The Lon AAA+ protease (LonA) is a ubiquitous ATP-dependent proteolytic machine, which selectively degrades damaged proteins or native proteins carrying exposed motifs (degrons). Here we characterize the structural basis for substrate recognition and discrimination by the N-terminal domain (NTD) of LonA. The results reveal that the six NTDs are attached to the hexameric LonA chamber by flexible linkers such that the formers tumble independently of the latter. Further spectral analyses show that the NTD selectively interacts with unfolded proteins, protein aggregates, and degron-tagged proteins by two hydrophobic patches of its N-lobe, but not intrinsically disordered substrate, α-casein. Moreover, the NTD selectively binds to protein substrates when they are thermally induced to adopt unfolded conformations. Collectively, our findings demonstrate that NTDs enable LonA to perform protein quality control to selectively degrade proteins in damaged states and suggest that substrate discrimination and selective degradation by LonA are mediated by multiple NTD interactions.
Keywords: Lon AAA+ protease; Meiothermus taiwanensis; N-terminal domain; biochemistry; chemical biology; degrons; misfolded proteins; molecular biophysics; protein aggregates; structural biology.
There are many different types of protein which each have different roles in biology. Most proteins are surrounded by water and are folded so that their water-attracting regions are on the outside and more fat-like regions, which repel water, are on the inside. When a protein becomes damaged or is assembled incorrectly, some of the fat-like regions end up on the outside of the protein and become exposed to water. This can prevent the protein from performing its role and harm the cell instead. LonA proteases are responsible for dismantling and recycling these harmful proteins, as well as proteins that have been labelled for destruction. They do this by unfolding the unwanted protein and transporting it into an enclosed chamber made of six LonA molecules. Once inside the chamber, the target protein is broken down into smaller fragments that can be used to build other structures. LonA proteases contain a region called the N-terminal domain, or NTD for short, which is thought to be responsible for identifying which proteins need degrading. Yet it remained unclear how the NTD recognizes and binds to these target proteins. To answer this question, Tzeng et al. studied the detailed structure of a LonA protease that had been purified from bacteria cells. This revealed that the NTD of LonA contains two water-repelling regions which bind to fat-like segments on the surface of proteins that have become unfolded or tagged for destruction. Further experiments showed that the NTD is bound to the main body of LonA via a ‘flexible linker’. This led Tzeng et al. to propose that the NTD sways around loosely at the end of LonA searching for proteins with exposed water-repelling regions. Once an NTD identifies and attaches to a target, the NTDs of the other LonA molecules then bind to the protein and help insert it into the chamber. Proteases are a vital component of all biological systems. Controlling protein destruction and recycling is a key factor in how cells divide and respond to a changing environment. This study provides new insights into how LonA operates in bacteria, which may apply to proteases more widely. This contributes to our knowledge of fundamental biology and may also be relevant in a range of diseases where protein recycling is defective or inefficient.
© 2021, Tzeng et al.