Sequence alignment to identify phage KP32 protein constituents was performed using BlastP (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA) [25]. We found that phage KP32 proteins share high sequence similarity with proteins of the E. coli T7 phage, whose composition and structure have been described [12]. The structure alignment was performed using DALI (Distance-matrix ALIgnment, http://ekhidna2.biocenter.helsinki.fi/dali) [26].

The three-dimensional structure models of seven proteins composing phage KP32 were obtained using AF3 modeling server (https://www.alphafoldserver.com) and the most up-to-date database available (as of February 3, 2025). The server produced five ranked models for each protein [27]. One phage protein, the tail spike depolymerase KP32gp38, is available from the PDB (PDB codes 6TKU, 9QRM) [10, 23]. The confidence of the models was evaluated on the basis of the predicted local distance difference test (pLDDT), a per-residue measure of model confidence. pLDDT is scaled between 0 and 100, with higher scores reflecting greater confidence in the structural model (very low: pLDDT $\le$50, low: 50 pLDDT $\le$ 70, confident: 70 pLDDT $\le$ 90, and very high: 90 $\le$ pLDDT $\le$ 100) [27]. The oligomeric state of the individual components was inferred from their homologs in the T7 phage. To assess the accuracy of the predicted assemblies, we used AF3 ipTM values. Indeed, ipTM measures the accuracy of the predicted relative positions of the subunits within the complex [27]. Values higher than 0.8 represent confident high-quality predictions, whereas values below 0.6 suggest likely a failed prediction. ipTM values between 0.6 and 0.8 are in the gray zone where predictions could be correct or incorrect. The capsid was modeled by AF3 in a heptameric arrangement, analogous to the T7 phage capsid, and superimposed onto the T7 phage capsid (PDB code 2XVR [16]) in ChimeraX (University of California, San Francisco (UCSF), San Francisco, CA, USA) [28, 29, 30] using the MatchMaker tool, which applies the Needleman–Wunsch algorithm with a BLOSUM-62 (BLOcks SUbstitution Matrix) substitution matrix. The symmetry matrix of the T7 phage capsid was then applied to the KP32 capsid heptamer using the sym command in ChimeraX to generate the icosahedral biological assembly. Similarly, the nozzle, the adaptor and the tail spike KP32gp37 were modeled in AF3 as hexameric, dodecameric and trimeric structures, respectively. To assemble the portal, nozzle, adaptor, and branched depolymerase systems, we superimposed each assembly on its homolog of the portal-tail complex (including gp11, gp12, gp8 and gp17) of the T7 phage (PDB code 9JYZ [17]) using MatchMaker in ChimeraX as described above. Finally, to assemble the portal–tail complex and the capsid and reconstruct the complete Kp32 phage, one vertex of the capsid was removed to accommodate the portal–tail adaptor. The generated assembly was subjected to 100 steps of steepest descent minimization using ChimeraX. Electrostatic potential surfaces were computed using ChimeraX. All the structures were visualized via PyMol (Schrödinger, Inc., New York, NY, USA) [31].

Given the low value of ipTM (0.5) for the AF3 prediction of the heptameric assembly of KP32gp30 and the failed attempts to model both the entire inner core of KP32 and the single proteins that constitute it (KP32gp34, KP32gp35, KP32gp36) using AF3, we additionally model them via homology modeling and the software SWISS-MODEL (Swiss Institute of Bioinformatics (SIB) and Biozentrum, University of Basel, Basel, Switzerland) [32]. A template search with BLAST [33] and HHblits [34] was performed against the SWISS-MODEL template library (SMTL, last update: 2025-07-02, last included PDB release: 2025-06-27). The target sequence was searched with BLAST against the primary amino acid sequence contained in the SMTL. An initial HHblits profile was built using the procedure outlined in Steinegger et al. [34], followed by iterations of HHblits against Uniclust30 [35]. The obtained profile was searched against all the profiles of the SMTL. For each identified template, the quality was predicted from features of the target-template alignment. The templates with the highest quality were selected for model building. Models were built on the basis of target–template alignment using ProMod3 (Swiss Institute of Bioinformatics (SIB) and Biozentrum, University of Basel, Basel, Switzerland) and geometry normalization [36]. The global and per-residue model quality was assessed using the Qualitative Model Energy Analysis (QMEAN) scoring function combined with a distance constraint (DisCo) score, which assesses the agreement of pairwise distances in the model and an ensemble of constraints derived from experimentally determined homologous protein structures [37]. The quaternary structure annotation of the template is used to model the target sequence in its oligomeric form. The method proposed by Bertoni et al. [38] is based on a supervised machine learning algorithm, support vector machines (SVMs), which combines interface conservation, structural clustering, and other template features to provide a quaternary structure quality estimate (QSQE). The QSQE score is a number between 0 and 1, reflecting the expected accuracy of the interchain contacts for a model built on the basis of a given alignment and template. This complements the Global Model Quality Estimation (GMQE) score, which estimates the accuracy of the tertiary structure of the resulting model.

By analyzing the phage KP32 genome, we identified a major capsid protein, constituting the phage capsid shell, the major head protein KP32gp30 (YP_003347548.1). A search in the PDB using the BlastP tool revealed that this protein has high sequence identity (79.5%) with the major capsid protein from phage T7, whose structure is reported in the PDB (PDB code 2XVR [16]). Phage T7 is a well-studied lytic virus that infects E. coli, making it a powerful model in molecular biology and a potential tool in biotechnology and phage therapy. Starting from this observation, we searched for all proteins composing phage KP32 on the basis of sequence identity with their homologs in phage T7. Overall, we identified ten protein components of phage KP32 that are homologous to T7 phage proteins, with sequence identities higher than 45% (Table 1, Ref. [10, 13, 16, 17, 23]). We have previously shown that the phage KP32 has the ability to carry on its phage portal two types of depolymerases, KP32gp37 and KP32gp38, with different serotype specificities [19]. The second KP32 protein with depolymerase activity, KP32gp38, has no homologs in phage T7.

Once we had identified all phage KP32 components, we modeled their 3D structures using AF3 and with the oligomeric state observed for the phage T7. As its name suggests, this phage presents a capsid with T7 symmetry, with icosahedral symmetry in its capsid structure [14, 15].

Most protein structures were modeled with high confidence (pLDDT values $>$77; Table 2). Lower pLDDT values were computed for the KP32gp26 and KP32gp27 proteins (65.8 and 68, respectively), indicating reduced reliability of the predicted models; therefore, these proteins were excluded from modeling. Although these proteins of the phage KP32 are not well characterized, we predict that its highly flexible nature may help enhance capsid integrity during DNA packaging. For protein assemblies, ipTM values were considered. These values are higher than 0.5 in all cases, except for the heptameric organization of the KP32gp30 mature capsid protein, for which ipTM is 0.5. Therefore, we modeled this assembly in parallel using homology modeling and the software SWISS-MODEL as a further control. The resulting model, which could be predicted with a qualitative model energy analysis scoring function combined with a distance constraint (QMEANDisCo) of 0.63 and a QSQE of 0.68, was fully superposable to the AF3 model (Root-Mean-Square Deviation (RMSD), computed on C$\alpha$ atoms of residues 29–221, of 1.7 Å). In the case of the tail spike depolymerase KP32gp38, the crystal structure is available in the PDB in both its free form (PDB code 6TKU [10]) and in complex with a capsular polysaccharide (CPS) degradation product (9QRM [23]). Once all the structure assemblies were modeled (Table 2), the phage KP32 was assembled using ChimeraX [28].

The KP32gp30 mature capsid protein was modeled as a heptamer, analogous to the T7 phage. The KP32gp30 mature capsid promoter has an alpha‒beta fold (Fig. 1A). Like T7gp10, KP32gp30 is composed of an A-domain, which is predominantly composed of $\beta$-strands, and a P-domain, which is rich in $\alpha$-helices (Fig. 1A). The N-terminal region 1–40 is predicted to have a lower pLDDT (76.1) by AF3 (Fig. 1A). This region is known to undergo a transition from an $\alpha$-helix to a $\beta$-hairpin during maturation to stabilize intercapsomeric joints [15, 39, 40]. Alignment of its AF3 structure with the available structures in the PDB using DALI revealed the major capsid protein of the Klebsiella phage Kp9 (RMSD = 1.3 Å) (PDB code 7Y23), albeit still unpublished. The KP32gp30 heptamer is composed of one skewed hexamer and one extra chain that composes the pentameric faces upon assembly of the entire capsid (Fig. 1B).

Fig. 1.

Structural model of the phage KP32 capsid. Cartoon representations of (A) the KP32gp30 capsid monomer colored according to the AF3 confidence intervals: dark blue, very high (pLDDT $>$90); light blue, confident (90 $>$ pLDDT $>$ 70); yellow, low (70 $>$ pLDDT $>$ 50); and orange, very low (pLDDT 50). (B) Cartoon representation of a composing heptamer and (C) of the whole capsid. Pentameric vertices are drawn in purple, whereas hexameric faces are drawn in light blue.

After full reconstruction, the phage KP32 capsid is composed of 60 KP32gp30 heptameric assemblies, with a total of 420 chains (Fig. 1C). This reconstruction of a phage T7 icosahedral capsid exposes 60 hexameric and 12 pentameric faces (Fig. 1C). Pentamers constitute the vertices of the icosahedron, which provide curvature and close the shell. The resulting capsid shell has a diameter of 616 Å (Fig. 1B). To build the mature phage, one pentameric vertex was removed to make room for the portal for genetic code entry and exit [41, 42, 43].

In addition to the capsid shell of phage KP32, an additional 104 protein chains constitute the phage portal-tail complex, with a total of over 500 protein chains (Table 2). As previously mentioned, the missing pentamer at one vertex of the phage KP32 capsid is replaced by the portal complex formed by the protein KP32gp28, which shares 78.8% sequence identity with gp8 of the phage T7, whose structure has been solved using cryo-EM (PDB code 7EY6) (Table 2). A reliable structural model of the KP32gp28 portal dodecamer was obtained using AF3 (pLDDT/ipTM: 77.2/0.7, Table 2, Fig. 2). The KP32gp28 monomer folds into an $\alpha$-$\beta$ structure (Fig. 2A) and is the sole building block of the portal, contributing to the formation of a 135 Å-long central tunnel in the mature phage (Fig. 2B,C). This DNA tunnel narrows to ~26 Å in the DNA channel (Fig. 2B,C). Consistent with its function, the analysis of the electrostatic potential of KP32gp28 revealed a fully negatively charged electrostatic potential, which is instrumental in preventing DNA leakage when the phage has not infected its host. A positively charged electrostatic potential is present solely at the tunnel, due to the presence of R293 (Fig. 3), to guide and retain negatively charged DNA.

Fig. 2.

Structural model of the phage KP32 adaptor. Cartoon representations of (A) the KP32gp28 portal monomer, colored according to the AF3 confidence intervals (dark blue, high (pLDDT $>$90); light blue, confident (90 $>$ pLDDT $>$ 70); yellow, low (70 $>$ pLDDT $>$ 50); orange, very low (pLDDT 50); and its dodecameric assembly in the phage in top (B) and side (C) views.

Fig. 3.

Electrostatic potential surface of the KP32gp28 portal dodecameric assembly. (A) Top view, (B) bottom view, (C) side view, and (D) inner section. The red- and blue-colored surfaces represent negative and positive electrostatic potentials, respectively.

Compared with phage T7, phage KP32 also possesses a similar inner core structure, which is expected to form a large cylindrical assembly located inside the capsid, right beneath the portal protein at one of the icosahedral vertices of the phage head. Indeed, the T7gp14, T7gp15, and T7gp16 ejection proteins are homologous to KP32gp34, KP32gp35 and KP32gp36, with sequence identities of 59%, 65.2% and 66.7%, respectively (Table 1). Additionally, the inner core proteins T7gp6.7 and T7gp7.3 have homologous sequences in KP32, sharing 47.7% and 51.3% with KP32gp26 and KP32gp27, respectively. Analogous to the T7 phage structure, the inner core of KP32 is expected to be composed of eight copies of KP32gp34, eight copies of KP32gp35, four copies of KP32gp36, 12 copies of KP32gp26 and 6 copies of KP32gp27, accounting for 15,154 amino acid residues overall [17]. An attempt to model the entire inner core of KP32 using AF3 failed. Given the large size of the inner core, we attempted to model smaller oligomers using AF3. However, these models did not produce reliable models (pLDDT 50, ipTM 0.5), likely because of the need for a heteromeric assembly for overall core stability. Given the high sequence identity, we used homology modeling and the software SWISS-MODEL to model the structure of the 20-mer formed by KP32gp34, KP32gp35 and KP32gp36 [32]. As expected, the best template was the structure of the inner core of the T7 phage (overall 64.5% sequence identity, PDB code 9JYY). The resulting model, which could be predicted with high confidence (QMEANDisCo 0.72), resulted in a large, almost cylindrical assembly with all proteins in a fully $\alpha$-helical conformation, where the four large tetrameric KP32gp36 sit on the top (Fig. 4A,B). On the bottom side, the KP32gp34 and KP32gp35 octamers form the inner and outer cores of an intricate toroidal structure (Fig. 4C). KP32gp26 and KP32gp27 could not be modeled by SWISS-MODEL, likely because of their intrinsic flexibility.

Fig. 4.

Structural model of the phage KP32 inner core KP32gp34-KP32gp35-KP32gp36 complex in the mature KP32 phage. Cartoon representations of the complex: (A) side, (B) top and (C) bottom views. The insets show details of the protein monomers composing the complex.

Notably, the described structure of the KP32 inner core refers to the mature form of the phage before infection. Indeed, cryo-electron microscopy of the T7 phage revealed the transient nature of the inner core, which changed conformation after infection. These characteristics are likely shared by the KP32 phage [44, 45, 46].

The structures of all the components of the phage KP32 portal–tail complex were modeled with high confidence (Table 2). The tail is formed by the hexameric assembly of the protein KP32gp32 (nozzle) and the dodecameric assembly of the protein KP32gp31 (adaptor) (Fig. 5). KP32gp32 is the tail nozzle protein, forming the final conduit through which the viral DNA exits during infection. The structure of the KP32gp32 monomer is composed of a large central $\beta$-propeller domain and three other domains: the platform and the fiber dock on one side, and the nozzle tip is on the opposite side (Fig. 5A,B). The nozzle tip presents an almost fully $\alpha$-helical structure; it has been reported for phage T7 that this region of the nozzle plays a role in regulating genome ejection during infection [44]. The hexameric assembly of the nozzle is 160 Å long and leaves an inner channel ~38 Å in diameter, likely regulating DNA flow during ejection (Fig. 5C,D). It acts as the final checkpoint in the DNA delivery pathway, ensuring that the viral genome is released only upon proper host recognition [44]. Consistently, analysis of the electrostatic potential of the KP32gp32 nozzle revealed a strongly negatively charged character (Fig. 6), which prevents premature DNA leakage by repelling the negatively charged phosphate backbone of the viral DNA.

Fig. 5.

Structural model of the KP32gp32 nozzle protein. Cartoon representations of a KP32gp32 monomer, displayed (A) colored according to the AF3 confidence intervals (dark blue, very high (pLDDT $>$90); light blue, confident (90 $>$ pLDDT $>$ 70); yellow, low (70 $>$ pLDDT $>$ 50); orange, very low (pLDDT 50), and (B) with differently colored and labeled domains; and (C) the top and (D) the side view of its hexameric assembly.

Fig. 6.

Electrostatic potential surface of the KP32gp32 nozzle hexameric assembly. (A) Top view, (B) bottom view, (C) side view, and (D) inner section. The red- and blue-colored surfaces represent negative and positive electrostatic potentials, respectively.

Another important element of phage KP32 is the KP32gp31 adaptor, which we have modeled with good confidence (Table 2). KP32gp31 is homologous (62.2% sequence identity) to the T7gp11 protein, a key structural component of the tail apparatus, acting as a gatekeeper and adaptor between the portal protein (KP32gp28) and the tail nozzle (KP32gp32). The KP32gp31 adaptor assembles into a dodecameric toroidal ring and acts as a structural bridge between the portal KP32gp28 proteins and the KP32gp32 nozzle proteins. It is formed by a core $\alpha$-helical domain that forms a rigid toroidal ring (Fig. 7A,B) and a loop and $\beta$-helical regions that form the external part of the adaptor (Fig. 7B,C). These flexible regions likely play a role in accommodating the symmetry mismatch between the dodecameric KP32gp28 portal and the hexameric nozzle [44]. Analysis of the electrostatic potential of the adaptor assembly revealed a fully negative electrostatic surface potential at the bottom side of KP32gp31 (Fig. 8). This allows KP32gp31 to interact with KP32gp32 through electrostatic interactions with the positively charged patch on the upper side of the KP32gp32 hexamer (Fig. 6A).

Fig. 7.

Structural model of the KP32gp31 adaptor protein. Cartoon representations of (A) the KP32gp31 monomer, colored according to the AF3 confidence intervals (dark blue, very high (pLDDT $>$90); light blue, confident (90 $>$ pLDDT $>$ 70); yellow, low (70 $>$ pLDDT $>$ 50); orange, very low (pLDDT 50); and (B) the top and (C) bottom views of its dodecameric assembly in the KP32 phage.

Fig. 8.

Electrostatic potential surface of the KP32gp32 adaptor dodecameric assembly. (A) Top view, (B) bottom view, (C) side view, and (D) inner section. The red- and blue-colored surfaces represent negative and positive electrostatic potentials, respectively.

Tail spike depolymerases of the KP32 bacteriophage are specialized enzymatic proteins located at the distal end of the phage’s tail structure [10, 47, 48, 49]. By enzymatically cleaving the glycosidic bonds within the capsule, they degrade the protective polysaccharide layer surrounding the bacterial cell, thereby facilitating phage adsorption and genome injection. Like other depolymerases, they play crucial roles in host recognition and infection [10, 47]. KP32 encodes two structurally related but functionally distinct tail-spike depolymerases, KP32gp37 and KP32gp38, each of which form a stable homotrimer [10, 19, 23]. We have previously shown that the KP32gp37 and KP32gp38 depolymerases exhibit remarkable specificity toward the K3 and K21 capsular serotypes, respectively [10, 19]. The crystal structure of the KP32gp38 depolymerase was determined in its unliganded form [10] and in complex with its degradation product, a pyruvated pentasaccharide, bound to its interchain catalytic site [23]. This depolymerase is formed by an N-terminal region, with a key role in the interaction with KP32gp37 [10, 23], a catalytic domain and two oligosaccharide binding domains (Fig. 9A). Compared with KP32gp38, KP32gp37 presents a more complex and elongated structure. Unlike KP32gp38, KP32gp37 includes an extra helical domain at its C-terminus (Fig. 9B) that displays a strong structural resemblance (DALI Z score = 12.8) to the intramolecular chaperone domain of the phage T5 L-shaped tail fiber, which undergoes autocleavage [23]. Additionally, it includes an extended N-terminal region formed by three coiled coil regions and two rings of lectin domains (Fig. 9B). The structure of the complex between the two depolymerases, modeled using AF3 [23], shows the sole involvement of the N-terminal helices of KP32gp38 in the binding of KP32gp37, in full agreement with our previous calorimetric studies [10].

Fig. 9.

Structural models of phage KP32 depolymerases. Cartoon representations of (A) the KP32gp38 crystal structure, (B) the KP32gp37 AF3 model and (C) the complex between KP32gp37 and KP32gp38 computed with AF3.

The N-terminal 150 residues of KP32gp37 have a strong sequence and structural similarity to the N-terminal region of the tail fiber gp17 from the mature E. coli phage T7 (PDB code 7EY9, Dali Z = 16.0; PDB code 8DSP, Dali Z = 18.8) (sequence identity 45%) [13]. Analogous to T7, we propose that the KP32 portal directly engages KP32gp37 through interactions with its N-terminal adaptor. In turn, KP32gp37 engages the KP32gp38 depolymerase through interactions with its N-terminus (Fig. 9) [23].

The entire phage KP32 was reconstructed on the basis of the AI-modeled structures of its components and the available structural information on the E. coli phage T7 [12, 13, 15, 18, 44]. A scheme of the organization of the entire phage KP32 virion is reported in Fig. 10A. From inside the icosahedral capsid of phage KP32 toward its tail, a vertex pentamer consisting of five KP32gp30 chains is replaced by the phage portal, formed by a dodecameric assembly of KP32gp28 proteins. This crown-like portal domain faces five KP32gp30 hexamers at the inner side of the capsid, adjacent to the missing pentamer (Fig. 10B). The portal connects to the phage tail through a tail adaptor made by a dodecameric assembly of KP32gp31 proteins. A tail tubular protein, KP32gp32, then stacks below KP32gp31 and extends the DNA translocation channel; there, KP32gp32 acts as the central nozzle that seals the DNA channel until infection begins (Fig. 10A). As previously mentioned, the tapered internal cylinder sitting on top of the portal and made by the ejection proteins KP32gp34, KP32gp35 and KP32gp36 could not be reliably modeled by AF3, likely due to both size limits and the different conformational states of these proteins depending on the functional state. Indeed, the homologous proteins in the T7 phage form a transient tunnel that spans the bacterial envelope from the outer membrane to the cytoplasm, allowing the phage genome to pass through without being degraded or exposed [46].

Fig. 10.

The phage KP32 virion structure. (A) Simplified sketch showing the position of each phage KP32 protein in the phage structure. (B) A 3D model of the entire phage KP32 virion, excluding the inner core/ejectosome.

In addition to being a gate for the KP32 genome, the nozzle is also the attachment site of the capsular depolymerases via noncovalent interactions and structural complementarity. Indeed, due to the sequence and structural similarity of the N-terminal 150 residues of the KP32gp37 tail spike depolymerase with the N-terminal region of the tail fiber gp17 from the mature E. coli phage T7 (PDB code 7EY9), we could model the interaction of KP32gp37 with the KP32gp32 central nozzle (Fig. 10). In turn, the KP32gp38 depolymerase does not directly interact with the KP32 central nozzle, but it hijacks KP32gp37 through its N-terminal region. Consistently, we have shown that a truncated form of KP32gp38, deprived of its N-terminal 29 amino acids, is unable to bind KP32gp37 [10]. In this branched model, the KP32 portal carries twelve depolymerase molecules on a single virion, which are arranged in six branches. This arrangement allows the depolymerases to extend outward, enabling them to bind and degrade the CPS on the bacterial surface (Fig. 10).