Deciphering the structural basis that guides the oxidative folding of leech-derived tryptase inhibitor.
Protein folding mechanisms have remained elusive mainly because of the transient nature of intermediates. Leech-derived tryptase inhibitor (LDTI) is a Kazal-type serine proteinase inhibitor that is emerging as an attractive model for folding studies. It comprises 46 amino acid residues with three disulfide bonds, with one located inside a small triple-stranded antiparallel beta-sheet and with two involved in a cystine-stabilized alpha-helix, a motif that is widely distributed in bioactive peptides. Here, we analyzed the oxidative folding and reductive unfolding of LDTI by chromatographic and disulfide analyses of acid-trapped intermediates. It folds and unfolds, respectively, via sequential oxidation and reduction of the cysteine residues that give rise to a few 1- and 2-disulfide intermediates. Species containing two native disulfide bonds predominate during LDTI folding (IIa and IIc) and unfolding (IIa and IIb). Stop/go folding experiments demonstrate that only intermediate IIa is productive and oxidizes directly into the native form. The NMR structures of acid-trapped and further isolated IIa, IIb, and IIc reveal global folds similar to that of the native protein, including a native-like canonical inhibitory loop. Enzyme kinetics shows that both IIa and IIc are inhibitory-active, which may substantially reduce proteolysis of LDTI during its folding process. The results reported show that the kinetics of the folding reaction is modulated by the specific structural properties of the intermediates and together provide insights into the interdependence of conformational folding and the assembly of native disulfides during oxidative folding.
Pantoja-Uceda
David
D
Departamento de Espectroscopía y Estructura Molecular, Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones Científicas, Serrano 119, E-28006 Madrid, Spain.
Arolas
Joan L
JL
Aviles
Francesc X
FX
Santoro
Jorge
J
Ventura
Salvador
S
Sommerhoff
Christian P
CP
D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D004220:Disulfides / Q000737:chemistry; D047709:Hirudo medicinalis / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*
D004220:Disulfides; D011506:Proteins; C090901:leech-derived tryptase inhibitor C