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

Abstract

The metalloproteinases TACE [tumour necrosis factor alpha-converting enzyme; also known as ADAM17 (a disintegrin and metalloproteinase 17)] and ADAM10 are the primary enzymes responsible for catalysing release of membrane-anchored proteins from the cell surface in metazoan organisms. Although the repertoire of protein substrates for these two proteases is partially overlapping, each one appears to target a subset of unique proteins in vivo. The mechanisms by which the two proteases achieve specificity for particular substrates are not completely understood. We have used peptide libraries to define the cleavage site selectivity of TACE and ADAM10. The two proteases have distinct primary sequence requirements at multiple positions surrounding the cleavage site in their substrates, which allowed us to generate peptide substrates that are highly specific for each of these proteases. The major difference between the two protease specificities maps to the P1' position (immediately downstream of the cleavage site) of the substrate. At this position, TACE is selective for smaller aliphatic residues, whereas ADAM10 can accommodate aromatic amino acids. Using mutagenesis we identified three residues in the S1' pockets of these enzymes that dramatically influence specificity for both peptide and protein substrates. Our results suggest that substrate selectivity of TACE and ADAM10 can be at least partly rationalized by specific features of their active sites.

Figure 1. TACE and ADAM10 display distinct cleavage site motifs

(A) Peptide cleavage selectivity for TACE and ADAM10. The distribution of amino acid residues at positions C-terminal to the ADAM cleavage site was determined by sequencing a partial digest of an N-terminally blocked random dodecamer library. Secondary and tertiary libraries used to determine specificity at the indicated positions upstream of the cleavage site had free amino termini and C-terminal biotin groups. The amino acid distribution of the substrate pool was determined by Edman sequencing of the digest following removal of the biotinylated C-termini using immobilized avidin. Positively selected residues for each primed and unprimed position are indicated by heat map according to the scale on the bottom of the panel. Data were normalized such that to a value of 1 (black) corresponds the average quantity per amino acid in a given sequencing cycle and would indicate no selectivity, while residues having a value greater than 1 (red) are positively selected by the protease. Due to interfering peaks on the sequencer, data is missing for the following residues at specific upstream positions: Arg at P5, Gly from P5 to P3, Gln from P4 to P1, and Lys at P2 and P1. Heat maps were generated using MapleTree using quantified data available online as Supplementary Table S1. (B) Comparison of P1′ selectivity of TACE and ADAM10. Selectivity values used to generate the heat map in panel A are depicted as a bar graph.

Figure 2. Cleavage kinetics for peptide substrates based on the predicted optimal cleavage motifs for TACE and ADAM10

(A) Consensus decapeptide substrates were designed and synthesized based on the data from Figure 1. Peptides were dually labeled with a fluorophores and quencher, allowing cleavage to be monitored by fluorescence increase as described under Experimental Procedures. The arrow indicates the site of cleavage. (B) The kinetics of cleavage (k_cat/K_M values) of each fluorescent peptide substrate by TACE and ADAM10 were determined directly from the initial rate at a single substrate concentration (2 μM) under conditions where K_M >> [S]. (C) Cleavage rates are shown as a percentage of the k_cat/K_M value of the peptide that was cleaved the fastest by a given protease (the “L” substrate is identical to ADAMtide from panel B). Error bars reflect the standard deviation from at least three separate experiments.

Figure 3. Determinants of TACE and ADAM10 P1′ specificity

(A) Structure of the TACE active site. Coordinates were taken from the X-ray crystal structure of in complex with a hydroxamic acid peptide-based inhibitor ([39], PDB code 1BKC). For clarity, only the P1′ leucine mimetic and hydroxamate group of the bound inhibitor are shown (in light green). The hydroxamate metal chelating group coordinates the active site zinc ion (dark gray). The seven amino-acid residues that comprise the S1′ pocket are labeled and shown in magenta in stick representation. The figure was prepared using Pymol. (B) Cleavage parameters for fluorescent peptide substrates by ADAM10 I380L/T381V (left panel) and ADAM10 T381V (right panel) mutants. (C) Cleavage parameters for TACE L401I/V402T (left panel), TACE V402T (middle panel) and TACE V402T/V440T (right panel). Values were determined from measuring initial cleavage rates at 2 μM substrate as for the data shown in Figure 2.

Figure 4. Active site contribution to protein substrate cleavage by TACE

(A) Time course for cleavage of TNF and its mutants by WT TACE (upper panel) and by TACE L401I/V402T (lower panel). Protein substrate was incubated with enzyme at 37 °C for the indicated times. Reactions were quenched and subjected to SDS-PAGE followed by immunoblotting with anti-His₆. (B) Cleavage rates of AP-fused substrates by WT TACE (black) or TACE L401I/V402T (grey) were measured after 60 min incubation at 37 °C. The amount of immobilized substrate released from FLAG beads was quantified using a colorimetric assay for AP. The results show a single representative of three independent experiments.