Crystal structure of the N domain of Lon protease from Mycobacterium avium complex

Abstract

Lon protease is evolutionarily conserved in prokaryotes and eukaryotic organelles. The primary function of Lon is to selectively degrade abnormal and certain regulatory proteins to maintain the homeostasis in vivo. Lon mainly consists of three functional domains and the N-terminal domain is required for the substrate selection and recognition. However, the precise contribution of the N-terminal domain remains elusive. Here, we determined the crystal structure of the N-terminal 192-residue construct of Lon protease from Mycobacterium avium complex at 2.4 å resolution，and measured NMR-relaxation parameters of backbones. This structure consists of two subdomains, the β-strand rich N-terminal subdomain and the five-helix bundle of C-terminal subdomain, connected by a flexible linker，and is similar to the overall structure of the N domain of Escherichia coli Lon even though their sequence identity is only 26%. The obtained NMR-relaxation parameters reveal two stabilized loops involved in the structural packing of the compact N domain and a turn structure formation. The performed homology comparison suggests that structural and sequence variations in the N domain may be closely related to the substrate selectivity of Lon variants. Our results provide the structure and dynamics characterization of a new Lon N domain, and will help to define the precise contribution of the Lon N-terminal domain to the substrate recognition.

Keywords: Mycobacterium avium complex; Lon protease; backbone dynamics; crystal structure; the N domain.

Figure 1

Structure and backbone dynamics of MacLon‐N192. (a) 2D ¹H—¹⁵N HSQC spectra of MacLon‐N192 (black) and MacLon‐N231 (orange). Assignments for MacLon‐N192 are indicated. (b) Crystal structure of MacLon‐N192 shown in cartoon representation with two different directions of views. Secondary structure elements are labeled on the structure. (c) ¹H—¹⁵N relaxation parameters (bottom) for MacLon‐N192. Secondary structures predicted by TALOS+ (α‐helix: red; β‐strand: black) based on assigned chemical shifts agree well with the crystal structure regardless of unassigned regions (top). The crystal secondary structure elements are labeled at the top for comparison

Figure 2

Comparison of MacLon and its homologs. (a) Superposition of MacLon‐N192 (green, PDB ID: 6IHG) and EcLon‐N245 (orange, PDB ID: 3LJC)/EcLon‐N119 (blue, PDB ID: 2ANE) in different directions of views. Part of the long C‐terminal helix of EcLon‐N245 are deleted in comparison. Arrows indicate the structural deviation of MacLon‐N192 from EcLon‐N. (b) Sequence alignment of MacLon‐N192 with the corresponding fragments of Escherichia coli Lon (EcLon; UniProt ID P0A9M0), Bacillus subtilis Lon (BsLon; UniProt ID P37945), Mycobacterium tuberculosis Lon (MtLon; UniProt ID A0A0T9M6Q7), and human Lon (HuLon; UniProt ID P36776). (c) Superposition of possible binding residues of MacLon‐N192 (green) and EcLon‐N245 (orange)/EcLon‐N119 (blue) to the potential hydrophobic substrates