Crystal structure of KLHL3 in complex with Cullin3

Area of Science:

• Biochemistry and Molecular Biology
• Structural Biology
• Genetics and Genomics

Background:

• Kelch-like protein 3 (KLHL3) is a key component of Cullin3 (Cul3) E3 ubiquitin ligase complexes.
• KLHL3 is crucial for electrolyte homeostasis and blood pressure regulation in distal nephron tubules.
• Mutations in KLHL3 are linked to inherited hypertension disorders, often affecting the Cul3 binding region.

Purpose of the Study:

• To elucidate the structural basis of the KLHL3-Cul3 interaction.
• To investigate the impact of disease-associated KLHL3 mutations on Cul3 binding.
• To understand the architecture of the dimeric CRL3 complex involving KLHL3 and Cul3.

Main Methods:

• X-ray crystallography to determine the structure of the KLHL3 BTB-BACK domain dimer complexed with Cul3.
• Isothermal titration calorimetry (ITC) to quantify the binding affinity between KLHL3 mutants and Cul3.
• Structural modeling to propose an arrangement of the dimeric CRL3 complex.

Main Results:

• The crystal structure of the KLHL3 BTB-BACK domain dimer bound to N-terminal Cul3 fragments was resolved.
• Disease-associated mutations in KLHL3 significantly disrupt its association with Cul3, as confirmed by ITC.
• Both BTB and BACK domains of KLHL3 contribute to the Cul3 interaction interface.
• A model suggests a suprafacial arrangement of E2 binding sites relative to substrate-binding sites in the dimeric CRL3 complex.

Conclusions:

• The structural and binding data provide a mechanistic explanation for how KLHL3 mutations cause hypertension.
• The study highlights the importance of the KLHL3-Cul3 interaction for maintaining blood pressure homeostasis.
• Understanding the CRL3 complex architecture offers insights into ubiquitin ligase function and substrate recognition.