Structural and biochemical analysis of a thermostable membrane‐bound stomatin‐specific protease

Membrane‐bound proteases are involved in various regulatory functions. The N‐terminal region of PH1510p (1510‐N) from the hyperthermophilic archaeon Pyrococcus horikoshii is a serine protease with a catalytic Ser–Lys dyad (Ser97 and Lys138), and specifically cleaves the C‐terminal hydrophobic region of the p‐stomatin PH1511p. In a form of human hemolytic anemia known as hereditary stomatocytosis, the stomatin protein is deficient in the erythrocyte membrane due to mis‐trafficking. In order to understand the catalytic mechanism of 1510‐N in more detail, here the structural and biochemical analysis of 1510‐N is reported. Two degraded products were produced via acyl‐enzyme intermediates. 1510‐N is a thermostable protease, and thus crystallization after heat treatment of the protease–peptide complex was attempted in order to understand the catalytic mechanism of 1510‐N. The structure after heat treatment is almost identical to that with no heat treatment. According to the superposition between the structures with heat treatment and with no heat treatment, the N‐terminal half of the peptide is superposed well, whereas the C‐terminal half of the peptide is slightly deviated. The N‐terminal half of the peptide binds to 1510‐N more tightly than the C‐terminal half of the peptide. The flexible L2 loops of 1510‐N cover the peptide, and are involved in the protease activity.