Structural Studies of the Amyloid State of Proteins

Amyloid diseases, including Alzheimer's, Parkinson's, and the prion conditions, are each associated with a particular protein in fibrillar form. At the morphological level, these fibers appear similar and are termed "amyloid." We found that the adhesive segments of amyloid fibers are short protein sequences which form pairs of interdigitated, in-register beta sheets. These amyloid fibrils were long suspected to be the disease agents, but evidence suggests that in the neurodegenerative diseases, smaller, often transient and polymorphic oligomers are the toxic entities. We have identified a segment of the amyloid-forming protein, alphaB crystallin, which forms an oligomeric complex exhibiting properties of other amyloid oligomers: beta-sheet-rich structure, cytotoxicity, and recognition by an anti-oligomer antibody. The X-ray-derived atomic structure of the oligomer reveals a cylindrical barrel, formed from six anti-parallel, out-of-register protein strands, which we term a cylindrin. The cylindrin structure is compatible with sequence segments from the Abeta protein of Alzheimer's disease and from other amyloid proteins. Cylindrins offer models for the hitherto elusive structures of amyloid oligomers, and are distinct in structure from amyloid fibrils.

SUNBIM a package for X-ray imaging of materials with SWAXS & GISWAXS techniques

SUNBIM (Supramolecular & SUbmolecular Nano & Bio Materials X-ray IMaging Project) is a suite of integrated programs developed, in collaboration with Rigaku Innovative Technologies, to treat Small and Wide Angle X-ray Scattering data, collected either in transmission geometry (SAXS/WAXS) or in reflection geometry (GISAXS/GIWAXS). In addition, a specific routine to collect and analyze data in SAXS scanning transmission microscopy has been developed as additional tool to investigate tissues or material science samples through a focused X-ray beam which is used to raster scan a specimen while acquiring SAXS scattering patterns with a 2D detector. Indeed, a first-generation-synchrotron-class FrE+ SuperBright Rigaku microsource, coupled to a three pinhole S-MAX3000 camera, was recently installed at the X-ray MicroImaging Laboratory (XMI-L@b) and used with success in SAXS/WAXS/GISAXS/GIWAXS experiments (De Caro et al, 2012; 2013)and for SAXS scanning microscopy (Altamura et al, 2012; Giannini et al, 2013). The SUNBIM package was developed and interfaced to the XMI-L@b to perform: i) q-scale calibration and 2D->1D folding on SAXS/GISAXS/WAXS data, including also possible eccentricity corrections studied in the case of WAXS/GIWAXS data (Cervellino et al., 2006; 2008); ii) background evaluation and subtraction, denoising and primary beam angular divergence deconvolution on SAXS/GISAXS profiles (De Caro et al, 2012; 2013); iii) indexing of 2D GISAXS frames (Tate et al., 2006) and extraction of 1D GISAXS profiles along specific cuts; iv) scanning SAXS microscopy: collection of SAXS data in a mesh across mm2 area, composite of the as-collected 2D SAXS frames into a single image, analysis of the composed data to derive the transmitted intensity microscopy as well as the distribution and orientation of nano-scale structures over the analyzed area (Bunk et al., 2009).

Complex structure of MERS-CoV spike glycoprotein with human receptor DPP4

Recently identified the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe and fatal acute respiratory illness in human. No prophylactic and therapeutic agents specifically against MERS-CoV are currently available. Entry of MERS-CoV into the target cells depends on binding of receptor-binding domain (RBD) on viral envelope spike glycoprotein to the cellular receptor dipeptidyl peptidase 4 (DPP4). We report the 3.0 angstrom-resolution crystal structure of MERS-CoV RBD bound to the extracellular domain of DPP4. The structure shows that MERS-CoV RBD consists of a core and a receptor binding subdomain. MERS-CoV RBD and related SARS-CoV RBD share a high degree of structural similarity in their core subdomains, but are notably divergent in the receptor binding subdomain. Structural and mutagenesis analyses identified several key residues in the receptor binding subdomain of RBD that are critical for viral binding to DPP4 and entry into the target cell. Two RBD-specific potent human neutralizing monoclonal antibodies were derived from single-chain variable region fragments (scFvs) of nonimmune human antibody library. They inhibited infection of both pseudotyped and live MERS-CoV with IC50 at nanomolar concentration. Biochemical analysis indicated that these two antibodies blocked RBD interaction with DPP4 on the cell surface.