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Cited by 13Protein Structure and DynamicsMachine Learning in BioinformaticsGeometric and Algebraic Topology

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Knot or not? Identifying unknotted proteins in knotted families with sequence‐based Machine Learning model

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Sikora, Maciej, Klimentova, Eva, Uchal, Dawid, Sramkova, Denisa, Perlinska, Agata P., Nguyen, Mai Lan, Korpacz, Marta, Malinowska, Roksana, Nowakowski, Szymon, Rubach, Pawel, Simecek, Petr, Sulkowska, Joanna I.·2024·Protein Science
DOI 10.1002/pro.4998PMC PMC11184937

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Abstract

Knotted proteins, although scarce, are crucial structural components of certain protein families, and their roles continue to be a topic of intense research. Capitalizing on the vast collection of protein structure predictions offered by AlphaFold (AF), this study computationally examines the entire UniProt database to create a robust dataset of knotted and unknotted proteins. Utilizing this dataset, we develop a machine learning (ML) model capable of accurately predicting the presence of knots in protein structures solely from their amino acid sequences. We tested the model's capabilities on 100 proteins whose structures had not yet been predicted by AF and found agreement with our local prediction in 92% cases. From the point of view of structural biology, we found that all potentially knotted proteins predicted by AF can be classified only into 17 families. This allows us to discover the presence of unknotted proteins in families with a highly conserved knot. We found only three new protein families: UCH, DUF4253, and DUF2254, that contain both knotted and unknotted proteins, and demonstrate that deletions within the knot core could potentially account for the observed unknotted (trivial) topology. Finally, we have shown that in the majority of knotted families (11 out of 15), the knotted topology is strictly conserved in functional proteins with very low sequence similarity. We have conclusively demonstrated that proteins AF predicts as unknotted are structurally accurate in their unknotted configurations. However, these proteins often represent nonfunctional fragments, lacking significant portions of the knot core (amino acid sequence).

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Figures

FIGURE 1 Scheme of the machine learning workflow. The entire approach consists of three main steps: 1. Obtaining data from PDB (with support of Knotprot) and AlphaFold (with support of AlphaKnot) predictions for UniProt; 2. Identifying nontrivial topology in the protein backbone using Topoly; 3. Training a machine learning model to predict nontrivial topology from the protein sequence.
FIGURE 1 Scheme of the machine learning workflow. The entire approach consists of three main steps: 1. Obtaining data from PDB (with support of Knotprot) and AlphaFold (with support of AlphaKnot) predictions for UniProt; 2. Identifying nontrivial topology in the protein backbone using Topoly; 3. Training a machine learning model to predict nontrivial topology from the protein sequence.CC BY · PubMed Central
FIGURE 2 Unknotted proteins aligned with their knotted homologs. The schematic on the left represents the sequential alignment between knotted (green) and unknotted (black) proteins. The region with the knot is marked with a gray area. The unknotted proteins that start or end prematurely in comparison to their knotted counterpart are discarded from further analysis (see the section about SPOUT superfamily for details). We focus on cases in which the unknotted protein can be globally aligned with the knotted homolog (even with a deletion in the knot core) or does not have any knotted homologs (the numbers of these proteins are bolded).
FIGURE 2 Unknotted proteins aligned with their knotted homologs. The schematic on the left represents the sequential alignment between knotted (green) and unknotted (black) proteins. The region with the knot is marked with a gray area. The unknotted proteins that start or end prematurely in comparison to their knotted counterpart are discarded from further analysis (see the section about SPOUT superfamily for details). We focus on cases in which the unknotted protein can be globally aligned with the knotted homolog (even with a deletion in the knot core) or does not have any knotted homologs (the numbers of these proteins are bolded).CC BY · PubMed Central
FIGURE 3 Unknotted membrane proteins from Sodium/calcium exchanger family superposed with their knotted homologs. Upper part: unknotted structure (light orange; UniProtKB ID: A0A5K0X7L3) without the entire knot core, unknotted structure (orange; UniProtKB ID: A0A397EWI1) structure lacking the C‐terminal part of the knot core, and unknotted structure (green; UniProtKB ID: A0A562DKV6) with low‐quality part of the knot core. Lower part: unknotted structure (cyan; UniProtKB ID: A0A453N937) without the N‐terminal and C‐terminal part of the knot core, unknotted structure (magenta; UniProtKB ID: A0A378FTV0) without the N‐terminal part of the knot core, and unknotted structure (light pink; UniProtKB ID: K1RUG8) without the entire knot core. All knotted homologs are shown with gray color (UniProtKB IDs: V4U0P6, A0A397A8U0, E6WVS2, A0A453N959, A0A0E1C8E8, and A0A0L8I7H8, respectively).
FIGURE 3 Unknotted membrane proteins from Sodium/calcium exchanger family superposed with their knotted homologs. Upper part: unknotted structure (light orange; UniProtKB ID: A0A5K0X7L3) without the entire knot core, unknotted structure (orange; UniProtKB ID: A0A397EWI1) structure lacking the C‐terminal part of the knot core, and unknotted structure (green; UniProtKB ID: A0A562DKV6) with low‐quality part of the knot core. Lower part: unknotted structure (cyan; UniProtKB ID: A0A453N937) without the N‐terminal and C‐terminal part of the knot core, unknotted structure (magenta; UniProtKB ID: A0A378FTV0) without the N‐terminal part of the knot core, and unknotted structure (light pink; UniProtKB ID: K1RUG8) without the entire knot core. All knotted homologs are shown with gray color (UniProtKB IDs: V4U0P6, A0A397A8U0, E6WVS2, A0A453N959, A0A0E1C8E8, and A0A0L8I7H8, respectively).CC BY · PubMed Central
FIGURE 4 Overview of protein families with the potential of having both knotted and unknotted topologies. (a) Family DUF4253. A deletion (in red) aligning to the knot loop in the nontrivial homolog (UniProtKB ID: A0A1F0KRL5) creates an unknotted structure (B0C9T3). (b) DUF2254 family. The C‐terminus (in red) instead of passing through the loop (in blue) as in the knotted structure (A0A848U8E4), turns back creating a slipknot—an unknotted structure (D5C1U6). (c) UCH family. Compared with the standard structure with 52 topology (A0A0E0DGQ1), either we observe a different arrangement of the loop committing one crossing (in red), which simplifies the topology to 31 (A0A0E0DGQ2) or the fragment of N‐terminus is missing, which simplifies the topology to an unknot (A0A178ZI05). (d) Sodium/calcium exchanger transmembrane protein family. In the unknotted protein (A0A2G1VBT6) we observe the deletion of the fragment (in red) corresponding to the loop knot in the nontrivial homolog (A0A2S5ZE01).
FIGURE 4 Overview of protein families with the potential of having both knotted and unknotted topologies. (a) Family DUF4253. A deletion (in red) aligning to the knot loop in the nontrivial homolog (UniProtKB ID: A0A1F0KRL5) creates an unknotted structure (B0C9T3). (b) DUF2254 family. The C‐terminus (in red) instead of passing through the loop (in blue) as in the knotted structure (A0A848U8E4), turns back creating a slipknot—an unknotted structure (D5C1U6). (c) UCH family. Compared with the standard structure with 52 topology (A0A0E0DGQ1), either we observe a different arrangement of the loop committing one crossing (in red), which simplifies the topology to 31 (A0A0E0DGQ2) or the fragment of N‐terminus is missing, which simplifies the topology to an unknot (A0A178ZI05). (d) Sodium/calcium exchanger transmembrane protein family. In the unknotted protein (A0A2G1VBT6) we observe the deletion of the fragment (in red) corresponding to the loop knot in the nontrivial homolog (A0A2S5ZE01).CC BY · PubMed Central
FIGURE 5 Unknotted SPOUT proteins (a) superposed with their knotted homologs. Upper part: unknotted structure (magenta; UniProtKB ID: A0A418H8R5) lacking the N‐terminal part of the knot core. Lower part: unknotted structure (blue; UniProtKB ID: A0A090WL68) without the entire knot core, and unknotted structure (orange; UniProtKB ID: A0A356CS99) without the C‐terminal part of the knot core. All knotted homologs are shown with gray color and their knotted regions are marked (UniProtKB IDs: A0A2X1NEC8, A0A090W861, and A0A3D0W667, respectively). Since all of these unknotted proteins lack at least a portion of the knot core, and the knot core is necessary for the SPOUT protein to function, we presume that these proteins are not truly unknotted. (b) Left: an example of a SPOUT protein with a typical 31 knotted structure (UniProtKB ID: R1DT92). Middle: structure with a 31 slipknot topology (UniProtKB ID: A0A424QRT9). The slipknot is formed by a C‐terminal end (colored blue) threaded back through the knot loop. The structure of the knot (colored green) remains typical for the SPOUT superfamily. Right: unknotted structure due to a deletion in the knot core (UniProtKB ID: R1FAN9). The presented structures are models from the AlphaFold database (4th version).
FIGURE 5 Unknotted SPOUT proteins (a) superposed with their knotted homologs. Upper part: unknotted structure (magenta; UniProtKB ID: A0A418H8R5) lacking the N‐terminal part of the knot core. Lower part: unknotted structure (blue; UniProtKB ID: A0A090WL68) without the entire knot core, and unknotted structure (orange; UniProtKB ID: A0A356CS99) without the C‐terminal part of the knot core. All knotted homologs are shown with gray color and their knotted regions are marked (UniProtKB IDs: A0A2X1NEC8, A0A090W861, and A0A3D0W667, respectively). Since all of these unknotted proteins lack at least a portion of the knot core, and the knot core is necessary for the SPOUT protein to function, we presume that these proteins are not truly unknotted. (b) Left: an example of a SPOUT protein with a typical 31 knotted structure (UniProtKB ID: R1DT92). Middle: structure with a 31 slipknot topology (UniProtKB ID: A0A424QRT9). The slipknot is formed by a C‐terminal end (colored blue) threaded back through the knot loop. The structure of the knot (colored green) remains typical for the SPOUT superfamily. Right: unknotted structure due to a deletion in the knot core (UniProtKB ID: R1FAN9). The presented structures are models from the AlphaFold database (4th version).CC BY · PubMed Central
FIGURE 6 Unknotted UCH proteins superposed with their knotted homologs. Upper part: unknotted structure (orange; UniProtKB ID: A0A7S3I8E7) lacking the C‐terminal part of the knot core. Lower part: unknotted structure (cyan; UniProtKB ID: Q06AV7) without the N‐terminal and C‐terminal part of the knot core, unknotted structure (magenta; UniProtKB ID: A0A1A7YJ99) without the N‐terminal part of the knot core, and unknotted structure (green; UniProtKB ID: A0A024WZA) with low‐quality part of the knot core. All knotted homologs are shown with gray color (UniProtKB IDs: A0A078B145, A0A6P6PV05, A0A1A7X9F0, and W7F0M6, respectively).
FIGURE 6 Unknotted UCH proteins superposed with their knotted homologs. Upper part: unknotted structure (orange; UniProtKB ID: A0A7S3I8E7) lacking the C‐terminal part of the knot core. Lower part: unknotted structure (cyan; UniProtKB ID: Q06AV7) without the N‐terminal and C‐terminal part of the knot core, unknotted structure (magenta; UniProtKB ID: A0A1A7YJ99) without the N‐terminal part of the knot core, and unknotted structure (green; UniProtKB ID: A0A024WZA) with low‐quality part of the knot core. All knotted homologs are shown with gray color (UniProtKB IDs: A0A078B145, A0A6P6PV05, A0A1A7X9F0, and W7F0M6, respectively).CC BY · PubMed Central
FIGURE 7 Important regions in SPOUT proteins based on machine learning model. (a) An example of how the model's prediction changes with respect to the patch location based on Nep1 protein (patches of length 10; UniProtKB ID: Q57977). Note the one significant drop inside the knot core. (b) Cumulative positions of the patches (with different lengths: 10, 20, and 40 amino acids) based on the knot core alignment. (c) Logo of the C‐terminal part of the knot core showing the conserved glycine motif. (d) Positions of patches in a TrmD structure (based on the N‐terminal domain of AlphaFold model of UniProtKB ID: B6YQ31). Colored regions represent from the left: knotted region (rainbow) and estimated location of the ligand (magenta); 10 (red), 20 (yellow), and 40 (green) amino acid‐long patches, respectively. Black dots show the position of the conserved glycine residues.
FIGURE 7 Important regions in SPOUT proteins based on machine learning model. (a) An example of how the model's prediction changes with respect to the patch location based on Nep1 protein (patches of length 10; UniProtKB ID: Q57977). Note the one significant drop inside the knot core. (b) Cumulative positions of the patches (with different lengths: 10, 20, and 40 amino acids) based on the knot core alignment. (c) Logo of the C‐terminal part of the knot core showing the conserved glycine motif. (d) Positions of patches in a TrmD structure (based on the N‐terminal domain of AlphaFold model of UniProtKB ID: B6YQ31). Colored regions represent from the left: knotted region (rainbow) and estimated location of the ligand (magenta); 10 (red), 20 (yellow), and 40 (green) amino acid‐long patches, respectively. Black dots show the position of the conserved glycine residues.CC BY · PubMed Central

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Metadata

Authors
Sikora, M., Klimentova, E., Uchal, D., Sramkova, D., Perlinska, A. P., Nguyen, M. L., Korpacz, M., Malinowska, R., Nowakowski, S., Rubach, P., Simecek, P., & Sulkowska, J. I.
Affiliations
  • University of Warsaw (PL)
  • Central European Institute of Technology, Masaryk University Brno Czech Republic
  • Centre of New Technologies, University of Warsaw Warsaw Poland
Corresponding author
Sulkowska, Joanna I.
ORCID
Venue
Protein Science
Publisher
Wiley
Published
2024-06-18
Volume
33
Issue
7
Pages
e4998-e4998
ISSN
  • 0961-8368 (ISSN-L)
  • 1469-896X
DOI
10.1002/pro.4998
PMC
PMC11184937
OpenAlex
W4399776685
Cited by
13
References
3
Citations by year
2026: 3 · 2025: 5 · 2024: 5
FWCI
1.77
Type
journal article
Language
en
Open access
Licence
Locations
  • Protein Science (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • ReIn UW (University of Warsaw) (submittedVersion, cc-by-nc-nd)
Funders
  • Narodowe Centrum Nauki (UMO‐2018/31/B/NZ1/04016, 2021/43/I/NZ1/03341)
  • Grantová Agentura České Republiky (23‐04260L)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Machine Learning in Bioinformatics
  • Physical Sciences › Mathematics › Geometry and Topology › Geometric and Algebraic Topology
Keywords
knotted proteins, sequence-based prediction, protein topology, AlphaFold, UniProt
MeSH terms
Machine Learning, Amino Acid Sequence, Models, Molecular, Protein Conformation, Proteins, Databases, Protein

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