Multiple domains of bacterial and human Lon proteases define substrate selectivity

Abstract

The Lon protease selectively degrades abnormal proteins or certain normal proteins in response to environmental and cellular conditions in many prokaryotic and eukaryotic organisms. However, the mechanism(s) behind the substrate selection of normal proteins remains largely unknown. In this study, we identified 10 new substrates of F. tularensis Lon from a total of 21 candidate substrates identified in our previous work, the largest number of novel Lon substrates from a single study. Cross-species degradation of these and other known Lon substrates revealed that human Lon is unable to degrade many bacterial Lon substrates, suggestive of a “organism-adapted” substrate selection mechanism for the natural Lon variants. However, individually replacing the N, A, and P domains of human Lon with the counterparts of bacterial Lon did not enable the human protease to degrade the same bacterial Lon substrates. This result showed that the “organism-adapted” substrate selection depends on multiple domains of the Lon proteases. Further in vitro proteolysis and mass spectrometry analysis revealed a similar substrate cleavage pattern between the bacterial and human Lon variants, which was exemplified by predominant representation of leucine, alanine, and other hydrophobic amino acids at the P(−1) site within the substrates. These observations suggest that the Lon proteases select their substrates at least in part by fine structural matching with the proteins in the same organisms.

Iron Limitation in Klebsiella pneumoniae Defines New Roles for Lon Protease in Homeostasis and Degradation by Quantitative Proteomics

“…We predict that the presence of Lon protease in the extracellular environment may play roles in degrading host proteins to release iron or other scarce metal ions into the surrounding environment. Given the substrate selectivity by multiple domains of bacterial and human Lon proteases, the enzyme's release by the bacteria may evolve to recognize host degradation targets as a mode of virulence (Karlowicz et al, 2017;He et al, 2018). We also propose that Lon protease may be degrading host proteins that are detrimental to the bacteria or degrading host proteins to promote invasion of the pathogen.…”

Crystal structure of the N domain of Lon protease from Mycobacterium avium complex

“…Structural variations of the N domains even being small may contribute to the specific substrate recognition in different species of Lon proteases. The substrate selection of Lon proteases is organism dependent, and three domains were all observed functioning in the substrate selectivity revealed by the domain swap experiments . The N domains of bacteria and human Lon proteases share the sequence identity less than 10% (Figure b), whereas the sequence identity of the AP domains is more than 30%, suggesting that the N domain may contribute more to the substrate selectivity.…”

“…The substrate selection of Lon proteases is organism dependent, and three domains were all observed functioning in the substrate selectivity revealed by the domain swap experiments. 17 The N domains of bacteria and human Lon proteases share the sequence identity less than 10% (Figure 2b), whereas the sequence identity of the AP domains is more than 30%, 19 suggesting that the N domain may contribute more to the substrate selectivity. Thus, the different conformations of the region between strands β3 and β4 probably indicate the different substrate binding for EcLon and MacLon although no substrates have been identified for MacLon so far.…”

“…The N‐terminal domain of bacterial Lon consists of ~300 amino acids and is further divided into two or more subdomains including the N domain (~200 amino acids) and the coiled‐coil (CC) domain (~100 amino acids) . Mutagenesis experiments have indicated that the N‐terminal domain is required for substrate selection and recognition, and the direct interaction between the N domain of the Escherichia coli Lon ( Ec Lon) and the sul20 degron has been detected, but the precise contribution of the N domain remains unclear …”

“…[13][14][15] Mutagenesis experiments have indicated that the N-terminal domain is required for substrate selection and recognition, and the direct interaction between the N domain of the Escherichia coli Lon (EcLon) and the sul20 degron has been detected, but the precise contribution of the N domain remains unclear. 12,16,17 The high resolution structure of the full-length Lon has not been determined, and the determined structures were focused on the Lon ATPase and proteolytic domains, including the α subdomain of the Lon ATPase domain from Brevibacillus thermoruber, 18 the Lon ATPase domains from Meiothermus taiwanensis and Bacillus subtilis, 14,19 and the Lon proteolytic domain from four organisms. 14,[19][20][21][22][23] Whereas structural information about the N-terminal domain is still scarce, only the N domains of B. subtilis and E. coli Lon proteases (BsLon-N, EcLon-N) have been structurally determined.…”