Abstract
Caspases are cysteine aspartate proteases that are major players in key cellular processes, including apoptosis and inflammation. Specifically, caspase-6 has also been implicated in playing a unique and critical role in neurodegeneration; however, structural similarities between caspase-6 and other caspase active sites have hampered precise targeting of caspase-6. All caspases can exist in a canonical conformation, in which the substrate binds atop a β-strand platform in the 130's region. This caspase-6 region can also adopt a helical conformation that has not been seen in any other caspases. Understanding the dynamics and interconversion between the helical and strand conformations in caspase-6 is critical to fully assess its unique function and regulation. Here, hydrogen/deuterium exchange mass spectrometry indicated that caspase-6 is inherently and dramatically more conformationally dynamic than closely related caspase-7. In contrast to caspase-7, which rests constitutively in the strand conformation before and after substrate binding, the hydrogen/deuterium exchange data in the L2' and 130's regions suggested that before substrate binding, caspase-6 exists in a dynamic equilibrium between the helix and strand conformations. Caspase-6 transitions exclusively to the canonical strand conformation only upon substrate binding. Glu-135, which showed noticeably different calculated pK a values in the helix and strand conformations, appears to play a key role in the interconversion between the helix and strand conformations. Because caspase-6 has roles in several neurodegenerative diseases, exploiting the unique structural features and conformational changes identified here may provide new avenues for regulating specific caspase-6 functions for therapeutic purposes.
Highlights
Caspases are cysteine proteases that recognize aspartatecontaining substrates and are major players in key cellular processes, including apoptosis and inflammation
Our results show that caspase-6 displays unique backbone dynamics in the 130’s region compared with caspase-7, consistent with the helix-strand interconversion upon substrate binding
This study reveals that caspase-6 has local backbone dynamics in L2Ј and in the 130’s region consistent with the helixstrand interconversion
Summary
Caspases are cysteine proteases that recognize aspartatecontaining substrates and are major players in key cellular processes, including apoptosis and inflammation. Structures of the procaspase-6 zymogen, in which the intersubunit linker occupies and orients the active site loops [12, 41], and mature caspase-6 bound to a substrate-like inhibitor [12, 42, 43] are similar to other caspases and show the canonical strand conformation. The mature unliganded caspase-6 is capable of adopting a distinctive and noncanonical conformation, with extended helices in the 60’s region (residues 57–70) and 130’s region (residues 125–142) [44, 45] These conformational changes are accompanied by a 21° outward rotation of the 90’s helix (Fig. 1). These structures underscore the remaining question: What is the physiologically relevant structural ensemble for unliganded caspase-6? The goal of this work is to understand the relative populations of the helical and strand conformations of mature caspase-6 before substrate binding
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