Enhancing protein dynamics analysis with hydrophilic polyethylene glycol cross-linkers.

Cross-linkers play a critical role in capturing protein dynamics in chemical cross-linking mass spectrometry techniques. Various types of cross-linkers with different backbone features are widely used in the study of proteins. However, it is still not clear how the cross-linkers' backbone affect their own structure and their interactions with proteins. In this study, we systematically characterized and compared methylene backbone and polyethylene glycol (PEG) backbone cross-linkers in terms of capturing protein structure and dynamics. The results indicate the cross-linker with PEG backbone have a better ability to capture the inter-domain dynamics of calmodulin, adenylate kinase, maltodextrin binding protein and dual-specificity protein phosphatase. We further conducted quantum chemical calculations and all-atom molecular dynamics simulations to analyze thermodynamic and kinetic properties of PEG backbone and methylene backbone cross-linkers. Solution nuclear magnetic resonance was employed to validate the interaction interface between proteins and cross-linkers. Our findings suggest that the polarity distribution of PEG backbone enhances the accessibility of the cross-linker to the protein surface, facilitating the capture of sites located in dynamic regions. By comprehensively benchmarking with disuccinimidyl suberate (DSS)/bis-sulfosuccinimidyl-suberate(BS3), bis-succinimidyl-(PEG)2 revealed superior advantages in protein dynamic conformation analysis in vitro and in vivo, enabling the capture of a greater number of cross-linking sites and better modeling of protein dynamics. Furthermore, our study provides valuable guidance for the development and application of PEG backbone cross-linkers.

Real-Time NMR-Based Drug Discovery to Identify Inhibitors against Fatty Acid Synthesis in Living Cancer Cells.

Abnormal fatty acid metabolism is recognized as a key driver of tumor development and progression. Although numerous inhibitors have been developed to target this pathway, finding drugs with high specificity that do not disrupt normal cellular metabolism remains a formidable challenge. In this paper, we introduced a novel real-time NMR-based drug screening technique that operates within living cells. This technique provides a direct way to putatively identify molecular targets involved in specific metabolic processes, making it a powerful tool for cell-based drug screening. Using 2-13C acetate as a tracer, combined with 3D cell clusters and a bioreactor system, our approach enables real-time detection of inhibitors that target fatty acid metabolism within living cells. As a result, we successfully demonstrated the initial application of this method in the discovery of traditional Chinese medicines that specifically target fatty acid metabolism. Elucidating the mechanisms behind herbal medicines remains challenging due to the complex nature of their compounds and the presence of multiple targets. Remarkably, our findings demonstrate the significant inhibitory effect of P. cocos on fatty acid synthesis within cells, illustrating the potential of this approach in analyzing fatty acid metabolism events and identifying drug candidates that selectively inhibit fatty acid synthesis at the cellular level. Moreover, this systematic approach represents a valuable strategy for discovering the intricate effects of herbal medicine.

Assessment of Two Restraint Potentials for Coarse-Grained Chemical-Cross-Link-Assisted Modeling of Protein Structures.

The influence of distance restraints from chemical cross-link mass spectroscopy (XL-MS) on the quality of protein structures modeled with the coarse-grained UNRES force field was assessed by using a protocol based on multiplexed replica exchange molecular dynamics, in which both simulated and experimental cross-link restraints were employed, for 23 small proteins. Six cross-links with upper distance boundaries from 4 Å to 12 Å (azido benzoic acid succinimide (ABAS), triazidotriazine (TATA), succinimidyldiazirine (SDA), disuccinimidyl adipate (DSA), disuccinimidyl glutarate (DSG), and disuccinimidyl suberate (BS3)) and two types of restraining potentials ((i) simple flat-bottom Lorentz-like potentials dependent on side chain distance (all cross-links) and (ii) distance- and orientation-dependent potentials determined based on molecular dynamics simulations of model systems (DSA, DSG, BS3, and SDA)) were considered. The Lorentz-like potentials with properly set parameters were found to produce a greater number of higher-quality models compared to unrestrained simulations than the MD-based potentials, because the latter can force too long distances between side chains. Therefore, the flat-bottom Lorentz-like potentials are recommended to represent cross-link restraints. It was also found that significant improvement of model quality upon the introduction of cross-link restraints is obtained when the sum of differences of indices of cross-linked residues exceeds 150.

ACR-Based Probe for the Quantitative Profiling of Histidine Reactivity in the Human Proteome.

The quantitative profiling of residue reactivity in proteins promotes the discovery of covalent druggable targets for precise therapy. Histidine (His) residues, accounting for more than 20% of the active sites in enzymes, have not been systematically characterized for their reactivity, due to lack of labeling probes. Herein, we report a chemical proteomics platform for the site-specific quantitative analysis of His reactivity by combination of acrolein (ACR) labeling and reversible hydrazine chemistry enrichment. Based on this platform, in-depth characterization of His residues was conducted for the human proteome, in which the rich content of His residues (>8200) was quantified, including 317 His hyper-reactive residues. Intriguingly, it was observed that the hyper-reactive residues were less likely to be the sites for phosphorylation, and the possible mechanism of this antagonistic effect still needs to be evaluated in further research. Based on the first comprehensive map of His residue reactivity, many more residues could be adopted as the bindable sites to disrupt the activities of a diverse number of proteins; meanwhile, ACR derivatives could also be used as a novel reactive warhead in the development of covalent inhibitors.

Improved Cross-Linking Coverage for Protein Complexes Containing Low Levels of Lysine by Using an Enrichable Photo-Cross-Linker.

Chemical cross-linking coupled with mass spectrometry (XL-MS) is an important technique for the structural analysis of protein complexes where the coverage of amino acids and the identification of cross-linked sites are crucial. Photo-cross-linking has multisite reactivity and is valuable for the structural analysis of chemical cross-linking. However, a high degree of heterogeneity results from this multisite reactivity, which results in samples with higher complexity and lower abundance. Additionally, the applicability of photo-cross-linking is limited to purified protein complexes. In this work, we demonstrate a photo-cross-linker, alkynyl-succinimidyl-diazirine (ASD) with the reactive groups of N-hydroxysuccinimide ester and diazirine, as well as the click-enrichable alkyne group. Photo-cross-linkers can provide higher site reactivity for proteins that contain only a small number of lysine residues, thereby complementing the more commonly used lysine-targeting cross-linkers. By systematically analyzing proteins with differing lysine contents and differing flexibilities, we demonstrated clear enhancement in structure elucidation for proteins containing less lysine and with high flexibility. In addition, enrichment approaches of alkynyl-azide click chemistry conjugated with biotin-streptavidin purification (coinciding with parallel orthogonal digestion) improved the identification coverage of cross-links. We show that this photo-cross-linking approach can be used for membrane proteome-wide complex analysis. This method led to the identification of a total of 14066 lysine-X cross-linked site pairs from a total of 2784 proteins. Thus, this cross-linker is a valuable addition to a photo-cross-linking toolkit and improves the identification coverage of XL-MS in functional structure analysis.

Dynamic geometry design of cyclic peptide architectures for RNA structure.

Designing inhibitors for RNA is still challenging due to the bottleneck of maintaining the binding interaction of inhibitor-RNA accompanied by subtle RNA flexibility. Thus, the current approach usually needs to screen thousands of candidate inhibitors for binding. Here, we propose a dynamic geometry design approach to enrich the hits with only a tiny pool of designed geometrically compatible scaffold candidates. First, our method uses graph-based tree decomposition to explore the complementarity rigid binding cyclic peptide and design the amino acid side chain length and charge to fit the RNA pocket. Then, we perform an energy-based dynamical network algorithm to optimize the inhibitor-RNA hydrogen bonds. Dynamic geometry-guided design yields successful inhibitors with low micromolar binding affinity scaffolds and experimentally competes with the natural RNA chaperone. The results indicate that the dynamic geometry method yields higher efficiency and accuracy than traditional methods. The strategy could be further optimized to design the length and chirality by adopting nonstandard amino acids and facilitating RNA engineering for biological or medical applications.

Decoding Protein Dynamics in Cells Using Chemical Cross-Linking and Hierarchical Analysis.

Protein dynamics play a crucial role in their diverse functions. The intracellular environment significantly influences protein dynamics, particularly for intrinsically disordered proteins (IDPs). To comprehensively capture structural information from various proteins within cells and characterize protein dynamics, chemical cross-linking mass spectrometry was employed. In this study, we introduce a hierarchical decoding strategy that enables the investigation of protein dynamics in vivo. Computational analysis based on distance restraints derived from cross-links is used to infer protein dynamics in cells. To facilitate this analysis, we leverage the prior structure obtained from AlphaFold2. By employing this strategy, we can characterize the full-length structure of multi-domain proteins taking into account their distinct dynamic features. Furthermore, by combining restraint sampling with an unbiased sampling and evaluation approach, we can provide a comprehensive description of the intrinsic motion of IDPs. Consequently, the hierarchical strategy we propose holds significant potential in advancing our understanding of the molecular mechanisms that undelie protein functions in cells.

Nucleobase Clustering Contributes to the Formation and Hollowing of Repeat-Expansion RNA Condensate.

RNA molecules with repeat expansion sequences can phase separate into gel-like condensate, which could lead to neurodegenerative diseases. Here, we report that, in the presence of Mg2+, RNA molecules containing 20× CAG repeats self-assemble into three morphologically distinct droplets. Using hyperspectral stimulated Raman microscopy, we show that RNA phase separation is accompanied by the clustering of nucleobases while forfeiting the canonical base-paired structure. As the RNA/Mg2+ ratio increases, the RNA droplets first expand and then shrink to adopt hollow vesicle-like structures. Significantly, for both large and vesicle-like RNA droplets, the nucleobase-clustered structure is more prominent at the rim, suggesting a continuously hardening process. This mechanism may be implicated in the general aging processes of RNA-containing membrane-less organelles.

In-Depth In Vivo Crosslinking in Minutes by a Compact, Membrane-Permeable, and Alkynyl-Enrichable Crosslinker.

Chemical crosslinking coupled with mass spectrometry (CXMS) has emerged as a powerful technique to obtain the dynamic conformations and interaction interfaces of protein complexes. Limited by the poor cell membrane permeability, chemical reactivity, and biocompatibility of crosslinkers, in vivo crosslinking to capture the dynamics of protein complexes with finer temporal resolution and higher coverage is attractive but challenging. In this work, a trifunctional crosslinker bis(succinimidyl) with propargyl tag (BSP), involving compact size, proper amphipathy, and enrichment capacity, was developed to enable better cell membrane permeability and efficient crosslinking in 5 min without obvious cellular interference. Followed by a two-step enrichment method based on click chemistry at the peptide level, 13,098 crosslinked peptides (5068 inter-crosslinked peptides and 8030 intra-crosslinked peptides) were identified under the data threshold of peptide-spectrum matches (PSMs) ≥2 on the basic of the FDR control of 1%, which was the most comprehensive dataset for homo species cells by a non-cleavable crosslinker. Besides, the interactome network comprising 1519 proteins connected by 2913 interaction edges in various intracellular compartments, as well as 80S ribosome structural dynamics, were characterized, showing the great potential of our in vivo crosslinking approach in minutes. All these results demonstrated that our developed BSP could provide a valuable toolkit for the in-depth in vivo analysis of protein-protein interactions (PPIs) and protein architectures with finer temporal resolution.

Alkynyl-Enrichable Carboxyl-Selective Crosslinkers to Increase the Crosslinking Coverage for Deciphering Protein Structures.

The coverage of chemical crosslinking coupled with mass spectrometry (CXMS) is of great importance to determine its ability for deciphering protein structures. At present, N-hydroxysuccinimidyl (NHS) ester-based crosslinkers targeting lysines have been predominantly used in CXMS. However, they are not always effective for some proteins with few lysines. Other amino acid residues such as carboxyl could be crosslinked to complement lysines and improve the crosslinking coverage of CXMS, but the low intrinsic chemical reactivity of carboxyl compromises the application of carboxyl-selective crosslinkers for complex samples. To enhance the crosslinking efficiency targeting acidic residues and realize in-depth crosslinking analysis of complex samples, we developed three new alkynyl-enrichable carboxyl-selective crosslinkers with different reactive groups such as hydrazide, amino, and aminooxy. The crosslinking efficiencies of the three crosslinkers were systematically evaluated, giving the best reactivity of the amino-functionalized crosslinker BAP. Furthermore, BAP was extended to the crosslinking analysis of Escherichia coli lysate in combination with efficient crosslink enrichment. A total of 1291 D/E-D/E crosslinks involved in 392 proteins were identified under a false discovery rate (FDR) of ≤1%. Obvious structural complementarity of BAP was exhibited to the lysine-targeting crosslinker, facilitating the capability of CXMS for protein structure elucidation. To the best of our knowledge, this was the first time for the carboxyl-selective crosslinker to achieve proteome-wide crosslinking analysis of the whole cell lysate. Collectively, we believe that this work not only expands on a promising toolkit of CXMS targeting acidic residues but also provides a valuable guideline to advance the performance of carboxyl-selective crosslinkers.

Suborganelle-Specific Protein Complex Analysis Enabled by in Vivo Cross-Linking Coupled with Proximal Labeling.

The identification of the structure of protein complexes in the subcellular niche of cells is necessary to understand their diverse functions. In this study, we developed a suborganelle proteome labeling assisted in vivo cross-linking (SubPiXL) strategy to identify regional protein conformations and interactions in living cells. Due to the mitochondria's functional importance and well-defined compartmental partitions, the specific conformations and interactome of protein complexes located in the mitochondrial matrix were identified. Compared to the commonly used approach of organelle isolation followed by intact mitochondria cross-linking, our method achieved a more refined spatial characterization for the subcompartment of the cellular organelle. Additionally, this approach avoided cross-contamination and cell microenvironment disruption during organelle isolation. As such, we achieved 73% selectivity for mitochondria and 98% specificity of known suborganelle annotation for the mitochondrial matrix and accessible inner membrane. Meanwhile, more protein-protein interactions (PPIs) with high dynamics were captured, resulting in a 1.67-fold increase in the number of PPI identifications in 1/11th of the time. On the basis of these structural cross-links and the specific characterization of the interactome and conformation, the structural dynamics targeted in the mitochondrial matrix were delineated. Mitochondrial matrix-restricted information for proteins with multisubcellular localizations was then clarified. In summary, SubPiXL is a promising technique for the investigation of suborganelle-resolved protein conformation and interaction analysis and contributes to a better understanding of structure-derived functions.

Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex.

Chemical modifications of RNA have essential roles in a vast range of cellular processes. N(6)-methyladenosine (m(6)A) is an abundant internal modification in messenger RNA and long non-coding RNA that can be dynamically added and removed by RNA methyltransferases (MTases) and demethylases, respectively. An MTase complex comprising methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14) efficiently catalyses methyl group transfer. In contrast to the well-studied DNA MTase, the exact roles of these two RNA MTases in the complex remain to be elucidated. Here we report the crystal structures of the METTL3-METTL14 heterodimer with MTase domains in the ligand-free, S-adenosyl methionine (AdoMet)-bound and S-adenosyl homocysteine (AdoHcy)-bound states, with resolutions of 1.9, 1.71 and 1.61 Å, respectively. Both METTL3 and METTL14 adopt a class I MTase fold and they interact with each other via an extensive hydrogen bonding network, generating a positively charged groove. Notably, AdoMet was observed in only the METTL3 pocket and not in METTL14. Combined with biochemical analysis, these results suggest that in the m(6)A MTase complex, METTL3 primarily functions as the catalytic core, while METTL14 serves as an RNA-binding platform, reminiscent of the target recognition domain of DNA N(6)-adenine MTase. This structural information provides an important framework for the functional investigation of m(6)A.

Pseudopotentials for coarse-grained cross-link-assisted modeling of protein structures.

Pseudopotentials for the chemical cross-links comprising the glutamic- and aspartic-acid side chains bridged with adipic- (ADH) or pimelic-acid hydrazide (PDH), and the lysine side chains bridged with glutaric (BS2 G) or suberic acid (BS3 ) for coarse-grained cross-link-assisted simulations were determined by canonical molecular dynamics with the Amber14sb force field. The potentials depend on the distance between side-chain ends and on side-chain orientation, this preventing from making cross-link contacts across the globule in simulations. The potentials were implemented in the UNRES coarse-grained force field and their effect on the quality of models was assessed with 11 monomeric and 1 dimeric proteins, using synthetic or experimental cross-link data. Simulations with the new potentials resulted in improvement of the generated models compared to unrestrained simulations in more instances compared to those with the statistical potentials.

Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch.

Ubiquitin (Ub) is an important signaling protein. Recent studies have shown that Ub can be enzymatically phosphorylated at S65, and that the resulting pUb exhibits two conformational states-a relaxed state and a retracted state. However, crystallization efforts have yielded only the structure for the relaxed state, which was found similar to that of unmodified Ub. Here we present the solution structures of pUb in both states obtained through refinement against state-specific NMR restraints. We show that the retracted state differs from the relaxed state by the retraction of the last ß-strand and by the extension of the second α-helix. Further, we show that at 7.2, the pKa value for the phosphoryl group in the relaxed state is higher by 1.4 units than that in the retracted state. Consequently, pUb exists in equilibrium between protonated and deprotonated forms and between retracted and relaxed states, with protonated/relaxed species enriched at slightly acidic pH and deprotonated/retracted species enriched at slightly basic pH. The heterogeneity of pUb explains the inability of phosphomimetic mutants to fully mimic pUb. The pH-sensitive conformational switch is likely preserved for polyubiquitin, as single-molecule FRET data indicate that pH change leads to quaternary rearrangement of a phosphorylated K63-linked diubiquitin. Because cellular pH varies among compartments and changes upon pathophysiological insults, our finding suggests that pH and Ub phosphorylation confer additional target specificities and enable an additional layer of modulation for Ub signals.

Theory and practice of using solvent paramagnetic relaxation enhancement to characterize protein conformational dynamics.

Paramagnetic relaxation enhancement (PRE) has been established as a powerful tool in NMR for investigating protein structure and dynamics. The PRE is usually measured with a paramagnetic probe covalently attached at a specific site of an otherwise diamagnetic protein. The present work provides the numerical formulation for probing protein structure and conformational dynamics based on the solvent PRE (sPRE) measurement, using two alternative approaches. An inert paramagnetic cosolute randomly collides with the protein, and the resulting sPRE manifests the relative solvent exposure of protein nuclei. To make the back-calculated sPRE values most consistent with the observed values, the protein structure is either refined against the sPRE, or an ensemble of conformers is selected from a pre-generated library using a Monte Carlo algorithm. The ensemble structure comprises either N conformers of equal occupancy, or two conformers with different relative populations. We demonstrate the sPRE method using GB1, a structurally rigid protein, and calmodulin, a protein comprising two domains and existing in open and closed states. The sPRE can be computed with a stand-alone program for rapid evaluation, or with the invocation of a module in the latest release of the structure calculation software Xplor-NIH. As a label-free method, the sPRE measurement can be readily integrated with other biophysical techniques. The current limitations of the sPRE method are also discussed, regarding accurate measurement and theoretical calculation, model selection and suitable timescale.

Characterizing Protein Dynamics with Integrative Use of Bulk and Single-Molecule Techniques.

A protein dynamically samples multiple conformations, and the conformational dynamics enables protein function. Most biophysical measurements are ensemble-based, with the observables averaged over all members of the ensemble. Though attainable, the decomposition of the observables to the constituent conformational states can be computationally expensive and ambiguous. Here we show that the incorporation of single-molecule fluorescence resonance energy transfer (smFRET) data resolves the ambiguity and affords protein ensemble structures that are more precise and accurate. Using K63-linked diubiquitin, we characterize the dynamic domain arrangements of the model system, with the use of chemical cross-linking coupled with mass spectrometry (CXMS), small-angle X-ray scattering (SAXS), and smFRET techniques. CXMS allows the modeling of protein conformational states that are alternatives to the crystal structure. SAXS provides ensemble-averaged low-resolution shape information. Importantly, smFRET affords state-specific populations, and the FRET distances validate the ensemble structures obtained by refining against CXMS and SAXS restraints. Together, the integrative use of bulk and single-molecule techniques affords better insight into protein dynamics and shall be widely implemented in structural biology.

Modeling Protein Excited-state Structures from "Over-length" Chemical Cross-links.

Chemical cross-linking coupled with mass spectroscopy (CXMS) provides proximity information for the cross-linked residues and is used increasingly for modeling protein structures. However, experimentally identified cross-links are sometimes incompatible with the known structure of a protein, as the distance calculated between the cross-linked residues far exceeds the maximum length of the cross-linker. The discrepancies may persist even after eliminating potentially false cross-links and excluding intermolecular ones. Thus the "over-length" cross-links may arise from alternative excited-state conformation of the protein. Here we present a method and associated software DynaXL for visualizing the ensemble structures of multidomain proteins based on intramolecular cross-links identified by mass spectrometry with high confidence. Representing the cross-linkers and cross-linking reactions explicitly, we show that the protein excited-state structure can be modeled with as few as two over-length cross-links. We demonstrate the generality of our method with three systems: calmodulin, enzyme I, and glutamine-binding protein, and we show that these proteins alternate between different conformations for interacting with other proteins and ligands. Taken together, the over-length chemical cross-links contain valuable information about protein dynamics, and our findings here illustrate the relationship between dynamic domain movement and protein function.

Carboxylate-Selective Chemical Cross-Linkers for Mass Spectrometric Analysis of Protein Structures.

Chemical cross-linking coupled with mass spectrometry (CXMS) facilitates structural analysis of proteins. As current CXMS applications are almost exclusively limited to lysine residues, they can only retrieve a small portion of the structural information theoretically accessible to CXMS. Chemical cross-linkers targeting the acidic residues Asp/Glu could greatly enhance the power of CXMS. However, it has been difficult to develop chemistries that offer selectivity and efficiency under physiological conditions. Here, we report a class of carboxylate-selective diazo-containing cross-linkers (Diazoker) of which Diazoker 1, with a spacer arm consisting of two ethan-1,2-diol units, is the best example. Unlike previously developed carboxylate-selective cross-linkers like pimelic acid dihydrazide (PDH), Diazoker 1 does not require a coupling reagent. We tested Diazoker 1 on nine model proteins and found that Diazoker 1 generated an average of 73 cross-linked peptide pairs per protein. Although this is 32% fewer than the number generated by PDH, the Diazoker 1 cross-links have a higher rate of compatibility with protein crystal structures. From a more complex protein mixture, Diazoker 1 and PDH identified 75 and 76 cross-linked peptide pairs, respectively. The Asp/Glu residues cross-linked by Diazoker 1 are not the same as those cross-linked by PDH. Diazoker 1 favors acidic residues that are less exposed to solvent. In conclusion, Diazoker 1 is complementary to existing cross-linkers and expands the toolkit of CXMS for structural analysis of proteins.

Crystal structure of WA352 provides insight into cytoplasmic male sterility in rice.

Plant cytoplasmic male sterility (CMS) is an important phenomenon and is widely utilized in hybrid crop breeding. The Wild Abortive CMS (CMS-WA), a well-known CMS type, has been successfully applied in the commercial production of hybrid rice seeds for more than 40 years. The CMS-WA causal gene WA352 encodes a novel transmenbrane protein and the interacts with the mitochondrial copper chaperone COX11, triggering reactive oxygen species production and resulting in male sterility in CMS-WA lines. However, the structure of WA352 is currently unknown, and the structural mechanism whereby WA352 perturbs COX11 function to cause CMS remains largely unknown. Here, we report the crystal structure of the C-terminal functional domain of WA352 at 1.3 Šresolution. This functional domain, consisting of five α helices, is spindle-shaped with a length of 42 Å, and a diameter of 28 Å. Notably, the absence of any structural similarity to a known protein structure suggests that the WA352 functional domain is a novel fold. In addition, surface conservation analysis and structural modeling of the WA352-COX11 complex revealed details about the WA352-COX11 interaction. Further structural analysis suggested that the WA352-COX11 interaction blocks the copper ion transportation activity of COX11, which is essential for the assembly of cytochrome c oxidase, resulting in male sterility in CMS-WA lines. Our study paves the way toward structural determination of the WA352-COX11 complex and provides new insight into the mechanism of plant CMS.

Structural basis of nonribosomal peptide macrocyclization in fungi.

Nonribosomal peptide synthetases (NRPSs) in fungi biosynthesize important pharmaceutical compounds, including penicillin, cyclosporine and echinocandin. To understand the fungal strategy of forging the macrocyclic peptide linkage, we determined the crystal structures of the terminal condensation-like (CT) domain and the holo thiolation (T)-CT complex of Penicillium aethiopicum TqaA. The first, to our knowledge, structural depiction of the terminal module in a fungal NRPS provides a molecular blueprint for generating new macrocyclic peptide natural products.