Are there double knots in proteins? Prediction and in vitro verification based on TrmD-Tm1570 fusion from C. nitroreducens

Abstract

We have been aware of the existence of knotted proteins for over 30 years-but it is hard to predict what is the most complicated knot that can be formed in proteins. Here, we show new and the most complex knotted topologies recorded to date-double trefoil knots (3₁ #3₁). We found five domain arrangements (architectures) that result in a doubly knotted structure in almost a thousand proteins. The double knot topology is found in knotted membrane proteins from the CaCA family, that function as ion transporters, in the group of carbonic anhydrases that catalyze the hydration of carbon dioxide, and in the proteins from the SPOUT superfamily that gathers 3₁ knotted methyltransferases with the active site-forming knot. For each family, we predict the presence of a double knot using AlphaFold and RoseTTaFold structure prediction. In the case of the TrmD-Tm1570 protein, which is a member of SPOUT superfamily, we show that it folds in vitro and is biologically active. Our results show that this protein forms a homodimeric structure and retains the ability to modify tRNA, which is the function of the single-domain TrmD protein. However, how the protein folds and is degraded remains unknown.

Keywords: SPOUT; composite knot; domain; evolution; methyltransferase.

FIGURE 1

Predicted single chain structures of the fusion proteins. (A) Carbonic anhydrase (PF00194-PF00194; UniProtKB ID: A0A0B7AKD5) (B) Protein from Ca²⁺: Cation Antiporter (CaCA) family (UniProtKB ID: A0A0L0BYW8). (C) Protein with PF00588-PF00588 architecture (UniProtKB ID: Q4DMW6). (D) Nep1-Nep1 protein (PF03587-PF03587 architecture; UniProtKB ID: A0A498KD62). (E) TrmD-Tm1570 protein (PF01746-PF09936 architecture; UniProtKB ID: E4THH1). All the models were predicted with AlphaFold 2. Knotted regions are shown with rainbow coloring and their reduced structures were obtained with Knot_pull package (Jarmolinska et al., 2020).

FIGURE 2

Arrangement of TrmD and Tm1570 genes in different organisms. From the top: Aquifex aeolicus—the genes are separated by thousands of nucleotides; Desulfobacter postgatei—the genes are 11 nucleotides apart; Thermotoga maritima—the genes are overlapping by 7 nucleotides; Calditerrivibrio nitroreducens—the genes are fused.

FIGURE 3

Predicted structure of CnTrmD-Tm1570 fusion protein based on AlphaFold and docking. This homodimeric (second chain is transparent) complex binds tRNA (green) with its TrmD domains. A single chain of this protein contains two 3₁ knots (marked in blue and red) (Niemyska et al. 2022). TrmD domain (light grey) interacts with its counterpart from the second chain in an antiparallel fashion, whereas Tm1570 (dark grey) in a perpendicular fashion. Details about the modeled complex are in the Methods section.

FIGURE 4

Comparison of sequence motifs in TrmD and Tm1570 with the fusion protein. WebLogo3 was used to construct the conserved residue logos at the SAM and tRNA binding sites. Cartoon representation shows the TrmD-Tm1570 protein (TrmD domain in green, Tm1570 in blue) with selected motifs marked on the structure.

FIGURE 5

Activity of CnTrmD-Tm1570. (A) Protein constructs used for the activity assessment. (B) Time-course of the reaction catalyzed by fusion TrmD-Tm1570 (50 nM) in the presence of SAM (30 μM) towards the 8 μM tRNA Leu (CAG) substrate (E. coli tRNA black triangles, C. nitroreducens tRNA black circles). (C) Relative activities of different TrmD-Tm1570 constructs. Data obtained for the mutated tRNA substrate and negative control are also included. Reaction conditions are the same as in (B).