Formation of a noncovalent serpin-proteinase complex involves no conformational change in the serpin. Use of 1H-15N HSQC NMR as a sensitive nonperturbing monitor of conformation.
A structural understanding of the nature and scope of serpin inhibition mechanisms has been limited by the inability so far to crystallize any serpin-proteinase complex. We describe here the application of [(1)H-(15)N]-HSQC NMR on uniformly and residue-selectively (15)N-labeled serpin alpha(1)-proteinase inhibitor (Pittsburgh variant with stabilizing mutations) to provide a nonperturbing and exquisitely sensitive means of probing the conformation of the serpin alone and in a noncovalent complex with inactive, serine 195-modified, bovine trypsin. The latter should be a good model both for the few examples of reversible serpin-proteinase complexes and for the initial Michaelis-like complex formed en route to irreversible covalent inhibition. Cleavage of the reactive center loop, with subsequent insertion into beta-sheet A, caused dramatic perturbation of most of the NMR cross-peaks. This was true for both the uniformly labeled and alanine-specifically labeled samples. The spectra of uniformly or leucine- or alanine-specifically labeled alpha(1)-proteinase inhibitor in noncovalent complex with unlabeled inactive trypsin gave almost no detectable chemical shift changes of cross-peaks, but some general increase in line width. Residue-specific assignments of the four alanines in the reactive center loop, at P12, P11, P9, and P4, allowed specific examination of the behavior of the reactive center loop. All four alanines showed higher mobility than the body of the serpin, consistent with a flexible reactive center loop, which remained flexible even in the noncovalent complex with proteinase. The three alanines near the hinge point for insertion showed almost no chemical shift perturbation upon noncovalent complex formation, while the alanine at P4 was perturbed, presumably by interaction with the active site of bound trypsin. Reporters from both the body of the serpin and the reactive center loop therefore indicate that noncovalent complex formation involves no conformational change in the body of the serpin and only minor perturbation of the reactive center loop in the region which contacts proteinase. Thus, despite the large size of serpin and serpin-proteinase complex, 45 and 69 kDa respectively, NMR provides a very sensitive means of probing serpin conformation and mobility, which should be applicable both to noncovalent and to covalent complexes with a range of different proteinases, and probably to other serpins.
Peterson
F C
FC
Department of Biochemistry and Molecular Biology, M/C 536,College of Medicine, University of Illinois at Chicago, 1853 West Polk Street, Chicago, Illinois 60612, USA.
Gordon
N C
NC
Gettins
P G
PG
D000409:Alanine / Q000737:chemistry; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D006801:Humans; D006868:Hydrolysis; D007930:Leucine / Q000737:chemistry; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D011522:Protons; D014357:Trypsin / Q000737:chemistry*; D000515:alpha 1-Antitrypsin / Q000737:chemistry*
D046911:Macromolecular Substances; D009587:Nitrogen Isotopes; D011522:Protons; D000515:alpha 1-Antitrypsin; C038915:alpha 1-antitrypsin Pittsburgh; C011661:anhydrotrypsin; D014357:Trypsin; D007930:Leucine; D000409:Alanine