Posttranslational modification of ribosomally synthesized peptides provides an elegant means for the production of biologically active molecules known as RiPPs (ribosomally synthesized and posttranslationally modified peptides). Although the leader sequence of the precursor peptide is often required for turnover, the exact mode of recognition by the modifying enzymes remains unclear for many members of this class of natural products. Here, we have used X-ray crystallography and computational modeling to examine the role of the leader peptide in the biosynthesis of a homolog of streptide, a recently identified peptide natural product with an intramolecular lysine-tryptophan cross-link, which is installed by the radical -adenosylmethionine (SAM) enzyme, StrB. We present crystal structures of SuiB, a close ortholog of StrB, in various forms, including apo SuiB, SAM-bound SuiB, and a complex of SuiB with SAM and its peptide substrate, SuiA. Although the N-terminal domain of SuiB adopts a typical RRE (RiPP recognition element) motif, which has been implicated in precursor peptide recognition, we observe binding of the leader peptide in the catalytic barrel rather than the N-terminal domain. Computational simulations support a mechanism in which the leader peptide guides posttranslational modification by positioning the cross-linking residues of the precursor peptide within the active site. Together the results shed light onto binding of the precursor peptide and the associated conformational changes needed for the formation of the unique carbon-carbon cross-link in the streptide family of natural products.
Thermostabilization represents a critical and often obligatory step toward enhancing the robustness of enzymes for organic synthesis and other applications. While directed evolution methods have provided valuable tools for this purpose, these protocols are laborious and time-consuming and typically require the accumulation of several mutations, potentially at the expense of catalytic function. Here, we report a minimally invasive strategy for enzyme stabilization that relies on the installation of genetically encoded, nonreducible covalent staples in a target protein scaffold using computational design. This methodology enables the rapid development of myoglobin-based cyclopropanation biocatalysts featuring dramatically enhanced thermostability (Δ = +18.0 °C and Δ = +16.0 °C) as well as increased stability against chemical denaturation [Δ (GndHCl) = 0.53 M], without altering their catalytic efficiency and stereoselectivity properties. In addition, the stabilized variants offer superior performance and selectivity compared with the parent enzyme in the presence of a high concentration of organic cosolvents, enabling the more efficient cyclopropanation of a water-insoluble substrate. This work introduces and validates an approach for protein stabilization which should be applicable to a variety of other proteins and enzymes.
Biophysical interactions between proteins and peptides are key determinants of molecular recognition specificity landscapes. However, an understanding of how molecular structure and residue-level energetics at protein−peptide interfaces shape these landscapes remains elusive. We combine information from yeast-based library screening, next-generation sequencing, and structure-based modeling in a supervised machine learning approach to report the comprehensive sequence−energetics−function mapping of the specificity landscape of the hepatitis C virus (HCV) NS3/4A protease, whose function—site-specific cleavages of the viral polyprotein—is a key determinant of viral fitness. We screened a library of substrates in which five residue positions were randomized and measured cleavability of ∼30,000 substrates (∼1% of the library) using yeast display and fluorescence-activated cell sorting followed by deep sequencing. Structure-based models of a subset of experimentally derived sequences were used in a supervised learning procedure to train a support vector machine to predict the cleavability of 3.2 million substrate variants by the HCV protease. The resulting landscape allows identification of previously unidentified HCV protease substrates, and graph-theoretic analyses reveal extensive clustering of cleavable and uncleavable motifs in sequence space. Specificity landscapes of known drug-resistant variants are similarly clustered. The described approach should enable the elucidation and redesign of specificity landscapes of a wide variety of proteases, including human-origin enzymes. Our results also suggest a possible role for residue-level energetics in shaping plateau-like functional landscapes predicted from viral quasispecies theory.
There is growing interest in designing spatiotemporal control over enzyme activities using noninvasive stimuli such as light. Here, we describe a structure-based, computation-guided predictive method for reversibly controlling enzyme activity using covalently attached photoresponsive azobenzene groups. Applying the method to the therapeutically useful enzyme yeast cytosine deaminase, we obtained a ∼3-fold change in enzyme activity by the photocontrolled modulation of the enzyme's active site lid structure, while fully maintaining thermostability. Multiple cycles of switching, controllable in real time, are possible. The predictiveness of the method is demonstrated by the construction of a variant that does not photoswitch as expected from computational modeling. Our design approach opens new avenues for optically controlling enzyme function. The designed photocontrolled cytosine deaminases may also aid in improving chemotherapy approaches that utilize this enzyme.
The biocatalytic toolbox has recently been expanded to include enzyme-catalyzed carbene transfer reactions not occurring in Nature. Herein, we report the development of a biocatalytic strategy for the synthesis of enantioenriched α-trifluoromethyl amines through an asymmetric N–H carbene insertion reaction catalyzed by engineered variants of cytochrome c 552 from Hydrogenobacter thermophilus . Using a combination of protein and substrate engineering, this metalloprotein scaffold was redesigned to enable the synthesis of chiral α-trifluoromethyl amino esters with up to >99% yield and 95:5 er using benzyl 2-diazotrifluoropropanoate as the carbene donor. When the diazo reagent was varied, the enantioselectivity of the enzyme could be inverted to produce the opposite enantiomers of these products with up to 99.5:0.5 er. This methodology is applicable to a broad range of aryl amine substrates, and it can be leveraged to obtain chemoenzymatic access to enantioenriched β-trifluoromethyl-β-amino alcohols and halides. Computational analyses provide insights into the interplay of protein- and reagent-mediated control on the enantioselectivity of this reaction. This work introduces the first example of a biocatalytic N–H carbenoid insertion with an acceptor–acceptor carbene donor, and it offers a biocatalytic solution for the enantioselective synthesis of α-trifluoromethylated amines as valuable synthons for medicinal chemistry and the synthesis of bioactive molecules.
Multispecificity–the ability of a single receptor protein molecule to interact with multiple substrates–is a hallmark of molecular recognition at protein-protein and protein-peptide interfaces, including enzyme-substrate complexes. The ability to perform structure-based prediction of multispecificity would aid in the identification of novel enzyme substrates, protein interaction partners, and enable design of novel enzymes targeted towards alternative substrates. The relatively slow speed of current biophysical, structure-based methods limits their use for prediction and, especially, design of multispecificity. Here, we develop a rapid, flexible-backbone self-consistent mean field theory-based technique, MFPred, for multispecificity modeling at protein-peptide interfaces. We benchmark our method by predicting experimentally determined peptide specificity profiles for a range of receptors: protease and kinase enzymes, and protein recognition modules including SH2, SH3, MHC Class I and PDZ domains. We observe robust recapitulation of known specificities for all receptor-peptide complexes, and comparison with other methods shows that MFPred results in equivalent or better prediction accuracy with a ~10-1000-fold decrease in computational expense. We find that modeling bound peptide backbone flexibility is key to the observed accuracy of the method. We used MFPred for predicting with high accuracy the impact of receptor-side mutations on experimentally determined multispecificity of a protease enzyme. Our approach should enable the design of a wide range of altered receptor proteins with programmed multispecificities.
As non-"self" macromolecules, biotherapeutics can trigger an immune response that can reduce drug efficacy, require patients to be taken off therapy, or even cause life-threatening reactions. To enable the flexible and facile design of protein biotherapeutics while reducing the prevalence of T-cell epitopes that drive immune recognition, we have integrated into the Rosetta protein design suite a new scoring term that allows design protocols to account for predicted or experimentally identified epitopes in the optimized objective function. This flexible scoring term can be used in any Rosetta design trajectory, can be targeted to specific regions of a protein, and can be readily extended to work with a variety of epitope predictors. By performing extensive design runs with varied design parameter choices for three case study proteins as well as a larger diverse benchmark, we show that the incorporation of this scoring term enables the effective exploration of an alternative, deimmunized sequence space to discover diverse proteins that are potentially highly deimmunized while retaining physical and chemical qualities similar to those yielded by equivalent nondeimmunizing sequence design protocols.
An accurate energy function is an essential component of biomolecular structural modeling and design. The comparison of differently derived energy functions enables analysis of the strengths and weaknesses of each energy function and provides independent benchmarks for evaluating improvements within a given energy function. We compared the molecular mechanics Amber empirical energy function to two versions of the Rosetta energy function (talaris2014 and REF2015) in decoy discrimination and loop modeling tests. In decoy discrimination tests, both Rosetta and Amber (ff14SBonlySC) energy functions performed well in scoring the native state as the lowest energy conformation in many cases, but several false minima were found in with both talaris2014 and Amber ff14SBonlySC scoring functions. The current default version of the Rosetta energy function, REF2015, which is parametrized on both small molecule and macromolecular benchmark sets to improve decoy discrimination, performs significantly better than talaris2014, highlighting the improvements made to the Rosetta scoring approach. There are no cases in Rosetta REF2015, and 8/140 cases in Amber, where a false minimum is found that is absent in the alternative landscape. In loop modeling tests, Amber ff14SBonlySC and REF2015 perform equivalently, although false minima are detected in several cases for both. The balance between dihedral, electrostatic, solvation and hydrogen bonding scores contribute to the existence of false minima. To take advantage of the semi-orthogonal nature of the Rosetta and Amber energy functions, we developed a technique that combines Amber and Rosetta conformational rankings to predict the most near-native model for a given protein. This algorithm improves upon predictions from either energy function in isolation and should aid in model selection for structure evaluation and loop modeling tasks.
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