Calcium-sensitive thermal transitions and domain structure of human complement subcomponent C1r

Area of Science:

• Biochemistry
• Protein Chemistry
• Enzymology

Background:

• The complement system plays a vital role in innate immunity.
• C1r is a serine protease within the classical complement pathway, crucial for initiating immune responses.
• Understanding the thermal stability of C1r is essential for elucidating its function and regulation.

Purpose of the Study:

• To investigate the thermal stability of activated C1r and its functional fragments.
• To determine the influence of metal ions, particularly Ca2+, on C1r's thermal transitions and activity.
• To characterize the structural and functional consequences of these transitions.

Main Methods:

• Utilized fluorescent probes to monitor thermal stability.
• Employed tryptic digestion to isolate functionally intact protein fragments.
• Assessed enzyme activity, protein polymerization, and binding interactions under varying conditions.

Main Results:

• Activated C1r exhibits two irreversible thermal transitions: a high-temperature transition (around 53°C) associated with loss of catalytic activity, and a low-temperature transition (26-40°C) leading to polymerization.
• The low-temperature transition is Ca2+-dependent, with polymerization occurring in the absence of Ca2+ and abolishment by micromolar Ca2+ concentrations.
• A purified N-terminal fragment retains Ca2+-dependent interaction with C1s, but Ca2+ removal induces a transition that disrupts C1s binding.

Conclusions:

• C1r possesses distinct thermal transitions that are differentially sensitive to metal ions and ionic strength.
• Calcium ions play a critical role in stabilizing C1r structure, preserving its ability to interact with C1s and reconstitute complement function.
• The observed polymerization and loss of specific interactions highlight the complex regulatory mechanisms of C1r activity.