8CD8

Ulilysin - C269A with AEBSF complex

8CD8 の概要

• エントリーDOI: 10.2210/pdb8cd8/pdb
• 分子名称: Ulilysin, GLY-SER-SER, CALCIUM ION, ... (7 entities in total)
• 機能のキーワード: inhibitor, complex, protease, serine protease, metalloprotease, metzincin, hydrolase
• 由来する生物種: Methanosarcina acetivorans C2A; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 82248.22

構造登録者

Rodriguez-Banqueri, A.,Eckhard, U.,Gomis-Ruth, F.X. (登録日: 2023-01-30, 公開日: 2023-03-22, 最終更新日: 2024-11-13)

主引用文献

Rodriguez-Banqueri, A.,Moliner-Culubret, M.,Mendes, S.R.,Guevara, T.,Eckhard, U.,Gomis-Ruth, F.X.
Structural insights into latency of the metallopeptidase ulilysin (lysargiNase) and its unexpected inhibition by a sulfonyl-fluoride inhibitor of serine peptidases.
Dalton Trans, 52:3610-3622, 2023

PubMed Abstract: Peptidases are regulated by latency and inhibitors, as well as compatibilization and cofactors. Ulilysin from , also called lysargiNase, is an archaeal metallopeptidase (MP) that is biosynthesized as a zymogen with a 60-residue N-terminal prosegment (PS). In the presence of calcium, it self-activates to yield the mature enzyme, which specifically cleaves before basic residues and thus complements trypsin in proteomics workflows. Here, we obtained a low-resolution crystal structure of proulilysin, in which 28 protomers arranged as 14 dimers form a continuous double helix of 544 Å pitch that parallels cell axis of the crystal. The PS includes two α-helices and obstructs the active-site cleft of the catalytic domain (CD) by traversing it in the opposite orientation of a substrate, and a cysteine blocks the catalytic zinc according to a "cysteine-switch mechanism". Moreover, the PS interacts through its first helix with an "S-loop" of the CD, which acts as an "activation segment" that lacks one of two essential calcium cations. Upon PS removal during maturation, the S-loop adopts its competent conformation and binds the second calcium ion. Next, we found that in addition to general MP inhibitors, ulilysin was competitively and reversibly inhibited by 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; = 4 μM). This is a compound that normally forms an irreversible covalent complex with serine peptidases but does not inhibit MPs. A high-resolution crystal structure of the complex revealed that the inhibitor penetrates the specificity pocket of ulilysin. A primary amine of the inhibitor salt-bridges an aspartate at the pocket bottom, thus mimicking the basic side chain of substrates. In contrast, the sulfonyl fluoride warhead is not involved and the catalytic zinc ion is freely accessible. Thus, the usage of inhibitor cocktails of peptidases, which typically contain AEBSF at ∼25-fold higher concentrations than the determined , should be avoided when working with ulilysin. Finally, the structure of the complex, which occurred as a crystallographic dimer recurring in previous mature ulilysin structures, unveiled an N-terminal product fragment that delineated the non-primed side of the cleft. These results complement prior structures of ulilysin with primed-side product fragments and inhibitors.

PubMed: 36857690
DOI: 10.1039/d3dt00458a

実験手法

X-RAY DIFFRACTION (1.65 Å)