Chemical Synthesis of Atomically Tailored SUMO E2 Conjugating Enzymes for the Formation of Covalently Linked SUMO–E2–E3 Ligase Ternary Complexes

Abstract

E2 conjugating enzymes are the key catalytic actors in the transfer of ubiquitin, SUMO, and other ubiquitin-like modifiers to their substrate proteins. Their high rates of transfer and promiscuous binding complicate studies of their interactions and binding partners. In order to access specific, covalently-linked conjugates of the SUMO E2 conjugating enzyme Ubc9, we prepared synthetic variants bearing site-specific non-native modifications including: 1) replacement of Cys93 to 2,3-diaminopropionic acid to form the amide-linked stable E2-SUMO conjugate, which is known to have high affinity for E3 ligases; 2) a photoreactive group (diazirine) to trap E3 ligases upon UV irradiation; and 3) an N-terminal biotin for purification and detection. To construct these Ubc9 variants in a flexible, convergent manner, we combined the three leading methods -native chemical ligation (NCL), α-ketoacid-hydroxylamine (KAHA) ligation, and serine/threonine ligation (STL). Using the synthetic proteins, we demonstrated the selective formation of Ubc9-SUMO conjugates and the trapping of an E3 ligase (RanBP2) to form the stable, covalently linked SUMO1-Ubc9-RanBP2 ternary complex. The powerful combination of ligation methods -which minimizes challenges of functional group manipulations -will enable chemical probes based on E2 conjugating enzymes to trap E3 ligases and facilitate the synthesis of other protein classes.

Targeting TRIM Proteins: A Quest towards Drugging an Emerging Protein Class

“…In the case of scaffolding proteins like TRIMs, the absence of the catalytic Cys in the design of ABPs might be partially overcome by the incorporation of photocrosslinking moieties (e. g., Bpa: p ‐benzoyl‐β‐phenylalanine) within the E2‐Ub conjugate. Such a strategy has been previously used for metalloenzymes [206] and more recently for SUMO E3 [207] and RING E3 s [208] …”

Development and application of ubiquitin-based chemical probes

“…UV light is used to induce a covalent bond between the probe and the interactor to capture the latter. [41][42][43] Common photoactive groups include aryl azide, diaziridine, and benzophenone. The aryl azide group can generate a nitrene intermediate, though may be reduced under the physiological conditions, resulting in low crosslinking efficiency.…”

Chemical Synthesis of Activity‐Based E2‐Ubiquitin Probes for the Structural Analysis of E3 Ligase‐Catalyzed Transthiolation

“…[11] However,fine structural tuning of the previously reported activitybased E2-Ub probes has been limited by the reliance of their syntheses on recombination technology and bioconjugation chemistry.C hemical protein synthesis is ap owerful platform to access proteins of almost any amino acid sequence,and has been widely used to obtain customized proteins for the study of biochemical and structural mechanisms. [12] We are therefore interested in the total chemical synthesis of E2 enzyme variants [13] to facilitate the construction of structurallydiverse,a tomically tailored activity-based E2-Ub probes. Herein, we report the expedient chemical synthesis of at ypical E2 enzyme-Ubch7 through hydrazide-based native chemical ligation, [14] which enables development of astructurally new E2-Ub probe containing a2,3-diaminopropionic acid (Dap) unit and dehydroalanine (Dha) warhead.…”