L2′ loop is critical for caspase‐7 active site formation

Abstract

The active sites of caspases are composed of four mobile loops. A loop (L2) from one half of the dimer interacts with a loop (L2 0 ) from the other half of the dimer to bind substrate. In an inactive form, the two L2 0 loops form a cross-dimer hydrogen-bond network over the dimer interface. Although the L2 0 loop has been implicated as playing a central role in the formation of the active-site loop bundle, its precise role in catalysis has not been shown. A detailed understanding of the active and inactive conformations is essential to control the caspase function. We have interrogated the contributions of the residues in the L2 0 loop to catalytic function and enzyme stability. In wild-type and all mutants, active-site binding results in substantial stabilization of the complex. One mutation, P214A, is significantly destabilized in the ligand-free conformation, but is as stable as wild type when bound to substrate, indicating that caspase-7 rests in different conformations in the absence and presence of substrate. Residues K212 and I213 in the L2 0 loop are shown to be essential for substrate-binding and thus proper catalytic function of the caspase. In the crystal structure of I213A, the void created by side-chain deletion is compensated for by rearrangement of tyrosine 211 to fill the void, suggesting that the requirements of substrate-binding are sufficiently strong to induce the active conformation. Thus, although the L2 0 loop makes no direct contacts with substrate, it is essential for buttressing the substrate-binding groove and is central to native catalytic efficiency.

Studies of the molecular mechanism of caspase-8 activation by solution NMR

“…22 Structural differences of the L2 loop between inhibitor-free and inhibitor-bound caspase-8 could explain the higher dimer affinity upon inhibitor binding, a common phenomenon for initiator caspases. 27 Previous experiments on caspase-7 revealed an increased overall protein stability upon inhibitor binding by 17.9 degrees, 32 which is in the same range as observed for caspase-8 and in case of dimeric caspase-7 must be independent of dimerization. We therefore speculate that the increased overall stability of caspase-8 upon addition of the inhibitor is due to rigidification of the loops around the active site and possibly may also involve loop 4 and 5, which in procaspase-8 undergo conformational exchange.…”

Structural and Enzymatic Insights into Caspase-2 Protein Substrate Recognition and Catalysis

“…The key residues involved in catalysis, including the catalytic dyad and the invariant Arg-378, superpose with each other regardless of the peptide inhibitor binding. Loop 2Ј, which is critical for active-site formation (29), also overlaps well with the loop 2Ј in the occupied subunit. Taken together, these observations indicate that the empty subunit is also in a catalytically active conformation.…”

Zinc-mediated Allosteric Inhibition of Caspase-6

“…We have previously observed how phosphorylation of caspase-6 at Ser-257 results in a steric clash of as few as three atoms in the side chain of Pro-201, which leads to allosteric inhibition by preventing the active conformation of one loop. In caspase-7 (45,63,64) or -1 (65,66), binding small synthetic allosteric inhibitors at the dimer interface leads to a series of side chain conformational changes that ultimately block the substrate-binding groove, preventing proteolytic activity. These examples and a great number of other examples from a large number of proteins are satisfying as it is possible to visualize the series of side chain conformational changes that lead to inactivation.…”

“…Induction of caspase-3 was at 30°C for 3 h; caspase-7 was at 18°C for 18 h. These proteins were purified as described previously for caspase-3 (29) and caspase-7 (45). The eluted proteins were stored at Ϫ80°C in the buffer in which they were eluted.…”