A Lon-like protease with no ATP-powered unfolding activity

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.

Figure 1. Phylogenetic tree calculated from amino acid sequence alignment of Lon-like proteases (LonC) with representative members of LonA and LonB subfamilies.

Three distinct clades can be recognized through phylogenetic reconstruction. Indicated are the names of species where at least one member of the corresponding subfamily of Lon proteases is present. Note that LonA proteases are ubiquitous in all species shown; thus some bacteria, including E. coli, possess more than one subfamilies of Lon. In addition, multiple paralogs related to LonA have been found in certain bacteria. The Lon genes are indicated by binomial names of the species. The accession codes for the genes can be found in Table S1.

Figure 2. Two LonC-specific insertions.

(A) Sequence alignment of selected Lon proteases (LonA and LonB members are indicated accordingly) centered on the loop between helix α3 (see text), which houses the catalytic lysine (K625 in M. taiwanensis; close triangle), and strand β9 in the protease domain. Open triangles mark the insertion region. (B) Alignment detail of the C-terminal region of Lon protease domain focused on helix α5. (C) Sequence alignment of MtaLonC and TonLonB. Regions corresponding to the Walker A and B motifs of TonLonB are outlined in blue and red boxes, respectively. The two LonC-specific insertions is in black box. The MtaLonC residues corresponding to the sensor-1, sensor-2, and Arg finger of TonLonB are in boxes of yellow, purple, and green colors, respectively, for comparison (see text). The transmembrane regions of TonLon are underlined. Conserved residues are highlighted in black blocks. Catalytic dyad residues are marked with asterisks. (D) Locations of the two LonC-specific insertions, in the α3-β9 loop (colored in magenta) and helix α5 (pink) of the Lon protease domain (green), mapped onto a surface representation of hexameric TonLon (PDB code 3K1J).

Figure 3. Hexameric assembly of MtaLonC.

(A) Molecular weight analysis of MtaLonC by analytical ultracentrifugation. Representative sedimentation equilibrium distribution of Mt-LonC suggested an average molecular weight of 462,476 Da, corresponding to hexamers in solution. (B) Representative class averages of negatively stained MtaLonC particles. Images such as 1, 15, 20, and 23 show the predominant top view of the hexameric complex. Images 3, 5, 7, and 27 may represent various side views of the assembly. The scale bar is 10 nm.

Figure 4. ATPase and ATP-binding activities of MtaLonC.

ATPase activity of MtaLonC were assayed at 37°C with EcLonA as a positive control (A) and at 55°C, the growth temperature of M. taiwanensis, using BtLonA as a positive control (B). Blank, no enzyme added. (C, D) ³¹P-NMR spectra of 10 mM ATP in the presence of EcLonA (2 µg) at 37°C for 5 days (C) and 1 hour (D). (E, F) ³¹P-NMR spectra of 10 mM ATP in the presence of MtaLonC (2 µg) at 37°C for 5 days (E) and 1 hour (F). The positions of chemical shifts for the phosphorus atoms of ATP and of inorganic phosphate (Pi) are indicated. Four peaks are shown in figure C indicates that EcLonA possesses ATPase activity whereas MtaLonC does not. (G) Binding isotherms of MtaLonC titrated with ATPγS showing a binding ratio (n) of 1.15 (fitted by one-site function). The association constant (K), ΔH, and ΔS was 1.28×10⁵ (M⁻¹), −5283 (cal/mol), and 5.65 (cal/mol/deg), respectively. ΔG was −6.97 (kcal/mol) indicating a spontaneous binding.

Figure 5. MtaLonC degrades unstructured polypeptide.

(A) Degradation of F-β20-Q in the absence or presence of ATP, ADP or AMPPNP as revealed by increased fluorescence upon cleavage. (B) Degradation of α_S2-Casein by MtaLonC at 55°C was assayed by SDS-PAGE. The sample also contained minor amount of α_S1-Casein.

Figure 6. MtaLonC degrades unfolded protein substrates.

(A) Degradation of Huβ by MtaLonC or EcLonA. The reactions were carried out at 37 or 55°C, with or without addition of ATP as indicated in each panel, and was analysed by SDS-PAGE. Left lanes were reaction mixtures without enzyme incubated for 360 min. (B) CD spectra of of Huβ at various temperatures. (C) Degradation of lysozyme by MtaLonC at 55°C.

Figure 7. MtaLonC cannot degrade well-folded protein.

(A) Degradation of POP1 by EcLonA at 37°C with or without ATP. (B) Degradation of POP1-β8 by MtaLonC or EcLonA. The reactions were carried out at 37 or 55°C, with or without addition of ATP as indicated in each panel, and was analysed by SDS-PAGE. Left lanes were reaction mixtures without enzyme incubated for 360 min. (C) CD spectra of of POP1-β8 at various temperatures.

Figure 8. Processive degradation of MtaLonC.

HPLC profiles of the degraded products from E. coli Huβ (A) and α_S2-casein (B) after indicated incubation times. The cutting sites of MtaLonC in E. coli Huβ analyzed by mass spectrometry are shown in supplementary data (Fig.S6). Molecular species of the major peaks of casein digestion were confirmed by mass spectrometry. Peak 1: AMKPWIQPK; peak 2: TKVIPYVR; peak 3: FALPQYLK.