Andrew D. Miranker

Global unfolding of a substrate protein by the Hsp100 chaperone ClpA

The bacterial protein ClpA, a member of the Hsp100 chaperone family, forms hexameric rings that bind to the free ends of the double-ring serine protease ClpP (refs 1, 2). ClpA directs the ATP-dependent degradation of substrate proteins bearing specific sequences, much as the 19S ATPase ‘cap’ of eukaryotic proteasomes functions in the degradation of ubiquitinated proteins. In isolation, ClpA and its relative ClpX can mediate the disassembly of oligomeric proteins; another similar eukaryotic protein, Hsp104, can dissociate low-order aggregates. ClpA has been proposed to destabilize protein structure, allowing passage of proteolysis substrates through a central channel into the ClpP proteolytic cylinder. Here we test the action of ClpA on a stable monomeric protein, the green fluorescent protein GFP, onto which has been added an 11-amino-acid carboxy-terminal recognition peptide, which is responsible for recruiting truncated proteins to ClpAP for degradation. Fluorescence studies both with and without a ‘trap’ version of the chaperonin GroEL, which binds non-native forms of GFP, and hydrogen-exchange experiments directly demonstrate that ClpA can unfold stable, native proteins in the presence of ATP.

Protein-induced photophysical changes to the amyloid indicator dye thioflavin T

The small molecule thioflavin T (ThT) is a defining probe for the identification and mechanistic study of amyloid fiber formation. As such, ThT is fundamental to investigations of serious diseases such as Alzheimer’s disease, Parkinson disease, and type II diabetes. For each disease, a different protein undergoes conformational conversion to a β-sheet rich fiber. The fluorescence of ThT exhibits an increase in quantum yield upon binding these fibers. Despite its widespread use, the structural basis for binding specificity and for the changes to the photophysical properties of ThT remain poorly understood. Here, we report the co-crystal structures of ThT with two alternative states of β-2 microglobulin (β2m); one monomeric, the other an amyloid-like oligomer. In the latter, the dye intercalates between β-sheets orthogonal to the β-strands. Importantly, the fluorophore is bound in such a manner that a photophysically relevant torsion is limited to a range of angles generally associated with low, not high, quantum yield. Quantum mechanical assessment of the fluorophore shows the electronic distribution to be strongly stabilized by aromatic interactions with the protein. Monomeric β2m gives little increase in ThT fluorescence despite showing three fluorophores, at two binding sites, in configurations generally associated with high quantum yield. Our efforts fundamentally extend existing understanding about the origins of amyloid-induced photophysical changes. Specifically, the β-sheet interface that characterizes amyloid acts both sterically and electronically to stabilize the fluorophore’s ground state electronic distribution. By preventing the fluorophore from adopting its preferred excited state configuration, nonradiative relaxation pathways are minimized and quantum yield is increased.

The Interplay of Catalysis and Toxicity by Amyloid Intermediates on Lipid Bilayers: Insights from Type II Diabetes

The dynamics, energies, and structures governing protein folding are critical to biological function. Amyloidoses are a class of disease defined, in part, by the misfolding and aggregation of functional protein precursors into fibrillar states. Amyloid fibers contribute to the pathology of many diseases, including type II diabetes, Alzheimers, and Parkinsons. In these disorders, amyloid fibers are present in affected tissues. However, it has become clear that intermediate states, rather than mature fibers, represent the cytotoxic species. In this review, we focus particularly on lipid bilayer-bound intermediates. Remarkably, the precursors of these fibers are intrinsically disordered, and yet catalysis of beta-sheet formation appears to be mediated by the stabilization of alpha-helical states. On the lipid bilayer, these intermediate species have been implicated as cytotoxic through elimination of ionic homeostasis. Recent advances are enabling insights at a molecular level that promise to provide meaningful targets for the development of therapeutics.

A native to amyloidogenic transition regulated by a backbone trigger.

Many polypeptides can self-associate into linear, aggregated assemblies termed amyloid fibers. High-resolution structural insights into the mechanism of fibrillogenesis are elusive owing to the transient and mixed oligomeric nature of assembly intermediates. Here, we report the conformational changes that initiate fiber formation by β-2-microglobulin (β2m) in dialysis-related amyloidosis. Access of β2m to amyloidogenic conformations is catalyzed by selective binding of divalent cations. The chemical basis of this process was determined to be backbone isomerization of a conserved proline. On the basis of this finding, we designed a β2m variant that closely adopts this intermediate state. The variant has kinetic, thermodynamic and catalytic properties consistent with its being a fibrillogenic intermediate of wild-type β2m. Furthermore, it is stable and folded, enabling us to unambiguously determine the initiating conformational changes for amyloid assembly at atomic resolution.

Direct detection of transient α‐helical states in islet amyloid polypeptide

The protein islet amyloid polypeptide (IAPP) is a glucose metabolism associated hormone cosecreted with insulin by the β‐cells of the pancreas. In humans with type 2 diabetes, IAPP deposits as amyloid fibers. The assembly intermediates of this process are associated with β‐cell death. Here, we examine the rat IAPP sequence variant under physiological solution conditions. Rat IAPP is mechanistically informative for fibrillogenesis, as it samples intermediate‐like states but does not progress to form amyloid. A central challenge was the development of a bacterial expression system to generate isotopically labeled IAPP without terminal tags, but which does include a eukaryotic post‐translational modification. While optical spectroscopy shows IAPP to be natively unfolded, NMR chemical shifts of backbone and β‐carbon resonances reveal the sampling of α‐helical states across a continuous stretch comprising ∼40% of the protein. In addition, the manifestation of nonrandom coil chemical shifts is confirmed by the relative insensitivity of the amide proton chemical shifts to alterations in temperature. Intriguingly, the residues displaying helical propensity are conserved with the human sequence, suggesting a functional role for this conformational bias. The inability of rat IAPP to self assemble can be ascribed, in part, to several slowly exchanging conformations evident as multiple chemical shift assignments in the immediate vicinity of three proline residues residing outside of this helical region.

Fiber-dependent amyloid formation as catalysis of an existing reaction pathway

A central component of a number of degenerative diseases is the deposition of protein as amyloid fibers. Self-assembly of amyloid occurs by a nucleation-dependent mechanism that gives rise to a characteristic sigmoidal reaction profile. The abruptness of this transition is a variable characteristic of different proteins with implications to both chemical mechanism and the aggressiveness of disease. Because nucleation is defined as the rate-limiting step, we have sought to determine the nature of this step for a model system derived from islet amyloid polypeptide. We show that nucleation occurs by two pathways: a fiber-independent (primary) pathway and a fiber-dependent (secondary) pathway. We first show that the balance between primary and secondary contributions can be manipulated by an external interface. Specifically, in the presence of this interface, the primary mechanism dominates, whereas in its absence, the secondary mechanism dominates. Intriguingly, we determine that both the reaction order and the enthalpy of activation of the two nucleation processes are identical. We interrogate this coincidence by global analysis using a simplified model generally applicable to protein polymerization. A physically reasonable set of parameters can be found to satisfy the coincidence. We conclude that primary and secondary nucleation need not represent different processes for amyloid formation. Rather, they are alternative manifestations of the same, surface-catalyzed nucleation event.

Helix stabilization precedes aqueous and bilayer catalyzed fiber formation in islet amyloid polypeptide

Islet amyloid polypeptide (IAPP) is an unstructured polypeptide hormone that is cosecreted with insulin. In patients with type 2 diabetes, IAPP undergoes a transition from its natively disordered state to a highly ordered, all-beta-strand amyloid fiber. Although predominantly disordered, IAPP transiently samples alpha-helical structure in solution. IAPP adopts a fully helical structure when bound to membrane surfaces in a process associated with catalysis of amyloid formation. Here, we use spectroscopic techniques to study the structure of full-length, monomeric IAPP under amyloidogenic conditions. We observe that the residues with helical propensity in solution (1-22) also form the membrane-associated helix. Additionally, reduction of the N-terminal disulfide bond (Cys2-Cys7) decreases the extent of helix formed throughout this region. Through manipulation of sample conditions to increase or decrease the amount of helix, we show that the degree of helix formed affects the rate of amyloid assembly. Formation of helical structure is directly correlated with enhanced amyloid formation both on the membrane surface and in solution. These observations support suggested mechanisms in which parallel helix associations bring together regions of the peptide that could nucleate beta-strand structure. Remarkably, stabilization of non-amyloid structure appears to be a key intermediate in assembly of IAPP amyloid.

Islet amyloid polypeptide demonstrates a persistent capacity to disrupt membrane integrity

Amyloid fiber formation is correlated with pathology in many diseases, including Alzheimer’s, Parkinson’s, and type II diabetes. Although β-sheet–rich fibrillar protein deposits define this class of disorder, increasing evidence points toward small oligomeric species as being responsible for cell dysfunction and death. The molecular mechanism by which this occurs is unknown, but likely involves the interaction of these species with biological membranes, with a subsequent loss of integrity. Here, we investigate islet amyloid polypeptide, which is implicated in the loss of insulin-secreting cells in type II diabetics. We report the discovery of oligomeric species that arise through stochastic nucleation on membranes and result in disruption of the lipid bilayer. These species are stable, result in all-or-none leakage, and represent a definable protein/lipid phase that equilibrates over time. We characterize the reaction pathway of assembly through the use of an experimental design that includes both ensemble and single-particle evaluations. Complexity in the reaction pathway could not be satisfied using a two-state description of membrane-bound monomer and oligomeric species. We therefore put forward a three-state kinetic framework, one of which we conjecture represents a non-amyloid, non-β-sheet intermediate previously shown to be a candidate therapeutic target.

Synthetic α-Helix Mimetics as Agonists and Antagonists of Islet Amyloid Polypeptide Aggregation

The development of small molecules that can modulate the damaging effects of protein aggregation processes remains a high priority goal in contemporary medicinal chemistry.[1] An important class of these aggregates, called amyloids, has been implicated in numerous degenerative diseases including Alzheimer’s, type II diabetes, senile systemic amyloidosis (SSA), prion diseases and rheumatoid arthritis. The attribute shared by these symptomatically unrelated diseases is that a normally soluble protein undergoes a conformational change resulting in self-assembly into cytotoxic forms culminating in a β-sheet rich fibrillar structure. Islet amyloid polypeptide (IAPP), or amylin, is one such protein that has been implicated in amyloidogenesis in type II diabetes.[2] IAPP is cosecreted with insulin by the β-cells of the islets of Langerhans and an aggregated form of IAPP is believed to play a role in β-cell toxicity in the pathology of type II diabetes.[3]

A regulatable switch mediates self-association in an immunoglobulin fold

β-2 microglobulin (β2m) is a globular protein that self-associates into fibrillar amyloid deposits in patients undergoing hemodialysis therapy. Formation of these β-sheet–rich assemblies is a fundamental property of polypeptides that can be triggered by diverse conditions. For β2m, oligomerization into pre-amyloidogenic states occurs in specific response to coordination by Cu2+. Here we report the basis for this self-association at atomic resolution. Metal is not a direct participant in the molecular interface. Rather, binding results in distal alterations enabling the formation of two new surfaces. These interact to form a closed hexameric species. The origins of this include isomerization of a buried and conserved cis-proline previously implicated in the β2m aggregation pathway. The consequences of this isomerization are evident and reveal a molecular basis for the conversion of this robust monomeric protein into an amyloid-competent state.