Probing Substrate-Loaded Carrier Proteins by Nuclear Magnetic Resonance.

Carrier proteins (CPs) are central actors in nonribosomal peptide synthetases (NRPSs) as they interact with all catalytic domains, and because they covalently hold the substrates and intermediates leading to the final product. Thus, how CPs and their partner domains recognize and engage with each other as a function of CP cargos is paramount to understanding and engineering NRPSs. However, rapid hydrolysis of the labile thioester bonds holding substrates challenges molecular and biophysical studies to determine the molecular mechanisms of domain recognition. In this chapter, we describe a protocol to counteract hydrolysis and study loaded carrier proteins at the atomic level with nuclear magnetic resonance (NMR) spectroscopy. The method relies on loading CPs in situ, with adenylation domains in the NMR tube, to reach substrate-loaded CPs at steady state. We describe controls and experimental readouts necessary to assess the integrity of the sample and maintain loading on CPs. Our approach provides a basis to conduct subsequent NMR experiments and obtain kinetic, thermodynamic, dynamic, and structural parameters of substrate-loaded CPs alone or in the presence of other domains.

Dynamics and mechanistic interpretations of nonribosomal peptide synthetase cyclization domains.

Ox-/thiazoline groups in nonribosomal peptides are formed by a variant of peptide-forming condensation domains called heterocyclization (Cy) domains and appear in a range of pharmaceutically important natural products and virulence factors. Recent cryo-EM, crystallographic, and NMR studies of Cy domains make it opportune to revisit outstanding questions regarding their molecular mechanisms. This review covers structural and dynamical findings about Cy domains that will inform future bioengineering efforts and our understanding of natural product synthesis.

High-resolution structures of a siderophore-producing cyclization domain from Yersinia pestis offer a refined proposal of substrate binding.

Nonribosomal peptide synthetase heterocyclization (Cy) domains generate biologically important oxazoline/thiazoline groups found in natural products, including pharmaceuticals and virulence factors such as some siderophores. Cy domains catalyze consecutive condensation and cyclodehydration reactions, although the mechanism is unknown. To better understand Cy domain catalysis, here we report the crystal structure of the second Cy domain (Cy2) of yersiniabactin synthetase from the causative agent of the plague, Yersinia pestis. Our high-resolution structure of Cy2 adopts a conformation that enables exploration of interactions with the extended thiazoline-containing cyclodehydration intermediate and the acceptor carrier protein (CP) to which it is tethered. We also report complementary electrostatic interfaces between Cy2 and its donor CP that mediate donor binding. Finally, we explored domain flexibility through normal mode analysis and identified small-molecule fragment-binding sites that may inform future antibiotic design targeting Cy function. Our results suggest how CP binding may influence global Cy conformations, with consequences for active-site remodeling to facilitate the separate condensation and cyclodehydration steps as well as potential inhibitor development.

15N-Detected TROSY NMR experiments to study large disordered proteins in high-field magnets.

Intrinsically disordered regions (IDRs) of proteins are critical in the regulation of biological processes but difficult to study structurally. Nuclear magnetic resonance (NMR) is uniquely equipped to provide structural information on IDRs at atomic resolution; however, existing NMR methods often pose a challenge for large molecular weight IDRs. Resonance assignment of IDRs using 15ND-detection was previously demonstrated and shown to overcome some of these limitations. Here, we improve the methodology by overcoming the need for deuterated buffers and provide better sensitivity and resolution at higher magnetic fields and physiological salt concentrations using transverse relaxation optimized spectroscopy (TROSY). Finally, large disordered regions with low sequence complexity can be assigned efficiently using these new methods as demonstrated by achieving near complete assignment of the 398-residue N-terminal IDR of the transcription factor NFAT1 harboring 18% prolines.

NMR as a readout to monitor and restore the integrity of complex chemoenzymatic reactions.

The non-invasive nature of NMR offers a means to monitor biochemical reactions in situ at the atomic level. We harness this advantage to monitor a complex chemoenzymatic reaction that sequentially modifies reagents and loads the product on a nonribosomal peptide synthetase carrier protein. We present a protocol including a pulse sequence that permits to assess both the integrity of reagents and the completion of each step in the reaction, thus alleviating otherwise time-consuming and costly approaches to debug and repeat inefficient reactions. This study highlights the importance of NMR as a tool to establish reliable and reproducible experimental conditions in biochemical studies.

Global protein dynamics as communication sensors in peptide synthetase domains.

Biological activity is governed by the timely redistribution of molecular interactions, and static structural snapshots often appear insufficient to provide the molecular determinants that choreograph communication. This conundrum applies to multidomain enzymatic systems called nonribosomal peptide synthetases (NRPSs), which assemble simple substrates into complex metabolites, where a dynamic domain organization challenges rational design to produce new pharmaceuticals. Using a nuclear magnetic resonance (NMR) atomic-level readout of biochemical transformations, we demonstrate that global structural fluctuations help promote substrate-dependent communication and allosteric responses, and impeding these global dynamics by a point-site mutation hampers allostery and molecular recognition. Our results establish global structural dynamics as sensors of molecular events that can remodel domain interactions, and they provide new perspectives on mechanisms of allostery, protein communication, and NRPS synthesis.

Using delayed decoupling to attenuate residual signals in editing filters.

Isotope filtering methods are instrumental in biomolecular nuclear magnetic resonance (NMR) studies as they isolate signals of chemical moieties of interest within complex molecular assemblies. However, isotope filters suppress undesired signals of isotopically enriched molecules through scalar couplings, and variations in scalar couplings lead to imperfect suppressions, as occurs for aliphatic and aromatic moieties in proteins. Here, we show that signals that have escaped traditional filters can be attenuated with mitigated sensitivity losses for the desired signals of unlabeled moieties. The method uses a shared evolution between the detection and preceding preparation period to establish non-observable antiphase coherences and eliminates them through composite pulse decoupling. We demonstrate the method by isolating signals of an unlabeled post-translational modification tethered to an isotopically enriched protein.

Minimizing Pervasive Artifacts in 4D Covariance Maps for Protein Side Chain NMR Assignments.

Nuclear magnetic resonance (NMR) is a mainstay of biophysical studies that provides atomic level readouts to formulate molecular mechanisms. Side chains are particularly important to derive mechanisms involving proteins as they carry functional groups, but NMR studies of side chains are often limited by challenges in assigning their signals. Here, we designed a novel computational method that combines spectral derivatives and matrix square-rooting to produce reliable 4D covariance maps from routinely acquired 3D spectra and facilitates side chain resonance assignments. Thus, we generate two 4D maps from 3D-HcccoNH and 3D-HCcH-TOCSY spectra that each help overcome signal overlap or sensitivity losses. These 4D maps feature HC-HSQCs of individual side chains that can be paired to assigned backbone amide resonances of individual aliphatic signals, and both are obtained from a single modified covariance calculation. Further, we present 4D maps produced using conventional triple resonance experiments to easily assign asparagine side chain amide resonances. The 4D covariance maps encapsulate the lengthy manual pattern recognition used in traditional assignment methods and distill the information as correlations that can be easily visualized. We showcase the utility of the 4D covariance maps with a 10 kDa peptidyl carrier protein and a 52 kDa cyclization domain from a nonribosomal peptide synthetase.

Conserved allosteric ensembles in disordered proteins using TROSY/anti-TROSY R2-filtered spectroscopy.

Defining the role of intrinsic disorder in proteins in the myriad of biological processes with which it is involved represents a significant goal in modern biophysics. Toward this end, NMR is uniquely suited for molecular studies of dynamic and disordered regions, but studying these regions in concert with their more structured domains and binding partners presents spectroscopic challenges. Here, we investigate the interactions between the structured and disordered regions of the human glucocorticoid receptor (GR). To do this, we developed an NMR strategy that relies on a novel relaxation filter for the simultaneous study of structured and unstructured regions. Using this approach, we conducted a comparative analysis of three translational isoforms of GR containing a folded DNA-binding domain (DBD) and two disordered regions that flank the DBD, one of which varies in size in the different isoforms. Notably, we were able to assign resonances that had previously been inaccessible because of the spectral complexity of the translational isoforms, which in turn allowed us to 1) identify a region of the structured DBD that undergoes significant changes in the local chemical environment in the presence of the disordered region and 2) determine differences in the conformational ensembles of the disordered regions of the translational isoforms. Furthermore, an ensemble-based thermodynamic analysis of the isoforms reveals conserved patterns of stability within the N-terminal domain of GR that persist despite low sequence conservation. These studies provide an avenue for further investigations of the mechanistic underpinnings of the functional relevance of the translational isoforms of GR while also providing a general NMR strategy for studying systems containing both structured and disordered regions.

Covariance NMR Processing and Analysis for Protein Assignment.

During NMR resonance assignment it is often necessary to relate nuclei to one another indirectly, through their common correlations to other nuclei. Covariance NMR has emerged as a powerful technique to correlate such nuclei without relying on error-prone peak peaking. However, false-positive artifacts in covariance spectra have impeded a general application to proteins. We recently introduced pre- and postprocessing steps to reduce the prevalence of artifacts in covariance spectra, allowing for the calculation of a variety of 4D covariance maps obtained from diverse combinations of pairs of 3D spectra, and we have employed them to assign backbone and sidechain resonances in two large and challenging proteins. In this chapter, we present a detailed protocol describing how to (1) properly prepare existing 3D spectra for covariance, (2) understand and apply our processing script, and (3) navigate and interpret the resulting 4D spectra. We also provide solutions to a number of errors that may occur when using our script, and we offer practical advice when assigning difficult signals. We believe such 4D spectra, and covariance NMR in general, can play an integral role in the assignment of NMR signals.

Molecular impact of covalent modifications on nonribosomal peptide synthetase carrier protein communication.

Nonribosomal peptide synthesis involves the interplay between covalent protein modifications, conformational fluctuations, catalysis, and transient protein-protein interactions. Delineating the mechanisms involved in orchestrating these various processes will deepen our understanding of domain-domain communication in nonribosomal peptide synthetases (NRPSs) and lay the groundwork for the rational reengineering of NRPSs by swapping domains handling different substrates to generate novel natural products. Although many structural and biochemical studies of NRPSs exist, few studies have focused on the energetics and dynamics governing the interactions in these systems. Here, we present detailed binding studies of an adenylation domain and its partner carrier protein in apo-, holo-, and substrate-loaded forms. Results from fluorescence anisotropy, isothermal titration calorimetry, and NMR titrations indicated that covalent modifications to a carrier protein modulate domain communication, suggesting that chemical modifications to carrier proteins during NRPS synthesis may impart directionality to sequential NRPS domain interactions. Comparison of the structure and dynamics of an apo-aryl carrier protein with those of its modified forms revealed structural fluctuations induced by post-translational modifications and mediated by modulations of protein dynamics. The results provide a comprehensive molecular description of a carrier protein throughout its life cycle and demonstrate how a network of dynamic residues can propagate the molecular impact of chemical modifications throughout a protein and influence its affinity toward partner domains.

Molecular Cross-Talk between Nonribosomal Peptide Synthetase Carrier Proteins and Unstructured Linker Regions.

Nonribosomal peptide synthetases (NRPSs) employ multiple domains separated by linker regions to incorporate substrates into natural products. During synthesis, substrates are covalently tethered to carrier proteins that translocate between catalytic partner domains. The molecular parameters that govern translocation and associated linker remodeling remain unknown. Here, we used NMR to characterize the structure, dynamics, and invisible states of a peptidyl carrier protein flanked by its linkers. We showed that the N-terminal linker stabilizes and interacts with the protein core while modulating dynamics at specific sites involved in post-translational modifications and/or domain interactions. The results detail the molecular communication between peptidyl carrier proteins and their linkers and could guide efforts in engineering NRPSs to obtain new pharmaceuticals.

Covariance nuclear magnetic resonance methods for obtaining protein assignments and novel correlations.

Protein NMR resonance assignment can be a tedious and error prone process, and it is often a limiting factor in biomolecular NMR studies. Challenges are exacerbated in larger proteins, disordered proteins, and often alpha-helical proteins, owing to an increase in spectral complexity and frequency degeneracies. Here, several multi-dimensional spectra must be inspected and compared in an iterative manner before resonances can be assigned with confidence. Over the last two decades, covariance NMR has evolved to become applicable to protein multi-dimensional spectra. The method, previously used to generate new correlations from spectra of small organic molecules, can now be used to recast assignment procedures as mathematical operations on NMR spectra. These operations result in multidimensional correlation maps combining all information from input spectra and providing direct correlations between moieties that would otherwise be compared indirectly through reporter nuclei. Thus, resonances of sequential residues can be identified and side-chain signals can be assigned by visual inspection of 4D arrays. This review highlights advances in covariance NMR that permitted to generate reliable 4D arrays and describes how these arrays can be obtained from conventional NMR spectra.

Effortless assignment with 4D covariance sequential correlation maps.

Traditional Nuclear Magnetic Resonance (NMR) assignment procedures for proteins rely on preliminary peak-picking to identify and label NMR signals. However, such an approach has severe limitations when signals are erroneously labeled or completely neglected. The consequences are especially grave for proteins with substantial peak overlap, and mistakes can often thwart entire projects. To overcome these limitations, we previously introduced an assignment technique that bypasses traditional pick peaking altogether. Covariance Sequential Correlation Maps (COSCOMs) transform the indirect connectivity information provided by multiple 3D backbone spectra into direct (H, N) to (H, N) correlations. Here, we present an updated method that utilizes a single four-dimensional spectrum rather than a suite of three-dimensional spectra. We demonstrate the advantages of 4D-COSCOMs relative to their 3D counterparts. We introduce improvements accelerating their calculation. We discuss practical considerations affecting their quality. And finally we showcase their utility in the context of a 52 kDa cyclization domain from a non-ribosomal peptide synthetase.

Solution Structure of a Nonribosomal Peptide Synthetase Carrier Protein Loaded with Its Substrate Reveals Transient, Well-Defined Contacts.

Nonribosomal peptide synthetases (NRPSs) are microbial enzymes that produce a wealth of important natural products by condensing substrates in an assembly line manner. The proper sequence of substrates is obtained by tethering them to phosphopantetheinyl arms of holo carrier proteins (CPs) via a thioester bond. CPs in holo and substrate-loaded forms visit NRPS catalytic domains in a series of transient interactions. A lack of structural information on substrate-loaded carrier proteins has hindered our understanding of NRPS synthesis. Here, we present the first structure of an NRPS aryl carrier protein loaded with its substrate via a native thioester bond, together with the structure of its holo form. We also present the first quantification of NRPS CP backbone dynamics. Our results indicate that prosthetic moieties in both holo and loaded forms are in contact with the protein core, but they also sample states in which they are disordered and extend in solution. We observe that substrate loading induces a large conformational change in the phosphopantetheinyl arm, thereby modulating surfaces accessible for binding to other domains. Our results are discussed in the context of NRPS domain interactions.

Assignment of methyl NMR resonances of a 52 kDa protein with residue-specific 4D correlation maps.

Methyl groups have become key probes for structural and functional studies by nuclear magnetic resonance. However, their NMR signals cluster in a small spectral region and assigning their resonances can be a tedious process. Here, we present a method that facilitates assignment of methyl resonances from assigned amide groups. Calculating the covariance between sensitive methyl and amide 3D spectra, each providing correlations to C(α) and C(ß) separately, produces 4D correlation maps directly correlating methyl groups to amide groups. Optimal correlation maps are obtained by extracting residue-specific regions, applying derivative to the dimensions subject to covariance, and multiplying 4D maps stemming from different 3D spectra. The latter procedure rescues weak signals that may be missed in traditional assignment procedures. Using these covariance correlation maps, nearly all assigned isoleucine, leucine, and valine amide resonances of a 52 kDa nonribosomal peptide synthetase cyclization domain were paired with their corresponding methyl groups.

A nuclear magnetic resonance method for probing molecular influences of substrate loading in nonribosomal peptide synthetase carrier proteins.

Carrier proteins (CPs) play a central role in nonribosomal peptide synthetases (NRPSs) as they shuttle covalently attached substrates between active sites. Understanding how the covalent attachment of a substrate (loading) influences the molecular properties of CPs is key to determining the mechanism of NRPS synthesis. However, structural studies have been impaired by substrate hydrolysis. Here, we used nuclear magnetic resonance spectroscopy to monitor substrate loading of a CP and to overcome hydrolysis. Our results reveal the spectroscopic signature of substrate loading and provide evidence of molecular communication between an NRPS carrier protein and its covalently attached substrate.

A 3D time-shared NOESY experiment designed to provide optimal resolution for accurate assignment of NMR distance restraints in large proteins.

Structure determination of proteins by solution NMR has become an established method, but challenges increase steeply with the size of proteins. Notably, spectral crowding and signal overlap impair the analysis of cross-peaks in NOESY spectra that provide distance restraints for structural models. An optimal spectral resolution can alleviate overlap but requires prohibitively long experimental time with existing methods. Here we present a time-shared 3D experiment optimized for large proteins that provides ¹5N and ¹³C dispersed NOESY spectra in a single measurement. NOESY correlations appear in the detected dimension and hence benefit from the highest resolution achievable of all dimensions without increase in experimental time. By design, this experiment is inherently optimal for non-uniform sampling acquisition when compared to current alternatives. Thus, ¹5N and ¹³C dispersed NOESY spectra with ultra-high resolution in all dimensions were acquired in parallel within about 4 days instead of 80 days for a 52 kDa monomeric protein at a concentration of 350 µM.

Facilitated assignment of large protein NMR signals with covariance sequential spectra using spectral derivatives.

Nuclear magnetic resonance (NMR) studies of larger proteins are hampered by difficulties in assigning NMR resonances. Human intervention is typically required to identify NMR signals in 3D spectra, and subsequent procedures depend on the accuracy of this so-called peak picking. We present a method that provides sequential connectivities through correlation maps constructed with covariance NMR, bypassing the need for preliminary peak picking. We introduce two novel techniques to minimize false correlations and merge the information from all original 3D spectra. First, we take spectral derivatives prior to performing covariance to emphasize coincident peak maxima. Second, we multiply covariance maps calculated with different 3D spectra to destroy erroneous sequential correlations. The maps are easy to use and can readily be generated from conventional triple-resonance experiments. Advantages of the method are demonstrated on a 37 kDa nonribosomal peptide synthetase domain subject to spectral overlap.

Practical aspects of NMR signal assignment in larger and challenging proteins.

NMR has matured into a technique routinely employed for studying proteins in near physiological conditions. However, applications to larger proteins are impeded by the complexity of the various correlation maps necessary to assign NMR signals. This article reviews the data analysis techniques traditionally employed for resonance assignment and describes alternative protocols necessary for overcoming challenges in large protein spectra. In particular, simultaneous analysis of multiple spectra may help overcome ambiguities or may reveal correlations in an indirect manner. Similarly, visualization of orthogonal planes in a multidimensional spectrum can provide alternative assignment procedures. We describe examples of such strategies for assignment of backbone, methyl, and nOe resonances. We describe experimental aspects of data acquisition for the related experiments and provide guidelines for preliminary studies. Focus is placed on large folded monomeric proteins and examples are provided for 37, 48, 53, and 81 kDa proteins.