All cells contain a network of molecular chaperones that help other proteins (often called “clients”) fold into functional three-dimensional conformations. Decades of research on chaperones has led to a detailed understanding of their mechanisms, especially for chaperones that depend on ATP binding and hydrolysis to drive the chaperone-client folding cycle. In a new paper published in Molecular Cell, we investigated how a bespoke, ATP-independent chaperone system uses the energy of GTP hydrolysis by its G protein client to drive the folding cycle. This unexpected new chaperone mechanism turns the ATP-dependent chaperone mechanism on its head and advances our understanding of the diverse ways that ATP-independent chaperones enable client folding.
In another recent work (Sabbarini et al., 2023) we discovered a highly conserved and essential chaperone called Zinc-finger protein 1 (Zpr1) that is dedicated to folding the cell’s amino acid deliveryman, translation elongation factor 1A (EF1A, or eEF1A in eukaryotes). The primary function of eEF1A is to deliver the correct amino acid-charged tRNAs to ribosomes during protein synthesis, and Zpr1 makes sure eEF1A folds into the correct structure to do so. Zpr1 facilitates eEF1A folding by cradling an eEF1A folding intermediate using extensive contacts tailored to the eEF1A structure but does not bind and hydrolyze ATP like many other chaperones. As the energy of ATP hydrolysis is not involved to help regulate the folding mechanism, this raised a key question that we addressed in the current paper: How is the Zpr1•eEF1A interaction regulated to allow for efficient release of folded eEF1A and recycling of Zpr1? A key insight came when we discovered that the energy of GTP hydrolysis, a molecule similar to ATP that eEF1A binds and hydrolyzes, is critical for Zpr1 to fold eEF1A. Further experiments led to the discovery that Zpr1 enables misfolded eEF1A to acquire its enzymatic GTPase activity. Without GTP hydrolysis Zpr1 becomes “clogged” with eEF1A folding intermediates. This discovery alone was exciting, because there are few definitive examples of folding proteins who drive their own exit from the chaperone pathway by directly harnessing the energy of nucleotide hydrolysis.
The story doesn’t end there. No chaperone is an island, as exemplified by the diverse co-chaperones that regulate the activity of core chaperones. Zpr1 is no exception. Using yeast genetics and cell microscopy, we also identified an uncharacterized protein family that functions as a Zpr1 co-chaperone. This co-chaperone is called Aim29 in budding yeast and is needed for efficient eEF1A folding. It was clear in early experiments that deleting the AIM29 gene caused severe eEF1A misfolding problems, but the biggest challenge was understanding the molecular mechanism of Aim29 function. This was in part because our initial efforts to isolate Aim29 associated with Zpr1•eEF1A folding complexes failed to detect any interactions. A key breakthrough came when we found that supplementing cell lysates with a non-hydrolyzable GTP analog stabilized a Zpr1•eEF1A•Aim29 complex. Structural modeling using the structural prediction tool ColabFold gave some additional insight into how Aim29 might function. The structural models suggested that Aim29 senses the GTP-bound conformation of the eEF1A “switch” region, a structural motif that is known to undergo switch-like conformations timed by GTP binding and hydrolysis. We went on to show that Aim29 and GTP hydrolysis both enable eEF1A release from Zpr1 in a reconstituted folding reaction, suggesting that Aim29 sensing of GTP-bound eEF1A folding intermediates and a GTP hydrolysis event both contribute to efficient eEF1A biogenesis.
Many mysteries remain. Does Aim29 mechanically couple eEF1A conformational changes induced by GTP hydrolysis to release from Zpr1’s grasp? We identified Zpr1 mutations that bypass the need for Aim29, but how do these mutations alter the dynamics of the Zpr1 chaperone mechanism? What captures a folded eEF1A molecule after it is released from Zpr1 to prevent unnecessary recapture? Future efforts to answer these questions will undoubtedly unravel more exciting mechanistic details that increase our understanding of molecular chaperones.