SAXS Analizi ile HERC5 HECT Alanının Trimerik Oluşumuna İlişkin Yapısal Bilgiler

Abstract

HERC5, antiviral yanıtları düzenleyen önemli bir post-translasyonel modifikasyon olan ISGylation yolağında yer alan interferon kaynaklı bir E3 ubikitin ligazdır. HERC5'in HECT alanı, substrat tanıma ve ubikitin transferinde önemli bir rol oynar. HERC protein ailesi yapısal olarak C-terminal HECT (Homologous to the E6-AP Carboxyl Terminus) alanı ve N-terminal RCC1 benzeri (The Regulator of Chromosome Condensation 1) alanları içermesiyle karakterize edilir. Buna rağmen, HERC5'in HECT alanına özgü oligomerizasyon kapasitesi tam olarak açıklığa kavuşturulmamıştır. Burada, HERC5 HECT alanının çözeltideki oligomerik durumunu, afinite ve boyut dışlama kromatografisi ile saflaştırılmasının ardından 2.0 mg/ml ve 10°C'de SAXS( Küçük Saçınımlı X-Ray) kullanarak araştırıyoruz. SAXS veri analizi, Guinier ve Kratky analizleri, mesafe dağılım fonksiyonları ve ab initio modelleme ile desteklenen trimerik bir birleşim ortaya koymuştur. Bu bulgular, trimerizasyonun HERC5 işlevinde düzenleyici bir rol oynayabileceğini ve HECT tipi E3 ligaz mekanizmalarının daha geniş bir şekilde anlaşılmasına katkıda bulunabileceğini göstermektedir.

Keywords

• HERC5
• HECT domain
• SAXS
• ISGylasyon
• ubiquitin ligaz
• protein yapısı

Structural Insights into the Trimeric Assembly of the HERC5 HECT Domain through SAXS Analysis

Abstract

HERC5 is an interferon-induced E3 ubiquitin ligase involved in ISGylation, a crucial post-translational modification regulating antiviral responses. The HECT domain of HERC5 plays a key role in substrate recognition and ubiquitin transfer. The HERC protein family is structurally characterized by containing the C-terminal HECT (Homologous to the E6-AP Carboxyl Terminus) domain and N-terminal RCC1-like ( The Regulator of Chromosome Condensation 1) domains. In spite of this, the HECT domain-specific oligomerization capacity of HERC5 is not fully clarified. Here, we investigate the oligomeric state of the HERC5 HECT domain in solution, using affinity and size-exclusion chromatography, followed by SAXS measurements at 2.0 mg/ml and 10°C. SAXS data analysis revealed a trimeric assembly, supported by Guinier and Kratky analyses, distance distribution functions, and ab initio modeling. These findings suggest that trimerization may play a regulatory role in HERC5 function, contributing to a broader understanding of HECT-type E3 ligase mechanisms.

Keywords

• HERC5
• HECT domain
• SAXS
• ISGylation
• ubiquitin ligase
• protein structure

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