Abstract
Lon proteases are a family of ATP-dependent proteases involved in protein quality control, with a unique proteolytic domain and an AAA+ (ATPases associated with various cellular activities) module accommodated within a single polypeptide chain. They were classified into two types as either the ubiquitous soluble LonA or membrane-inserted archaeal LonB. In addition to the energy-dependent forms, a number of medically and ecologically important groups of bacteria encode a third type of Lon-like proteins in which the conserved proteolytic domain is fused to a large N-terminal fragment lacking canonical AAA+ motifs. Here we showed that these Lon-like proteases formed a clade distinct from LonA and LonB. Characterization of one such Lon-like protease from Meiothermus taiwanensis indicated that it formed a hexameric assembly with a hollow chamber similar to LonA/B. The enzyme was devoid of ATPase activity but retained an ability to bind symmetrically six nucleotides per hexamer; accordingly, structure-based alignment suggested possible existence of a non-functional AAA-like domain. The enzyme degraded unstructured or unfolded protein and peptide substrates, but not well-folded proteins, in ATP-independent manner. These results highlight a new type of Lon proteases that may be involved in breakdown of excessive damage or unfolded proteins during stress conditions without consumption of energy.
Highlights
Proteases play a crucial role in cellular protein quality control to degrade aberrant or unwanted proteins, which may be prone to toxic aggregate formation, into short peptides
We conducted BLAST searches of the protein databases and identified a large number of bacterial sequences with BLAST Evalues suggesting homology with MtaLonC. Most of these Lon-like proteases are found in the proteomes of Gram-negative bacteria, of which many belong to the medically important class of cproteobacteria, which includes the genera of Acinetobacter, Klebsiella, Pseudomonas, Salmonella, Vibrio, and Yersinia
Similar to TTC1975, we found no ATPase activity associated with MtaLonC at either 37uC or 55uC, the optimal growth temperature for the thermophilic bacteria, using a colorimetric assay [21,22]; by contrast, the activity was detected for EcLonA, as well as a LonA protease from the thermophilic Brevibacillus thermoruber (BtLonA)(Fig. 4A and B)
Summary
Proteases play a crucial role in cellular protein quality control to degrade aberrant or unwanted proteins, which may be prone to toxic aggregate formation, into short peptides. The two subfamilies are associated with two distinct consensus sequences of the Walker motifs in the AAA+ domains and specific domain organization features in which an N-terminal domain is attached to the AAA+ modules of all LonA but not LonB members, the latters are found only in archaebacteria and possess instead additional membrane-spanning segments integrated into their AAA+ domains that anchors the proteins to the membrane [7] Such proteolytic site-based classification approach, has left out a large group of Lon-like proteases encoded in the genomes of many Gram-negative bacteria ( in certain Gram-positive bacteria and archaea), which contain LonB-like consensus sequences at the proteolytic sites but have no transmembrane regions nor detectable AAA+ consensus motifs [6,8]. The lack of ATPase activity has been confirmed for a Lon-like protease from Thermus thermophilus [9]; the biochemical properties and functional roles of these Lon-like proteases have remained largely uncharacterized
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