Sequential NMR resonance assignment and structure determination of the Kunitz-type inhibitor domain of the Alzheimer's beta-amyloid precursor protein.
Certain precursor proteins (APP751 and APP770) of the amyloid beta-protein (AP) present in Alzheimer's disease contain a Kunitz-type serine protease inhibitor domain (APPI). In this study, the domain is obtained as a functional inhibitor through both recombinant (APPIr) and synthetic (APPIs) methodologies, and the solution structure of APPI is determined by 1H 2D NMR techniques. Complete sequence-specific resonance assignments (except for P13 and G37 NH) for both APPIr and APPIs are achieved using standard procedures. Ambiguities arising from degeneracies in the NMR resonances are resolved by varying sample conditions. Qualitative interpretation of short- and long-range NOEs reveals secondary structural features similar to those extensively documented by NMR for bovine pancreatic trypsin inhibitor (BPTI). A more rigorous interpretation of the NOESY spectra yields NOE-derived interresidue distance restraints which are used in conjunction with dynamic simulated annealing to generate a family of APPI structures. Within this family, the beta-sheet and helical regions are in good agreement with the crystal structure of BPTI, whereas portions of the protease-binding loops deviate from those in BPTI. These deviations are consistent with those recently described in the crystal structure of APPI (Hynes et al., 1990). Also supported in the NMR study is the hydrophobic patch in the protease-binding domain created by side chain-side chain NOE contacts between M17 and F34. In addition, the NMR spectra indicate that the rotation of the W21 ring in APPI is hindered, unlike Y21 in BPTI, showing a greater than 90% preference for one orientation in the hydrophobic groove.
Heald
S L
SL
Miles Research Center, Miles, Incorporated, West Haven, Connecticut 06516.
Tilton
R F
RF
Hammond
L J
LJ
Lee
A
A
Bayney
R M
RM
Kamarck
M E
ME
Ramabhadran
T V
TV
Dreyer
R N
RN
Davis
G
G
Unterbeck
A
A
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry*; D007611:Aprotinin / Q000235:genetics*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015842:Serine Proteinase Inhibitors / Q000235:genetics*
D016564:Amyloid beta-Protein Precursor; D015842:Serine Proteinase Inhibitors; D007611:Aprotinin