Structure of the N-terminal fragment of Escherichia coli Lon protease.

The structure of a recombinant construct consisting of residues 1-245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 A resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal alpha-helix. The structure of the first subdomain (residues 1-117), which consists mostly of beta-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.

Limited proteolysis of E. coli ATP-dependent protease Lon - a unified view of the subunit architecture and characterization of isolated enzyme fragments.

We carried out chymotryptic digestion of multimeric ATP-dependent Lon protease from Escherichia coli. Four regions sensitive to proteolytic digestion were located in the enzyme and several fragments corresponding to the individual structural domains of the enzyme or their combinations were isolated. It was shown that (i) unlike the known AAA(+) proteins, the ATPase fragment (A) of Lon has no ATPase activity in spite of its ability to bind nucleotides, and it is monomeric in solution regardless of the presence of any effectors; (ii) the monomeric proteolytic domain (P) does not display proteolytic activity; (iii) in contrast to the inactive counterparts, the AP fragment is an oligomer and exhibits both the ATPase and proteolytic activities. However, unlike the full-length Lon, its AP fragment oligomerizes into a dimer or a tetramer only, exhibits the properties of a non-processive protease, and undergoes self-degradation upon ATP hydrolysis. These results reveal the crucial role played by the non-catalytic N fragment of Lon (including its coiled-coil region), as well as the contribution of individual domains to creation of the quaternary structure of the full-length enzyme, empowering its function as a processive protease.

Slicing a protease: structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains.

ATP-dependent Lon proteases are multi-domain enzymes found in all living organisms. All Lon proteases contain an ATPase domain belonging to the AAA(+) superfamily of molecular machines and a proteolytic domain with a serine-lysine catalytic dyad. Lon proteases can be divided into two subfamilies, LonA and LonB, exemplified by the Escherichia coli and Archaeoglobus fulgidus paralogs, respectively. The LonA subfamily is defined by the presence of a large N-terminal domain, whereas the LonB subfamily has no such domain, but has a membrane-spanning domain that anchors the protein to the cytoplasmic side of the membrane. The two subfamilies also differ in their consensus sequences. Recent crystal structures for several individual domains and sub-fragments of Lon proteases have begun to illuminate similarities and differences in structure-function relationships between the two subfamilies. Differences in orientation of the active site residues in several isolated Lon protease domains point to possible roles for the AAA(+) domains and/or substrates in positioning the catalytic residues within the active site. Structures of the proteolytic domains have also indicated a possible hexameric arrangement of subunits in the native state of bacterial Lon proteases. The structure of a large segment of the N-terminal domain has revealed a folding motif present in other protein families of unknown function and should lead to new insights regarding ways in which Lon interacts with substrates or other cellular factors. These first glimpses of the structure of Lon are heralding an exciting new era of research on this ancient family of proteases.

Crystal structure of the N-terminal domain of E. coli Lon protease.

We report here the first crystal structure of the N-terminal domain of an A-type Lon protease. Lon proteases are ubiquitous, multidomain, ATP-dependent enzymes with both highly specific and non-specific protein binding, unfolding, and degrading activities. We expressed and purified a stable, monomeric 119-amino acid N-terminal subdomain of the Escherichia coli A-type Lon protease and determined its crystal structure at 2.03 A (Protein Data Bank [PDB] code 2ANE). The structure was solved in two crystal forms, yielding 14 independent views. The domain exhibits a unique fold consisting primarily of three twisted beta-sheets and a single long alpha-helix. Analysis of recent PDB depositions identified a similar fold in BPP1347 (PDB code 1ZBO), a 203-amino acid protein of unknown function from Bordetella parapertussis, crystallized as part of a structural genomics effort. BPP1347 shares sequence homology with Lon N-domains and with a family of other independently expressed proteins of unknown functions. We postulate that, as is the case in Lon proteases, this structural domain represents a general protein and polypeptide interaction domain.

Atomic-resolution crystal structure of the proteolytic domain of Archaeoglobus fulgidus lon reveals the conformational variability in the active sites of lon proteases.

The atomic-resolution crystal structure of the proteolytic domain (P-domain, residues 415-621) of Archaeoglobus fulgidus B-type Lon protease (wtAfLonB) and the structures of several mutants have revealed significant differences in the conformation of the active-site residues when compared to other known Lon P-domains, despite the conservation of the overall fold. The catalytic Ser509 is facing the solvent and is distant from Lys552, the other member of the catalytic dyad. Instead, the adjacent Asp508 forms an ion pair with the catalytic lysine residue. Glu506, an analog of the putative third catalytic residue from a related Methanococcus jannaschii LonB, also faces the solvent and does not interact with the catalytic dyad. We have established that full-length wtAfLonB is proteolytically active in an ATP-dependent manner. The loss of enzymatic activity of the S509A mutant confirms the functional significance of this residue, while retention of considerable level of activity by the D508A and E506A mutants rules out their critical involvement in catalysis. In contrast to the full-length enzymes, all individually purified P-domains (wild-type and mutants) were inactive, and the mutations had no influence on the active-site structure. These findings raise the possibility that, although isolated proteolytic domains of both AfLonB and E.coli LonA are able to assemble into expected functional hexamers, the presence of the other domains, as well as substrate binding, may be needed to stabilize the productive conformation of their active sites. Thus, the observed conformational variability may reflect the differences in the stability of active-site structures for the proteolytic counterparts of single-chain Lon versus independently folded proteolytic subunits of two-chain AAA+ proteases.

Classification of ATP-dependent proteases Lon and comparison of the active sites of their proteolytic domains.

ATP-dependent Lon proteases belong to the superfamily of AAA+ proteins. Until recently, the identity of the residues involved in their proteolytic active sites was not elucidated. However, the putative catalytic Ser-Lys dyad was recently suggested through sequence comparison of more than 100 Lon proteases from various sources. The presence of the catalytic dyad was experimentally confirmed by site-directed mutagenesis of the Escherichia coli Lon protease and by determination of the crystal structure of its proteolytic domain. Furthermore, this extensive sequence analysis allowed the definition of two subfamilies of Lon proteases, LonA and LonB, based on the consensus sequences in the active sites of their proteolytic domains. These differences strictly associate with the specific characteristics of their AAA+ modules, as well as with the presence or absence of an N-terminal domain.

Crystal structure of the AAA+ alpha domain of E. coli Lon protease at 1.9A resolution.

The crystal structure of the small, mostly helical alpha domain of the AAA+ module of the Escherichia coli ATP-dependent protease Lon has been solved by single isomorphous replacement combined with anomalous scattering and refined at 1.9A resolution to a crystallographic R factor of 17.9%. This domain, comprising residues 491-584, was obtained by chymotrypsin digestion of the recombinant full-length protease. The alpha domain of Lon contains four alpha helices and two parallel strands and resembles similar domains found in a variety of ATPases and helicases, including the oligomeric proteases HslVU and ClpAP. The highly conserved "sensor-2" Arg residue is located at the beginning of the third helix. Detailed comparison with the structures of 11 similar domains established the putative location of the nucleotide-binding site in this first fragment of Lon for which a crystal structure has become available.

The catalytic domain of Escherichia coli Lon protease has a unique fold and a Ser-Lys dyad in the active site.

ATP-dependent Lon protease degrades specific short-lived regulatory proteins as well as defective and abnormal proteins in the cell. The crystal structure of the proteolytic domain (P domain) of the Escherichia coli Lon has been solved by single-wavelength anomalous dispersion and refined at 1.75-A resolution. The P domain was obtained by chymotrypsin digestion of the full-length, proteolytically inactive Lon mutant (S679A) or by expression of a recombinant construct encoding only this domain. The P domain has a unique fold and assembles into hexameric rings that likely mimic the oligomerization state of the holoenzyme. The hexamer is dome-shaped, with the six N termini oriented toward the narrower ring surface, which is thus identified as the interface with the ATPase domain in full-length Lon. The catalytic sites lie in a shallow concavity on the wider distal surface of the hexameric ring and are connected to the proximal surface by a narrow axial channel with a diameter of approximately 18 A. Within the active site, the proximity of Lys(722) to the side chain of the mutated Ala(679) and the absence of other potential catalytic side chains establish that Lon employs a Ser(679)-Lys(722) dyad for catalysis. Alignment of the P domain catalytic pocket with those of several Ser-Lys dyad peptide hydrolases provides a model of substrate binding, suggesting that polypeptides are oriented in the Lon active site to allow nucleophilic attack by the serine hydroxyl on the si-face of the peptide bond.