Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands.

With the great progress on determining protein structures over the last decade comes a renewed appreciation that structures must be combined with dynamics and energetics to understand function. Fluorescence spectroscopy, specifically Förster resonance energy transfer (FRET), provides a great window into dynamics and energetics due to its application at physiological temperatures and ability to measure dynamics on the ångström scale. We have recently advanced transition metal FRET (tmFRET) to study allosteric regulation of maltose binding protein and have reported measurements of maltose-dependent distance changes with an accuracy of ∼1.5 Å. When paired with the noncanonical amino acid Acd as a donor, our previous tmFRET acceptors were useful over a working distance of 10 to 20 Å. Here, we use cysteine-reactive bipyridyl and phenanthroline compounds as chelators for Fe2+ and Ru2+ to produce novel tmFRET acceptors to expand the working distance to as long as 50 Å, while preserving our ability to resolve even small maltose-dependent changes in distance. We compare our measured FRET efficiencies to predictions based on models using rotameric ensembles of the donors and acceptors to demonstrate that steady-state measurements of tmFRET with our new probes have unprecedented ability to measure conformational rearrangements under physiological conditions.

Measuring conformational equilibria in allosteric proteins with time-resolved tmFRET.

Proteins are the workhorses of biology, orchestrating a myriad of cellular functions through intricate conformational changes. Protein allostery, the phenomenon where binding of ligands or environmental changes induce conformational rearrangements in the protein, is fundamental to these processes. We have previously shown that transition metal Förster resonance energy transfer (tmFRET) can be used to interrogate the conformational rearrangements associated with protein allostery and have recently introduced novel FRET acceptors utilizing metal-bipyridyl derivatives to measure long (>20 Å) intramolecular distances in proteins. Here, we combine our tmFRET system with fluorescence lifetime measurements to measure the distances, conformational heterogeneity, and energetics of maltose-binding protein, a model allosteric protein. Time-resolved tmFRET captures near-instantaneous snapshots of distance distributions, offering insights into protein dynamics. We show that time-resolved tmFRET can accurately determine distance distributions and conformational heterogeneity of proteins. Our results demonstrate the sensitivity of time-resolved tmFRET in detecting subtle conformational or energetic changes in protein conformations, which are crucial for understanding allostery. In addition, we extend the use of metal-bipyridyl compounds, showing that Cu(phen)2+ can serve as a spin label for pulse dipolar electron paramagnetic resonance (EPR) spectroscopy, a method that also reveals distance distributions and conformational heterogeneity. The EPR studies both establish Cu(phen)2+ as a useful spin label for pulse dipolar EPR and validate our time-resolved tmFRET measurements. Our approach offers a versatile tool for deciphering conformational landscapes and understanding the regulatory mechanisms governing biological processes.

Modeling of Cu(II)-based protein spin labels using rotamer libraries.

The bifunctional spin label double-histidine copper-(II) capped with nitrilotriacetate [dHis-Cu(II)-NTA], used in conjunction with electron paramagnetic resonance (EPR) methods can provide high-resolution distance data for investigating protein structure and backbone conformational diversity. Quantitative utilization of this data is limited due to a lack of rapid and accurate dHis-Cu(II)-NTA modeling methods that can be used to translate experimental data into modeling restraints. Here, we develop two dHis-Cu(II)-NTA rotamer libraries using a set of recently published molecular dynamics simulations and a semi-empirical meta-dynamics-based conformational ensemble sampling tool for use with the recently developed chiLife bifunctional spin label modeling method. The accuracy of both the libraries and the modeling method are tested by comparing model predictions to experimentally determined distance distributions. We show that this method is accurate with absolute deviation between the predicted and experimental modes between 0.0-1.2 Å with an average of 0.6 Å over the test data used. In doing so, we also validate the generality of the chiLife bifunctional label modeling method. Taken together, the increased structural resolution and modeling accuracy of dHis-Cu(II)-NTA over other spin labels promise improvements in the accuracy and resolution of protein models by EPR.

Measuring conformational equilibria in allosteric proteins with time-resolved tmFRET.

Proteins are the workhorses of biology, orchestrating a myriad of cellular functions through intricate conformational changes. Protein allostery, the phenomenon where binding of ligands or environmental changes induce conformational rearrangements in the protein, is fundamental to these processes. We have previously shown that transition metal Förster resonance energy transfer (tmFRET) can be used to interrogate the conformational rearrangements associated with protein allostery and have recently introduced novel FRET acceptors utilizing metal-bipyridyl derivatives to measure long (>20 Å) intramolecular distances in proteins. Here, we combine our tmFRET system with fluorescence lifetime measurements to measure the distances, conformational heterogeneity, and energetics of maltose binding protein (MBP), a model allosteric protein. Time-resolved tmFRET captures near-instantaneous snapshots of distance distributions, offering insights into protein dynamics. We show that time-resolved tmFRET can accurately determine distance distributions and conformational heterogeneity of proteins. Our results demonstrate the sensitivity of time-resolved tmFRET in detecting subtle conformational or energetic changes in protein conformations, which are crucial for understanding allostery. In addition, we extend the use of metal-bipyridyl compounds, showing Cu(phen)2+ can serve as a spin label for pulse dipolar electron paramagnetic resonance (EPR) spectroscopy, a method which also reveals distance distributions and conformational heterogeneity. The EPR studies both establish Cu(phen)2+ as a useful spin label for pulse dipolar EPR and validate our time-resolved tmFRET measurements. Our approach offers a versatile tool for deciphering conformational landscapes and understanding the regulatory mechanisms governing biological processes.

Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands.

With the great progress on determining protein structures over the last decade comes a renewed appreciation that structures must be combined with dynamics and energetics to understand function. Fluorescence spectroscopy, specifically Förster resonance energy transfer (FRET), provides a great window into dynamics and energetics due to its application at physiological temperatures and ability to measure dynamics on the ångström scale. We have recently advanced transition metal FRET (tmFRET) to study allosteric regulation of maltose binding protein and have reported measurements of maltose-dependent distance changes with an accuracy of ~1.5 Å. When paired with the noncanonical amino acid Acd as a donor, our previous tmFRET acceptors were useful over a working distance of 10 Å to 20 Å. Here, we use cysteine-reactive bipyridyl and phenanthroline compounds as chelators for Fe2+ and Ru2+ to produce novel tmFRET acceptors to expand the working distance to as long as 50 Å, while preserving our ability to resolve even small maltose-dependent changes in distance. We compare our measured FRET efficiencies to predictions based on models using rotameric ensembles of the donors and acceptors to demonstrate that steady-state measurements of tmFRET with our new probes have unprecedented ability to measure conformational rearrangements under physiological conditions.

Design of stimulus-responsive two-state hinge proteins.

In nature, proteins that switch between two conformations in response to environmental stimuli structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Designing proteins with two distinct but fully structured conformations is a challenge for protein design as it requires sculpting an energy landscape with two distinct minima. Here we describe the design of "hinge" proteins that populate one designed state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, double electron-electron resonance spectroscopy, and binding measurements demonstrate that despite the significant structural differences the two states are designed with atomic level accuracy and that the conformational and binding equilibria are closely coupled.

A novel approach to modeling side chain ensembles of the bifunctional spin label RX.

We introduce a novel approach to modeling side chain ensembles of bifunctional spin labels. This approach utilizes rotamer libraries to generate side chain conformational ensembles. Because the bifunctional label is constrained by two attachment sites, the label is split into two monofunctional rotamers which are first attached to their respective sites, then rejoined by a local optimization in dihedral space. We validate this method against a set of previously published experimental data using the bifunctional spin label, RX. This method is relatively fast and can readily be used for both experimental analysis and protein modeling, providing significant advantages over modeling bifunctional labels with molecular dynamics simulations. Use of bifunctional labels for site directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy dramatically reduces label mobility, which can significantly improve resolution of small changes in protein backbone structure and dynamics. Coupling the use of bifunctional labels with side chain modeling methods allows for improved quantitative application of experimental SDSL EPR data to protein modeling.

chiLife: An open-source Python package for in silico spin labeling and integrative protein modeling.

Here we introduce chiLife, a Python package for site-directed spin label (SDSL) modeling for electron paramagnetic resonance (EPR) spectroscopy, in particular double electron-electron resonance (DEER). It is based on in silico attachment of rotamer ensemble representations of spin labels to protein structures. chiLife enables the development of custom protein analysis and modeling pipelines using SDSL EPR experimental data. It allows the user to add custom spin labels, scoring functions and spin label modeling methods. chiLife is designed with integration into third-party software in mind, to take advantage of the diverse and rapidly expanding set of molecular modeling tools available with a Python interface. This article describes the main design principles of chiLife and presents a series of examples.

Structural studies of human fission protein FIS1 reveal a dynamic region important for GTPase DRP1 recruitment and mitochondrial fission.

Fission protein 1 (FIS1) and dynamin-related protein 1 (DRP1) were initially described as being evolutionarily conserved for mitochondrial fission, yet in humans the role of FIS1 in this process is unclear and disputed by many. In budding yeast where Fis1p helps to recruit the DRP1 ortholog from the cytoplasm to mitochondria for fission, an N-terminal "arm" of Fis1p is required for function. The yeast Fis1p arm interacts intramolecularly with a conserved tetratricopeptide repeat core and governs in vitro interactions with yeast DRP1. In human FIS1, NMR and X-ray structures show different arm conformations, but its importance for human DRP1 recruitment is unknown. Here, we use molecular dynamics simulations and comparisons to experimental NMR chemical shifts to show the human FIS1 arm can adopt an intramolecular conformation akin to that observed with yeast Fis1p. This finding is further supported through intrinsic tryptophan fluorescence and NMR experiments on human FIS1 with and without the arm. Using NMR, we observed the human FIS1 arm is also sensitive to environmental changes. We reveal the importance of these findings in cellular studies where removal of the FIS1 arm reduces DRP1 recruitment and mitochondrial fission similar to the yeast system. Moreover, we determined that expression of mitophagy adapter TBC1D15 can partially rescue arm-less FIS1 in a manner reminiscent of expression of the adapter Mdv1p in yeast. These findings point to conserved features of FIS1 important for its activity in mitochondrial morphology. More generally, other tetratricopeptide repeat-containing proteins are flanked by disordered arms/tails, suggesting possible common regulatory mechanisms.

Comparative evaluation of spin-label modeling methods for protein structural studies.

Site-directed spin-labeling electron paramagnetic resonance spectroscopy is a powerful technique for the investigation of protein structure and dynamics. Accurate spin-label modeling methods are essential to make full quantitative use of site-directed spin-labeling electron paramagnetic resonance data for protein modeling and model validation. Using a set of double electron-electron resonance data from seven different site pairs on maltodextrin/maltose-binding protein under two different conditions using five different spin labels, we compare the ability of two widely used spin-label modeling methods, based on accessible volume sampling and rotamer libraries, to predict experimental distance distributions. We present a spin-label modeling approach inspired by canonical side-chain modeling methods and compare modeling accuracy with the established methods.

Dipolar pathways in dipolar EPR spectroscopy.

Dipolar electron paramagnetic resonance (EPR) experiments such as double electron-electron resonance (DEER) measure distributions of nanometer-scale distances between unpaired electrons, which provide valuable information for structural characterization of proteins and other macromolecular systems. To determine these distributions from the experimental signal, it is critical to employ an accurate model of the signal. For dilute samples of doubly spin-labeled molecules, the signal is a product of an intramolecular and an intermolecular contribution. We present a general model based on dipolar pathways valid for dipolar EPR experiments with spin-1/2 labels. Our results show that the intramolecular contribution consists of a sum and the intermolecular contribution consists of a product over individual dipolar pathway contributions. We examine several commonly used dipolar EPR experiments in terms of dipolar pathways and show experimental results confirming the theoretical predictions. This multi-pathway model makes it possible to analyze a wide range of dipolar EPR experiments within a single theoretical framework.

Perspectives on the Pseudomonas aeruginosa Type III Secretion System Effector ExoU and Its Subversion of the Host Innate Immune Response to Infection.

Pseudomonas aeruginosa is an opportunistic, Gram-negative pathogen and an important cause of hospital acquired infections, especially in immunocompromised patients. Highly virulent P. aeruginosa strains use a type III secretion system (T3SS) to inject exoenzyme effectors directly into the cytoplasm of a target host cell. P. aeruginosa strains that express the T3SS effector, ExoU, associate with adverse outcomes in critically ill patients with pneumonia, owing to the ability of ExoU to rapidly damage host cell membranes and subvert the innate immune response to infection. Herein, we review the structure, function, regulation, and virulence characteristics of the T3SS effector ExoU, a highly cytotoxic phospholipase A2 enzyme.

Characterization of the ExoU activation mechanism using EPR and integrative modeling.

ExoU, a type III secreted phospholipase effector of Pseudomonas aeruginosa, serves as a prototype to model large, dynamic, membrane-associated proteins. ExoU is synergistically activated by interactions with membrane lipids and ubiquitin. To dissect the activation mechanism, structural homology was used to identify an unstructured loop of approximately 20 residues in the ExoU amino acid sequence. Mutational analyses indicate the importance of specific loop amino acid residues in mediating catalytic activity. Engineered disulfide cross-links show that loop movement is required for activation. Site directed spin labeling EPR and DEER (double electron-electron resonance) studies of apo and holo states demonstrate local conformational changes at specific sites within the loop and a conformational shift of the loop during activation. These data are consistent with the formation of a substrate-binding pocket providing access to the catalytic site. DEER distance distributions were used as constraints in RosettaDEER to construct ensemble models of the loop in both apo and holo states, significantly extending the range for modeling a conformationally dynamic loop.

Achromobacter xylosoxidans Cellular Pathology Is Correlated with Activation of a Type III Secretion System.

Achromobacter xylosoxidans is increasingly recognized as a colonizer of cystic fibrosis (CF) patients, but the role that A. xylosoxidans plays in pathology remains unknown. This knowledge gap is largely due to the lack of model systems available to study the toxic potential of this bacterium. Recently, a phospholipase A2 (PLA2) encoded by a majority of A. xylosoxidans genomes, termed AxoU, was identified. Here, we show that AxoU is a type III secretion system (T3SS) substrate that induces cytotoxicity to mammalian cells. A tissue culture model was developed showing that a subset of A. xylosoxidans isolates from CF patients induce cytotoxicity in macrophages, suggestive of a pathogenic or inflammatory role in the CF lung. In a toxic strain, cytotoxicity is correlated with transcriptional activation of axoU and T3SS genes, demonstrating that this model can be used as a tool to identify and track expression of virulence determinants produced by this poorly understood bacterium.

Rapid Simulation of Unprocessed DEER Decay Data for Protein Fold Prediction.

Despite advances in sampling and scoring strategies, Monte Carlo modeling methods still struggle to accurately predict de novo the structures of large proteins, membrane proteins, or proteins of complex topologies. Previous approaches have addressed these shortcomings by leveraging sparse distance data gathered using site-directed spin labeling and electron paramagnetic resonance spectroscopy to improve protein structure prediction and refinement outcomes. However, existing computational implementations entail compromises between coarse-grained models of the spin label that lower the resolution and explicit models that lead to resource-intense simulations. These methods are further limited by their reliance on distance distributions, which are calculated from a primary refocused echo decay signal and contain uncertainties that may require manual refinement. Here, we addressed these challenges by developing RosettaDEER, a scoring method within the Rosetta software suite capable of simulating double electron-electron resonance spectroscopy decay traces and distance distributions between spin labels fast enough to fold proteins de novo. We demonstrate that the accuracy of resulting distance distributions match or exceed those generated by more computationally intensive methods. Moreover, decay traces generated from these distributions recapitulate intermolecular background coupling parameters even when the time window of data collection is truncated. As a result, RosettaDEER can discriminate between poorly folded and native-like models by using decay traces that cannot be accurately converted into distance distributions using regularized fitting approaches. Finally, using two challenging test cases, we demonstrate that RosettaDEER leverages these experimental data for protein fold prediction more effectively than previous methods. These benchmarking results confirm that RosettaDEER can effectively leverage sparse experimental data for a wide array of modeling applications built into the Rosetta software suite.

Conformational Changes and Membrane Interaction of the Bacterial Phospholipase, ExoU: Characterization by Site-Directed Spin Labeling.

Numerous pathogenic bacteria produce proteins evolved to facilitate their survival and dissemination by modifying the host environment. These proteins, termed effectors, often play a significant role in determining the virulence of the infection. Consequently, bacterial effectors constitute an important class of targets for the development of novel antibiotics. ExoU is a potent phospholipase effector produced by the opportunistic pathogen Pseudomonas aeruginosa. Previous studies have established that the phospholipase activity of ExoU requires non-covalent interaction with ubiquitin, however the molecular details of the mechanism of activation and the manner in which ExoU associates with a target lipid bilayer are not understood. In this review we describe our recent studies using site-directed spin labeling (SDSL) and EPR spectroscopy to elucidate the conformational changes and membrane interactions that accompany activation of ExoU. We find that ubiquitin binding and membrane interaction act synergistically to produce structural transitions that occur upon ExoU activation, and that the C-terminal four-helix bundle of ExoU functions as a phospholipid-binding domain, facilitating the association of ExoU with the membrane surface.

Identification and Verification of Ubiquitin-Activated Bacterial Phospholipases.

ExoU is a potent type III secretion system effector that is injected directly into mammalian cells by the opportunistic pathogen Pseudomonas aeruginosa As a ubiquitin-activated phospholipase A2 (PLA2), ExoU exhibits cytotoxicity by cleaving membrane phospholipids, resulting in lysis of the host cells and inhibition of the innate immune response. Recently, ExoU has been established as a model protein for a group of ubiquitin-activated PLA2 enzymes encoded by a variety of bacteria. Bioinformatic analyses of homologous proteins is a powerful approach that can complement and enhance the overall understanding of protein structure and function. To conduct homology studies, it is important to have efficient and effective tools to screen and to validate the putative homologs of interest. Here we make use of an Escherichia coli-based dual expression system to screen putative ubiquitin-activated PLA2 enzymes from a variety of bacteria that are known to colonize humans and to cause human infections. The screen effectively identified multiple ubiquitin-activated phospholipases, which were validated using both biological and biochemical techniques. In this study, two new ExoU orthologs were identified and the ubiquitin activation of the rickettsial enzyme RP534 was verified. Conversely, ubiquitin was not found to regulate the activity of several other tested enzymes. Based on structural homology analyses, functional properties were predicted for AxoU, a unique member of the group expressed by Achromobacter xylosoxidansIMPORTANCE Bacterial phospholipases act as intracellular and extracellular enzymes promoting the destruction of phospholipid barriers and inflammation during infections. Identifying enzymes with a common mechanism of activation is an initial step in understanding structural and functional properties. These properties serve as critical information for the design of specific inhibitors to reduce enzymatic activity and ameliorate host cell death. In this study, we identify and verify cytotoxic PLA2 enzymes from several bacterial pathogens. Similar to the founding member of the group, ExoU, these enzymes share the property of ubiquitin-mediated activation. The identification and validation of potential toxins from multiple bacterial species provide additional proteins from which to derive structural insights that could lead to paninhibitors useful for treating a variety of infections.

Identification of a ubiquitin-binding interface using Rosetta and DEER.

ExoU is a type III-secreted cytotoxin expressing A2 phospholipase activity when injected into eukaryotic target cells by the bacterium Pseudomonas aeruginosa The enzymatic activity of ExoU is undetectable in vitro unless ubiquitin, a required cofactor, is added to the reaction. The role of ubiquitin in facilitating ExoU enzymatic activity is poorly understood but of significance for designing inhibitors to prevent tissue injury during infections with strains of P. aeruginosa producing this toxin. Most ubiquitin-binding proteins, including ExoU, demonstrate a low (micromolar) affinity for monoubiquitin (monoUb). Additionally, ExoU is a large and dynamic protein, limiting the applicability of traditional structural techniques such as NMR and X-ray crystallography to define this protein-protein interaction. Recent advancements in computational methods, however, have allowed high-resolution protein modeling using sparse data. In this study, we combine double electron-electron resonance (DEER) spectroscopy and Rosetta modeling to identify potential binding interfaces of ExoU and monoUb. The lowest-energy scoring model was tested using biochemical, biophysical, and biological techniques. To verify the binding interface, Rosetta was used to design a panel of mutations to modulate binding, including one variant with enhanced binding affinity. Our analyses show the utility of computational modeling when combined with sensitive biological assays and biophysical approaches that are exquisitely suited for large dynamic proteins.

Structure and Dynamics of Type III Secretion Effector Protein ExoU As determined by SDSL-EPR Spectroscopy in Conjunction with De Novo Protein Folding.

ExoU is a 74 kDa cytotoxin that undergoes substantial conformational changes as part of its function, that is, it has multiple thermodynamically stable conformations that interchange depending on its environment. Such flexible proteins pose unique challenges to structural biology: (1) not only is it often difficult to determine structures by X-ray crystallography for all biologically relevant conformations because of the flat energy landscape (2) but also experimental conditions can easily perturb the biologically relevant conformation. The first challenge can be overcome by applying orthogonal structural biology techniques that are capable of observing alternative, biologically relevant conformations. The second challenge can be addressed by determining the structure in the same biological state with two independent techniques under different experimental conditions. If both techniques converge to the same structural model, the confidence that an unperturbed biologically relevant conformation is observed increases. To this end, we determine the structure of the C-terminal domain of the effector protein, ExoU, from data obtained by electron paramagnetic resonance spectroscopy in conjunction with site-directed spin labeling and in silico de novo structure determination. Our protocol encompasses a multimodule approach, consisting of low-resolution topology sampling, clustering, and high-resolution refinement. The resulting model was compared with an ExoU model in complex with its chaperone SpcU obtained previously by X-ray crystallography. The two models converged to a minimal RMSD100 of 3.2 Å, providing evidence that the unbound structure of ExoU matches the fold observed in complex with SpcU.

Cooperative Substrate-Cofactor Interactions and Membrane Localization of the Bacterial Phospholipase A2 (PLA2) Enzyme, ExoU.

The ExoU type III secretion enzyme is a potent phospholipase A2 secreted by the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa Activation of phospholipase activity is induced by protein-protein interactions with ubiquitin in the cytosol of a targeted eukaryotic cell, leading to destruction of host cell membranes. Previous work in our laboratory suggested that conformational changes within a C-terminal domain of the toxin might be involved in the activation mechanism. In this study, we use site-directed spin-labeling electron paramagnetic resonance spectroscopy to investigate conformational changes in a C-terminal four-helical bundle region of ExoU as it interacts with lipid substrates and ubiquitin, and to examine the localization of this domain with respect to the lipid bilayer. In the absence of ubiquitin or substrate liposomes, the overall structure of the C-terminal domain is in good agreement with crystallographic models derived from ExoU in complex with its chaperone, SpcU. Significant conformational changes are observed throughout the domain in the presence of ubiquitin and liposomes combined that are not observed with either liposomes or ubiquitin alone. In the presence of ubiquitin, two interhelical loops of the C-terminal four-helix bundle appear to penetrate the membrane bilayer, stabilizing ExoU-membrane association. Thus, ubiquitin and the substrate lipid bilayer act synergistically to induce a conformational rearrangement in the C-terminal domain of ExoU.