Structure of the potato inhibitor complex of carboxypeptidase A at 2.5-A resolution.

Abstract

The structure of the complex between the proteolytic enzyme carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and the 39-amino-acid carboxypeptidase A inhibitor from potatoes has been determined at 2.5-A resolution. A combination of multiple isomorphous replacement, molecular replacement, and noncrystallographic symmetry averaging techniques was used to solve the structure. The chain trace of the inhibitor and details of the binding interactions in the complex are described. A surprising aspect of the complex is that the carboxy-terminal peptide bond of the inhibitor has been hydrolyzed, and the carboxy-terminal glycine is trapped in the binding pocket of carboxypeptidase A. Consequently, the complex resembles a stage in the catalytic mechanism after hydrolysis of the peptide bond. The ring of tyrosine-248, which is known to undergo large conformational changes upon substrate binding, is in the "down" position and interacts with the inhibitor in the complex.

A common structural motif incorporating a cystine knot and a triple‐stranded β‐sheet in toxic and inhibitory polypeptides

“…This is probably due to the proximity of the first halfcystine residue to the N-terminus in 0-CgTx and CMTI-I, allowtent with the results of Chiche et al (1993) and the previously mentioned structural similarity between CMTI-I and EETI-11. On the basis of these superpositions and the similar hydrogen bonding patterns in all 4 molecules, the extended segments of CPI can be described as a small, triple-stranded &sheet as in EETI-I1 and w-CgTx, as was suggested by Chiche et al (1993) but not commented on in the original X-ray crystallographic structure determination (Rees & Lipscomb, 1982).…”

X-ray crystallographic investigation of substrate binding to carboxypeptidase A at subzero temperature.

“…Other binding interactions observed are typical of those found in CPA-inhibitor complexes (8)(9)(10)(11)(12)(13)(14)(15): the hydroxybenzyl group of tyrosine resides in the hydrophobic pocket, or specificity pocket, of the Sj' subsite, and the terminal carboxylate is salt linked with the guanidinium moiety of arginine-145. The phenolic residue of tyrosine-248 is in the "down" conformation, and its phenolic oxygen is 2.8 A away from one of the terminal carboxylate oxygens of GY.…”

“…Either the zinc ion of the active site or the positively charged guanidinium moiety of arginine-127, or both, may serve to polarize the substrate carbonyl prior to its attack (regardless of the general mechanism). Mechanistic analyses have beenaided in part by structural studies of the native enzyme (5)(6)(7) and its complexes with different inhibitors (8)(9)(10)(11)(12)(13)(14)(15), including the slowly hydrolyzed (16) substrate glycyl-L-tyrosine (GY; Fig. 1) (6, 7), which is a competitive inhibitor of more rapidly cleaved substrates.…”

Tissue-Specific Expression of Head-to-Tail Cyclized Miniproteins in Violaceae and Structure Determination of the Root CyclotideViola hederacearoot cyclotide1[W]

“…The cystine knot motif is found not only in cyclotides and two circular trypsin inhibitors from the seeds of bitter melon, M. cochinchinensis (Hernandez et al, 2000), but is also present in a carboxypeptidase A inhibitor from potato (Solanum tuberosum; Rees and Lipscomb, 1980), squash trypsin inhibitors (Bode et al, 1989;Heitz et al, 1989;Le Nguyen et al, 1990), as well as growth factors (McDonald and Hendrickson, 1993) and miscellaneous inhibitor molecules (Pallaghy et al, 1994). All cystine knot proteins share the general disulfide connectivity pattern of I-IV, II-V, and III-VI, with numbers following the N-to-C chemical direction.…”