The Active Site of a Lon Protease from Methanococcus jannaschii Distinctly Differs from the Canonical Catalytic Dyad of Lon Proteases

Abstract

ATP-dependent Lon proteases catalyze the degradation of various regulatory proteins and abnormal proteins within cells. Methanococcus jannaschii Lon (MjLon) is a homologue of Escherichia coli Lon (Ec-Lon) but has two transmembrane helices within its N-terminal ATPase domain. We solved the crystal structure of the proteolytic domain of Mj-Lon using multiwavelength anomalous dispersion, refining it to 1.9-Å resolution. The structure displays an overall fold conserved in the proteolytic domain of Ec-Lon; however, the active site shows uniquely configured catalytic Ser-Lys-Asp residues that are not seen in Ec-Lon, which contains a catalytic dyad. In Mj-Lon, the C-terminal half of the ␤4-␣2 segment is an ␣-helix, whereas it is a ␤-strand in Ec-Lon. Consequently, the configurations of the active sites differ due to the formation of a salt bridge between Asp-547 and Lys-593 in Mj-Lon. Moreover, unlike Ec-Lon, Mj-Lon has a buried cavity in the region of the active site containing three water molecules, one of which is hydrogen-bonded to catalytic Ser-550. The geometry and environment of the active site residues in Mj-Lon suggest that the charged Lys-593 assists in lowering the pK a of the Ser-550 hydroxyl group via its electrostatic potential, and the water in the cavity acts as a proton acceptor during catalysis. Extensive sequence alignment and comparison of the structures of the proteolytic domains clearly indicate that Lon proteases can be classified into two groups depending on active site configuration and the presence of DGPSA or (D/E)GDSA consensus sequences, as represented by Ec-Lon and Mj-Lon.In all cells, energy-dependent proteolysis plays a key role in the rapid turnover of short-lived regulatory proteins and in the elimination of defective and denatured proteins (1). Bacterial cells possess a number of ATP-dependent proteases, which are complex enzymes containing both ATPase and proteolytic activity as separate domains within a single polypeptide or as individual subunits within complex assemblies. Escherichia coli, for example, express five different ATP-dependent proteases: Lon, ClpAP, ClpXP, HslUV (ClpYQ), and FtSH (2). Homologous proteases have also been identified in archaea and eubacteria, as well as in numerous eukaryotes. Some archaeal Lons have one or two putative transmembrane regions, suggesting that they are membrane-associated (3). The proteolytic components of ATP-dependent proteases include several different types of active sites. For instance, ClpP is a classical serine protease (4), whereas HslV has a catalytic N-terminal Thr residue (5).Lon was the first ATP-dependent protease to be described (6). Similar to the molecular chaperon, Lon recognizes a broad range of proteins and mediates their turnover of abnormal and short-lived normal proteins. Indeed, through degradation of various specialized proteins, Lon is involved in the regulation of a number of biological functions (7). Moreover, it also reportedly acts as a DNA-binding protein, influencing the regulation of DNA replication and gene...

Identification of the Amino Acid Residues Essential for Proteolytic Activity in an Archaeal Signal Peptide Peptidase

“…In addition to the bacterial type I signal peptidase, Lon protease, LexA, UmuDЈ, and the repressor C-terminal domain mentioned above, other examples include the archaeal Lon protease from Methanocaldococcus jannaschii (32), PH1510 from Pyrococcus horikoshii (33), the periplasmic tail-specific protease from E. coli (34), and the C-terminal endoprotease from cyanobacteria (35). This study has revealed that the SPPs from Euryarchaeota are also members of this enzyme family.…”

Structure of the catalytic domain of the human mitochondrial Lon protease: Proposed relation of oligomer formation and activity

“…4(A)]. It was previously suggested for MjLon that this aspartate made up a pseudo-catalytic triad 24 ; however, mutation of the equivalent glutamate (E506A) in AfLon does not abolish the ATP-dependent catalytic activity and has no effect on the ATP-independent activity, making it appear that the aspartate does not participate directly in catalysis. 25 Furthermore, this helix blocks one end of the shallow groove seen in EcLonP that is postulated to participate in substrate binding.…”

“…The active site was open to the surface suggesting it was accessible to substrates. This was followed by the structures from two archaea Methanocaldococcus jannaschii 24 and Archaeoglobus fulgidus, 25 henceforth…”

Crystal Structures of Bacillus subtilis Lon Protease

“…A fragment consisting of amino acids 8-117 of the N-terminal domain of E. coli Lon, 28 the α subdomain of the E. coli AAA+ module, 29 and the protease domain from four different organisms have been successfully crystallised and their structures have been solved. 20,27,30,31 In this study, we present two crystal structures of truncated forms of Lon from B. subtilis. BsLon-AP (ATPase and protease domains) is the first structure of a Lon fragment consisting of both the AAA+ and the protease domains, revealing their arrangement within a Lon monomer.…”