Cryo-EM structure of Nma111p, a unique HtrA protease composed of two protease domains and four PDZ domains

Apoptosis, a physiological form of programmed cell death, is essential for the maintenance of normal cellular homeostasis¹. In the unicellular eukaryote Saccharomyces cerevisiae, a number of evolutionarily conserved apoptosis-regulatory proteins have been identified, one of which is nuclear mediator of apoptosis 111 kDa protein (Nma111p), a protease targeting Bir1p, the sole inhibitor-of-apoptosis protein (IAP) in yeast². Nma111p is a serine protease of the HtrA family, of which a common structural feature is the presence of the trypsin-like protease domain and the post-synaptic density 95, Drosophila discs large, zona-occludens-1 (PDZ) domain³. Based on the domain organization, the HtrA family proteins can be divided into distinct groups. Group 1 proteins (e.g., DegS in E. coli and Htra2 in mammal) contain one protease domain and one PDZ domain^4,5, and group 2 proteins (e.g., DegP and DegQ in E. coli) contain one protease domain and two PDZ domains^6,7. Nma111p-like proteases in group 3 are distinctively characterized by possessing two protease domains and four PDZ domains, of which the N-terminal protease domain exhibits protease activity, whereas the C-terminal protease domain is supposed to be degenerated in protease activity. All members in group 3 have the length twice that of group 2 HtrA proteins and harbor an internal duplication sequence. They are widely present in various organisms and possess diverse functions⁸.

The correct positioning of the catalytic triad and the main chain interactions between loop L2 and the substrate are essential features of active trypsin-like serine proteases, and may be used to define whether a protease is active or inactive¹¹. In proteolytically competent trypsin, the active loop L2 directly forms three hydrogen bonds with the substrate (PDB:1OX1¹¹; Supplementary information, Figure S2C). In the protease1 domain of Nma111p, the 1L2 (1 represents the protease1 domain) blocks the substrate-binding groove, thus preventing substrate binding and hydrogen bond formation (Figure 1C). This feature renders Nma111p in an inactive state. In addition, the loops of the protease1 domain (i.e., 1LD, 1L1, and 1L2) have weak density, suggesting a flexible conformation (Supplementary information, Figure S2D), reminiscent of many HtrA family proteins in inactive states, in which these loops are also too flexible to be traced^12,13. The catalytic triad of trypsin is composed of His, Asp, and Ser¹¹. In the active state, interactions between His and Asp or Ser facilitate proteolytic activity. In the protease1 domain, the His121, Asp152, and Ser236 (mutated to Ala) catalytic residues can be clearly observed (Figure 1C). His121 has two conformations; however, in either conformation, His121 fails to form hydrogen bonds with Asp152 or Ser236 due to the distances between them (Supplementary information, Figure S2E), further suggesting that the protease1 domain is in an inactive state.

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