Study of a Major Intermediate in the Oxidative Folding of Leech Carboxypeptidase Inhibitor: Contribution of the Fourth Disulfide Bond Joan L. Arolas 1 , Grzegorz M. Popowicz 2 , Sı ´lvia Bronsoms 1 , Francesc X. Aviles 1 * , Robert Huber 2 , Tad A. Holak 2 * and Salvador Ventura 1 1 Institut de Biotecnologia i de Biomedicina and Departament de Bioquı ´mica i Biologia Mol- ecular, Universitat Auto `noma de Barcelona, 08193 Bellaterra Spain 2 Abteilung fu ¨r Struktur- forschung, Max-Planck-Institut fu ¨ r Biochemie, Am Klopferspitz 18a, D-82152 Martinsried Germany The oxidative folding pathway of leech carboxypeptidase inhibitor (LCI; four disulfide bonds) proceeds through the formation of two major intermediates (III-A and III-B) that contain three native disulfide bonds and act as strong kinetic traps in the folding process. The III-B intermediate lacks the Cys19–Cys43 disulfide bond that links the b-sheet core with the a-helix in wild-type LCI. Here, an analog of this intermediate was constructed by replacing Cys19 and Cys43 with alanine residues. Its oxidative folding follows a rapid sequential flow through one, two, and three disulfide species to reach the native form; the low accumulation of two disulfide intermediates and three disulfide (scrambled) isomers accounts for a highly efficient reaction. The three-dimensional structure of this analog, alone and in complex with carboxypeptidase A (CPA), was determined by X-ray crystallography at 2.2 A ˚ resolution. Its overall structure is very similar to that of wild-type LCI, although the residues in the region adjacent to the mutation sites show an increased flexibility, which is strongly reduced upon binding to CPA. The structure of the complex also demonstrates that the analog and the wild-type LCI bind to the enzyme in the same manner, as expected by their inhibitory capabilities, which were similar for all enzymes tested. Equilibrium unfolding experiments showed that this mutant is destabilized by w1.5 kcal mol K1 (40%) relative to the wild-type protein. Together, the data indicate that the fourth disulfide bond provides LCI with both high stability and structural specificity. q 2005 Elsevier Ltd. All rights reserved. Keywords: carboxypeptidase inhibitor; oxidative folding; folding inter- mediates; crystal structures; protein stability *Corresponding authors Introduction Protein folding commonly proceeds through a series of intermediates that define the folding landscape from the unfolded polypeptide to the native structure. 1,2 Understanding the fundamental relationship between the amino acid sequence and the three-dimensional structure of a native protein is advanced by structural analysis of those folding intermediates. Although characterizing the inter- mediates is usually a difficult task due to their short half-life, studies of proteins stabilized by disulfide bonds have provided considerable insight into the field of protein folding. 3 In disulfide-rich proteins, the coupling of the covalent chemistry of disulfide bond formation to the non-covalent folding pro- cesses makes it feasible to trap, isolate and 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: BPTI, bovine pancreatic trypsin inhibitor; CD, circular dichroism; CP, carboxypeptidase; DTT, dithiothreitol; GSH, reduced glutathione; GSSG, oxidized glutathione; GdnHCl, guanidine hydrochloride; IGF-1, insulin-like growth factor 1; K i , inhibition constant; LCI, leech carboxypeptidase inhibitor; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; PCI, potato carboxypeptidase inhibi- tor; r.m.s.d., root-mean-square deviation; RNase A, ribonuclease A; RP, reversed-phase; TAFI, thrombin- activatable fibrinolysis inhibitor; TFA, trifluoroacetic acid; TAP, tick anticoagulant peptide. E-mail addresses of the corresponding authors: fxaviles@einstein.uab.es; holak@biochem.mpg.de doi:10.1016/j.jmb.2005.07.065 J. Mol. Biol. (2005) 352, 961–975