NMR characterizations of an amyloidogenic conformational ensemble of the PI3K SH3 domain

Abstract

Amyloid formation is associated with structural changes of native polypeptides to monomeric intermediate states and their self-assembly into insoluble aggregates. Characterizations of the amyloidogenic intermediate state are, therefore, of great importance in understanding the early stage of amyloidogenesis. Here, we present NMR investigations of the structural and dynamic properties of the acid-unfolded amyloidogenic intermediate state of the phosphatidylinositol 3-kinase (PI3K) SH3 domain-a model peptide. The monomeric amyloidogenic state of the SH3 domain studied at pH 2.0 (35°C) was shown to be substantially disordered with no secondary structural preferences.15 N NMR relaxation experiments indicated that the unfolded polypeptide is highly flexible on a subnanosecond timescale when observed under the amyloidogenic condition (pH 2.0, 35°C). However, more restricted motions were detected in residues located primarily in the b-strands as well as in a loop in the native fold. In addition, nonnative long-range interactions were observed between the residues with the reduced flexibility by paramagnetic relaxation enhancement (PRE) experiments. These indicate that the acid-unfolded state of the SH3 domain adopts a partly folded conformation through nonnative longrange contacts between the dynamically restricted residues at the amyloid-forming condition.Keywords: amyloids; PI3K SH3; NMR; dynamics; amyloidogenic intermediate; long-range interactions; PRE Interest in the unfolded and partly folded states of proteins has been growing because of their important roles in a variety of biological processes, including cellular signaling and transcriptional activation (Shortle 1993;Dyson and Wright 2005). In addition, it is widely accepted that partly structured states of proteins are involved in early events in the formation of amyloid, which underlies a number of debilitating human diseases such as Alzheimer's and Parkinson's diseases (Dobson 2003). Thus, characterization of the unfolded and partially folded states of proteins is of central importance in understanding the molecular mechanism of the biological processes as well as the amyloid diseases. Multinuclear, multidimensional NMR spectroscopy has proven to be a powerful technique to investigate site-specific structural and dynamic properties of nonnative states of proteins (Shortle 1996;Dyson and Wright 2004;Redfield 2004 Abbreviations: NMR, nuclear magnetic resonance; HSQC, heteronuclear single-quantum coherence; PI3K SH3, phosphatidylinositol 3-kinase Src homology 3; R 2 , transverse relaxation rate; NOE, nuclear Overhauser effect; R 1r , relaxation rate in the rotating frame; H/D, hydrogen/deuterium; PRE, paramagnetic relaxation enhancement; MTSL, (1-oxyl-2,2,5,5-tetramethyl-D3-pyrroline-3-methyl) methanethiosulfonate; UV, ultraviolet; CD, circular dichroism.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

Electrostatic Effects in the Folding of the SH3 Domain of the c-Src Tyrosine Kinase: pH-Dependence in 3D-Domain Swapping and Amyloid Formation

“…Both proteins form amyloids at acid pHs (PI3K at pH 2.0 and α-spectrin at pH 3.0) and moderate temperatures (25–37°C). NMR studies performed with the PI3K SH3 domain indicate the presence of partly folded conformations in the acid-unfolded state [69] , but not the presence of domain-swapped forms. In the α-spectrin SH3 domain, the presence of dimers and other higher order oligomers have been described [28] , but not domain-swapped forms.…”

“…Moreover, solid-state NMR experiments show that, in many cases, the amyloid fibers formed by domain-swapped proteins lack of native structure and adopt the β-arcade amyloid motif (for a review see ref [66] ). In fact, the formation of domain-swapped oligomers has not been found in well-characterized amyloid-forming proteins, as it is the case of the PI3K [69] and the α-spectrin SH3 domains [26] . Several NMR studies on the PI3K-SH3 domain show that the backbone conformation of the protein in the fibril form is different from that of the native one [62] , [63] .…”

Residual Structures in the Acid-Unfolded States of Vλ6 Proteins Affect Amyloid Fibrillation

“…22,25,26,37 To determine the pH at which the three rVλ6 proteins were unfolded, we analyzed the pH dependence of secondary structures by circular dichroism (CD) spectroscopy. The far-UV CD spectra of the three rVλ6 proteins at pH 7.5 showed the extension of a single negative peak at around 215 nm, which is characteristic of a β-structure (Fig.…”

Fibril formation from the amyloid-β peptide is governed by a dynamic equilibrium involving association and dissociation of the monomer

“…The PRE effect is caused by an unpaired electron, which has a high magnetogyric ratio (γ e /γ H ∼ 600), resulting in a significant decrease in NMR signal intensity of nearby (within ∼ 25 Å) nuclear spins. This method has been successfully used to detect long-range intra-or intermolecular interactions present in low-populated species (Ahn et al 2006;Bertoncini et al 2005;Iwahara and Clore 2006;Wu et al 2008).…”