Structural Basis of Leader Peptide Recognition in Lasso Peptide

Articles Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Structural Basis of Leader Peptide Recognition in Lasso Peptide Biosynthesis Pathway Tomomi Sumida,†,‡,§ Svetlana Dubiley,*,∥,⊥ Brendan Wilcox,∥ Konstantin Severinov,∥,⊥,# and Shunsuke Tagami*,†,‡

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†

Center for Biosystems Dynamics Research and ‡Center for Life Science Technologies, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan § Research Center for Bioscience and Nanoscience, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan ∥ Center for Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia ⊥ Institute of Gene Biology, Russian Academy of Science, Moscow 119334, Russia # Waksman Institute for Microbiology, 190 Frelinghuysen Road, Piscataway, New Jersey 08854, United States S Supporting Information *

ABSTRACT: Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique 3Dinterlocked structure, in which an N-terminal macrolactam ring is threaded by a linear C-terminal part. The unique structure of lasso peptides is introduced into ribosomally translated precursor peptides by lasso peptide synthetase encompassing proteins B and C or B1, B2, and C when the B enzyme is split into two distinct proteins. The B1 protein recognizes the leader sequence of the precursor peptide, and then the B2 protein cleaves it. The C protein catalyzes the formation of the macrolactam ring. However, the detailed mechanism of lasso peptide maturation has remained elusive, due to the lack of structural information about the responsible proteins. Here we report the crystal structure of the B1 protein from the thermophilic actinobacteria, Thermobifida f usca (TfuB1), complexed with the leader peptide (TfuA-Leader), which revealed the detailed mechanism of leader peptide recognition. The structure of TfuB1 consists of an N-terminal β-sheet and three C-terminal helices. The leader peptide is docked on one edge of the N-terminal β-sheet of TfuB1, as an additional β strand. Three conserved amino acid residues of the leader peptide (TfuA Tyr-17, Pro-14, and Leu-12) fit well on the hydrophobic cleft between the β-sheet and adjacent helices. Biochemical analysis demonstrated that these conserved residues are essential for affinity between TfuB1 and the TfuA-Leader. Furthermore, we found that TfuB1 and the leader peptide jointly form a hydrophobic patch on the β-sheet, which includes the highly conserved TfuA Phe-6 and TfuB1 Tyr33. Homology modeling and mutational analysis of the B1 protein from a firmicute, Bacillus pseudomycoides (PsmB1), revealed that the hydrophobic patch is conserved in a wide range of species and involved in the cleavage activity of the B2 protein, indicating it forms the interaction surface for the B2 protein or the core part of the precursor peptide.

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Lasso peptides are a class of RiPPs with a unique 3-D interlocked rotaxane structure and are found in diverse bacterial lineages (Figure 1A).4,5 The N-terminal amino group of the lasso peptide is linked to the side chain of an acidic amino acid residue at position 7−9 by an isopeptide bond to form an N-terminal macrolactam ring, and the Cterminal linear part passes through the N-terminal ring. This “threaded” conformation of lasso peptides is stabilized by bulky side chains in the linear part (class II) and/or by disulfide bonds (classes I and III).4,5 This unique structure of the lasso

eptides are now regarded as potent materials for nextgeneration drugs, due to their amenability to engineering. Ribosomal translation of DNA libraries with randomized sequences allows huge numbers of peptide variants to be tested in one experiment (e.g., 1012−14 peptides in the case of mRNA display).1 Rigid structures have also been introduced to peptide libraries to enhance their functionality.2 Such strategies to functionalize ribosomally translated peptides by structuration are also employed by natural organisms. Ribosomally synthesized and post-translationally modified peptides (RiPPs) contain diverse classes of peptides produced by all three domains of life.3 In the biosynthetic processes of RiPPs, their linear precursors are first synthesized by ribosomes and then matured by enzymes that are usually encoded on the same gene clusters as the precursor peptides. © XXXX American Chemical Society

Received: May 3, 2019 Accepted: June 12, 2019 Published: June 12, 2019 A

DOI: 10.1021/acschembio.9b00348 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Figure 1. Biosynthesis of lasso peptides. (A) Schematic depiction of the lasso peptide general structure. (B) Biosynthetic gene cluster and maturation pathway of a lasso peptide, fusilassin/fuscanodin.37,38 (C) Amino acid sequence of TfuA, the precursor peptide of fusilassin/fuscanodin.

Figure 2. Crystal structure of the TfuB1·TfuA-Leader complex. (A) Overall structure and (B) topology diagram of the TfuB1·TfuA-Leader complex. TfuB1 and TfuA-Leader are colored yellow and cyan, respectively. (B) TfuA-Leader Met-22, Glu-21, Thr-2, and Gly-1 are not fixed in the crystal and shown as dashed lines.

peptides confers thermostability6 and resistance to proteolysis.7 Although the antibacterial activities to kill other bacteria in the same biological niches are regarded as the most common roles of lasso peptides,5 various functions and applications have been reported. Antibacterial lasso peptides include inhibitors for RNA polymerases from Gram-negative bacteria (microcin J25 and capistruin)8,9 and inhibitors of ClpC1P1P2-catalyzed proteolysis.10 Some bacterial lasso peptides reportedly have anti-HIV1 activities or inhibitory activity for cell invasion by a human lung cancer cell line, although their natural functions are not known.11−15 Microcin J25 has been used as a stable scaffold to develop an artificial peptide that binds to integrin αvβ3, a potent target for cancer therapy.16,17 Recently, lasso peptides have been shown to serve as building blocks for larger catenane structures.18 Therefore, lasso peptides are regarded as potential scaffolds to develop new-generation drugs and nanomaterials. While the sequences and functions of lasso peptides vary markedly, the systems for their biosynthesis are mostly conserved across species (Figure 1B). A typical lasso peptide biosynthetic gene cluster encodes a linear precursor peptide (A), three conserved proteins for lasso peptide maturation (B1, B2, and C), and a transporter to secrete the matured lasso peptide (D). In the beginning of the biosynthetic process, a 40−50 amino acid residue linear precursor peptide composed of an N-

terminal leader sequence (A-Leader) and a C-terminal core sequence (A-Core) is synthesized by the ribosome. This precursor peptide is then processed by the maturation proteins, B1, B2, and C (Figure 1B). First, the leader sequence is recognized by the B1 protein and cleaved from the core peptide by the protease activity of B2. The B1 and B2 proteins are translated as one fused protein in some gene clusters (e.g., McjB for microcin J25 biosynthesis19,20). The N-terminal amino group of the core peptide formed after B2-mediated cleavage is linked to the side chain carboxy group of an acidic amino residue (Asp/Glu) at position 7−9 by the C protein. The B and C proteins reportedly work interdependently and are suggested to form a structural complex.21 It has been also suggested that the core peptide is prefolded in a lasso-like conformation before the macrolactam ring is closed, as most of the lasso peptides have a few bulky amino acids on the linear part to prevent their “rethreading/unthreading”.10,22−26 Although extensive in vitro and in vivo experiments have been performed, the detailed mechanism of lasso peptide biosynthesis has remained elusive because of the lack of structural information about the responsible enzymes. The B1 protein is predicted to be structurally homologous to other RiPP recognition elements (RREs), which are mostly used to recognize leader peptides in the biosyntheses of various RiPPs. 27 Although several crystal structures of RREs complexed with their target peptides have been reported,28−34 they revealed that different RREs adopt various conformations B


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Figure 3. Recognition of TfuA-Leader by TfuB1. (A) Sequence alignment of representative members of the precursor peptides and B1 proteins of lasso peptide biosynthesis systems from actinobacteria and furmicutes. LarA and LarE are from the biosynthesis system of lariatin produced by Rhodococcus jostii K01−B0171.36 StmA and StmE are from the biosynthesis system of streptomonomicin from Streptomonospora alba.46 PsmA and PsmB1 are from the lasso peptide synthesis gene cluster from Bacillus pseudomycoides (unpublished). PadeA and PadeB1 are from the biosynthesis system for paeninodin produced by Paenibacillus dendritiformis C454.47 The amino acid numberings for TfuA and TfuB are indicated. (B−E) Detailed views of the interactions between conserved residues of TfuB1 and TfuA-Leader TfuB1 and TfuA-Leader are colored yellow and cyan, respectively.

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RESULTS AND DISCUSSION Crystallization and Structure Determination. TfuB1 was cocrystallized with the leader sequence of TfuA (Met-22− Gly-1, TfuA-Leader) under conditions containing Zn2+ ions, and its structure was determined at 1.7 Å resolution (Figure 2A, Table S1, Figure S1A) by the single-wavelength anomalous diffraction (SAD) technique, utilizing the anomalous scatterings from the zinc ions bound on the protein surface (Figure S1B−D). The asymmetric unit of the crystal contains one complex of TfuB1·TfuA-Leader, which apparently forms a symmetric dimer with another complex related by a 2-fold crystallographic axis (Figure S2). To investigate if this apparent dimer is a functional dimer or a crystallographic artifact, we performed ultracentrifugation analyses and confirmed the monomeric states of free TfuB1 and the TfuB1·TfuA-Leader complex in aqueous solution (Figure S3). The estimated molecular masses in solution (TfuB1, 10.4 kDa; TfuB1·TfuALeader, 13.6 kDa) are consistent with the theoretical molecular weights of their monomers (TfuB1, 10.9 kDa; TfuB1· TfuALeader, 13.3 kDa) and indicated that TfuB1 remains in a monomeric state both in the presence and in the absence of the leader peptide. Therefore, the dimer observed in the crystal is probably a crystallographic artifact. However, the interaction surface of the crystallographic dimer is composed of conserved hydrophobic residues from both TfuB1 and TfuA-Leader (Figure S2B), which may be involved in interactions with other members of the lasso peptide biosynthesis system (see below). Overall Structure of the TfuB1·TfuA-Leader Complex. TfuB1 is a single domain protein with an N-terminal β-sheet (β1−β3) and a C-terminal α-helical region (α1−α3) (Figure 2A,B). The overall structure of TfuB1 has the same winged helix-turn-helix (wHTH) topology as other reported structures

and different binding modes to recognize their target peptides. Therefore, the structural determination of the B1 protein is needed to understand the details of its leader peptide recognition mechanism. In this paper, we report the crystal structure of the B1 protein from a moderate thermophile, Thermobifida f usca (TfuB1), complexed with the leader peptide, TfuA-Leader (Met-22−Gly-1, Figure 1C), of which Lys-20−Ala-3 are observed in the electron density. The lasso peptide biosynthesis system of T. f usca is one of the most well-characterized lasso peptide biosynthesis systems. It was previously mined by a bioinformatical analysis,35,36 and the production of the mature lasso peptide, either termed fusilassin or fuscanodin, has recently been demonstrated both in heterologous in vivo and in in vitro systems.37,38 The structure of the TfuB1·TfuALeader has revealed how the maturation process of the lasso peptide is initialized by TfuB1. The leader peptide is bound on one edge of the N-terminal β-sheet of TfuB1, expanding the sheet by one strand. Three conserved residues in the leader peptide (TfuA Tyr-17, Pro-14, and Leu-12) fit snugly on a cleft formed by the well-conserved residues of the B1 protein. The importance of these conserved residues in the leader peptide for the affinity to TfuB1 was confirmed by an isothermal titration calorimetry (ITC) analysis. Furthermore, we observed a hydrophobic patch formed by conserved hydrophobic residues from both TfuA-Leader and TfuB1 on the intermolecular β-sheet. A mutational analysis of the B1 protein from Bacillus pseudomycoides (PmsB1) revealed that some residues on the β-sheet contribute to leader peptide cleavage by the B2 protein, indicating that the intermolecular β-sheet is conserved in a wide range of species and involved in the interaction with B2 or the core part of the precursor peptide. C


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Table 1. Affinity between TfuB1 and TfuA-Leadera

of RREs.27−34 However, their structures are significantly different in terms of the lengths and relative angles of the secondary structure elements (Figure S4), which explains why our phase determination trials by molecular replacement did not work for the crystal of the TfuB1·TfuA-Leader complex. TfuA-Leader adopts an extended β-conformation and is aligned with the β3 strand of TfuB1, forming an intermolecular antiparallel β-sheet. The formation of such an intermolecular β-sheet was also observed in some other RRE structures complexed with their target peptides.29,30,32,33 However, the conformations of the peptides from the various systems significantly differ, indicating that their recognition mechanisms are not strictly conserved (Figure S4). Mechanism of Leader Peptide Recognition by TfuB1. The crystal structure of the TfuB1·TfuA-Leader complex revealed the detailed leader peptide recognition mechanism by the B1 protein (Figure 3). Three amino acid residues of the leader peptide (TfuA Tyr-17 and Pro-14 from the conserved YxxP motif,39,40 and another well conserved residue, Leu-12) snugly fit on the cleft between β3 and α1−α3 of TfuB1 (Figure 2A, Figure 3B−D, Figure S5). TfuA Tyr-17 is surrounded by hydrophobic residues from α2, α3, and the loop between them (TfuB1 Leu60, Tyr64, Val66) (Figure 3B). TfuA Tyr-17 also forms a hydrogen bond with a well-conserved aspartic acid residue (TfuB1 Asp74). In a previous report, this aspartic acid residue and TfuA Tyr-17 were shown to be under coevolutionary pressure,37 and TfuB1 D74A substitution caused an 8fold reduction in the binding affinity to TfuA-Leader as determined by a direct fluorescence polarization analysis.37 The corresponding mutation of the B1 protein for paeninodin biosynthesis (PadeB1 D79A) reportedly decreased the binding affinity to the leader peptide by more than 10-fold,41 indicating that the observed interaction between Tyr-17 and Asp74 is essential and conserved in a wide range of species. TfuA Pro-14 is another conserved residue of the YxxP motif.39,40 In the TfuB1·TfuA-Leader complex, it resides at the N-terminal edge of the extended β part of the leader peptide and introduces a sharp kink into the leader peptide (Figure 2A). The proline residue fits on the hydrophobic surface formed by TfuB1 Ala40, Ile43, and Leu78 (Figure 3C). Furthermore, the kinked conformation at this proline seems to be stabilized by hydrogen bonds between the backbone of TfuA-Leader and TfuB1 Asn37, which is well conserved in the B1 proteins from actinobacteria (Figure 3A). Recent research has confirmed the contribution of the conserved YxxP motif for the interactions between the B1 proteins and leader peptides in the biosynthetic systems for a few lasso peptides, including lariatin,39 paeninodin,42 chaxapeptin,43 and fusilassin/fuscanodin.37 We analyzed the alanine mutants of TfuA Y-17 and P-14 by isothermal titration calorimetry (Table 1 and Figure S6). The TfuA Y-17A substitution abolished the binding affinity to TfuB1, whereas the wild type TfuA showed nanomolar binding affinity to TfuB1. TfuA P-14A also showed significantly (more than 10fold) reduced affinity to TfuB1. Therefore, the interactions between the YxxP motif and the conserved residues of the B1 protein play critical roles in the leader peptide recognition across a wide variety of lasso peptide biosynthesis systems. In the crystal structure of the TfuB1·TfuA-Leader complex, another conserved residue, TfuA Leu-12, also binds within the hydrophobic cleft between β3 and α3 of TfuB1 (Figure 3D). The alanine mutant of this leucine residue significantly decreased the affinity to TfuB1 (more than 10-fold) in our

TfuB1

TfuA-Leader

KD (nM)

WT WT WT WT WT V24A/L26A/Y33A

WT Y-17A P-14A L-12A F-6A WT

6.0 ± 2.1 N/A 81.3 ± 21.5 82.6 ± 15.9 8.5 ± 2.5 11.9 ± 3.7

a Affinities of variants were measured by isothermal titration calorimetry (ITC).

ITC experiment (Table 1). Its binding surface on TfuB1 consists of TfuB1 Trp34, Leu36, Ala40, Leu78, Leu82, and Met87. Most of these hydrophobic residues are well conserved (Figure 3A), and a few of them (Ala40 and Leu78) also contact TfuA Pro-14 as mentioned above. The alanine mutation of TfuB1 Leu78 reportedly lowered the affinity to TfuA-Leader by roughly 10-fold,37 indicating the importance of the hydrophobic interactions in the leader peptide recognition mechanism. We also found a hydrogen bond network between TfuALeader and TfuB1, around TfuA Lys-20 (Figure 3E). Although these hydrogen bonds are mostly formed by the main chains, continuous positively charged residues are observed at the Nterminus of the leader peptide (TfuA Lys-20, Lys-19, Lys-18, Figure 3A). A few acidic side chains were also observed on the TfuB1 side of this area (Glu63, Glu65, Glu67). Although these charged side chains do not directly interact with each other in the crystal, TfuA Lys-19 and TfuB1 Glu63 are under coevolutionary pressure37 and the TfuB1 E63A mutant showed a ∼2-fold reduction in the affinity to TfuA-Leader.37 Therefore, the charged residues around this position may contribute to the initial interactions between TfuB1 and TfuA by electrostatic attraction over a long distance, as previously discussed.37 Intermolecular β-Sheet and a Hydrophobic Patch Jointly Formed by TfuB1 and TfuA-Leader. TfuB1 and TfuA-Leader form an intermolecular β-sheet (Figure 2). The β part of the leader peptide (TfuA Leu-12−Phe-6) is one residue longer in actinobacteria than in firmicutes, because of an additional glycine residue (TfuA Gly-8, Figure 3A). Although the glycine residue introduces a short twist in the β strand (Figure 4A), the β-type hydrogen bonds between TfuB1 β3 and the leader peptide allow a proper alignment in the form of an antiparallel β-sheet (Figure 4B). In the crystal structure of TfuB1·TfuA-Leader, a hydrogen bond network among TfuA Lys-10, Glu-7, and TfuB1 Glu32 is observed (Figure 4C). However, it probably has only a minor contribution to the binding affinity between TfuA and TfuB1, since in a previous study, the alanine mutant of TfuB1 Glu32 did not show a significant difference in the affinity to TfuA-Leader.37 Interestingly, we found a hydrophobic patch formed on the surface of the intermolecular β-sheet (Figure 4D, Figure S5). TfuA Phe-6 is highly conserved in actinobacteria and is accommodated between TfuB1 Gly31 and Tyr33, which are also broadly conserved in the B1 proteins (Figure 3A). Other amino acid residues on this hydrophobic patch (TfuA Val-9, TfuB1 Val24, Leu26) are also well conserved, suggesting that this hydrophobic surface plays an important role in lasso peptide biosynthesis. Although this hydrophobic patch was utilized in the crystal packing to form the symmetric dimer of the TfuB1·TfuA-Leader complex (Figure S2), TfuB1·TfuAD


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bacteria aligned well, except for the N-terminal loop and β1 strand. Therefore, we expected the homology model to have relatively high accuracy from β2 to α3. In this model, the conserved amino acid residues of PsmA (PsmA Trp-15, Pro12, Leu-10) are recognized by PsmB1 in mostly the same way as in the TfuB1·TfuA-Leader complex (Figure 5A). The positions of the hydrophobic residues on the intermolecular βsheet are also predicted to be conserved between actinobacteria and firmicutes. To compare the functions of the conserved residues in PsmB1 and PsmA-Leader to the corresponding residues in TfuB1 and TfuA-Leader, we performed a copurification analysis of PsmB1 and PsmA combined with in vivo leader peptide cleavage by PsmB2 (Figure 5B). In this analysis we first coexpressed an MBP-PsmA-TrxA conjugate, His-tagged PsmB1, and PsmB2 and then purified the His-tagged PsmB1 with Talon Co2+ resin to capture the PsmA bound to PsmB1. When we coexpressed wild-type PsmA and wild-type PsmB1, the MBP-PsmA-TrxA was cleaved by PsmB2 and the MBPPsmA-Leader was copurified with PsmB1 (Figure 5C,D, lane WT). Cleavage accuracy was confirmed with MALDI MS analysis of tryptic peptides derived from the MBP-PsmALeader. In the absence of PsmA, the PsmB1 protein did not copurify with an MBP-TrxA fusion protein (Figure 5C,D, lane ΔA). In the case where a catalytic mutation of PsmB2 (PsmB2 C83A) was introduced, the intact MBP-PsmA-TrxA conjugate was copurified with PsmB1 (Figure 5C,D, lane B2mut). Therefore, we can evaluate both the binding affinity between PsmA and PsmB1 and the cleavage activity of PsmB2 simultaneously in this system. Alanine mutants of the YxxP motif (PsmA W-15A and P12A) did not copurify with His-tagged PsmB1 (Figure 5C, lanes W-15A and P-12A), confirming the importance of this motif in leader-peptide recognition by the B1 protein. Although TfuA L-12A showed significantly decreased binding affinity to TfuB1, the corresponding mutant of PsmA (L-10A) copurified well with PsmB1 (Figure 5C, lane L-10A). However, the leader peptide cleavage by PsmB2 was decreased, suggesting that PsmA L-10A substitution may distort the conformation of the leader peptide required for cleavage, even though it does not eliminate the affinity to the B1 protein. The alanine mutation of PsmB1 Phe-83, which contacts PsmA Leu10 (Figure 5A), eliminated the interaction between PsmA and PsmB1, indicating that the interaction between these residues is still important for their binding in firmicutes. Although the interaction between PsmA Asp-6 and PsmB1 Lys37 was observed in the homology model of PsmB1·PsmA-Leader (Figure 5A), PsmA D-6A did not impair the binding affinity, while PsmB1 K37A showed slightly decreased copurification with the MBP-PsmA-Leader (Figure 5C, lane D-6A and Figure 5D, lane K37A), demonstrating that the interaction is not critical for the B1/B2 functions. Mutations in the hydrophobic patch on the intermolecular β-sheet (PsmA I-5A, PsmB1 V29A, M31A, and Y38A) were also tested with this system. The PsmA I-5A mutation abolished the binding with B1 (Figure 5C, lane I-5A), indicating that this residue is still important for the affinity to the B1 protein, unlike TfuA F-6A, which had almost no effect on the binding affinity to TfuB1. All of the PsmB1 mutants (V29A, M31A, and Y38A) copurified with PsmA (Figure 5D, lanes V29A, M31A, and Y38A). However, Y38A abolished the B2 activity, as almost no cleavage product was copurified with the mutant. A small amount of intact MBP-

Figure 4. Intermolecular β-strand formed between TfuB1 and TfuALeader. (A−D) Detailed views of the β parts of TfuB1 and TfuALeader TfuB1 and TfuA-Leader are colored yellow and cyan, respectively. Alternative conformations for TfuB1 Val24 are shown.

Leader remains in a monomeric state in aqueous solution (Figure S3). The ITC analysis revealed that the alanine substitution of TfuA Phe-6 did not remarkably change the affinity to TfuB1 (Table 1). We also prepared a triple alanine mutant of the B1 protein (TfuB1 V24A/L26A/Y33A) and confirmed that its binding affinity to the leader peptide was only slightly (∼2-fold) decreased (Table 1). In a previous study, mutations of the residues on this hydrophobic patch (TfuB1 L26A, G31V, Y33A) also did not lower the affinity to the leader peptide (less than 2-fold) but rather abolished the activity of the B2 protein (TfuB2) in the leader peptide cleavage assay containing TfuA, TfuB1, and TfuB2.37 Therefore, the hydrophobic patch on the intermolecular β-sheet is probably an interaction site with the B2 protein and/or the core part of the precursor peptide, as discussed below. Homology Model and Biochemical Analysis of a Lasso Biosynthesis System from a Firmicute. To investigate if the binding mechanism between the B1 protein and the leader peptide observed in the crystal structure of the TfuB1·TfuA-Leader complex from an actinobacterium, T. f usca, is conserved in a lasso peptide biosynthesis system from another bacterial phylum, we built a homology model of the B1·A-Leader complex from a firmicute, Bacillus pseudomycoides (PsmB1·PsmA-Leader), and performed mutational analyses of the conserved amino acids. To build the homology model of the PsmB1·PsmA-Leader complex, we first replaced the side chains of the structure of TfuB1·TfuA-Leader with the sequence of B. pseudomycoides and manually remodeled the β-strand part of the leader peptide to remove its twisting caused by the glycine insertion specific to actinobacteria (Figure 3A) and then relaxed the model by the ROSETTA program suites44,45 (Figure 5A). Most of the amino acid sequences of the B1 proteins from these two E


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Figure 5. Homology model and mutational analysis of PsmB1 and PsmA based on the crystal structure of the TfuB1·TfuA-Leader complex. (A) Homology model of the PsmB1·PsmA-Leader complex. PsmB1 and PsmA-Leader are colored yellow and cyan, respectively. (B) Schematic depiction of the in vivo cleavage/pull down analysis of PsmA and PsmB1 mutants. (C, D) Results of the in vivo cleavage/pull down analysis with mutations in (C) PsmA and (D) PsmB1. The lane ΔA was performed with an MBP-TrxA fusion without PsmA. The lane B2mut was performed with an inactive mutant of PsmB2 (C83A).

Potential Docking Surface for the B2 Protein and Core Peptide. The hydrophobic surface on the β-sheet, jointly formed by the B1 protein and the leader peptide, is not pivotal for their binding affinity to each other (Table 1). In other published structures of the RRE·peptide complexes,28−34 the β-sheets in this position form interaction surfaces for other domains/proteins (LynD, NisB, CteB, SuiB, McbB) or to the target peptide (MccB) (Figure S4). A coevolutionary analysis suggested that TfuB1 Gly31 and Tyr33 from the hydrophobic patch interact with the B2 protein.37 Furthermore, TfuB1 was required in in vitro maturation of fusilassin/fuscanodin even in the case the reaction was performed with TfuA-Core without the leader sequence.38 Therefore, the hydrophobic patch on the β-sheet probably contributes to the subsequent processes performed by the B2 protein. In another report, a cross-linking analysis between LarA and LarB1 from the lariatin biosynthesis system was performed to identify amino acids located on interaction surfaces.39 The results suggested that LarB1 Tyr16 (TfuB1 Tyr21) on the β1/ β2 loop and LarB1 Val19 (TfuB1 Val24) from the hydrophobic patch contact LarA Thr-3−Ala-1 (TfuA Ala-3−Gly-1) at the C-terminal end of the leader peptide. Although the β1/ β2 loop and the C-terminus of the leader do not contact each other in the crystal of the TfuB1·TfuA-Leader complex (Figure S7A), they both may relocate in aqueous solution or in the presence of the B2 protein/core peptide. The loop between β1 and β2 has weak electron density and high B factors that indicate its flexibility, whereas most of the modeled regions of TfuB1·TfuA-Leader were stably fixed in the crystal (Figure

PsmA-TrxA was left in copurified samples containing PsmB1 V29A and M31A, indicating that these substitutions reduced the activity of the B2 protein. The copurified sample with PsmB1 carrying N40A substitution located close to the hydrophobic patch also contained uncut substrate (Figure 5D, lane N40A). PadeB1 Y38A (TfuB1 Y33A) reportedly could bind to the leader peptide with a similar affinity to the wild-type PadeB1, but PadeB2 could not cleave the leader peptide when mixed with PadeB1 Y38A.41 Therefore, the overall conformation and role of the intermolecular β-sheet to support the B2 activity are conserved between actinobacteria and firmicutes. A few C-terminal residues of PsmA-Leader (PsmA Asn-4− Met-1) were excluded from our homology model, because the corresponding residues are not fixed or merely stabilized by the crystal packing interactions in the structure of the TfuB1·TfuALeader complex. Two alanine mutants of the C-terminal residues of PsmA-Leader (PsmA T-2A and M-1A) did not affect the copurification with PsmA (Figure 5C, lanes T-2A and M-1A), confirming that they are unnecessary to form a stable complex with the B1 protein. However, PsmA T-2A significantly impaired cleavage by B2 protease. In previous studies, it was also shown that the corresponding mutants of the precursor peptides of Microcin J25 (McjA T-2A) and paeninodin (PadeA T-2A) inhibit in vitro proteolysis by McjB and PadeB2, respectively.21,42 Therefore, this conserved threonine residue is probably required for the interaction with the B2 protein. F


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Although almost the entire leader peptide was well fixed in the crystal structure of the TfuB1·TfuA-Leader complex, the most conserved residue in the leader sequence (TfuA Thr-2) was not observed. This residue is near the cleavage site between the leader and the core peptide and is possibly important for the B2 function, as the alanine mutation of the corresponding residue in PsmA (PsmA T-2A) significantly inhibited its cleavage by PsmB2 (Figure 5C). A structural analysis of the B2 protein complexed with the precursor peptide and the B1 protein will be required to reveal the cleavage mechanism and how the precursor peptide and B1 protein are involved in more detail.

S7B). Furthermore, the position of the C-terminus of the leader peptide in the crystal (TfuA Lys-5−Ala-3) seems to be merely fixed by the crystal packing, because the residues are mainly interacting with the TfuB1 molecule in the adjacent TfuB1·TfuA-Leader complex related by the 2-fold symmetry axis (Figure S2). Therefore, the β1/β2 loop and the Cterminus of the leader peptide may adopt different conformations from those observed in the crystal structure and contact each other on the intermolecular β-sheet formed by the B1 protein and the leader peptide.

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CONCLUSION Here we have reported the crystal structure of the B1 protein complexed with the leader peptide from T. f usca. In the structure, the leader peptide binds to one edge of the β-sheet of TfuB1 to jointly form an intermolecular β-sheet. Previously, homology models of B1 proteins from a few different bacteria were built based on crystal structures of the RRE domains from other RiPP biosynthesis systems.37,39,41 Although formation of the intermolecular β-sheet was predicted from these homology models, the positions of amino acid residues were not precisely modeled as the structures of RRE domains from various RiPP biosynthesis systems cannot be well aligned (Figure S4). In this study, the crystal structure of the TfuB1·TfuA complex has revealed conclusive structural details of the B1 protein and how it recognizes the leader peptide for the first time. Three well-conserved residues of the leader peptide (TfuA Tyr-17, Pro-14, and Leu-12) fit onto a hydrophobic cleft formed between β3 and α1−3 of TfuB1 (Figure 3, Figure S5). Mutational analyses of TfuA-Leader and TfuB1 revealed that these residues are necessary for the affinity between TfuALeader and TfuB1 (Table 1). Interestingly, the intermolecular β-sheet formed by TfuB1 and TfuA-Leader has a conspicuous hydrophobic patch on its surface (Figure 4, Figure S5). This hydrophobic patch is not required for the binding affinity between TfuB1 and TfuA-Leader (Table 1) and is possibly used in the interaction with TfuA-Core or TfuB2. Although this hydrophobic patch is composed of well-conserved amino acid residues, it was not observed on the previous homology models based on structures of the RRE domains from other RiPP biosynthesis systems,37,39,41 where some of the hydrophobic amino acid residues on the β-sheet were mistakenly faced to the opposite side of the β-sheet or positioned on the loop between the β-strands. Homology modeling and mutant analyses of PsmB1 and PsmA based on the TfuB1·TfuA-Leader structure confirmed the mechanism of leader peptide recognition by the B1 proteins and the role of the hydrophobic patch are conserved in B. pseudomycoides (Figure 5). The residues involved in leader peptide recognition and the hydrophobic patch are well conserved in gene clusters of other lasso peptides including lariatin,36 streptomonomicin,46 and paeninodin47 (Figure 3A), indicating the structural features observed in the TfuB1·TfuALeader complex are also conserved in a wide range of species. Furthermore, crystal structures of the B1·A-Leader complexes of the putative lasso peptide gene cluster from Thermobaculum terrenum have recently been released in the PDB (PDB ID 5V1U and 5V1V), although the report for the structures has not been published. The crystal structures of the B1·A-Leader complexes from T. f usca and T. terrenum can be well aligned (Cα RMSD = 1.5 Å), suggesting the leader peptide recognition mechanism is also conserved in Chloroflexi, the bacterial phylum to which T. terrenum belongs.48,49

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.9b00348. Methods details, data collection and refinement statistics, synthetic genes, DNA primers, electron density maps of the TfuB1·TfuA-Leader crystal, crystallographic dimer of the TfuB1·TfuA-Leader complex, ultracentrifugation of free TfuB1 and the TfuB1·TfuA-Leader complex, structures of RRE domains from different RiPP biosynthesis proteins complexed with their target peptides, hydrophobic surface of the TfuB1·TfuALeader complex, binding curves between TfuB1 and TfuA, and potential flexibility of the TfuB1·TfuA-Leader structure (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Svetlana Dubiley: 0000-0001-9225-5534 Shunsuke Tagami: 0000-0002-1720-3627 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is based on experiments performed at KEK (Project Number 2017G591) and SPring-8. We thank the beamline staff scientists at KEK and SPring-8, especially K. Hirata, for collecting the diffraction data at the SPring-8 beamline BL32XU. We thank M. Metelev for advice on the design of the peptide/protein sequences used in our experiments. We also thank M. Moriya and N. Sakai for helping with the protein expression and crystallography, respectively. The authors are grateful to M. Kikuchi and S. Sato for their help with the ITC experiment. We also thank R. Akasaka for assistance with the ultracentrifugation measurement. We thank M. Serebryakova for performing MALDI MS analysis. S.D. was supported by Grant RSF 19-14-00266. B.W. was supported by a graduate fellowships from Skoltech and funds from Skoltech Center for Life Sciences. S.T. was supported by the Nakajima Foundation, Astellas Foundation for Research on Metabolic Disorders, Inamori Foundation, and Koyanagi Foundation. G


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Structural Basis of Leader Peptide Recognition in Lasso Peptide

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