Key feature of the catalytic cycle of TNF-α converting enzyme involves communication between distal protein sites and the enzyme catalytic core

Abstract

Despite their key roles in many normal and pathological processes, the molecular details by which zinc-dependent proteases hydrolyze their physiological substrates remain elusive. Advanced theoretical analyses have suggested reaction models for which there is limited and controversial experimental evidence. Here we report the structure, chemistry and lifetime of transient metal-protein reaction intermediates evolving during the substrate turnover reaction of a metalloproteinase, the tumor necrosis factor-␣ converting enzyme (TACE). TACE controls multiple signal transduction pathways through the proteolytic release of the extracellular domain of a host of membrane-bound factors and receptors. Using stopped-flow x-ray spectroscopy methods together with transient kinetic analyses, we demonstrate that TACE's catalytic zinc ion undergoes dynamic charge transitions before substrate binding to the metal ion. This indicates previously undescribed communication pathways taking place between distal protein sites and the enzyme catalytic core. The observed charge transitions are synchronized with distinct phases in the reaction kinetics and changes in metal coordination chemistry mediated by the binding of the peptide substrate to the catalytic metal ion and product release. Here we report key local charge transitions critical for proteolysis as well as long sought evidence for the proposed reaction model of peptide hydrolysis. This study provides a general approach for gaining critical insights into the molecular basis of substrate recognition and turnover by zinc metalloproteinases that may be used for drug design.dynamics ͉ matrix metalloproteinases ͉ proteolysis ͉ x-ray absorption ͉ stopped flow Z inc-dependent metalloproteinases comprise a large superfamily of enzymes possessing key biological roles. These enzymes use zinc (Fig. 1a) to catalyze the hydrolysis of peptide bonds in a wide variety of substrates in both normal and pathological processes (1, 2). Over the last five decades, substantial efforts have been aimed at understanding the molecular basis by which the catalytic zinc machinery executes such enzymatic reactions (3, 4). Protein crystallography, proteomic, and theoretical studies have been instrumental in proposing reaction mechanisms (3,5,6). However, the molecular details that link the catalytic chemistry to key kinetic, electronic, and structural events have remained elusive because of the difficulties associated with probing time-dependent structure-function aspects of such reactions in the presence of peptide substrates.Here we used a strategy based on dynamic structural spectroscopy to elucidate in detail the molecular mechanisms at work during substrate turnover by TACE. TACE is a disintegrin and metalloproteinase family member (also known as ADAM 17) with key roles in the regulation of the proteolytic release of cytokines, chemokines, growth factors, and receptors from cellular membranes (a process known as ectodomain shedding) (7). We have used stopped-flow freeze-quench x-ray absorption spectros...

Cross-domain inhibition of TACE ectodomain

“…Although most ADAM catalytic domains appear to share homologous structural topology, significant sequence variation is common throughout the noncatalytic Dis-Cys domains [especially in the "hyper-variable" region (HVR)] (13). Despite their divergent nomenclature, accumulating evidence suggests the catalytic domain and Dis-Cys domain are spatially associated within the complete ADAM ectodomain (14,15). Specifically, the crystal structures of ADAM homologs VAP1 (13), VAP2B (16), RVVX (17), and the nonproteolytic ectodomain of human ADAM22 (18) support the notion that ADAM proteins are "C-shaped" (Fig.…”

“…S8), it is logical to presume that D1(A12) inhibits TACE proteolysis by simply blocking macromolecular substrate access to the catalytic site. However, as the catalytic TACE zinc ion is thought to undergo dynamic charge transitions mediated by noncatalytic regions (15), D1(A12) could also allosterically disrupt crucial communication between distal protein sites by simultaneously binding both noncatalytic and catalytic regions of TACE. Monitoring TACE behavior in the presence of D1(A12) using stopped-flow X-ray spectroscopy could help distinguish between steric hindrance and allosteric inhibition models.…”

Active site dynamics in the zinc-dependent medium chain alcohol dehydrogenase superfamily

“…This work also provides a structural framework around which the catalytic events of this enzyme family can be investigated and which may provide a more complete description of the roles of the various functional groups than has hitherto been possible. Indeed, such changes in zinc coordination during the catalytic cycle have also been observed in other metalloenzymes, such as the matrix metlloproteinases (27,28) and TNF-␣ converting enzyme (29), perhaps suggesting that active site dynamic coordination chemistry is an ubiquitous process in zinc metalloenzymes.…”

The Functional Maturation of A Disintegrin and Metalloproteinase (ADAM) 9, 10, and 17 Requires Processing at a Newly Identified Proprotein Convertase (PC) Cleavage Site

“…The degradation patterns were analyzed using SDS-PAGE, and ADAM17 activity was measured by the proteolysis of a fluorogenic peptide mimicking the cleavage site of TNF␣ (41) (Fig. 4A, WTϩ).…”