High-resolution MAS NMR analysis of PI3-SH3 amyloid fibrils: backbone conformation and implications for protofilament assembly and structure .
The SH3 domain of the PI3 kinase (PI3-SH3 or PI3K-SH3) readily aggregates into fibrils in vitro and has served as an important model system in the investigation of the molecular properties and mechanism of formation of amyloid fibrils. We describe the molecular conformation of PI3-SH3 in amyloid fibril form as revealed by magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy. The MAS NMR spectra of these fibrils display excellent resolution, with narrow (13)C and (15)N line widths, representing a high degree of structural order and the absence of extensive molecular motion for the majority of the polypeptide chain. We have identified the spin systems of 82 of the 86 residues in the protein and obtained sequential resonance assignments for 75 of them. Chemical shift analysis indicates that the protein subunits making up the fibril adopt a compact conformation consisting of four well-defined beta-sheet regions and four random-coil elements with varying degrees of local dynamics or disorder. The backbone conformation of PI3-SH3 in fibril form differs significantly from that of the native state of the protein, both in secondary structure and in the location of dynamic or disordered segments. The site-specific MAS NMR analysis of PI3-SH3 fibrils we report here is compared with previously published mechanistic and structural data, resulting in a detailed interpretation of the factors that mediate fibril formation by PI3-SH3 and allowing us to propose a possible model of the core structure of the fibrils. Our results confirm the structural similarities between PI3-SH3 fibrils and amyloid assemblies directly related to degenerative and infectious diseases.
Bayro
Marvin J
MJ
Francis Bitter Magnet Laboratory and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Maly
Thorsten
T
Birkett
Neil R
NR
Macphee
Cait E
CE
Dobson
Christopher M
CM
Griffin
Robert G
RG
D000682:Amyloid / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D018909:src Homology Domains
D000682:Amyloid; D019869:Phosphatidylinositol 3-Kinases