Internal Motions Prime cIAP1 for Rapid Activation

Abstract

Cellular inhibitor of apoptosis 1 (cIAP1) is an E3 ubiquitin ligase with roles in both apoptosis and NF-κB signaling. Recent crystallographic and SAXS data have shown that the ligase activity of cIAP1 is autoinhibited by occlusion of the RING dimerization interface through several inter-domain interactions, most critically in the BIR3:RING interface. This interaction is disrupted by SMAC and SMAC mimetics, initiating cIAP1 dimerization through its newly exposed RING domain and activation of its E3 ligase activity. Activation of cIAP1's ligase activity leads to cIAP1 autoubiquitination and proteasomal degradation. Intriguingly, we observed that although the binding site for cIAP1's E2 partner enzyme is occluded in the crystal structure, E2 binds to both the monomeric and dimeric forms of cIAP1 with nearly the same affinity. This unexpected result indicated that there were likely significant residual motions occurring within monomeric cIAP1. Through NMR relaxation dispersion measurements and resonance broadening we have been able to detect significant motions occurring on relatively fast, microsecond-millisecond timescales at specific domain interfaces present in the autoinhibited form of cIAP1. In addition, time-resolved SAXS measurements conducted in line with a stopped flow injection system have allowed us to measure the opening rate of cIAP1, which occurs over hundreds of milliseconds. The tight regulation of apoptosis is essential in multicellular organisms and the proteins involved in transducing pro-apoptotic and anti-apoptotic signals are no doubt under stringent evolutionary control. The internal motions present in cIAP1 likely arose from the competing pressures to activate apoptosis as quickly as possible while maintaining the fidelity of the pro-apoptotic signal.