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.
AT P-dependentL on proteasesa re multi-domain enzymesf ound in alll ivingo rganisms.A ll Lonp roteases containa nA TPased omainb elonging to theA AA + superfamilyo fm olecularm achinesa nd ap roteolytic domain with as erine-lysine catalyticd yad. Lonp roteases canb ed ivided into twos ubfamilies,L onAa nd LonB,e xemplified by the Escherichiac oli and Archaeoglobusf ulgidus paralogs,r espectively. TheL onA subfamily is definedbythe presence of alarge N-terminal domain,wh ereasthe LonB subfamilyhas no such domain,b ut hasamembrane-spanningd omaint hata nchors thep rot eint ot he cytoplasmics ideo ft he membrane.The twosubfamilies also differ in theirconsensus sequences. Recent crystalstructuresfor several individual domainsand sub-fragmentsofLon proteaseshavebegun to illuminate similarities anddifferences in structure-function relationshipsb etween thet wo subfamilies. Differencesi no rientation of thea ctives ite residues in severali solatedL on protease domainsp oint to possible rolesf or theA AA + domainsa nd/or substrates in positioningt he catalyticr esiduesw ithin thea ctives ite. Structures of thep roteolytic domains have also indicatedapossible hexamerica rrangement of subunits in then atives tate of bacterialL on proteases. Thes tructure of al arge segmento ft he N-terminal domain hasr evealedafoldingm otif present in otherprotein families of unknownfunctionand should lead to newinsightsregarding ways in whichLon interactswith substrates or othercellularfactors.These firstglimpsesofthe structureofLon areheraldingan exciting newe ra of research on this ancientf amilyo fp roteases.Keywords: AAA + protein; Lon protease; AT P-dependent protease; Ser-Lys dyad; LonA and LonB subfamilies; domains; crystal structure
The Lon family of peptidasesThe Lonp rotease family (MEROPS [Rawlings et al. 2004] clan SJ,f amily S16),w hich is conserved in the prokaryotes andi ne ukaryotico rganelles such as mitochondria and peroxisomes, is themostwidespread family of AT P-dependent proteases. Prokaryotic Lons are key enzymes responsible for intracellularp roteolysis, contributing to protein quality and cellular homeostasis by
The structure of a recombinant construct consisting of residues 1–245 ofEscherichia coliLon 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 Å 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 α-helix. The structure of the first subdomain (residues 1–117), which consists mostly of β-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 fromBordetella 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.
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