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

Abstract

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.

Figure 1

The final 2F _o − F _c electron-density maps of Lon-N245. (a) Representative density in the N-­terminal subdomain. (b) Representative density in the C-terminal subdomain. The maps were contoured at the 1.5σ level and selected residues are labeled.

Figure 2

An anomalous difference map showing the experimentally determined location of the Se atoms in the crystals of Lon-N245 superimposed on the backbone trace of the protein, with SeMet residues shown as sticks. The map was calculated with the phases calculated from the final model after deletion of Se atoms and was contoured at the 3.0σ level.

Figure 3

Stereoview of the crystal structure of Lon-N245. The elements of the secondary structure are colored magenta for β-strands, blue for α-helices and brown for coils.

Figure 4

Comparison of the backbone trace of Lon-N245 (blue) with the hypothetical B. parapertussis protein BB1347 (yellow).

Figure 5

Conservation of the amino-acid sequence among the N domains of LonA proteins. The two views of the cudgel-shaped Lon-N245 are related by ∼180° rotation around the vertical axis. A surface rendering shows highly conserved residues around a putative substrate-binding cleft in the pommel and along the long helical shaft extending toward the C-­terminus. The color gradient shows a descending order of conservation: from darkest blue (identical) through light blue, green, yellow and orange to darkest red (nonconserved). Conservation scores were obtained with the program ConSurf 2005 (Glaser et al., 2003; Landau et al., 2005 ▶) using a multiple sequence alignment of 250 Lon sequences as input. The alignment was generated with the ClustalW program at the EMBL–EBI website (http://www.ebi.ac.uk/Tools/clustalw/). This figure was prepared with PyMOL (DeLano, 2002 ▶).