Stabilization of the autoproteolysis of TNF-alpha converting enzyme (TACE) results in a novel crystal form suitable for structure-based drug design studies

Abstract

The crystallization of TNF-alpha converting enzyme (TACE) has been useful in understanding the structure-activity relationships of new chemical entities. However, the propensity of TACE to undergo autoproteolysis has made enzyme handling difficult and impeded the identification of inhibitor soakable crystal forms. The autoproteolysis of TACE was found to be specific (Y352-V353) and occurred within a flexible loop that is in close proximity to the P-side of the active site. The rate of autoproteolysis was found to be proportional to the concentration of TACE, suggesting a bimolecular reaction mechanism. A limited specificity study of the S(1)' subsite was conducted using surrogate peptides and suggested substitutions that would stabilize the proteolysis of the loop at positions Y352-V353. Two mutant proteases (V353G and V353S) were generated and proved to be highly resistant to autoproteolysis. The kinetics of the more resistant mutant (V353G) and wild-type TACE were compared and demonstrated virtually identical IC(50) values for a panel of competitive inhibitors. However, the k(cat)/K(m) of the mutant for a larger substrate (P6 - P(6)') was approximately 5-fold lower than that for the wild-type enzyme. Comparison of the complexed wild-type and mutant structures indicated a subtle shift in a peripheral P-side loop (comprising the mutation site) that may be involved in substrate binding/turnover and might explain the mild kinetic difference. The characterization of this stabilized form of TACE has yielded an enzyme with similar native kinetic properties and identified a novel crystal form that is suitable for inhibitor soaking and structure determination.

Active-site determinants of substrate recognition by the metalloproteinases TACE and ADAM10

“…The S1′ pocket of TACE has been defined through X-ray crystallography of the catalytic domain in complex with peptidomimetic inhibitors (Figure 3A) [39, 40]. Interestingly, despite the differences in selectivity, ADAM10 and TACE have a high degree of sequence conservation within their S1′ pockets (Table 3).…”

The Canonical Notch Signaling Pathway: Unfolding the Activation Mechanism

“…Thus, LNR-A, the LNR-AB linker, and LNR-B must all be removed to produce molecules with constitutive signaling activity. Given the deep active site pocket in ADAM17/TACE (Ingram et al, 2006; Maskos et al, 1998; Wasserman et al, 2003), Gordon et al speculate that not only does the receptor activation mechanism forcibly lift at least two of the three LNR repeats, but the process must also disengage the stabilizing helix within the HD domain from the S2 containing strand (perhaps by partially unfolding the helix) to permit access to the scissile bond at S2 (a model the authors dub “lift and cut”). The NRR crystal structure reveals that Ca ++ chelation will most likely unfold the LNR repeats and not just the HD domain (Figure 3B), suggesting that “lift” could simultaneously induce complete receptor dissociation.…”

ADAM-17: the enzyme that does it all

“…At the same time, there was intense work to identify and develop selective ADAM-17 inhibitors (reviewed in (Newton and Decicco, 1999) either by changing the structure of broad spectrum MMP inhibitors or by designing new compounds (Xue et al, 2001, Rabinowitz et al, 2001, Duan et al, 2002). Improvement of the structure (Ingram et al, 2006) and description of the crystal structure of the ADAM-17 catalytic domain in complex with its naturally occurring inhibitor tissue inhibitor of metalloenzymes-3 (TIMP-3) (Wisniewska et al, 2008) was useful for the recent development of several highly selective, potent ADAM-17 inhibitors targeted against the active center (Gilmore et al, 2006, Gilmore et al, 2007, Huang et al, 2007, Venkatesan et al, 2004). Orally active, selective ADAM inhibitors were developed and tested in combination with ErbB2 receptor antibodies for the treatment of breast cancer (Fridman et al, 2007).…”