Structural basis for the dynamic chaperoning of disordered clients by Hsp90.

Molecular chaperone heat shock protein 90 (Hsp90) is a ubiquitous regulator that fine-tunes and remodels diverse client proteins, exerting profound effects on normal biology and diseases. Unraveling the mechanistic details of Hsp90's function requires atomic-level insights into its client interactions throughout the adenosine triphosphate-coupled functional cycle. However, the structural details of the initial encounter complex in the chaperone cycle, wherein Hsp90 adopts an open conformation while engaging with the client, remain elusive. Here, using nuclear magnetic resonance spectroscopy, we determined the solution structure of Hsp90 in its open state, bound to a disordered client. Our findings reveal that Hsp90 uses two distinct binding sites, collaborating synergistically to capture discrete hydrophobic segments within client proteins. This bipartite interaction generates a versatile complex that facilitates rapid conformational sampling. Moreover, our investigations spanning various clients and Hsp90 orthologs demonstrate a pervasive mechanism used by Hsp90 orthologs to accommodate the vast array of client proteins. Collectively, our work contributes to establish a unified conceptual and mechanistic framework, elucidating the intricate interplay between Hsp90 and its clients.

Dynamic interactions between E-cadherin and Ankyrin-G mediate epithelial cell polarity maintenance.

E-cadherin is an essential cell‒cell adhesion protein that mediates canonical cadherin-catenin complex formation in epithelial lateral membranes. Ankyrin-G (AnkG), a scaffold protein linking membrane proteins to the spectrin-based cytoskeleton, coordinates with E-cadherin to maintain epithelial cell polarity. However, the molecular mechanisms governing this complex formation and its relationships with the cadherin-catenin complex remain elusive. Here, we report that AnkG employs a promiscuous manner to encapsulate three discrete sites of E-cadherin by the same region, a dynamic mechanism that is distinct from the canonical 1:1 molar ratio previously described for other AnkG or E-cadherin-mediated complexes. Moreover, we demonstrate that AnkG-binding-deficient E-cadherin exhibited defective accumulation at the lateral membranes and show that disruption of interactions resulted in cell polarity malfunction. Finally, we demonstrate that E-cadherin is capable of simultaneously anchoring to AnkG and ß-catenin, providing mechanistic insights into the functional orchestration of the ankyrin-spectrin complex with the cadherin-catenin complex. Collectively, our results show that complex formation between E-cadherin and AnkG is dynamic, which enables the maintenance of epithelial cell polarity by ensuring faithful targeting of the adhesion molecule-scaffold protein complex, thus providing molecular mechanisms for essential E-cadherin-mediated complex assembly at cell‒cell junctions.

Sandwiched-fusion strategy facilitates recombinant production of small labile proteins.

Efficient production of large quantities of soluble, properly folded proteins is of high demand in modern structural and functional genomics. Despite much advancement toward improving recombinant protein expression, many eukaryotic proteins especially small peptides often fail to be recovered due to rapid proteolytic degradation. Here we show that the sandwiched-fusion strategy, which is based on two protein tags incorporated both at the amino- and carboxyl-terminus of target protein, could be employed to overcome this obstacle. We have exploited this strategy on heterologous expression in Escherichia coli of eight small degradation-prone eukaryotic proteins, whose successful recombinant productions have yet to be achieved. These include seven mitochondria-derived peptides (MDPS), a class of unique metabolic regulators of human body, and a labile mosquito transcription factor, Guy1. We show here that the sandwiched-fusion strategy, which provides robust protection against proteolysis, affords an economical method to obtain large quantities of pure five MDPs and the transcription factor Guy1, in sharp contrast to otherwise unsuccessful recovery using the traditional amino-fusion method. Further biophysical characterization and interaction studies by NMR spectroscopy confirmed that the proteins produced by this novel approach are properly folded into their biologically active structures. We anticipate this strategy could be widely utilized in production of other labile protein systems.