Cell-Free Mutant Analysis Combined with Structure Prediction of a Lasso Peptide Biosynthetic Protease B2

Abstract

Biochemical and structural analyses of purified proteins are essential for the understanding of their properties. However, many proteins are unstable and difficult to purify, hindering their characterization. The B2 proteins of the lasso peptide biosynthetic pathways are cysteine proteases that cleave precursor peptides during the maturation process. The B2 proteins are poorly soluble, and no experimentally solved structures are available. Here, we performed a rapid semicomprehensive mutational analysis of the B2 protein from the thermophilic actinobacterium, Thermobifida f usca (FusB2), using a cell-free transcription/translation system, and compared the results with the structure prediction by AlphaFold2. Analysis of 34 FusB2 mutants with substitutions of hydrophobic residues confirmed the accuracy of the predicted structure, and revealed a hydrophobic patch on the protein surface, which likely serves as the binding site of the partner protein, FusB1. Our results suggest that the combination of rapid cell-free mutant analyses with precise structure predictions can greatly accelerate structure−function research of proteins for which no structures are available.

Peptidase Activation by a Leader Peptide-Bound RiPP Recognition Element

“…These data agree with an earlier study of the paeninodin pathway in which the variant equivalent to FusE-Y33A (i.e., PadeB1-Y33A) abolished leader peptidase activity . These data experimentally established the critical residues of the domain interface and serve to enforce previous studies that explored the RRE and leader peptidase domains individually. ,, In general, the β-sandwich-like interaction consists of hydrophobic contacts and several notable complementary “bump–hole” interactions (e.g., residues of FusE and FusB: Ser15 and Ile135, Leu26 and Gly111, Gly28 and Ile135, and Tyr33 and Gly111, respectively).…”

“…48,51 An identical structure was reported during the preparation of our manuscript. 25 The predicted structure of FusE was nearly identical to the crystal structure (PDB entry 6JX3) 6 with a root-mean-square deviation (RMSD) of 0.9 Å. AlphaFold2 was further used to predict structures for several fused RRE-leader peptidases, and high confidence scores (pLDDT > 0.80) were obtained for SubB (subterisin) and KorB (koreesin, WP_157926355.1) (Figure S12 and Supplemental Data Set 3). 50,52 The FusE:FusB, SubB, and KorB predicted structures provide a unifying view of the putative RRE-leader peptidase interface: three β-strands from the RRE and leader peptidase domains face each other, forming an extended interaction surface analogous to a β-sandwich.…”

“…Given the qualitative nature of MS-based end point assays, we developed a more quantitative in vitro leader peptidase assay inspired by a previous report. 25 MBP-tagged FusA conjugated to mCherry (Figure S28) was used as the proteolytic substrate, which would result in the products MBP-FusA[leader peptide] and FusA[core]-linker-mCherry. The leader peptidase activity of MBP-FusE and MBP-FusB variants was then monitored by a fluorescence gel shift assay (Figure S29 and Table S12).…”

“… ,,, Proteins from the lasso peptide BGC encoded by Thermobifida fusca (fusilassin/fuscanodin) have been the focus of several recent reports, given that the proteins are a rare example that can be expressed and purified from Escherichia coli while retaining catalytic activity. However, despite significant published and unpublished efforts, no experimentally determined structures are available for any lasso leader peptidase. While the readily identifiable Cys/His catalytic residues for the leader peptidase have been confirmed, details of how the leader peptidase interacts with the RRE domain and the lasso leader peptide remain unknown.…”

Tail-tape-fused virion and non-virion RNA polymerases of a thermophilic virus with an extremely long tail

“…For structural analysis of gp96 vRNAP, the shortest gp96 fragment possessing the RNAP activity was delineated using a fast cell-free assay system ( Extended Data Fig. 3 ) 27 . The shortest fragment of gp96 that retained RNAP activity encompassed residues 327-1124 ( Extended Data Fig.…”