Structure, dynamics and thermodynamics of the human centrin 2/hSfi1 complex

Area of Science:

• Structural biology
• Biochemistry
• Molecular dynamics

Background:

• Centrin (HsCen2) is an EF-hand calcium-binding protein crucial for centrosome duplication.
• Sfi1 (Suppressor of fermentation-induced loss of stress resistance protein 1) is a centrosomal target of centrin, with multiple repeats.
• Previous studies characterized HsCen2 binding to a peptide from xeroderma pigmentosum complementation group C (XPC) protein.

Purpose of the Study:

• To investigate the structural, dynamic, and affinity impacts of a reversed L8L4W1 motif in the R17-hSfi1-20 peptide compared to the XPC motif.
• To determine the binding mode of HsCen2 to R17-hSfi1-20 and analyze its structural and dynamic consequences.

Main Methods:

• X-ray crystallography to determine the structure of the C-terminal domain of HsCen2 (C-HsCen2) in complex with R17-hSfi1-20.
• Nuclear Magnetic Resonance (NMR) spectroscopy to monitor the dynamics of the C-HsCen2/R17-hSfi1-20 complex.
• Verification that the N-terminal domain of HsCen2 does not interact with the peptide.

Main Results:

• The crystal structure revealed a binding mode similar to HsCen2/P17-XPC, but with a 2 Å translation of the R17-hSfi1-20 helix.
• This translation resulted in reduced protein anchorage and disrupted a key tryptophan-glutamate hydrogen bond.
• NMR dynamics studies indicated an unusual calcium secondary binding mode in loop III, dependent on calcium concentration and ligand type.

Conclusions:

• The reversed motif in R17-hSfi1-20 alters the binding interaction with HsCen2, affecting structural stability and dynamics.
• An uncommon residue composition in calcium-binding loop III enables a secondary calcium-binding site, suggesting novel regulatory mechanisms for centrin.
• These findings provide insights into the molecular basis of centrin-Sfi1 interactions and calcium-dependent regulation.