Structural Basis for Protein Antiaggregation Activity of the Trigger Factor Chaperone

Introduction Molecular chaperones prevent aggregation and misfolding of proteins in the cellular environment and are thus central to maintaining protein homeostasis. Molecular chaperones are thought to recognize and bind to exposed hydrophobic regions of the unfolded proteins, thereby shielding these regions from the solvent. If unprotected, the proteins would likely aggregate or misfold to bury the hydrophobic residues. Despite the central importance of the binding of chaperones to unfolded proteins, the structural basis of their interaction remains poorly understood. The scarcity of structural data on complexes between chaperones and unfolded proteins is primarily due to technical challenges originating in the size and dynamic nature of these complexes. Structural basis of PhoA binding by TF. PhoA (blue/gray) is captured in an unfolded state by three TF chaperone molecules (orange). Complex formation is mediated by multivalent binding of hydrophobic surfaces, which are shielded from water, thereby preventing folding and, at the same time, aggregation of the substrate protein.Structural basis of PhoA binding by TF. PhoA (blue/gray) is captured in an unfolded state by three TF chaperone molecules (orange). Complex formation is mediated by multivalent binding of hydrophobic surfaces, which are shielded from water, thereby preventing folding and, at the same time, aggregation of the substrate protein. Rationale Recent advances in nuclear magnetic resonance (NMR) and isotope labeling approaches make it possible to study large, dynamic complexes. We used NMR spectroscopy to characterize the binding of the 48-kD unfolded alkaline phosphatase (PhoA) to the 50-kD trigger factor (TF) chaperone. We obtained atomic insight into the dynamic binding and determined the solution structure of PhoA captured in an extended, unfolded state by three TF molecules. Based on our NMR studies, we gained insight into how TF rescues an aggregation-prone protein and how it exerts its unfoldase activity. Results We show that TF uses multiple sites, which are located in two different domains and extend over a distance of ~90 Å, to bind to several regions of the unfolded PhoA that are dispersed throughout its entire length. Three TF molecules are required to interact with the entire length of PhoA, giving rise to a ~200-kD complex in solution. The TF-PhoA interactions are mediated primarily by hydrophobic contacts. TF interacts with PhoA in a highly dynamic fashion, giving rise to a rugged landscape for the free energy of interaction. As the number and length of the PhoA regions engaged by TF increases, a more stable complex gradually emerges. The multivalent binding keeps PhoA in an extended, unfolded conformation. Crucially, even the lowest-energy TF-PhoA complex remains rather dynamic with a lifetime of ~20 ms. The structural data of the three TF molecules in complex with different regions of PhoA reveal how the same binding sites within a molecular chaperone can recognize and interact with a large number of substrates with unrelated primary sequences. This promiscuous recognition is further enabled by the notable plasticity of the substrate-binding sites in TF. We finally show that TF in the cytosol prevents aggregation by interacting transiently with the low-populated, aggregation-prone unfolded state of the substrate but acts as a powerful unfoldase when it is bound at the ribosome and thus is colocalized with translating substrate. Conclusion The structural data reveal a multivalent binding mechanism between the chaperone and its protein substrate. This mechanism of binding presents several advantages as it enables chaperones to function as holdases and unfoldases by exerting forces to retain proteins in the unfolded state and at the same time protect them from aggregation by shielding their exposed hydrophobic regions. Given the existence of multiple binding sites in other molecular chaperones, this may present a general mechanism for the action of molecular chaperones. The fast kinetics of substrate binding enables chaperones to interact with transiently exposed, aggregation-prone regions of unstable proteins in the cytosol, thereby preventing their aggregation and increasing their solubility. Nuclear magnetic resonance data show how molecular chaperones recognize and prevent aggregation and misfolding of unfolded proteins. [Also see Perspective by Gamerdinger and Deuerling] Molecular chaperones prevent aggregation and misfolding of proteins, but scarcity of structural data has impeded an understanding of the recognition and antiaggregation mechanisms. We report the solution structure, dynamics, and energetics of three trigger factor (TF) chaperone molecules in complex with alkaline phosphatase (PhoA) captured in the unfolded state. Our data show that TF uses multiple sites to bind to several regions of the PhoA substrate protein primarily through hydrophobic contacts. Nuclear magnetic resonance (NMR) relaxation experiments show that TF interacts with PhoA in a highly dynamic fashion, but as the number and length of the PhoA regions engaged by TF increase, a more stable complex gradually emerges. Multivalent binding keeps the substrate protein in an extended, unfolded conformation. The results show how molecular chaperones recognize unfolded polypeptides and, by acting as unfoldases and holdases, prevent the aggregation and premature (mis)folding of unfolded proteins. Recognize and Protect Molecular chaperones play a key role in maintaining protein homeostasis in the cell by preventing protein aggregation and misfolding. Chaperone-substrate complexes tend to be large and dynamic, making structure determination challenging. Saio et al. (10.1126/science.1250494; see the Perspective by Gamerdinger and Deuerling) used advanced NMR spectroscopy techniques to determine the structure of three trigger factor (TF) chaperone molecules in complex with the unfolded substrate, alkaline phosphatase (PhoA), and of each of the TFs in complex with the relevant region of PhoA. TF binds at multiple sites on PhoA through hydrophobic contacts, thus shielding these residues from solvent and preventing aggregation. The stability of the complex increases as longer PhoA regions are engaged by TF, and the multivalent binding keeps the substrate in an extended conformation.