1ca0 Citations

Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): engineering of inhibitors with altered specificities.

Protein Sci 6 1806-24 (1997)
Related entries: 1cbw, 1taw

Cited: 88 times
EuropePMC logo PMID: 9300481

Abstract

The crystal structures of the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) complexed to bovine chymotrypsin (C-APPI) and trypsin (T-APPI) and basic pancreatic trypsin inhibitor (BPTI) bound to chymotrypsin (C-BPTI) have been solved and analyzed at 2.1 A, 1.8 A, and 2.6 A resolution, respectively. APPI and BPTI belong to the Kunitz family of inhibitors, which is characterized by a distinctive tertiary fold with three conserved disulfide bonds. At the specificity-determining site of these inhibitors (P1), residue 15(I)4 is an arginine in APPI and a lysine in BPTI, residue types that are counter to the chymotryptic hydrophobic specificity. In the chymotrypsin complexes, the Arg and Lys P1 side chains of the inhibitors adopt conformations that bend away from the bottom of the binding pocket to interact productively with elements of the binding pocket other than those observed for specificity-matched P1 side chains. The stereochemistry of the nucleophilic hydroxyl of Ser 195 in chymotrypsin relative to the scissile P1 bond of the inhibitors is identical to that observed for these groups in the trypsin-APPI complex, where Arg 15(I) is an optimal side chain for tryptic specificity. To further evaluate the diversity of sequences that can be accommodated by one of these inhibitors, APPI, we used phage display to randomly mutate residues 11, 13, 15, 17, and 19, which are major binding determinants. Inhibitors variants were selected that bound to either trypsin or chymotrypsin. As expected, trypsin specificity was principally directed by having a basic side chain at P1 (position 15); however, the P1 residues that were selected for chymotrypsin binding were His and Asn, rather than the expected large hydrophobic types. This can be rationalized by modeling these hydrophilic side chains to have similar H-bonding interactions to those observed in the structures of the described complexes. The specificity, or lack thereof, for the other individual subsites is discussed in the context of the "allowed" residues determined from a phage display mutagenesis selection experiment.

Reviews - 1ca0 mentioned but not cited (1)

  1. Alzheimer's disease--a panorama glimpse. Zhao LN, Lu L, Chew LY, Mu Y. Int J Mol Sci 15 12631-12650 (2014)

Articles - 1ca0 mentioned but not cited (11)



Reviews citing this publication (8)

  1. Mechanisms of macromolecular protease inhibitors. Farady CJ, Craik CS. Chembiochem 11 2341-2346 (2010)
  2. Structural and energetic determinants of the S1-site specificity in serine proteases. Czapinska H, Otlewski J. Eur J Biochem 260 571-595 (1999)
  3. Protease inhibitors from marine venomous animals and their counterparts in terrestrial venomous animals. Mourão CB, Schwartz EF. Mar Drugs 11 2069-2112 (2013)
  4. Surface loops of trypsin-like serine proteases as determinants of function. Goettig P, Brandstetter H, Magdolen V. Biochimie 166 52-76 (2019)
  5. Structural Studies Providing Insights into Production and Conformational Behavior of Amyloid-β Peptide Associated with Alzheimer's Disease Development. Urban AS, Pavlov KV, Kamynina AV, Okhrimenko IS, Arseniev AS, Bocharov EV. Molecules 26 2897 (2021)
  6. Phage display as a powerful tool to engineer protease inhibitors. Zani ML, Moreau T. Biochimie 92 1689-1704 (2010)
  7. Protein recognition using synthetic surface-targeted agents. Jain R, Ernst JT, Kutzki O, Park HS, Hamilton AD. Mol Divers 8 89-100 (2004)
  8. The Anemonia viridis Venom: Coupling Biochemical Purification and RNA-Seq for Translational Research. Nicosia A, Mikov A, Cammarata M, Colombo P, Andreev Y, Kozlov S, Cuttitta A. Mar Drugs 16 E407 (2018)

Articles citing this publication (68)



Related citations provided by authors (1)