The NMR Structures of the Major Intermediates of the Two-domain Tick Carboxypeptidase Inhibitor Reveal Symmetry in Its Folding and Unfolding Pathways

Abstract

There is a lack of experimental structural information about folding intermediates of multidomain proteins. Tick carboxypeptidase inhibitor (TCI) is a small, disulfide-rich protein consisting of two domains that fold and unfold autonomously through the formation of two major intermediates, IIIa and IIIb. Each intermediate contains three native disulfide bonds in one domain and six free cysteines in the other domain. Here we have determined the NMR structures of these two intermediates trapped and isolated at acidic pH in which they are stable and compared their structures with that of the native protein analyzed under the same conditions. Both IIIa and IIIb were found to contain a folded region that corresponds to the N-and C-terminal domains of TCI, respectively, with structures very similar to the corresponding regions of the native protein. The remainder of the polypeptide chains of the intermediates was shown to be unfolded in a random coil conformation. Solvent exchange measurements further indicated that the two protein domains are not completely independent, but affect each other in terms of dynamics and stability, in agreement with reported inhibitory activity data. The derived results provide structural evidence for symmetric TCI folding and unfolding mechanisms that converge in IIIa and IIIb and reveal the structural basis that accounts for the strong and simultaneous accumulation of both intermediates. Altogether, this work has important implications for a better understanding of the folding mechanisms of multidomain, disulfide-rich proteins.Understanding the sequence of folding events that lead to a biologically active protein from its amino acid sequence is one of the major challenges in structural and molecular biology. The initial theoretical efforts devoted to the study of protein folding have been reinforced in the last several years by the discovery of a wide range of pathological diseases associated with protein misfolding and the increasing pharmacological interest in protein drug design (1, 2). The structural characterization of partially folded intermediates and the analysis of the interactions that stabilize them are of fundamental importance to unveil the folding mechanisms (3, 4). But these studies are hampered by the rapid and cooperative nature of protein folding, and therefore, the short half-life of the intermediates arising during the folding reaction. It is however possible to trap and isolate discrete intermediates from the oxidative folding of disulfide-rich proteins because of the particular chemistry of disulfide bond formation that takes place as an integral part of disulfide folding (5, 6).A large set of single domain, disulfide-rich proteins has been investigated to date in terms of oxidative folding; among them, outstanding examples such as bovine pancreatic trypsin inhibitor (BPTI), 5 ribonuclease A (RNase A), hirudin, insulin-like growth factor-1 (IGF-1), or conotoxins (7-11). These folding studies have involved the trapping (either by acidification or alkylation), isolat...

Protease Inhibitors as Models for the Study of Oxidative Folding

“…The oxidative folding pathway of MCoTI-II and other cyclotides has been studied and reviewed by Craik and coworkers (19,20). The folding pathway of MCoTI-II is extremely simple, with a predominant 2S intermediate (des [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]; see structural discussion later) arising from the oxidation of an initially heterogeneous 1S population. This intermediate has the two native Cys8-Cys20 and Cys14-Cys26 disulfide bonds and lacks the Cys1-Cys18 disulfide that is formed by direct oxidation leading to the native protein.…”

“…The stability of the N 0 structure makes unfolding reactions energetically expensive so that the rate of this rearrangement is slow. In addition, the formation of N* and N SH-SH from N 0 is not a direct reaction but implies the creation of a transient population of two intermediates containing nonnative disulfide bonds, [5][6][7][8][9][10][11][12][13][14] and , which correspond to the productive nonnative intermediates in Creighton's scheme. Once N SH-SH is formed, as described above, the flexibility and accessibility of the free Cys14 and Cys34 thiols allow rapid oxidation to form the native protein.…”

“…In principle, the oxidation of the two free cysteines in any of these species would result in the formation of a native and functional LDTI. However, when the intermediates were trapped and isolated and their folding was re-initiated at neutral pH, it became clear that neither des nor des [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] can form directly native LDTI but rather reshuffle their disulfides to form des , which turns out to be the only productive intermediate in the folding pathway. The convergence of all 2S intermediates into des is due to both the relative conformational properties of these species and the different reactivity of their free cysteines.…”

“…In the des structure, Cys29 at the C-terminus of the a-helix is fully exposed and can react with Cys4 at the highly flexible N-terminus to produce the native inhibitor. The des [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] intermediate has a very short half-life and rapidly reshuffles into des . Cys6 has a highly reactive thiolate group.…”

“…AROLAS AND VENTURA [5][6][7][8][9][10][11][12][13][14], and , among which only the last one contains two native disulfide bonds; that is, it may be termed des . Although the formation of the Cys5-Cys55 disulfide bond in des would lead to native BPTI, this step finds a high energy barrier for its completion due to the highly folded structure of this intermediate.…”