The N‐terminal region of cystatin A (stefin A) binds to papain subsequent to the two hairpin loops of the inhibitor. Demonstration of two‐step binding by rapid‐kinetic studies of cystatin A labeled at the N‐terminus with a fluorescent reporter group

Abstract

The three-dimensional structures of cystatins, and other evidence, suggest that the flexible N-terminal region of these inhibitors may bind to target proteinases independent of the two rigid hairpin loops forming the remainder of the inhibitory surface. In an attempt to demonstrate such two-step binding, which could not be identified in previous kinetics studies, we introduced a cysteine residue before the N-terminus of cystatin A and labeled this residue with fluorescent probes. Binding of AANS-and AEDANS-labeled cystatin A to papain resulted in ;4-fold and 1.2-fold increases of probe fluorescence, respectively, reflecting the interaction of the N-terminal region with the enzyme. Observed pseudo-first-order rate constants, measured by the loss of papain activity in the presence of a fluorogenic substrate, for the reaction of the enzyme with excess AANS-cystatin A increased linearly with the concentration of the latter. In contrast, pseudo-first-order rate constants, obtained from measurements of the change of probe fluorescence with either excess enzyme or labeled inhibitor, showed an identical hyperbolic dependence on the concentration of the reactant in excess. This dependence demonstrates that the binding occurs in two steps, and implies that the labeled N-terminal region of cystatin A interacts with the proteinase in the second step, subsequent to the hairpin loops. The comparable affinities and dissociation rate constants for the binding of labeled and unlabeled cystatin A to papain indicate that the label did not appreciably perturb the interaction, and that unlabeled cystatin therefore also binds in a similar two-step manner. Such independent binding of the N-terminal regions of cystatins to target proteinases after the hairpin loops may be characteristic of most cystatin-proteinase reactions.Keywords: cystatin; fluorescence labeling; N-terminal region; papain; stefin; stopped-flow fluorescence; two-step binding Reprint requests to: Ingemar Björk, Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Box 575, SE-751 23 Uppsala, Sweden; e-mail: Ingemar.Bjork@vmk.slu.se.Abbreviations: AANS,~2-~49-acetamido!anilino!naphthalene-6-sulfonic acid; AANS-cystatin A, cystatin A labeled with an AANS group at a Cys residue introduced in the N-terminus; AEDANS, 5-~~~~2-acetyl!amino!ethyl!amino!naphthalene-1-sulfonic acid; AEDANS-cystatin A, cystatin A labeled with an AEDANS group introduced in the N-terminus; app, subscript denoting an apparent equilibrium or rate constant measured in the presence of an enzyme substrate; ϩCys-cystatin A, a variant of cystatin A with an extra Cys residue introduced before Met-1 of the wild-type inhibitor; DTT, dithiothreitol; His-tag, 10 consecutive histidine residues fused to an expressed protein; IAANS,~2-~49-iodoacetamido!anilino!naphthalene-6-sulfonic acid; IAEDANS, 5-~~~~2-iodoacetyl!amino!ethyl!amino!naphthalene-1-sulfonic acid; k ass , bimolecular association rate constant; K d , dissociation equilibrium constant; k diss , dissociation...

Fundamental Aspects of Protein−Protein Association Kinetics

“…A somewhat different mechanism involving a pre-equilibrium complex followed by a reorganization step was suggested for cystatin A binding papain. , The biphasic behavior of cystatin A/papain interaction was observed by following independently the inhibition of the catalytic activity of papain (where a linear dependence of k obs on protein concentration was observed), while the spectroscopic probe (which monitors the accumulation of the native complex) showed a hyperbolic dependence of k obs on protein concentration. This example demonstrates that characterization of the association behavior may depend on the probe used to monitor it.…”

The Occluding Loop of Cathepsin B Prevents Its Effective Inhibition by Human Kininogens

“…61,62 This is in contrast to papain and cathepsins L and S, which bind family 1 and 2 cystatins according to a one-step mechanism. [63][64][65][66] Likewise, the present experimental kinetics values, corresponding to the tight-binding inhibition of His110Ala cathepsin B by both kininogens, strongly suggest that the disruption of the salt bridge between His110 and Asp22 results in increased mobility of the occluding loop and a subsequent improved accessibility to the active site as previously discussed in detail by Pavlova et al 36 and Redzynia et al, 35 leading to a single, one-step reaction mechanism similar to that observed for occluding loop-lacking cathepsins interacting with stefins and cystatins. Human stefin B, similarly to kininogens and their isolated inhibitory domains, binds tightly to papain and cathepsin L and more weakly to cathepsin B. Stefin B also has a lower affinity to cathepsin B than ovocystatin or stefin A.…”

Selective targeting of a laccase from Stachybotrys chartarum covalently linked to a carotenoid‐binding peptide

“…Insertion of cyclic consensus sequence containing motifs or the replacement of existing amino acids by these cyclic consensus elements in protein α helices or β‐turns should also provide important structural information, provided that the resulting modifications do not impair function. Likewise, the impact of the attachment of these consensus containing elements to the N‐terminus, another region considered accessible and flexible in some proteins (32,34,35), might be another potential region of interest.…”