Slipknotted and unknotted monovalent cation-proton antiporters evolved from a common ancestor.

While the slipknot topology in proteins has been known for over a decade, its evolutionary origin is still a mystery. We have identified a previously overlooked slipknot motif in a family of two-domain membrane transporters. Moreover, we found that these proteins are homologous to several families of unknotted membrane proteins. This allows us to directly investigate the evolution of the slipknot motif. Based on our comprehensive analysis of 17 distantly related protein families, we have found that slipknotted and unknotted proteins share a common structural motif. Furthermore, this motif is conserved on the sequential level as well. Our results suggest that, regardless of topology, the proteins we studied evolved from a common unknotted ancestor single domain protein. Our phylogenetic analysis suggests the presence of at least seven parallel evolutionary scenarios that led to the current diversity of proteins in question. The tools we have developed in the process can now be used to investigate the evolution of other repeated-domain proteins.

Introduction

Application of mathematical methods in the structural biology revealed that backbones of some proteins are entangled [1, 2]—they tie and form knots when pulled by their termini (Fig 1). These knots can be found both in globular and in membrane proteins [3]. Another type of an entanglement are slipknots, described by Todd Yeates, who originally detected them, as so: “Slipknots occur when the path of some part of the chain forms a knot, which is then effectively undone when the terminus doubles back on itself, like a tied shoelace.” [4].

Fig 1

Schematic representation of the knotted (A) and slipknotted (B) chains.

(A) The knotted chain (left) and protein backbone (PDBID: 4kpp) (right) with a knotted core marked with blue color. This knot is called open trefoil. (B) The slipknotted chain (left) and its representation in the sodium-dependent citrate symporter (KpCitS), a protein with slipknotted backbone identified in this paper (PDBID: 5xar). The knotted core is shown in blue, the slipknot loop in orange and the slipknot tail in green.

The biological significance of a slipknot topology is not yet known, but it has been found previously in active sites of globular proteins [4, 5], and can be involved in transport mechanisms of transmembrane proteins [6]. Mechanical investigation of such proteins has shown that slipknot topology can give them a very high mechanical resistance [7], although such properties strongly depend on the slipknot geometry and the amino acid sequence motifs [8, 9]. The folding process of slipknotted proteins also remains unclear, and the question of whether its topology is a rate-limiting factor is unanswered [10, 11]. Theoretical investigations have shown that a slipknot could be formed either by flipping of a twisted loop (the blue loop, Fig 1) above a knotted core, or by pushing a slipknot loop (the orange loop, Fig 1) through a knotted core [10]. On the other hand, it was shown that the slipknot topology can appear as an intermediate step facilitating the entanglement in the case of the knotted proteins in the bulk [1, 12–18], in the confinement [19, 20], and in the case of a direct folding on the ribosome [21, 22]. The slipknot intermediate was also observed during unknotting [17, 23]. Finally, the slipknot topology has been observed as a step during the folding of other proteins with non-trivial topology such as lassos [24-27] and links [28, 29].

There is an increasing number of known proteins that form a slipknot or a knot in their native folded structure, however, their fraction is still very small [1]. Is it because they being are eliminated due to inefficient folding or structural complexity, and yet they make a sufficient contribution to protein structure that they are being preserved [30-32]? It was shown that both globular and transmembrane slipknotted proteins can provide an advantage in some extreme conditions [27], or increase the resistance to antibiotics [33, 34]. Still, the evolutionary origin of non-trivial topology in proteins remains unknown [35]. In this work we bring the field one step closer to answering these questions, by finding the first unknotted homologues to slipknotted membrane proteins.

Slipknotted transmembrane (TM) proteins were first discovered in 2007 [4], and since then many new structures with this topology have been discovered [6]. There are 16 different families with very different slipknot topology [36], but none of them have unknotted homologues. In consequence, the evolution of the slipknot could not be investigated. Therefore, we conducted a comprehensive analysis of all known protein structures from the KnotProt 2.0 database [36]. We identified proteins from sodium-dependent citrate symporter family (CitS, Fig 1B) as slipknotted, which remained unnoticed until now. A look at the fold of these proteins shows that they are made up of two structurally similar inverted domains, connected by a linker (S2 Fig). Other known membrane transporters with the slipknot topology are also composed of two inverted domains which, while very diverse in the sequence, are well conserved structurally. It was also shown that the majority of large transmembrane proteins are composed of duplicated domains positioned within membranes either parallel or anti-parallel to each other [37]. It was hypothesized that such proteins evolved from a one-domain ancestor gene via gene duplication and fusion [38, 39]. Thus, one could ask if the similarity between domains of slipknotted and unknotted proteins could be used to understand the evolution of the slipknot topology.

Based on the newly identified family of slipknotted proteins (sodium-dependent citrate symporter (CitS)), we aim to answer a long-standing question of how the transmembrane slipknotted structures appeared during evolution. Did they evolve from the unknotted proteins or vice versa? To test this possibility, we study the similarity between the domains of slipknotted and unknotted structures, and seek key elements that lead to the emergence of the slipknotted topology. Thus, we perform a thorough sequential and structural characterization of all homologous families. To study the characteristics of the domains, we utilize information from Pfam database [40], which recognizes proteins with related regions or domains as families enabling the research into evolutionary origin of these proteins. Based on a combination of known and new computational methods that we have developed, we analyze both sequence and structure of membrane transporters. This study provides evidence that several transmembrane protein families with different folds and topology types (slipknotted and unknotted) evolved from a common ancestor (schematically presented in Fig 2).

Fig 2

Conserved helical region (core) found in the monovalent cation-proton antiporter superfamily.

Conservation of this region suggests that three different fold types, including one possessing a non-trivial topology (a slipknot), evolved from a common, single-domain ancestor. The putative ancestor is shown in light green box in the middle. Three arrows from the ancestor navigate to three proteins with different folds: 1) left—two-domain slipknotted protein; 2) middle—one-domain unknotted protein; 3) right—two-domain unknotted protein. On the bottom left of the figure is shown a schematic diagram of the entangled region of the slipknotted protein colored from blue (N-terminal) to red (C-terminal). On the bottom right there is a similar schematic diagram that shows the topology of the unknotted protein backbone.

Results and discussion

New slipknotted family and the definition of the slipknot topology

Without going into much detail about entanglement detection methods, which have been extensively described in the literature [41-43], we introduce two terms important for the description of a slipknot: a knot core and a slipknot loop, as shown in Fig 1B. A slipknot can be imagined as knot of which one end doubles back through the loop leading to an ultimately unknotted structure. With that in mind, the knot core is the minimal part of the structure that would tie if pulled by its ends, and the slipknot loop is the “doubling back” part of the chain, as shown in Figs 1 and 2. In general, slipknots in proteins can be very complicated, they can be formed based on different types of knots along a single chain [6].

In the case of newly identified proteins with PDBID 5a1s (SeCitS) and 5xar (KpCitS) discussed in the next section, we found that amino acids between 166–411/115-401 (respectively) form a knot core (the blue loop) and amino acids between 412–415/402-422 (respectively) form a slipknot loop (the orange loop), as shown in Figs 1 and 3. The knot core forms the knot called a trefoil which is denoted 31 (it possesses three crossings when projected on the plane). Altogether, this protein forms a 31 slipknot, however, for simplicity in this paper we will just call it a slipknot.

Fig 3

Structure of the slipknotted transporter KpCitS.

(A) Location of the knotted core (N115-N401) starts at TM4 of domain A, loop-linker and TM8-TM11 and half of TM12 hairpin-like helix of domain B. Slipknot loop (R402-S421) is formed by half of TM12 hairpin-like helix and part of TM13. The rest of TM13 (residues Y422-I446) is slipknot tail. (B) Schematic representation of the slipknot topology. (C, D) Structure of the knotted core and slipknot’s loop and tail based on PDBID 5xar. (E) Knot fingerprint calculated based on the KpCitS structure (PDBID: 5xar).

Slipknotted family of sodium-dependent citrate symporters (CitS) belongs to the monovalent cation-proton antiporter superfamily

By using the KnotProt 2.0 database we spotted a slipknot topology in two proteins (PDBID: 5xar and 5a1s) from a family never before identified as entangled. In both proteins (sharing 92% similarity) the slipknot is formed by a single trefoil knot (the blue loop in the Fig 3) where the C-terminus doubles back on itself forming the orange loop, as shown in the Fig 3. These proteins are found in the cell membranes acting as transporters (sodium-dependent citrate symporter—CitS). They belong to the 2-hydroxycarboxylate transporter family (2HCT) as recognized by Pfam database (PF03390). A careful examination of the structures shows that they are composed of two structurally similar domains (S2 Fig).

In order to find all the proteins related to this slipknotted family, we used HMMER web server [44]. Altogether, we found 17 protein families, which are all parts of the same monovalent cation-proton antiporter superfamily according to the OPM database [45] (Table 1). Four of these families are represented by proteins with known 3D structures, and thus topology. Three unknotted families are Sodium/hydrogen exchanger family (represented by PDBID: 4bwz), Sodium/hydrogen antiporter 1 (represented by PDBID: 1zcd) and Sodium Bile acid symporter family (represented by PDBID: 3zuy). The fourth family is represented by the newly identified slipknotted proteins with PDBID 5a1s and 5xar. According to our knowledge this is the first clan where proteins with different topology were identified. This finding allows tracing and investigation of possible evolutionary steps to forming the slipknotted family. Proteins from the remaining families have undetermined topology since no structure was experimentally resolved.

Table 1

Known protein families from monovalent cation-proton antiporter superfamily investigated toward identification of possible evolution of the slipkotted topology.

IDs of families and clans are from Pfam database. ND—not determined.

Family ID	Clan ID	Name	Topology	Number of domains
PF03390	-	2-hydroxycarboxylate transporter family	slipknotted	2
PF00999	CL0064	Sodium/hydrogen exchanger family	unknotted	2
PF06965	CL0064	Sodium–hydrogen antiporter 1	unknotted	2
PF01758	CL0064	Sodium Bile acid symporter family	unknotted	2
PF03547	CL0064	Membrane transport protein	ND	2
PF03601	CL0064	Conserved hypothetical protein 698	ND	2
PF03812	CL0064	2-keto-3-deoxygluconate permease	ND	2
PF03977	CL0064	Na+-transporting oxaloacetate decarboxylase beta subunit	ND	2
PF03956	CL0064	Lysine exporter LysO	ND	2
PF05684	CL0064	Protein of unknown function (DUF819)	ND	2
PF05982	CL0064	Na+-dependent bicarbonate transporter superfamily	ND	2
PF03616	CL0064	Sodium/glutamate symporter	ND	2
PF13593	CL0064	SBF-like CPA transporter family (DUF4137)	ND	2
PF06826	CL0064	Predicted Permease Membrane Region	ND	2
PF05145	CL0142	Transition state regulatory protein AbrB	ND	2
PF03788	-	LrgA family	ND	1
PF04172	-	LrgB-like family	ND	1

Structural comparison shows a common motif in both slipknotted and unknotted transporters

In order to characterize the relationship between slipknotted and unknotted transmembrane proteins we have conducted their structural analysis. Both slipknotted and unknotted transporters are made up of two inverted structurally similar domains. Therefore, for the analysis, we extracted the domains (A and B) and compared them. As the representative structures, we have chosen unknotted structure of sodium-proton antiporter NhaA (PDBID: 4bwz, from PF00999 family) and slipknotted structure of 2-hydroxycarboxylate transporter SeCitS (PDBID: 5a1s). In both proteins the domains are created by 6 transmembrane helices (TM)—TM2-TM7 and TM8-TM13 in the slipknotted structure (domain A and B, respectively; Fig 4A) and TM1-TM6 and TM8-TM13 in the unknotted protein (domain A and B, respectively; Fig 4B).

Fig 4

Identified structural differences between the slipknotted and unknotted proteins.

(A) Schematic representation of the full length two-domain slipknotted protein. Two domains are connected by the long loop. The conserved core region is colored in green. TM helices are enumereted as in the structure (PDBID: 5a1s). (B) Schematic representation of the unknotted protein. Similarly, unknotted protein is composed of two domains. Conserved core is colored in green. TM helices are enumereted as in the structure (PDBID: 4bwz). (C) Structure of a single domain of the slipknotted protein (PDBID: 5a1s). (D) Superposition of the domains A of the slipknotted and unknotted proteins. (E) Structure of a single domain of the unknotted protein (PDBID: 4bwz). (F) Superposition of the fragments of domains A of the slipknotted and unknotted proteins.

First, we analyzed the level of structural similarity of the domains from the same structure. Domains from the slipknotted CitS slightly differ in length—domain A is 189 amino acid long (from T49 to Y237) and domain B is shorter by 5 residues (H265 to E448). The domains of the unknotted protein are slightly shorter than those of its slipknotted counterparts since both of them are 172 amino acid long (domain A—G3-G174 and domain B—P215-E386). Superimposition of the domains from the same protein shows that the structural similarity between domain A and B is high for both proteins (RMSD of 2.2 Å over 74 aligned residues for slipknotted and 68 for unknotted protein). However, comparison of their sequences shows that they are substantially different. The domains of slipknotted SeCitS are identical only in 15.2% (similar in 27.2%). The results for the unknotted protein show a comparable level of identity (14.5%) and similarity (27.3%). Therefore, for both proteins, regardless of their topology, we observe high structural similarity of the domains with low sequence conservation.

Next, we analyzed whether the structural similarity of the domains is present also between the proteins. We found that in general, the domain of the slipknotted protein can be easily superimposed on the domain of the unknotted one (Fig 4D). Although spatial structures of the domains differ, there is a TM-helical region (core) which is structurally similar in both domains (RMSD of approx. 2Å over 80 aligned residues; Fig 4D). This region consists of 3 transmembrane helices. In the slipknotted structure it is located between TM3 and TM5 for domain A (G80-C165) and between TM9 and TM11 (H299-G385) for domain B. For unknotted protein the core is placed between TM2-TM4 (P30-G110) for domain A and TM9-TM11 (P237-T315) for domain B.

The difference between the domains of slipknotted and unknotted proteins is manifested in the conformation of TM6 from the slipknotted protein and the corresponding TM5 from the unknotted one (Fig 4). TM5 from the unknotted structure goes straight through the membrane, while TM6 from slipknotted protein bends in the middle because of its GGxG motif and creates a hairpin-like structure (Figs 3C and 4F). Therefore, it changes the direction of the polypeptide chain. Most importantly, the motif is conserved in the slipknotted family in both domains and is absent in unknotted proteins (S1 Fig). Consequently, the following C-terminal TM helices end up at opposite sides of the membrane.

The difference in the topology between slipknotted and unknotted structures is most probably caused by the aforementioned transmembrane helices (TM5 and TM6), as well as by the linker between the domains (Fig 4 and S2 Fig). In the structure of the unknotted protein, there is an additional TM7 helix and a short loop which connects the two domains. On the other hand, in the slipknotted structure, domains are connected by a long (30 amino acids) cytoplasmic loop, which makes a turn around the structure (S2 Fig). This long flexible loop most likely plays an important role in forming the slipknot topology, as it wraps like a lasso around the structure to form the knot.

Comparison of sequence profiles reveals complex connections within the superfamily

In order to trace the evolution of the slipknotted transporter and related proteins, we broadened our investigation and performed sequence analysis based on all protein families. We took all the protein sequences from 17 families and for each domain we calculated multiple sequence alignment (MSA) and a profile (Fig 5A, S3 and S4 Figs and S2 File). Next, based on the profile-profile comparison of separated domains of each family, we traced the evolutionary events that resulted in the present day diversity (see Fig 5B and S3 Fig). Domains within the slipknotted family showed weak similarity to each other. Domain B is most closely related to domain A from PF03601, to domain B from PF05684 and to LrgA (PF03788), a one-domain family. In contrast, a sequence of domain A from the slipknotted protein differs significantly from all other domains in our dataset, including its sister domain. The same lack of similarity between two domains belonging to the same family is also seen for several other families (PF01758, PF03547 and PF013593; Fig 5A and S3 Fig). Interestingly, these families have high sequence similarity based on the full-sequence clustering (S10 Fig). When divided, domains A and B are clustered separately, which indicates that the two regions diverged over time (Fig 5A and S3 Fig, yellow, magenta and pink circles, respectively). Moreover, domains from PF03547 are clustered with their counterparts from other families (domain A in a group with domains B and vice versa; Fig 5A and S3 Fig). This indicates that proteins belonging to this family are products of a reverse-order fusion.

Fig 5

Sequence and phylogenetic analysis of the domains suggests a common evolutionary origin.

Panel A shows clustering of domain profiles. Each family shown in a different color, connections indicate similarity found with e-value below 1e−5. Two domains of the slipknotted protein are shown as orange stars. All families from CL0062 are shown as circles colored according to the family. The family IDs with known unknotted topology are highlighted in blue font. The one-domain proteins (PF03788 and PF04172) are shown as triangles and family ID are colored in magenta font. Two domains of PF05145 (CL0142) are shown as red squares. Panel B shows the phylogenetic tree of the domains. Color-coding of tree branches is the same as for domains clustering on the panel A. Additionally, colored background areas serve to group the tree branches according to some properties: 1) families with known unknotted topology are highlighted with blue; 2) the slipknotted family is highlighted in orange; 3) the families with unknown topology are highlighted in light gray; 4) the families of one-domain proteins are highlighted with magenta.

It is worth noting that the lack of sequence similarity between domains does not occur in all protein families—the largest unknotted family (PF00999) has some of the domains A and B well connected in sequence based clustering, thus significantly similar (in sequence) to domains from other families and to themselves. Additionally, we also found outlaying protein families, that show significant similarity only between their own domains (PF06826, PF03601, and PF05145; Fig 5A and S3 Fig).

Multiple sequence alignment shows a highly conserved core region

In order to directly compare the sequences of all the domains, we created a tool that generates a Multiple Profile Alignment (a multiple sequence alignment of the profiles). Only profile-profile alignments with e-value lower than 10−3 were used in creating it. Because of that, domain A from PF05982, as well as both domains from PF03977, were removed from the analysis, as they showed no statistically significant similarity to any other domain.

Multiple alignment (S4 Fig) shows that all of the remaining domains align well. The best conserved region of the alignment corresponds precisely to the transmembrane helical core we discussed above, found in all 3D structures (Fig 4). It is feasible that the core serves as the functional unit and dates back to the common ancestor of all the analyzed proteins. Moreover, we found that N- and C-termini of the domains are the most diverse parts of the sequences. The N-terminal part often differs in the number of transmembrane helices between the families—from no helices in the structure from PF01758 family (PDBID: 1zuy), to one (PDBID: 4bwz) and two (PDBID: 4czb) in structures from PF00999. C-terminal part may be the region of subfunctionalization since it contains the helices located at the internal pore of transporters.

Phylogeny shows seven distinct evolutionary paths resulting in internally repeated proteins

Fig 5B presents a diagram showing the evolution of the repeated regions found in membrane transporters (four versions of the tree are available in Supplementary Materials, Figs 5–8). In principle, all the families from our analysis could be represented in the same organism (our taxonomical analysis has shown up to 13 found in the same organism family; see S9 Fig).

In most cases, an ancestral gene duplicated and later, in the second event, the two genes fused to encode the two-domain protein. Between the events, the genes individually evolved, therefore we observe substantial differences between their sequences. However, another possible path that we observed is a single internal duplication event or a fast fusion. In this case, the duplication occurred within the gene, or the duplication and fusion were nearly simultaneous, thus leading to a protein with two domains with highly similar sequences.

Overall, our data shows seven evolutionary scenarios within the studied group of transporters (shown in Figs 5 and 6):

Fig 6

Seven evolutionary scenarios found within the transporters.

1) No fusion—diversification of the ancestor gene leading to one-domain protein. 2) Long diversification—gene duplication followed by an extended period of diversification which lead to two domains with low sequence similarity (shown with red and blue). 3) Duplication of already fused protein (PF13593 speciated from PF01758, PF06965 from PF00999). 4) Reverse-order fusion—gene duplication and fusion in a reverse order. 5) Fusion of the domains from different lineages (shown with blue and green). 6) Fast fusion—gene duplication followed by an instant fusion (in families PF05145, PF06826 and PF03956). 7) Fusion of unrelated lineages (PF05982).

No fusion—diversification of the ancestor gene leading to one-domain protein. Based on the profile analysis (Fig 5A) we found that one-domain families LrgA (PF03788) and LrgB (PF04172) are distantly related to two-domain slipknotted and unknotted proteins. Therefore, these families should share a common ancestor. The single-domain LrgA and LrgB families did not fuse, but interestingly, a LrgA-LrgB fusion protein was found in plants.

Fast fusion—gene duplication followed by an instant fusion (in families PF05145, PF06826 and PF03956). In these families domains A and B have very similar sequences. Domains A and B of the families PF05145 (red squares), PF06826 (cyan circles) and PF03956 (dark green circles) are located next to each other on the profile comparison figure (Fig 5A and S3 Fig). This indicates that domains duplicated and fused very soon after the duplication, therefore they are still very similar, since they did not have time to diverge.

Reverse-order fusion—gene duplication and fusion in a reverse order. Unknotted families PF03547 and PF01758 are closely related based on full sequence similarity (S10 Fig). However, when sequences are divided into two domains, another picture emerges—domain A of PF03547 is more similar to domain B of PF01758 than to its sister domain B and also to domain A of PF01758 (Fig 5A and S3 Fig). It means that after gene duplication domains A and B fused in different order than in other families (not AB but BA).

Long diversification—gene duplication followed by an extended period of diversification (subfunctionalization). In slipknotted family PF03390 and unknotted PF01758, most of PF00999 and PF03812, the two domains are not similar in sequence. This means that after gene duplication there was a long period of diversification before the domains A and B fused.

Duplication of already fused protein (PF13593 speciated from PF01758). Full sequences of families PF13593 and PF01758 are very similar, partly identical (S10 Fig). The same is true for the separate domains—domain A of PF13593 is most similar to domain A of PF01758, domain B of PF13593 is most similar to domain B of PF01758 based on profile-profile comparison (Fig 5A and S3 Fig). In this case, a full two-domain protein got duplicated and further evolved into two families PF01758 and PF13593. Therefore, these families share a common recent two-domain ancestor.

Fusion of the domains from different lineages formed (PF03601, PF05684, PF03616, PF06965). Here two domains of one protein do not share significant similarity to each other but, instead, are more similar to the domains from another family. For example, in family PF03601 domain A is more sequentially similar to domains from families PF05684, PF03812 and PF03390 than to its sister domain B.

Fusion of unrelated lineages formed (PF05982). In this family domain B is not similar to any other domain from our dataset. It is not clear whether the second domain originated from the same common ancestor or not. One possible scenario is that domain B strongly diverged and sequence similarity is not detectable anymore. Another possible scenario is that domain B evolved from a different unrelated clan. It would be possible to determine which scenario is correct when the structure from this family is available.

In order to understand which families appeared earlier in the course of evolution, we projected the superfamily tree on the Tree of Life (S9 Fig). The results show that PF00999 and PF01758 families are the most widely distributed across the species. They are present in all three domains of life (Bacteria, Archaea and Eukaryota). Therefore, these two families are perhaps the oldest among the superfamily. On the other hand, families PF05145 from the clan CL0142 and slipknotted PF03390 are not found in Archaea, which indicates that these families evolved later. This is also supported by the fact that PF05145 and PF03390 represent two different Pfam clans, outside of the main CL0064. Therefore, our data suggest that the family with slipknot topology (PF03390) emerged later in evolution. Family PF03812 has only been found in Bacteria (however this might be due to lack of data). The fusion of LrgA and LrgB must have happened later in evolution since it is found only in plants.

Interestingly, according to identified phylogenetic tree and sequence profiles, one-domain LrgA is most closely related to domain B of the slipknotted family. Due to the fact that LrgA family is widely present on the tree of life while the slipknotted one is not, LrgA might evolved earlier than domain B. This suggests that the family with slipknot and one-domain LrgA share a common recent ancestor (Fig 5B and S5–S8 Figs).

Our profile analysis shows that there are also two-domain families that are significantly similar to the family of slipknotted proteins—PF03616, PF03601 and PF05684. Due to the lack of experimental data and resolved structures, the topology of these families’ proteins is not known. Based on our results we suggest that proteins homologous to slipknotted PF03390 could possess non-trivial topology as well. The same conclusion was derived earlier based on experimental studies and sequence similarity for one of the families (PF03616) [46, 47].

Potential function of slipknot topology

It has been shown that the position of the active site very often overlaps with the location of the knot [6]. The universal role of such topological constraints is not known, however, in the case of some families its direct influence on biological function has been observed [48-50].

There is no recent and up to date review article describing slipknotted proteins. Our search through the slipknotted proteins deposited in the KnotProt database shows that slipknot topology, and more precisely the slipknot loop, is located directly in the active site of many globular and transmembrane proteins [36]. Even though, the role of the slipknot loop is unknown for the family studied here, we can say that it is directly involved in the transport mechanism (Fig 3). From the available structures, captured in different conformations (inward and outward open), it can be seen that the movement of the hairpins is connected with the transport of molecules across the membrane [51]. Hairpin formed by TM12 is also a slipknot loop and contains the conserved GGxG motif (Figs 3 and 4). Both hairpins TM6 and TM12 are involved in the coordination of sodium ions and substrates. Thus the slipknot constraints could be responsible for strapping together the transmembrane helices to form a flexible but stable channel similarly as was suggested in [6]. Therefore, slipknot topology is likely important for the transport mechanism of the PF03390 family of transporters.

Possible mechanism of slipknotted membrane protein folding

Proteins with complex topology such as slipknot pose a challenge for folding theories [6, 52]. In general, there are at least three ways to form a simple slipknot topology. First, it could be formed randomly during protein folding and then be stabilized and moved to the native position. In the case of knotted proteins, it was shown that such a scenario is very unlikely. However, theoretically one could imagine that such random slipknot could be stabilized when packed inside a membrane.

The second mechanism is based on the formation of the knot first and then one terminal would have to be threaded back the knotted loop to form the slipknot loop. This mechanism involves crossing the topological barrier twice, thus it is very unlikely for both globular and membrane proteins.

Third, the slipknot topology can be created by the formation of a twisted loop through which slipknot loop passes, thus bypassing the knotted structure stage altogether. Indeed such flips of the loop over the protein’s core to form a slipknot motif were observed in the case of globular slipknotted proteins. Moreover such flipping (called also a mousetrap) was recognized as the key mechanism in knotting a protein with a Stevedore knot (61 type) [53], the Gordian knot (52 type) [54-56], as well as one of possible ways to form trefoil knot (31 type) [10, 17, 31, 57, 58].

Comparing the described mechanism with our slipknotted membrane protein one could see some similarity. The loop that flips over a portion of the protein in other slipknotted proteins such as in a thymidine kinase [10] can be equated with the linker found in slipknotted protein family studied here (CitS; Fig 3). Even though we are not sure when, during the folding process, such a flipping could happen, one possibility is to first form the twisted core of the protein and then push the slipknot loop through it during insertion into the membrane. The linker is missing in unknotted families, suggesting that these proteins fold in a different way.

As the question of slipknot folding remains open, other pathways are being proposed [11]. However, this pathway is the most complex one and further investigations are necessary to understand how it could be applied for transmembrane proteins.

Conclusions

We identified the family of transmembrane proteins (2HCT transporters, PF03390) as possessing non-trivial slipknotted topology, which was not reported before. Moreover, we found that this family is related to several other families that contain unknotted proteins. This unique case allowed us to conduct a qualitative and quantitative investigation of the evolutionary relationship between slipknotted and unknotted proteins from the monovalent cation-proton antiporters superfamily and to understand how the slipknot topology appeared. Based on our sequential and structural analysis of the proteins’ repeated domains, we established that both slipknotted and unknotted proteins evolved from a common one-domain ancestor (Fig 2). The distant homology is further supported by the existence of a common core region of three transmembrane helices found in all known structures from the studied families (regardless of the topology; Fig 4).

Intriguingly, we found that domains of three two-domain families (PF03616, PF03601 and PF05684) are similar in sequence to the slipknotted domain (Fig 5A and S3 Fig). Therefore, these proteins might possess non-trivial topology as well.

Our analysis shows that the current diversity of membrane transporters was achieved through several evolutionary scenarios that allowed for diversification from a common, one-domain ancestor. Therefore, other transmembrane proteins with slipknot and knot topology could have followed similar paths. Our analysis indicates that the evolution of two-domain slipknotted family 2HCT started with gene duplication, and after a long diversification period the two genes merged. It was only the fusion of two genes (coding unknotted proteins) that made this slipknot protein possible.

It has been shown that the knotted proteins, both membrane and globular, can also consist of inverted repeats [3]. It is worth noting that the first artificially knotted protein was constructed based on two inverted repeats, thus our finding, supported by newly developed bioinformatics methods, could be used to identify and design artificially knotted proteins [59].

We also briefly discuss possible folding of slipknotted membrane proteins, suggesting that identified flexible linker could facilitate transition from unknotted to slipknotted topology. We also point out a potential role of slipknot topology in discussed family.

To our knowledge this is the first research devoted to the homologous slipknotted and unknotted transmembrane proteins. The tools we have developed can now be used to investigate the evolution of other proteins.

Methods

Data set

We used profile search HHMER (Jackhmmer [44]) to find homologues of the protein families from Monovalent cation-proton antiporter superfamily. Our final data set comprised of 17 Pfam [60] families: PF03390 (slipknotted, two-domain), 13 (two-domain) families from the CPA_AT clan (CL0064: unknotted PF00999, PF06965 and PF01758; families of unknown structure/topology: PF03547, PF03601, PF03812, PF03977, PF03956, PF05684, PF05982, PF03616, PF13593, PF06826), PF05145 from CL0142 (structure unknown), and two families with only one domain—LrgA (PF03788) and LrgB (PF04172) (structure unknown), and sequences of the fusion LrgA/LrgB.

Sequence analysis

All sequences in the data set were filtered at 90% similarity, and only sequences (for two-domain proteins) with lengths deviating by at most half of their length from the average length for a given group were kept. This left us with 28 717 sequences. The families differ significantly in the number of sequences, as can be seen in the S10 Fig. For example, the largest family PF00999 counts 11708 sequences (in the final dataset), slipknotted family PF03390 counts 295 sequences (in the final dataset). After filtering, the sequences were clustered by similarity (using CLANS [61]) and aligned within each cluster (using PROMALS3D [62]). Each resulting alignment was then separated into domains based on the alignment to one of the known protein structures (representatives from PF00999, PF06965, PF01758 and PF03390 families). Each of the single-domain-alignments was used to create a sequence profile (output files from clustering of domain profiles can be found in the S3 File; due to the file size, the output showing full-length sequences can be obtained upon request).

Sequence profiles for each domain—that is a combination of protein family, domain within the sequence (N- or C-terminal if applicable) and CLANS cluster within that family, were used to create a Multiple Profile Alignment (MPA). As, to our knowledge, no software with this capability is available, we have created an in-house script [63] (source code at https://github.com/ilbsm/HHsearch-results-aligner) based on the principle of the maximum weight trace. As an input, we used pairwise profile-profile alignments generated by HHsearch, with the final MPA optimizing the agreement between them. Additionally, this approach simplifies the resulting alignment by removing sequence fragments that cannot be gainfully aligned to anything (thus expand the alignment without providing any additional data). The MPA was then transformed into a multiple sequence alignment by replacing the profile states with corresponding residues of a representative sequence from each domain (selected based on its agreement with the profile).

Phylogenetic tree construction

Finally, the multiple sequence alignment of the separated (i.e. treated as unrelated) domains was used to create a phylogenetic tree (using MrBayes; Fig 5B and S4 File) [64], with LG model and a characteristics matrix coding additional information from profile clustering results (S11 Fig) and N- and C-terminal regions similarity—for more details see Supplementary Materials (S5–S8 Figs and S1 File).

Sequence logos of both slipknotted and unknotted protein families.

Sequence logo of slipknot family PF03390 showing that multiple glycines are highly conserved across the whole family. The logos were generated from families multiple sequence alignments (available in Pfam) with WebLogo3.

(TIFF)

Click here for additional data file.

The linkers connecting two domains in slipknotted and unknotted structures.

Figure shows that slipknotted and unknotted proteins are composed of two inverted domains which are connected by the linker. Panel A shows slipknotted structure (PDBID: 5a1s). From left to right: domain A, linker and domain B are shown. Similarly, panel B-C shows the linkers between the domains in unknotted structures.

(TIFF)

Click here for additional data file.

Profile-profile comparison of the domains.

Comparison of domains profiles, shown at cut-off 1e-5. Every domain profile is shown as one point with different shapes: star, circle, triangle, square. The connections between domains are shown as straight lines. The connection indicates that profile-profile alignment of these domains has significance value 1e-5 or less. Every family is colored in unique color, same as in the main Fig 5A. Two domains of the slipknotted family are shown as orange stars. All families from CL0062 are shown as circles colored according to the family. The families IDs with known unknotted topology are highlighted in blue font. The one-domain proteins (PF03788 and PF04172) are shown as triangles and families IDs are colored in magenta font. Two domains of PF05145 (CL0142) are shown as red squares. Families (PF00999 (blue), PF01758 (yellow), PF3547 (pink), PF03616 (olive), PF06826 (dark cyan) were divided into several subgroups based on full sequence clustering (S10 Fig), therefore there are more than two domains in these families. Domain A and B of PF00999 have separated into two clear clusters.

(TIFF)

Click here for additional data file.

Multiple sequence alignment of the domains profiles.

(A) Multiple alignment of domains profiles revealed the conserved core region. Y-axis lists all families which pass through the alignment threshold 1e-3. Every line in the alignment represents family profile. On the X-axis sequence length colored from blue to red. White spaces in the alignment are present when no significant similarity was found between the profiles. The borders of the domain and conserved 3 TM helical core are indicated below the plot. The turn in TM12 forming the slipknot loop is highlighted by black rectangle and next to it the multiple sequence alignment with conserved GGxG region is shown. (B) Schematic representation of individual domains of slipknotted and unknotted proteins.

(TIFF)

Click here for additional data file.

Bayesian phylogenetic tree 2.

The tree was generated from multiple sequence alignment of the domains using the characteristics matrix multiplied 10 times. The characteristics matrix (S11 Fig) was generated based on profile-profile connections (S3 Fig). For the tree calculation three representative sequences of each family were used. The tree shows several main branches: 1) Both domains of the slipknotted family PF03390 and one-domain family PF03788 are located on the same branch; 2) Another separated branch joins closely related families PF01758, PF013593 and PF3547; 3) Domains A and B of the unknotted family PF00999 were separated into two branches. Domains B of PF00999 are grouped together with the domain B of unknotted family PF06965 and with domain A of PF05684 (unknown topology). Domain A and B of families PF03956, PF06826, PF05145, domain B of PF05982 are located in one branch.

(TIFF)

Click here for additional data file.

Bayesian phylogenetic tree 3.

The tree was generated from multiple sequence alignment of the domains using the characteristics matrix multiplied 5 times. The characteristics matrix was generated based on profile-profile connections (Fig 5A and S3 Fig). The characteristics matrix is shown on S11 Fig. For the tree calculation 10 representative sequences of slipknotted PF03390 and one-domain families (PF03788, PF04172) and three representative sequences of remaining families were used. The tree shows three main branches: 1) domains A and B of slipknotted family PF03390 and one-domain family PF03788 are located together on one branch; 2) Another separated branch joins closely related families (domains A and B) PF01758, PF013593 and PF3547; 3) Domains A and B of the unknotted family PF00999 are located on different branches. Domain A of another unknotted family PF06965 is located together with domains A of PF00999 and domain B of PF06965 is placed together with domain B of PF00999.

(TIFF)

Click here for additional data file.

Bayesian phylogenetic tree 4.

The tree was generated from multiple sequence alignment of the extended conserved core (S4 Fig) which includes the conserved 3-TM helical core + 1 next TM helix (hairpin in slipknotted family). The characteristics matrix based on the profile-profile connections was used as in trees S5 and S6 Figs. Additionally, removed N- and C-terminal regions of the domains were introduced into tree calculation as N/C matrices. Characteristics matrix and N/C matrices were multiplied 10 times. For the tree calculation 10 representative sequences of slipknotted PF03390 and one-domain families (PF03788, PF04172) and three representative sequences of remaining families were used. The tree shows three main branches: 1) slipknotted family PF03390 is placed together with both one-domain families PF03788 and PF04172; 2) Also, as previously, a separated branch joins closely related families PF01758, PF013593 and PF3547; 3) Domains A and B of the unknotted family PF00999 are separated on the tree. However, evolution of other families is not resolved in this tree. PF04172 is placed on the same branch with PF03788 and according to profile analysis these families are distantly related. Also, PF03812(B) and PF03601(A) are together with PF00999(B) which is also in agreement with profile analysis.

(TIFF)

Click here for additional data file.

Parsimony phylogenetic tree.

The parsimony tree was generated from the extended core region (conserved core + 1 next TM helix (hairpin in slipknotted family)) same as in the Bayesian phylogenetic tree (S7 Fig). Eight families were used for this analysis: PF00999 (unknotted), PF01758 (unknotted), PF03547 (unknown topology), PF13593 (unknown topology), PF03390 (slipknotted), PF03788 (one-domain), PF04172 (one-domain) and two-domain family PF05145 from the clan CL0142. Five representative sequences of all eight families were used for tree construction. The tree shows four main branches with duplication events producing families: 1) slipknotted family PF03390 and one-domain family PF03788; 2) two-domain closely related families PF01758, PF013593 and PF3547; 3) unknotted PF00999 with two subbranches separating domains A and B; 4) two-domain family PF05145 from another clan (CL0142) was placed on the separate branch. In this tree one-domain family PF04172 is placed together with families PF03547, PF1758 and PF13593 that is in agreement with our profile analysis. The connections between these families are found at e-value 1e-3.

(TIFF)

Click here for additional data file.

Projection of the phylogenetic tree of all 17 families studied here on the Tree of Life.

The phylogenetic tree (Fig 5B) is projected on the Tree of Life. Each protein family is colored by unique color similarly as in CLANS (Fig 5A). Presence of the protein family in a particular group of organism is shown by a cross sign “X”.

(TIFF)

Click here for additional data file.

Comparison of unaligned full-length sequences in CLANS.

Full set of unaligned sequences of all 17 families were submitted into CLANS all-against-all BLAST search. A single sequence is represented by one “dot”, circle. The sequences clustered in 3D based on pairwise similarity. Mostly, sequences clustered well into families similarly as was assigned by Pfam. Each Pfam family is shown in unique color. The same colors are used in profile-profile analysis (Fig 5A and S3 Fig). The largest (and also unknotted) family PF00999 was clustered into seven groups, all groups colored in blue. The sequences of slipknotted family PF03390 (colored in orange) clustered into one group. The families IDs with unknotted topologies are highlighted with blue font (PF00999, PF06965, PF01758). The family PF05145 from the clan CL0142 is colored red. The one-domain families IDs (LrgA—PF03788, LrgB—PF04172) are highlighted with magenta font. The fusion LrgA/LrgB that partially co-localize with PF04172 (LrgB) is shown in magenta. Some of one-domain LrgA (PF03788) sequences are located in between fusion protein and LrgA, marked with the star sign “*”.

(TIFF)

Click here for additional data file.

Clustering-based characteristics matrix.

Characteristics matrix used for Bayesian phylogenetic trees generations. The matrix was generated based on the profile-profile connections at the lowest cut-off 1e-5 (Fig 5A and S3 Fig).

(TIFF)

Click here for additional data file.

Methods.

Sequence search. Sequence analysis. Procedure to identify the domains. Sequence profiles. Multiple sequence alignment. Phylogeny tree reconstruction. Visualization and figures preparation.

(PDF)

Click here for additional data file.

Multiple sequence alignment of domain sequences.

(TXT)

Click here for additional data file.

Output from clustering of the domain sequences.

(TXT)

Click here for additional data file.

Phylogenetic tree from MrBayes.

(TXT)

Click here for additional data file.

15 Sep 2020

Dear DR Sulkowska,

Thank you very much for submitting your manuscript "Slipknotted and unknotted monovalent cation-proton antiporters evolved from a common ancestor" for consideration at PLOS Computational Biology.

As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by three independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Please note that all reviewers (but specially reviewer 1) pointed out deficiencies regarding the structure of your manuscript and presentation of the results that should be fully taken into account in the revised version. Note as well that there is missing information and data, which must be provided in the revised version, as well as the source code, which is not available.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

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Guest Editor

PLOS Computational Biology

Stefano Allesina

Deputy Editor

PLOS Computational Biology

***********************

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: In the manuscript “Slipknotted and unknotted monovalent cation-proton antiporters evolved from a common ancestor” by Zayats et al. the authors study the evolutionary origin of the slipknot topology in proteins. After identifying several protein families, which show (slip)knotted and unknotted topologies, their phylogenetic relationship and evolutionary history is analysed and evolutionary scenarios, leading to the slipknot topology coming from an unknotted ancestor, are inferred.

In its current form, the manuscript is unfortunately not suited to provide new insights into the origin(s) of this special slipknot topology, due to deficiencies in the presentation of the results.

The following comments about the, in my opinion, major issues and shortcomings of the manuscript hopefully help to clarify several points to make the manuscript finally comprehensible and the approach reproducible for a broader audience:

Language:

The manuscript needs excessive proof reading (preferably by a native speaker), since not only many spelling mistakes and wrong words or grammar hinder comprehension, but also lead to ambiguous meanings of sentences or render the understanding of presented results or conclusions impossible.

Additionally, many used words have specific meanings in the field of evolutionary biology (e.g. pathway), which differ from the context in which they are used here. This can lead to confusion and should be revised (see also Consistency and Structure paragraphs).

Consistency and definition of terms:

Many terms are not used consistently throughout the manuscript and make an understanding very difficult. Most of these terms are also never explained or just after they were used already multiple times (see also Structure paragraph). Especially the terms or IDs related to the Pfam database (Pfam, pfam, PFAM, PFam etc. all exist btw → consistency) are likely not known (with their definitions) by most people and should be explained properly to provide guidance to interpretation and comprehension of the findings. PFxxxx and CLxxxx are used for example extensively in sentences, but if I wouldn’t know that these are IDs of Pfam entries or corresponding Pfam clan IDs, respectively (which is never explained), the findings or conclusions based on this are not understandable at all. The same with the use of family/repeat/domain – I have the feeling here the HMM-types from the Pfam database are meant, but it is not common knowledge how these are defined within the Pfam database. Especially, the use of “repeat” should be carefully re-evaluated, since it is often not clear if a (tandem-)repeated domain in a certain HMM/family/protein is meant or a single instance of that “Repeat” (type in the Pfam database).

Colour-coding in and across figures could be improved to ensure consistency as well.

Structure:

Although most information is provided somewhere in the manuscript, it takes multiple readthroughs to find the according explanations or used methods and understand afterwards the related sentences.

If the Methods are at the very end of the manuscript, some simple explanations what has been done and how, needs to go into the Results section. The Results section contains furthermore at the beginning parts that are introduction and throughout the section parts, which are Discussion. Especially the presentation of the Results should be restructured completely (with consistency and definition of terms at first appearance in mind; see above).

Missing information:

Some information is missing completely and should be provided to make the conclusions comprehensible and reproducible. Especially in the beginning of the Results section: where have how many sequences been taken from and analysed/how big are the analysed families/what are their properties? Description and explanation of the methodology (how were the 7 evolutionary scenarios inferred?). The gitlab repo linked in the manuscript is not existent, therefore I couldn’t have a look at the source code and see what has been done. In many figures information or explanation is missing (double-check also Supplementary Info, S12 e.g. does not provide any information of what is shown (headers of the columns or description and meaning of the character states) etc. etc.). The role of repeats (and potential tandem repeats formed by tandem duplications rather than “fusion” of two single-domain proteins) should be explained and visualised. The provided Pfam-IDs can represent HMMs for example, which span already multiple instances of one repeat. How many repeat instances are included in the single-coloured bars in the figures or the according mentioned Pfam-IDs in the text does not become clear. Also, it should be clearly indicated in the text when conservation/similarity is discussed/considered from a structural or sequence perspective. Most conclusions made in the Results section are not comprehensible (probably because the methods behind it are missing) and should be elaborated. One of the conclusions is that observable patterns are biased “due to lack of data”; has any quality assessment been conducted to estimate this bias?

Finally, it should be made very clear what has been found for the first time in this study and what is actually the achievement of the authors in this study. The text reads in its current version very cryptic regarding the contribution (identification of a “new” slipknotted family, but all that based on existent Pfam-IDs and clans and PDB structures?), which makes it hard to distinguish between discussed former findings and the findings of this study.

Reviewer #2: In this manuscript, Zayats et al. investigated the evolution of a family of Slipknotted proteins, i.e., 2-hydroxy carboxylate transporter family. Importantly, they found distantly related protein families with unknotted topology and used this divergence to identify the scenarios behind the evolution of Slipknotted proteins. Overall, this is an interesting study and provides important details for the evolution of protein topology in a specific case. The manuscript, though, can benefit from a revision to enhance clarity and presentation. Here is the list of my major and minor comments:

Major comments:

1) The evolution of membrane transporters from one-domain proteins is not surprising and thoroughly discussed in the literature. I encourage authors to focus on the mechanisms of the divergence of Slipknotted and unknotted proteins and the fact that they could identify different scenarios that explain this divergence.

2) The major assumption behind this work is that the difference in the topology of slipknotted and unknotted proteins is caused by differences between TM helices and loops. Although this assumption sounds plausible based on previous studies (cited by the authors), authors should elaborate and justify why this assumption likely holds. They may use the current dataset and employ statistical analyses to see whether TMs and the loops contribute the most to such divergence.

Minor comments:

1) The manuscript is well-written but can greatly benefit from the substantiation of important concepts. For example, the authors should explain why knotted proteins are interesting and cover the most important literature on this problem.

2) Authors write "It was shown, that globular, as well as transmembrane, slipknotted proteins often appear in new organisms" in Lines 13-14. This is an overstated claim as the cited paper is only talking about the one protein family (2-Hydroxycarboxylate Transporter Family).

3) The domains and regions that were used for evolutionary analyses should be clarified in the text. The current writing can confuse the readers. For example, authors write in lines 48-49 "thus we used a single repeat from each structure for comparison". The sequence position should be stated clearly.

4) I found figures S2 and S3 (of the supplementary information) quite informative. Why don't authors bring up these figure to the main text and discuss them when they write about the superposition of knotted and unknotted proteins (Lines 48-63).

5) In constructing figures S5 and S6 and Fig. 3, it appears that the authors used the full sequence of N- and C-repeats. Would their clustering change if they only look at TMs and the connecting loops?

Reviewer #3: The article scrutinizes evolutionary relations between unknotted and slipknotted protein domains by using the sodium-citrate transporter as an example of a protein with a slipknotted domain. The topic of the article is very interesting, and it deserves publication in PLOC Computational Biology after revision.

Before the publication, several points should be addressed.

General Comments:

The manuscript should be amended to make it understandable to the general reader of PLOS Computational Biology.

Specifically, the difference between the unknotted, knotted and slipiknotted domains should be clearly explained in the Introduction section of the main text. The Fig. 1 is not quite successful. It does not explain how a knot is formed from a flat sequence profile shown on the top. Perhaps, intermediate steps of the knot formation should be shown. The Fig. S1 can be used in the main text to make the explanation clearer.

Also, the topology of the citrate transporter should be explained in more detail. This protein consists of functionally distinct scaffold and elevator domains. From the current text it is not clear whether all the analysis is related to the whole protein or only to its elevator domain. Therefore, the overall topology of the protein should be described and shown. Only after that, the evolutionary relation of the slipknotted (elevator?) domain to other proteins (domains?) should be analyzed in detail. Fig. S2 could be also used in the main text as an explanatory image albeit after modification. Currently there is hardly any difference between the panels S2A and S2C (left).

Generally, the authors should imagine a biologist without any idea about slipknotted domains and write for such a reader.

Specific Comments:

1. In the given family of transporters, the authors describe proteins with unknotted and slipknotted domains. It remains, however, unclear whether it is possible – topologically - to get to a slipknotted domain directly from an unknotted domain or the evolutionary transition should go via a knotted intermediate. Discussion of this point is highly desirable.

2. Line 104 and after: the complete multiple alignment with all amino acid sequences should be provided in Supplementary materials.

3. Fig. 3. Something is wrong with the color code in Fig. 3B. There are colors that are not mentioned in the figure caption.

4. Line 152: a word appears to miss after “widely”.

5. Line 167 and after: PDB IDs of the four known structures of relevant proteins should be explicitly given somewhere in the manuscript. The reference to Fig. 3B is not enough; neither the figure not the caption contains the PDB IDs.

6. The Discussion section is very brief. It might be interesting to discuss whether the slipknotting brings any functional advantages.

7. The discussion of PF03616, PF03601 and PF05684 should be moved from Conclusions into Discussion.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: No: The gitlab repo with the source code provided as a link in the manuscript does not exist.

Reviewer #2: Yes

Reviewer #3: No: The multiple amino acid sequence alignment is absent

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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15 Apr 2021

Submitted filename: Reviewer_response.docx

Click here for additional data file.

3 May 2021

Dear DR Sulkowska,

Thank you very much for submitting your manuscript "Slipknotted and unknotted monovalent cation-proton antiporters evolved from a common ancestor" for consideration at PLOS Computational Biology. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Patrícia F. N. Faísca, Ph.D

Guest Editor

PLOS Computational Biology

Stefano Allesina

Deputy Editor

PLOS Computational Biology

***********************

A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact ploscompbiol@plos.org immediately:

[LINK]

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #2: I appreciate the authors' efforts to answer all the raised concerns. The manuscript is substantially improved and merits publication in PLoS Computational Biology. The figures' quality, however, could still be improved and I suggest authors and the editorial office to consider this issue.

Reviewer #3: General comments

1. The problem with the manuscript is still that some of its sections are incomprehensibly written. Apparently, experts in topology of knots, while applying their science to proteins, have developed their own terminology, which differs from that generally accepted in protein science. Since the manuscipt deals not only with the topology but also with the evolution of a large family of membrane transporters, the article should be understandable to researchers who work on these transporters. There is no reason to expect these researchers to know the mathematical theory of knots.

The terms "entlargement", "closed knot", "open knot" and "subchain of the backbone" are unusual in protein science and should be strictly defined in the text or replaced by more traditional and understandable analogues.

2. This reviewer recommends that the first paragraph of the results (or better, the whole text) be given to some biochemist to read and then edited according to the recommendations received.

3. The structures shown in Figures 3A and 3C are superposed in Fig. 3B. However, the green structure from Fig. 3A is painted blue in Fig. 3B, and the blue structure from Fig. 3C is painted green in Fig. 3B. The colouring of the structures in Fig. 3B should be changed.

Reviewer #4: This manuscript describes a broad family of transmembrane proteins where a slipknot has been found, along with homologues that are unknotted. Much of the paper deals with ideas about gene duplication and fusion for evolution in this family. The strength of the study is that the observation is intriguing, raising interesting evolutionary and functional questions. The limitation is that whether knotting features are important in proteins remains mainly speculative. Nonetheless, the evolutionary arguments are interesting, and the findings should be useful to those interested in transmembrane transporter function and evolution.

The revisions noted appear to have been largely effective at improving the original paper. Two issues for further improvement, one regarding substance and clarity and the other regarding presentation are:

1) The topological features that distinguish a slipknot from an unknot in a protein chain are hard to grasp even for experts and certainly for newcomers. The figures provided go part way to explaining things, but my sense is that most readers will remain somewhat befuddled. I suggest that the authors augment figure 4 with a simplified curve drawing for the knotted and unknotted curves, with a focus on explaining how two structurally similar cases can be topologically different. For example, can a diagram be drawn that might show that the difference arises from one structure having a curve crossing one way while the other structure has a curve crossing the other way. A diagram like this would go along way towards drawing in more readers with a connection between the sequence/structure evolution and the topological difference being reported.

2) The overall clarity of the paper is ok, and the mechanics are generally good. But some further editing is required. Maybe this can be done safely at the technical editor level. One point to focus on is the use of commas, which are not placed well in many instances (lines 188, 220, 308, and others). The negative use of “for none of these” in line 39 should be reworded. In line 143, ‘spacial’ should be ‘spatial’. In line 315, ‘constrains’ should be ‘constraints’. Words appear to be missing in line 327.

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Have the authors made all data and (if applicable) computational code underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data and code underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data and code should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data or code —e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #3: No

Reviewer #4: No

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24 Sep 2021

Submitted filename: Review_response_27_08_21.docx

Click here for additional data file.

28 Sep 2021

Dear DR Sulkowska,

We are pleased to inform you that your manuscript 'Slipknotted and unknotted monovalent cation-proton antiporters evolved from a common ancestor' has been provisionally accepted for publication in PLOS Computational Biology.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Computational Biology.

Best regards,

Patrícia F. N. Faísca, Ph.D

Guest Editor

PLOS Computational Biology

Stefano Allesina

Deputy Editor

PLOS Computational Biology

***********************************************************

11 Oct 2021

PCOMPBIOL-D-20-01125R2

Slipknotted and unknotted monovalent cation-proton antiporters evolved from a common ancestor

Dear Dr Sulkowska,

I am pleased to inform you that your manuscript has been formally accepted for publication in PLOS Computational Biology. Your manuscript is now with our production department and you will be notified of the publication date in due course.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript.

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Thank you again for supporting PLOS Computational Biology and open-access publishing. We are looking forward to publishing your work!

With kind regards,

Andrea Szabo

PLOS Computational Biology | Carlyle House, Carlyle Road, Cambridge CB4 3DN | United Kingdom ploscompbiol@plos.org | Phone +44 (0) 1223-442824 | ploscompbiol.org | @PLOSCompBiol