Structural Basis for Ufm1 Processing by UfSP1

摘要

Ubiquitin-fold modifier 1 (Ufm1) is a newly identified ubiquitin-like protein. Like ubiquitin and other ubiquitin-like proteins, Ufm1 is synthesized as a precursor that needs to be processed to expose the conserved C-terminal glycine prior to its conjugation to target proteins. Two novel proteases, named UfSP1 and UfSP2, have been shown to be responsible for the release of Ufm1 from Ufm1-conjugated cellular proteins as well as for the processing of its precursor. They show no sequence homology with known proteases. Here, we describe the 1.7Å resolution crystal structure of mouse UfSP1, consisting of 217 amino acids. The structure reveals that it is a novel cysteine protease having a papain-like fold, with Cys53, Asp175, and His177 that form a catalytic triad, and Tyr41 that participates in the formation of the oxyanion hole. This differs from the canonical catalytic triad of papain-like proteases in that the aspartate and the histidine residues are from the “Asp-Pro-His” box. The Asp-Pro-His configuration seen in UfSP1, together with Atg4B and M48USP, seem to form a new subfamily of the cysteine protease superfamily. The mutagenesis study of the active site residues confirms structural basis for catalysis. The interaction between UfSP1 and Ufm1 appears quite substantial, since the KD value was estimated to be 1.6 μm by the isothermal titration calorimetry analysis. Furthermore, the NMR data shows that the loop between β3 and α2 in addition to the C-terminal region of Ufm1 plays a role in binding to UfSP1. Ubiquitin-fold modifier 1 (Ufm1) is a newly identified ubiquitin-like protein. Like ubiquitin and other ubiquitin-like proteins, Ufm1 is synthesized as a precursor that needs to be processed to expose the conserved C-terminal glycine prior to its conjugation to target proteins. Two novel proteases, named UfSP1 and UfSP2, have been shown to be responsible for the release of Ufm1 from Ufm1-conjugated cellular proteins as well as for the processing of its precursor. They show no sequence homology with known proteases. Here, we describe the 1.7Å resolution crystal structure of mouse UfSP1, consisting of 217 amino acids. The structure reveals that it is a novel cysteine protease having a papain-like fold, with Cys53, Asp175, and His177 that form a catalytic triad, and Tyr41 that participates in the formation of the oxyanion hole. This differs from the canonical catalytic triad of papain-like proteases in that the aspartate and the histidine residues are from the “Asp-Pro-His” box. The Asp-Pro-His configuration seen in UfSP1, together with Atg4B and M48USP, seem to form a new subfamily of the cysteine protease superfamily. The mutagenesis study of the active site residues confirms structural basis for catalysis. The interaction between UfSP1 and Ufm1 appears quite substantial, since the KD value was estimated to be 1.6 μm by the isothermal titration calorimetry analysis. Furthermore, the NMR data shows that the loop between β3 and α2 in addition to the C-terminal region of Ufm1 plays a role in binding to UfSP1. In eukaryotes, the posttranslational modification of cellular proteins by ubiquitin (Ub) 4The abbreviations used are: Ub, ubiquitin; UBL, ubiquitin-like protein; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; DUB, deubiquitinating enzymes; ULP, UBL-specific protease; ITC, isothermal titration calorimetry; MAD, multiple wavelength anomalous diffraction. 4The abbreviations used are: Ub, ubiquitin; UBL, ubiquitin-like protein; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; DUB, deubiquitinating enzymes; ULP, UBL-specific protease; ITC, isothermal titration calorimetry; MAD, multiple wavelength anomalous diffraction. is an essential step in regulatory mechanisms of many cellular processes. These include cell cycle progression, signal transduction, and targeting of misfolded proteins for degradation by the proteasome (1Glickman M.H. Ciechanover A.