ABSTRACT
Many proteins are composed of independently-folded domains connected by flexible linkers. The primary sequence and length of such linkers can set the effective concentration for the tethered domains, which impacts rates of association and enzyme activity. The length of such linkers can be sensitive to environmental conditions, which raises questions as to how studies in dilute buffer relate to the highly-crowded cellular environment. To examine the role of linkers in domain separation, we measured Fluorescent Protein-Fluorescence Resonance Energy Transfer (FP-FRET) for a series of tandem FPs that varied in the length of their interdomain linkers. We used discrete molecular dynamics to map the underlying conformational distribution, which revealed intramolecular contact states that we confirmed with single molecule FRET. Simulations found that attached FPs increased linker length and slowed conformational dynamics relative to the bare linkers. This makes the CLYs poor sensors of inherent linker properties. However, we also showed that FP-FRET in CLYs was sensitive to solvent quality and macromolecular crowding making them potent environmental sensors. Finally, we targeted the same proteins to the plasma membrane of living mammalian cells to measure FP-FRET in cellulo. The measured FP-FRET when tethered to the plasma membrane was the same as that in dilute buffer. While caveats remain regarding photophysics, this suggests that the supertertiary conformational ensemble of these CLY proteins may not be affected by this specific cellular environment.
Subject(s)
Bacterial Proteins/chemistry , Green Fluorescent Proteins/chemistry , Luminescent Proteins/chemistry , Molecular Dynamics Simulation , Recombinant Fusion Proteins/chemistry , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , CHO Cells , Cricetulus , Fluorescence Resonance Energy Transfer , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Models, Molecular , Polyethylene Glycols/chemistry , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Single Molecule Imaging , Sodium Chloride/chemistry , Urea/chemistryABSTRACT
Clostridium difficile is an opportunistic pathogen that establishes in the colon when the gut microbiota are disrupted by antibiotics or disease. C. difficile infection (CDI) is largely caused by two virulence factors, TcdA and TcdB. Here, we report a 3.87-Å-resolution crystal structure of TcdB holotoxin that captures a unique conformation of TcdB at endosomal pH. Complementary biophysical studies suggest that the C-terminal combined repetitive oligopeptides (CROPs) domain of TcdB is dynamic and can sample open and closed conformations that may facilitate modulation of TcdB activity in response to environmental and cellular cues during intoxication. Furthermore, we report three crystal structures of TcdB-antibody complexes that reveal how antibodies could specifically inhibit the activities of individual TcdB domains. Our studies provide novel insight into the structure and function of TcdB holotoxin and identify intrinsic vulnerabilities that could be exploited to develop new therapeutics and vaccines for the treatment of CDI.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Clostridioides difficile/chemistry , Amino Acid Sequence , Antibodies, Neutralizing/immunology , Antigen-Antibody Complex/chemistry , Bacterial Proteins/immunology , Bacterial Toxins/immunology , Conserved Sequence , Crystallography, X-Ray , Endosomes/chemistry , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Liposomes , Membrane Potentials , Models, Molecular , Peptide Fragments/chemistry , Protein Binding , Protein Conformation , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Previous studies of the N-terminal PDZ tandem from PSD-95 produced divergent models and failed to identify interdomain contacts stabilizing the structure. We used ensemble and single-molecule FRET along with replica-exchange molecular dynamics to fully characterize the energy landscape. Simulations and experiments identified two conformations: an open-like conformation with a small contact interface stabilized by salt bridges, and a closed-like conformation with a larger contact interface stabilized by surface-exposed hydrophobic residues. Both interfaces were confirmed experimentally. Proximity of interdomain contacts to the binding pockets may explain the observed coupling between conformation and binding. The low-energy barrier between conformations allows submillisecond dynamics, which were time-averaged in previous NMR and FRET studies. Moreover, the small contact interfaces were likely overridden by lattice contacts as crystal structures were rarely sampled in simulations. Our hybrid approach can identify transient interdomain interactions, which are abundant in multidomain proteins yet often obscured by dynamic averaging.