wolbachia-infected insects, it has been proposed that CI is governed by a modification-rescue system15,16. Specifically, a modification activity from Wolbachia is postulated to modify sperm during spermatogenesis, and a rescue activity in the infected egg reverses or neutralizes the original sperm modification following fertilization. The existence of both unidirectional and bidirectional incompatibilities in infected insect populations has led to agreement that Wolbachia may carry multiple modification and rescue factors12,13, but the molecular identity of these factors remained unknown for nearly half a century17,18,19,20.

Recently, the key factors involved in CI induction and rescue were identified21,22,23,24. They are known as CI factors or Cifs, which are encoded by pairs of linked genes, cifA and cifB. Transgenic expression of both cifA and cifB genes, or cifB alone, in the germline of male Drosophila melanogaster or Anopheles gambiae, induces sterility that is highly similar to CI induced by Wolbachia21,22,25,26,27. This embryonic lethality can be rescued by crossing transgenic males with Wolbachia-infected females or those with a transgenic cifA gene expressed in the germline22,23,24. The cif genes have diverged into at least five distinct phylogenetic groups, which are named types I-V22,28,29,30. The degree of similarity and the presence/absence of cif gene homologs between Wolbachia strains correlates with known patterns of bidirectional incompatibility. For instance, Wolbachia strain wRi is able to rescue wMel-induced CI in same-species crosses, probably because these two strains share highly related type-I homologs of the cifA gene (99% amino acid identity); however, the reverse is not true: wMel cannot rescue wRi-induced CI, likely due to wRi also encoding a type II gene pair that is much more divergent12,13,24. In general, the cif genes from bidirectionally incompatible Wolbachia pairs are highly divergent, with only 29–68% amino-acid identity22. Recent analysis of transgenic cif gene expression in D. melanogaster is largely consistent with this view31.

The cifA and cifB genes are coevolving22,30,32. Protein-binding experiments and expression of cif genes in yeast have shown that CifA binds its cognate CifB specifically and rescues CifB-induced yeast growth defects21,23. Although the specific roles of cifA and cifB in CI induction and rescue are still not fully understood, the co-divergence of the cifA and cifB genes22,32,33 has been proposed to be partially responsible for bidirectional incompatibility, possibly by modulating CifA-CifB binding34,35.

Among the five types of cif genes, only type I and certain type V cifB genes encode a deubiquitylase (DUB) domain21. (Hereafter, we will use “Cif” for the entire set of CI factors and “Cid” for the deubiquitylase family and primarily focus on the type I Cid protein family.) The cidA and cidB genes from Wolbachia strains wMel (hereafter cidAwMel and cidBwMel) and wPip (hereafter cidAwPip and cidBwPip) are among the most well studied cif genes (Fig. 1a). These Wolbachia strains are expected to be incompatible, as shown by crosses between trans-infected Aedes albopictus mosquitoes36. Unlike the wMel strain, Wolbachia wPip strains found in different populations of Culex pipiens are highly diversified. Many of them carry several polymorphic copies of the cidAwPip and cidBwPip genes, which may function as independent modification and rescue factors and associate with the diverse CI phenotypes in wPip-infected C. pipiens32,37. Thus, the Cid proteins can be used as a model to study how the diversification of cidAwPip and cidBwPip affects protein-protein interactions and how it is related to CI induction and rescue.

Fig. 1: Crystal structures of CidAwMel and CidBwMelDUB, and a model of CidBwMelND1-ND2 reveal the molecular basis for CI.
figure 1

a CidAwMel and CidBwMel, and CidAwPip and CidBwPip form alternative two-gene CI systems. Each domain is assigned a unique color. b CidAwMel contains mostly α-helices. The C-terminal domain contains six HEAT repeats (HR1-HR6). Residues 111-154 and 158-165 are disordered. c CidBwMelDUB consists of a five-stranded β sheet flanked by α helices on both sides. The active center residues are labeled and shown as balls and sticks. d A model for CidBwMelND1-ND2 was built with AlphaFold. e The crystal structure of the CidAwPip(Tunis)-CidBwPip(Tunis)ND1-ND2 complex. NTD: N-terminal domain; CTD C-terminal domain; ND nuclease domain; DUB deubiquitylase domain; AA amino acids; HR HEAT repeats.

Here, we report crystal structures for CidAwMel and the DUB domain of CidBwMel and build a model of the CidAwMel-CidBwMel complex using AlphaFold-Multimer38, which is validated and confirmed by comparison to a crystal structure of a homologous CidAwPip-CidBwPip complex. Substituting the CidAwMel residues at the CidB-binding interfaces with those of CidAwPip enables it to bind CidBwPip and rescues CidBwPip-induced yeast growth defects, which suggests a role of cid gene co-evolution in modulating CidA-CidB binding specificity. Analysis using the crystal structures of the CidAwPip-CidBwPip complex further shows that sequence divergence in Wolbachia wPip may result in complex binding patterns between CidwPip protein variants. These results provide a solid base for future investigations of CI mechanism and shed light on how co-evolution of CidA-CidB pairs modulates their interactions and bidirectional CI.


Crystal structures of CidAwMel and CidBwMel DUB, and a model of CidBwMel ND1-ND2

We determined the crystal structure of CidAwMel to 2.75 Å resolution (Supplementary Table 1). CidAwMel is made up largely of α-helices (Fig. 1b). Residues 111-154 and 158-165 are disordered and cannot be modeled, which divide CidAwMel into an N-terminal domain (NTD, residues 1 to 161) (see below for why we include these disordered residues as part of the N-terminal domain) and a C-terminal domain (CTD, residues 162 to 422) (Fig. 1b). The C-terminal domain folds into a twisted set of six HEAT repeats, a structural motif that primarily mediates protein-protein interactions39 (Fig. 1b).

CidBwMel was expressed poorly in Escherichia coli, making direct structure determination difficult. We used a “divide-and-conquer” strategy to obtain structural information for each of its domains separately. CidBwMel consists of a region, which is predicted to contain two PD-(D/E)XK (pseudo) nuclease domains (residue 1-751; hereafter CidBwMelND1-ND2, ND: nuclease domain) and a C-terminal deubiquitylase (DUB) domain. An active DUB domain is required for inducing a CI-like phenotype in transgenic D. melanogaster for cid operons21. We determined the crystal structure of the CidBwMel DUB domain to 1.85 Å resolution (Supplementary Table 1 and Fig. 1c). Similar to the structures of other proteases in the CE clan/Ulp1-like protease family40,41, the DUB core consists of a five-stranded β sheet flanked by α helices on both sides. His957, Asp976, and Cys1016 form the catalytic triad (Fig. 1c). Three variable regions (VRs), a constant region (CR) and a C-terminal accessory domain may account for the S1 ubiquitin-binding interface41 (Supplementary Fig. 1a-d).

A model for CidBwMelND1-ND2 (Fig. 1d) was built with the AlphaFold42 program. Although the predicted structural model has an average pLDDT score as high as 83.190, which confirms its quality (pLDDT > 70 indicates the backbone prediction is correct43), we wanted to validate this model further.

CidBwMelND1-ND2 shares sequence homology with other CifB molecules34. Of note, the ND1-ND2 region of CidBwPip_I(b/2) (a CidBwMel ortholog from the Wolbachia strain wPip Tunis line30, hereafter CidBwPip(Tunis)ND1-ND2) expressed well in E. coli and shares a sequence identity of 71% with CidBwMelND1-ND2 (Supplementary Fig. 2). We successfully determined a crystal structure of CidBwPip(Tunis)ND1-ND2 in complex with CidAwPip(Tunis) (specifically CidAwPip_I(γ/2)32) (Supplementary Table 1, Figs. 1e and 2a). The structure of CidBwPip(Tunis)ND1-ND2 could be used for comparison with CidBwMelND1-ND2 modeled by AlphaFold42.

Fig. 2: CidA and CidB interact through a large conserved tripartite interface.
figure 2

a The structure of the CidAwPip(Tunis)-CidBwPip(Tunis)ND1-ND2 complex. CidAwPip(Tunis) binds CidBwPip(Tunis)ND1-ND2 through three regions (Interface I, II, and III). b A structural model of CidAwMel in complex with CidBwMelND1-ND2 was generated by AlphaFold-Multimer. The tripartite interface between CidAwMel and CidBwMelND1-ND2 is shown in yellow, magenta and cyan for Interface I, II, and III, respectively. ce Each interface of the CidAwMel-CidBwMelND1-ND2 complex involves a pair of structural motifs. Representative residues directly involving in the interaction are labeled and shown as balls and sticks. fh The structural motifs at Interface I, II and III of the CidAwPip(Tunis)-CidBwPip(Tunis)ND1-ND2 complex are shown, with residues directly involving in the interaction labeled. NTD N-terminal domain; CTD C-terminal domain; ND nuclease domain.

The structure of CidBwPip(Tunis)ND1-ND2 is very similar to the model for CidBwMelND1-ND2 (Supplementary Fig. 3a). Moreover, the residues that are varied among CidB homologs are mainly located at the surfaces of the protein (Supplementary Fig. 4a), which should not affect protein folding. Thus, the model for CidBwMelND1-ND2 is likely to be accurate. Since the cif genes from wMel have been well characterized, we will mainly focus on the structures and functional analysis of the CidAwMel-CidBwMelND1-ND2 pair in the following sections. The results from the CidAwPip(Tunis)-CidBwPip(Tunis) system will be mentioned when necessary.

CidBwMelND1-ND2 consists of two NDs; ND1 comprises residues 1-391 and ND2 comprises residues 392-751 (Fig. 1d). The ND1 and ND2 domains of CidBwMel interact through an extensive interface burying a surface area of ~2200 Å2. This interface is highly similar to that of CidBwPip(Tunis) (Supplementary Fig. 3b-c) and the residues involving in ND1-ND2 interaction are highly conserved among CidB homologs (Supplementary Fig. 3d-f). Interestingly, in addition to the (inactive) PD-(D/E)XK modules predicted by sequence homology analysis (residues 277-375 and 607-730)21,28,29,30, the structural model of CidBwMel shows that each ND has an additional more divergent PD-(D/E)XK module (residues 30-137 and 431-523) (Supplementary Fig. 5). None of these modules present the key canonical catalytic residues E-D-E-K23 (the putative corresponding residues are P33, K77, S92, S94 in module I, K279, Y311, N330, T332 in module II, F435, K475, I491, D493 in module III and K609, G663, V680, G682 in module IV based on structural analysis (Supplementary Fig. 5)). These modules may play structural roles or support an alternative catalytic activity.

CidAwMel binds CidBwMel through a large distinct interface

We built a model for the CidAwMel-CidBwMelND1-ND2 complex using AlphaFold-Multimer38 (Fig. 2b). The predicted end-to-end structure has a ranking score (TM-Score) as high as 0.8568 with a ptm score (intra-chain quality) of 0.86436 and an iptm score (interface score) of 0.85498. This indicates our prediction is highly reliable both for the protein themselves and the interface between them. The obtained end-to-end model was further optimized by Molecular Dynamics (MD) simulations. Two independent trajectories were performed and both achieved equilibrium within the first 100 ns and were then trapped in optima in the next 100 ns. The Root-Mean-Square Deviation (RMSD) for one of the simulation trajectories was recorded (Supplementary Fig. 6a). Trajectory cluster analysis was performed and the most stable binding conformation was extracted from the largest cluster of the trajectory as the final binding complex of CidAwMel-CidBwMelND1-ND2. The complex also predicts many hydrogen bonds and salt bridges between CidAwMel and CidBwMelND1-ND2 at the three interfaces (Fig. 2c–e) and only 0.57% of the residues are Ramachandran outliers (Supplementary Fig. 6b), which demonstrated the side chains of the predicted model have been reliably predicted. Therefore, we believe that the model of the CidBwMelND1-ND2 and its complex with CidAwMel is reasonable.

The interface between CidAwMel and CidBwMelND1-ND2 can be divided into three regions (Fig. 2b); each region mainly involves a pair of structural motifs. At the first region (Interface I), the helices consisting of the residues 93-158 of CidAwMel interact with a loop in CidBwMelND1-ND2 (residues 448-462) through a network of hydrogen bonds and salt bridges (Fig. 2c). Interestingly, the region of CidAwMel which is disordered in the absence of CidBwMel (residue 111-154 and 158-165) is modeled to form a helix bundle upon complex formation (Figs. 1b and 2c), consistent with its role in mediating CidAwMel-CidBwMelND1-ND2 interaction. These residues are part of the N-terminal domain. Interface II is formed by the helices at the N-terminus of CidAwMel (residues 2-60) and helices from both NDs of CidBwMelND1-ND2 (residues 337-353 and 395-418) (Fig. 2d). Interface III involves multiple HEAT repeats in CidAwMel and a cross-cutting helix (residues 241-264) in CidBwMelND1-ND2 that is bolstered by two β strands and their connecting loop (residues 365-387) to stabilize the interaction (Fig. 2e). Interestingly, although the model of CidAwMel-CidBwMelND1-ND2 complex is very similar to the structure of CidAwPip(Tunis)-CidBwPip(Tunis)ND1-ND2 (Fig. 2a, b), the residues involving in interaction at the three interfaces are very different between the two complexes (Fig. 2c–h), which could help explain their cognate-specific binding. (The residues at the interface are well resolved in CidAwPip(Tunis)-CidBwPip(Tunis)ND1-ND2 with good electron density (Supplementary Fig. 7)).

Pulldown experiments and yeast growth assays were used to validate these structural models. As a model system, it has been demonstrated that expression of cidB genes in the yeast Saccharomyces cerevisiae inhibits cell growth, but growth is at least partially restored if the cognate cidA gene is coexpressed, resembling CI induction and rescue in insects21,23. Substituting the CidAwPip(Tunis) residues at its CidB-binding interface with the corresponding ones from CidAwMel abolished its ability to bind CidBwPip(Tunis)ND1-ND2 and neutralize the CidBwPip(Tunis)-induced growth defect in yeast (Supplementary Fig. 8). Thus, the structure of the CidAwPip(Tunis)-CidBwPip(Tunis)ND1-ND2 complex accurately captures the CidA-CidB interaction mode and is biologically relevant. However, due to the poor expression of CidBwMelND1-ND2 in E. coli and the low toxicity of CidBwMel in yeast, we could not directly validate the model of the complex by pulldown or yeast growth analysis. To test the reliability of this model, we rationally designed a set of mutations based on the model to demonstrate that the interaction mode is conserved between the wPip and wMel CidA-CidB pairs (see below).

CidA-CidB binding specificity is determined by residues at their interfaces

Until now, only CifA and CifB proteins expressed from the same operon have been shown to interact21,23,35. The specific interactions between CifA and CifB have been proposed to play a role in CI induction and/or rescue, depending on different CI models. We substituted multiple residues at the binding interface of CidAwMel with the corresponding region of CidAwPip(Tunis) (Fig. 3a–c) and investigated if the chimeric construct (named CidAwMel(ST)) could now bind CidBwPip(Tunis)ND1-ND2. Their interaction, while detectable, was weak, probably because the residues substituted are not the major binding determinants for interaction between CidAwPip(Tunis) and CidBwPip(Tunis)ND1-ND2 (Fig. 3d). Interestingly, CidAwMel(ST) could bind a closely related CidBwPipND1-ND2 from Wolbachia strain wPip(Pel) (hereafter CidBwPip(Pel)ND1-ND2) to an extent similar to the binding of the cognate CidAwPip(Pel) (Fig. 3d, lane 2 vs. lane 4). By contrast, CidAwMel had been shown previously to be unable to bind CidBwPip(Pel) (Ref. 21). CidBwPip(Pel)ND1-ND2 only differs from CidBwPip(Tunis)ND1-ND2 by 30 residues (Supplementary Fig. 9). We used CidBwPip(Pel) for further biochemical investigation.

Fig. 3: Mutagenesis with binding and yeast growth assays reveal how residues at the three interfaces determine CidA binding specificity.
figure 3

a, b CidAwMel(ST) is a chimera with the body of CidAwMel (pink) and interfacial residues from CidAwPip(Tunis). The locations of the mutated residues are shown in orange on the CidAwMel structure. c The substituted residues in CidAwMel(ST) are divided into nine regions (R) and reversed back to those of CidAwMel, individually, to create CidAwMel(ST-1) through CidAwMel(ST-9). d CidAwMel(ST) does not bind wild-type CidBwMel but binds CidBwPip(Pel) to a similar extent as CidAwPip(Pel). Regions 4, 7, and 9 play important roles in binding. The experiment was repeated three times independently with similar results obtained. One representative is shown. e CidAwMel(ST) is able to rescue yeast from CidBwPip(Pel)-induced lethality. f CidAwMel(ST-4), CidAwMel(ST-7) and CidAwMel(ST-9), which do not bind CidBwPip(Pel), also fail to suppress CidBwPip(Pel)-induced yeast growth defects. g The crystal structure of the CidAwMel(ST)-CidBwPip(Pel)ND1-ND2 complex is similar to the model of the CidAwMel-CidBwMelND1-ND2 complex. Representative residues directly involved in the CidAwMel(ST) and CidBwPip(Pel)ND1-ND2 interactions at Interface (h) I and (i) III are labeled. ST substituted; ND nuclease domain. Source data for panels (d) and (e) are provided as a Source Data file.

Agreeing with the pulldown experiments, yeast growth defects induced by CidBwPip(Pel) could be rescued by both CidAwMel(ST) and CidAwPip(Pel), but not CidAwMel (Fig. 3e) even though the expression levels for CidAwMel and CidAwMel(ST) were similar (Supplementary Fig. 10). These data clearly show the importance of the interfacial residues (Fig. 3e) in determining binding specificity. They also support the accuracy of our CidAwMel-CidBwMelND1-ND2 model.

To locate the critical regions that determine binding specificity, the substituted interfacial residues of CidAwMel(ST) were divided into nine groups/regions based on their location in the primary sequence and three-dimensional structure. Each region was individually reverted back to the original sequence in CidAwMel (Fig. 3b, c), resulting in CidAwMel(ST-1) to CidAwMel(ST-9). (Regions 1, 2 are at Interface II. Regions 3, 4, 5, 6 are at Interface I. Regions 7, 8, 9 are at Interface III.) CidAwMel(ST-4), CidAwMel(ST-7) and CidAwMel(ST-9) showed reduced binding affinity to CidBwPip(Pel)ND1-ND2 (Fig. 3d). They also failed to rescue CidBwPip(Pel)-induced yeast growth defects (Fig. 3f). (These CidA variants were expressed at similar levels. Interestingly, however, co-expression of CidAwMel variants with CidBwPip(Pel) in yeast did strongly enhance levels of the latter protein but only if they were able to bind to it. (Supplementary Fig. 10)) Thus, these specific residues at the binding interface are particularly important in determining the binding specificity of CidAwMel.

Crystal structure of CidAwMel(ST)-CidBwPip(Pel) ND1-ND2 explains interaction specificity

We next determined the crystal structure of the CidAwMel(ST)-CidBwPip(Pel)ND1-ND2 complex (Supplementary Table 1 and Fig. 3g). The structure of the complex was very similar to that of CidAwPip(Tunis)-CidBwPip(Tunis)ND1-ND2 (RMSD: 0.8 Å over 691 Cα atoms). This structure revealed the interacting residues at the CidAwMel(ST)-CidBwPip(Pel)ND1-ND2 interface (Supplementary Fig. 11; electron density for representative residues shown in Supplementary Fig. 7) and explained why certain CidAwMel(ST) variants show reduced binding to CidBwPip(Pel)ND1-ND2. For example, at region 4, residues R114 and K121 of CidAwMel(ST) interact electrostatically with residues E460 and D459 of CidBwPip(Pel)ND1-ND2, respectively (Fig. 3h). In CidAwMel(ST-4), residues 114 and 121 were reverted back to the corresponding residues of CidAwMel, which are both glutamines (Fig. 3c). These residues are not charged and may lead to the reduced binding of CidAwMel(ST-4) to CidBwPip(Pel)ND1-ND2 (Fig. 3d). Similarly, at region 7, residues R288 and D299 of CidAwMel(ST) have charge complementarity with D246 and R252 of CidBwPip(Pel)ND1-ND2, respectively (Fig. 3i). In the CidAwMel(ST-7) revertant, residues 288 and 299 are glutamine and asparagine, respectively (Fig. 3c), which can no longer form salt bridges with D246 and R252 of CidBwPip(Pel)ND1-ND2. Finally, at region 9, residue R393 of CidAwMel(ST) interacts with residues E257 and D261 of CidBwPip(Pel)ND1-ND2 through salt bridges (Fig. 3i). In CidAwMel(ST-9), residue 393 is glutamic acid (Fig. 3c), which cannot bind to E257 and D261 of CidBwPip(Pel)ND1-ND2 due to electrostatic repulsion. The crystal structure clearly explains the reduced binding of CidAwMel(ST-4), CidAwMel(ST-7) and CidAwMel(ST-9) to CidBwPip(Pel)ND1-ND2 (Fig. 3d), which closely parallels their loss of function in yeast growth rescue assays (Fig. 3e, f).

Natural sequence variations affect CidAwPip(Tunis)-CidBwPip(Tunis) binding

The wPip cidA-cidB operon is duplicated and diversified extensively among CI-inducing wPip strains. Interestingly, however, only residues at certain positions are different among the CidAwPip and CidBwPip variants32 (Fig. 4a, b). If the varied residues are at the CidAwPip-CidBwPip binding interface, they may affect the interaction of these proteins and play a role in CI induction and/or rescue specificity. The wPip Tunis line belongs to group I of wPip (wPipI, grouping of wPip strains is based on phylogenetic analysis of seven Wolbachia genes)32,44, so we focused on the CidAwPip and CidBwPip variants from this group (hereafter, CidAwPip_I and CidBwPip_I). (As mentioned before, CidAwPip(Tunis) and CidBwPip(Tunis) are CidAwPip_I(γ/2) and CidBwPip_I(b/2), respectively.)

Fig. 4: Sequence variations modulate interactions between natural CidAwPip and CidBwPip alleles.
figure 4

a Sequence alignment shows that residues at certain positions are different among the CidAwPip and CidBwPip variants. The residues that are the same among the variants are not shown. The varied residues which are located at the binding interfaces are boxed. The numbers above sequence alignment were based on the sequences of CidA and CidB from wPip Tunis. b The locations of the varied residues are shown as spheres on the structure of the CidAwPip(Tunis)-CidBwPip(Tunis)ND1-ND2 complex. c The varied residues which are located at the binding interfaces are colored and labeled. Other residues at the binding interfaces are shown in gray. d, e His-tagged CidA variants were used to pull down GST-tagged CidB variants. The proteins were detected by Coomassie Blue staining. These experiments were repeated three times independently with similar results obtained. One representative is shown. ND nuclease domain. Source data for panels (d) and (e) are provided as a Source Data file.

Among the positions that show divergence (Fig. 4b), residues 118, 125, 149, 150, 341, 342, 399, and 400 of CidAwPip and residues 242, 243, 253, 254, 257, 451, 452, 460, 461, and 462 of CidBwPip map to the binding interface of the two proteins (Fig. 4c). These variations create distinct interaction surfaces for each of the four known CidAwPip_I variants (Fig. 4a). Specifically, CidAwPip variants I(α/1) and I(β/2) have the same residues A118, G125, G149 and G150; the corresponding residues in variants I(γ/1) and I(γ/2) are R118, K125, D149, and S150 (Fig. 4a). CidAwPip variants I(α/1) and I(γ/1) both share residues G342, R343, K400, and N401, which correspond to residues Y341, T342, R399, and D400 in variants I(β/2) and I(γ/2). Similarly, each of the four CidBwPip_I variants has a unique combination of residues at the CidA-binding interface (Fig. 4a). Pulldown experiments were used to investigate the interactions between the CidAwPip variants and the CidBwPip variants. His-tagged CidA variants I(α/1), I(γ/1), I(β/2), and I(γ/2) cannot pull down GST-tagged CidB variants I(b/2), I(b/1), I(a/2), and I(a/1), respectively (Figs. 4d, e), which supports the hypothesis that natural sequence variations affect binding interactions between CidAwPip and CidBwPip from different strains. If binding is required for CidA to rescue CidB-induced defects, this sequence diversity may at least partially explain the complex bidirectional incompatibility patterns in crosses between different C. pipiens lines (discussed below).


Here, we have presented structural, biochemical and functional studies on the Wolbachia CI factors CidAwMel and CidBwMel, providing a solid foundation for investigation of the molecular functions of the Cid proteins in CI. These proteins are used here as a model to investigate the evolution of CidA-CidB interactions and their biological impact. Our structure-based mutagenesis has led to a clear demonstration of noncognate CifA-CifB binding, which suggests how sequence changes specifically at the interfaces between these proteins could lead to the complex incompatibility relationships that can be observed in the wild32.

The Cid systems from Wolbachia strains wMel and wPip are evolutionarily related, but these strains reside in different clades or supergroups (supergroups A and B, respectively). The two strains are expected to be incompatible based on crosses with trans-infected Ae. albopictus mosquitoes36. Here, we show that the CidA-CidB pairs from these Wolbachia strains share the same interaction mode but have different residues at their binding interfaces. These residues are critical for determining their binding specificity. CidAwMel(ST), a rationally designed chimeric construct containing the scaffold of CidAwMel and the interfacial residues of CidAwPip, binds CidBwPip(Pel) and rescues CidBwPip(Pel)-induced growth defects in yeast. It proves that CidA variants from different Wolbachia strains use the same pathway to carry out their function.

To further evaluate the conservation and evolutionary relationship of CidA and CidB found in different Wolbachia strains, the conservation scores of CidA and CidB residues were calculated by the ConSurf server and mapped onto their structures45,46,47. The residues in the core of the proteins tend to be highly conserved. In contrast, the residues located at the surface are more variable (Supplementary Fig. 4a-b). Many of the varied residues are located at the CidA-CidB binding interfaces (Supplementary Fig. 4c). (Although several key residues of CidA at interface III are identical among its homologs, their interaction with CidB cannot be maintained due to residue variations at interface III of CidB.) This agrees with previous hypotheses that the CidA and CidB interfaces are co-evolving32,35, which could modulate binding between CidA and CidB proteins and lead to incompatibility.

Wolbachia strains infecting C. pipiens usually contain more than one copy of the cidA and cidB genes32, which has been proposed to be responsible for the very complex incompatible patterns documented in C. pipiens infected with these strains. We showed here that the natural sequence variations in CidAwPip and CidBwPip proteins affects their binding specificity. The expression of multiple CidAwPip and CidBwPip variants in one Wolbachia strain could lead to a mix-and-match type of binding and may be one reason for the very complex crossing results. The presence of cidB_IV(2) variants was associated with the incompatible phenotype of mosquitoes infected with group IV wPip when crossing with those infected with wPip from other groups32,37. Indeed, the CidB_IV(2) variants have unique residues at interface I (including residues 451, 452, 460, 461 and 462) compared to the CidB of the other groups. These differences may impair the binding of the CidB_IV(2) variants to the CidA variants of the other groups and lead to incompatibility.

The crystal structure of CidBwMelDUB provides a solid structural basis for further investigation of the molecular targets and function of CidB deubiquitylase family proteins. Given that bacteria do not possess a ubiquitin-conjugation system, the presence of a DUB domain in CidB strongly suggests that this Wolbachia protein functions within the host21,40. Indeed, CidB proteins have been proposed to target nuclear-protein import and protamine-histone exchange factors48. CidBwMelDUB has three variable regions and a C-terminal region different from other bacterial proteases in the CE-clan/Ulp1-like protease family, which could be responsible for its substrate preference for ubiquitin over other ubiquitin-like molecules and for Lys63-linked ubiquitin chains21,40,41.

In different models that try to account for how Cif proteins induce and rescue CI, CifA-CifB interaction is suggested to play different roles. In one, which is named the “2-by-1” model, CifB is suggested to have an ancillary role by modulating CifA stability or activity in the male germline25. By contrast, in the “toxin-antidote” model, specific CifA-CifB interaction in the egg has been proposed to be essential for CifA to rescue CifB-induced CI32,35. Two major differences between these models are where CifA and CifB interact (in male or female) and whether the interaction is involved in CI induction or rescue. Our structures of CidA-CidB complexes and attempts to modulate their binding specificity provide a way to rationally design CifA and CifB mutants with desired binding attributes. Analysis of these variants will help distinguish between these and other CI models.


DNA manipulation

The genes coding for CidA, the variants of CidA, and CidB of wPip(Tunis), wPip(Pel) and wMel were synthesized at the Beijing Genomics Institute (BGI China). These sequences were codon-optimized for expression in E. coli by BGI. For crystallization, CidAwPip(Tunis), CidBwPip(Tunis)ND1-ND2 (residues 1-761), CidBwPip(Pel)ND1-ND2 (residues 1-761) and CidAwMel genes were subcloned into the pET-22b(+) vector using restriction sites NdeI and XhoI, encoding proteins with a C-terminal His-tag. CidBwMelDUB (residues 797-1128) gene was subcloned into the pGEX-6p-1 vector possessing an N-terminal glutathione-S-transferase (GST) tag using restriction sites BamHI and XhoI.

For in vitro pull-down experiments, CidA variants were the same as those used for crystallization. A pET28a-GST vector was made where the GST coding sequence was inserted between restriction sites NcoI and BamHI on pET28a. The CidBwPip(Tunis)ND1-ND2 and CidBwPip(Pel)ND1-ND2 genes were cloned into the pET28a-GST expression vector between restriction sites EcoRI and XhoI so that the proteins expressed had an N-terminal GST-tag.

For yeast growth analysis, DNA fragments were subcloned from E. coli vectors by restriction digest or PCR amplification and ligated into yeast expression vectors. The 2-micron plasmid pRS425GAL1 (LEU2) utilizing a GAL1 promoter was used for galactose-induced CifA expression in yeast and the low-copy vector pRS416GAL1 (URA3) was used for galactose-induced CifB expression.

Primers used to generate CifA or CifB expression plasmids are summarized in Supplementary Table 2. All plasmids were verified by sequencing (Sangon Biotech, China).

Protein expression and purification

All proteins were expressed in E. coli (BL21 (DE3) strain). Briefly, E. coli transformed with an expressing plasmid was cultured in Luria broth (LB) at 37 °C to an optical density (OD600) of 0.6. Overexpression of the recombinant proteins was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM at 16 °C for 16–18 h.

The harvested bacteria overexpressing CidAwMel (or CidA variants) were resuspended in a lysis buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole, 10% glycerol) and lysed via a high-pressure homogenizer at 4 °C. The lysate was centrifugated at 26500 × g for 30 min at 4 °C. After centrifugation, the supernatant was loaded onto a Ni-NTA column (GE Healthcare, USA). The column was washed using a lysis buffer supplemented with 50 mM imidazole and eluted using a lysis buffer supplemented with 500 mM imidazole. The eluted protein was diluted with buffer A (20 mM Tris-HCl, pH 8.0, 5 mM DTT) and further purified by anion-exchange chromatography (Hi Trap Q HP 5 mL, GE Healthcare, USA), using a linear gradient of 0%-40% mixture of buffer A and buffer B (20 mM Tris-HCl, pH 8.0, 1 M NaCl, pH 8.0, 5 mM DTT). Finally, the protein was purified by gel-filtration chromatography (Superdex 200 10/300 GL, GE Healthcare, USA), using a buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM DTT.

To obtain the CidAwPip(Tunis)-CidBwPip(Tunis)ND1-ND2 and CidAwMel(ST)-CidBwPip(Pel)ND1-ND2 complexes, bacterial cells expressing each component of the specific complex were mixed and co-lysed in the lysis buffer containing 20 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 10 mM imidazole, 10 μg/mL DNase 1, 10 μg/mL RNase A and 10% glycerol. The complex were further purified by running cleared lysate sequentially through Ni-NTA affinity, anion-exchange and size exclusion chromatography using the same columns and buffers as described for CidAwMel.

The CidBwMelDUB protein was purified following a similar procedure, using affinity, anion-exchange and size exclusion chromatography. CidBwMelDUB has a GST-tag. Instead of using Ni-NTA affinity chromatography, GST affinity chromatography was used. The harvested bacteria overexpressing CidBwMelDUB were resuspended in a lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5% glycerol) and lysed via a high-pressure homogenizer at 4 °C. The lysate was centrifugated at 26500 × g for 30 min at 4 °C. After centrifugation, the supernatant was loaded onto a glutathione-Sepharose column (GE Healthcare, USA). The GST tag was removed by incubating the loaded column with PreScission protease overnight at 4 °C. The CidBwMelDUB protein was further purified through anion-exchange and size exclusion chromatography using the same columns and buffers as described above.

Selenomethionine (SeMet)-derivatized proteins were expressed by bacteria growing in M9 SeMet medium supplemented with 100 mg/L L-selenomethionine. The purification procedure of SetMet-derivated proteins was the same as mentioned above for the native.

Crystallization, data collection, and structure determination

All crystals were grown by the microbatch-under-oil method unless otherwise specified49. CidAwMel was crystallized at 16 °C by mixing 1 µL protein (5 mg/mL) with 1 µL crystallization buffer containing 0.2 M Sodium phosphate monobasic monohydrate, 20% w/v Polyethylene glycol 3350, pH 4.7. The crystals were cryoprotected by Parabar 10312 (previously known as Paratone oil, Hampton Research, USA). X-ray diffraction data were collected on beamline BL18U1 at the Shanghai Synchrotron Radiation Facility at 100 K and at a wavelength of 0.97852 Å. Data integration and scaling were performed using HKL300050. The structure was determined by SeMet single-wavelength anomalous dispersion (SAD) method with the AutoSol program in PHENIX51. The CidAwMel model was initially built by the Autobuild program in PHENIX and subsequently subjected to iterative cycles of manual building in Coot52 and refinement in PHENIX.

The crystals of the CidAwPip(Tunis)-CidBwPip(Tunis)ND1-ND2 complex were grown at 16 °C from a mixture of 1 µL protein (5 mg/mL) and 1 µL crystallization buffer containing 0.1 M BICINE, pH 8.5 and 15% w/v Polyethylene glycol 1500. The crystals were cryoprotected by Parabar 10312. X-ray diffraction data were collected on beamline BL18U1 at the Shanghai Synchrotron Radiation Facility. The structure was determined by molecular replacement using the structure of the CidAwPip(Pel)-CidBwPip(Pel)ND1-ND2 complex as the search model53.

The crystals of the CidAwMel(ST)-CidBwPip(Pel)ND1-ND2 complex were grown at 16 °C from a mixture of 1 µL protein (5 mg/mL) and 1 µL crystallization buffer containing 5% v/v (+/-)-2-Methyl-2,4-pentanediol, 0.1 M HEPES pH 7.5, 10% w/v Polyethylene glycol 10,000. The crystals were cryoprotected by Parabar 10312. X-ray diffraction data were collected on beamline BL18U1 at the Shanghai Synchrotron Radiation Facility. The structure was determined by molecular replacement using the structure of the CidAwPip(Tunis)-CidBwPip(Tunis)ND1-ND2 complex as the search model.

CidBwMelDUB was crystallized by hanging drop method at 18 °C, with the crystallization reservoir solution containing 10 mM Nickel (II) Chloride hexahydrate, 100 mM TRIS pH 8.5 and 20% w/v Polyethylene Glycol Monomethyl Ether 2000. The crystals were directly flash frozen in liquid nitrogen using reservoir solution supplemented with 10% glycerol as cryoprotectant. X-ray diffraction data were collected at beamline BL17U1 at the Shanghai Synchrotron Radiation Facility. The structure was determined by SeMet SAD method as described above.

Data collection and structure refinement statistics are summarized in Supplementary Table 1. All Molecular graphics were created using UCSF ChimeraX54.

In vitro pull-down experiments

E. coli BL21(DE3) expressing either His-tagged CidA mutants or GST-tagged CidBND1-ND2 variants were harvested by centrifugation and resuspended separately in a buffer containing 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1 mM PMSF and 5% glycerol. The expression levels of CidA variants were approximated by the BCA Protein Assay (Tiangen Biotech (Beijing) Co., Ltd., China). Equal amount of CidA variants were mixed with a large and fixed volume of CidBND1-ND2 variants to ensure that CidBND1-ND2 variants were in excess to CidA. The mixture was co-lysed by sonication. Cleared lysates were incubated with 50 μL Ni-NTA resin at 4 °C for 1 h. The resin was washed eight times with 800 μL buffer containing 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 0.01% Tween-20, 50 mM imidazole each time. Proteins were eluted by adding 3 resin volumes of 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 300 mM imidazole. The samples were analyzed using the SDS-PAGE and Coomassie stain.

Yeast growth assays

Growth was analyzed in the BY4741 strain as previously described55. Briefly, yeast cultures were grown overnight at 30 °C in Yeast Extract Peptone Dextrose (YPD) media or synthetically defined (SD) raffinose media lacking uracil, leucine or both. Yeast were pelleted by centrifugation, washed with sterile water, and spotted in six-fold serial dilution from an initial OD600 0.2 concentration on solid minimal synthetic media containing either 2% galactose or glucose and lacking either uracil, leucine, or both. Plates were placed at 30 or 36 °C for 2 to 3 days.

Western blot analysis

For immunoblotting, co-expression culture in raffinose minimal medium (SD) lacking uracil and leucine were diluted to 0.2 OD600 in galactose (inducing) minimal medium (SD) lacking uracil and leucine, kept 12–16 h at 30 °C until reaching 0.8-1.0 OD600 at which point the equivalent of 2.5 OD600 units of cells were harvested, washed and resuspended in 1 mL dH2O followed by the addition of 200 μL dH2O and 200 μL 0.2 M NaOH, incubated for 5 min at room temperature. Cells were vortexed intermittently for 20 s and pelleted at 10,000 x g, 1 min. Pellets were stored at −80 °C for at least 15 min, resuspended in 100 μL 1 x SDS-PAGE sample buffer and 4% β-mercaptoethanol then heated at 95 °C for 3 min, centrifuged and 20 μL supernatant were loaded in the 10% SDS-PAGE gel and transferred to PVDF Immobilon-P transfer membranes (0.45 μM pore size) (Sigma-Aldrich) under 70 V, 2.5 h used for immunoblot analyses. Antibodies used for immunoblotting were as following: mouse anti-FLAG M2 (Sigma, 1:10,000), secondary antibody used was sheep anti-mouse NXA931V (GE Healthcare, 1:5,000); and mouse anti-PGK1 (yeast phosphoglycerate kinase; Abcam, 1:10,000), secondary antibody used was sheep anti-mouse NXA931V (GE Healthcare, 1:10,000). All immunoblot analyses used 5% milk for blocking. All serial dilution and Western blot data are representative of at least two biological experiments. Proteins were visualized by HRP-based chemiluminescence56.

AlphaFold modeling

In this study, AlphaFold was used to predict the monomer structure of CidBwMelND1-ND2, and AlphaFold-Multimer was used to predict the binding complex of CidAwMel-CidBwMelND1-ND2 with multiple sequence alignments (MSA) set as the all genetics database used at CASP14. The prediction of complexes was run twice with different random seeds and 10 models were obtained. Beginning with visual inspection, four of them were selected to perform protein structural quality check for the side chain conformations using prime module of Schrödinger2021-3. Eventually, the one complex with the highest quality score was selected for further optimization with subsequent MD simulations.

Molecular dynamics (MD) simulations

MD simulations were performed by using Desmond package of Schrödinger2021-357 using the OPLS458 force field. The binding model of CidAwMel-CidBwMelND1-ND2 obtained in the last step was explicitly solvated with TIP3P59 water molecules under cubic periodic boundary conditions for a 15 Å buffer region. The overlapping water molecules are deleted and 0.15 M KCl is added, and the systems were neutralized by adding K+ as counter ions. Brownian motion simulation was used to relax these systems into local energy minimum states separately. An ensemble (NPT) was then applied to maintain the constant temperature (300 K) and pressure (1.01325 bar) of the systems, and the simulations were started with different random initial velocities. The results were visually analyzed by using Maestro graphical interfaces and the RMSD was calculated based on C-alpha atoms. Produced trajectories were clustered using the Desmond trajectory cluster analysis panel. Finally, the most energetically stable binding complex from the largest cluster of conformation in MD trajectory was selected and minimized again with backbone constraints using the prime module of Schrödinger2021-3. The Ramachandran plot of the eventual model was generated with Schrödinger2021-3.

Evolutionary conservation analysis

The conservation score per amino acid of CidA and CidB was calculated using the ConSurf server ( based on their homologous sequences (specifically, CidA homologs from WOPip1, WOBol1-b, WOPipJHB, WOHa1, WOSol, WORecB, WOMelB, WOSuziB, and WORiB and CidB homologs from WOPip1, WOBol1-b, WOHa1, WOSol, WOMelB, WOMelPop, and WOSuziB). The structures of CidAwPip(Tunis) and CidBwPip(Tunis)ND1-ND2 served as templates. “Maximun likelihood” and “best model” were selected as the calculation method and the evolutionary substitution model, respectively, for this analysis.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.