The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease.

Sami Kereïche,Ľuboš Ambro,Nina Kunová,Eva Kutejová,Lubomír Kováčik,Jan Bednár,Ivan Raška,Jana Bellová,Vladimír Pevala,Jacob Bauer,Gabriela Ondrovičová

doi:10.1038/srep33631

Abstract

Lon is an essential, multitasking AAA+ protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon’s N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning.

Highlights

Binding studies[14,15], and Su et al.[16] have very recently found that binding of Mg2+ ions to Lon protease from M. taiwanensis (MtaLon) induces conformational changes in Lon’s AP-domain that accompany ATP-independent partial proteolysis of unfolded proteins and cleavage of specific peptides
The most complete structural study reported the determination of two sub-structures of B. subtilis Lon (BsLon), which covered part of its N-terminal domain (PDB ID:3M65) and both its ATPase and proteolytic domains (PDB ID: 3M6A)[20] (Fig. 1a)
Using cryo-electron microscopy, we studied the structure of a proteolytically inactive hLon S855A mutant, which retains near wild-type levels of ATPase activity

Summary

Introduction

Binding studies[14,15], and Su et al.[16] have very recently found that binding of Mg2+ ions to Lon protease from M. taiwanensis (MtaLon) induces conformational changes in Lon’s AP-domain that accompany ATP-independent partial proteolysis of unfolded proteins and cleavage of specific peptides. The most complete structural study reported the determination of two sub-structures of B. subtilis Lon (BsLon), which covered part of its N-terminal domain (PDB ID:3M65) and both its ATPase and proteolytic domains (PDB ID: 3M6A)[20] (Fig. 1a) The region linking these two domains was not determined, the amino acid sequence analysis suggested that it was likely to be formed by coiled-coils. The deletion includes the 114 amino acids of the mitochondrial targeting pre-sequence and 156 amino acids from the N-terminal domain (Fig. 1a) Since both the enzymatic activities of hLonΔ2 70 were severely disturbed and its structure showed high variability, the missing 156 N-terminal residues are essential for the stability and proper functioning of the hLon hexamer

Methods

Results

Conclusion