Visualization of Sparsely-populated Lower-order Oligomeric States of Human Mitochondrial Hsp60 by Cryo-electron Microscopy.

Human mitochondrial Hsp60 (mtHsp60) is a class I chaperonin, 51% identical in sequence to the prototypical E. coli chaperonin GroEL. mtHsp60 maintains the proteome within the mitochondrion and is associated with various neurodegenerative diseases and cancers. The oligomeric assembly of mtHsp60 into heptameric ring structures that enclose a folding chamber only occurs upon addition of ATP and is significantly more labile than that of GroEL, where the only oligomeric species is a tetradecamer. The lability of the mtHsp60 heptamer provides an opportunity to detect and visualize lower-order oligomeric states that may represent intermediates along the assembly/disassembly pathway. Using cryo-electron microscopy we show that, in addition to the fully-formed heptamer and an "inverted" tetradecamer in which the two heptamers associate via their apical domains, thereby blocking protein substrate access, well-defined lower-order oligomeric species, populated at less than 6% of the total particles, are observed. Specifically, we observe open trimers, tetramers, pentamers and hexamers (comprising ∼4% of the total particles) with rigid body rotations from one subunit to the next within ∼1.5-3.5° of that for the heptamer, indicating that these may lie directly on the assembly/disassembly pathway. We also observe a closed-ring hexamer (∼2% of the particles) which may represent an off-pathway species in the assembly/disassembly process in so far that conversion to the mature heptamer would require the closed-ring hexamer to open to accept an additional subunit. Lastly, we observe several classes of tetramers where additional subunits characterized by fuzzy electron density are caught in the act of oligomer extension.

A weakened interface in the P182L variant of HSP27 associated with severe Charcot-Marie-Tooth neuropathy causes aberrant binding to interacting proteins.

HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot-Marie-Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C-terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient-derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V-containing proteins. We identified multiple IxI/V-bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V-binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein-protein interactions.

Probing the Interaction of Huntingtin Exon-1 Polypeptides with the Chaperonin Nanomachine GroEL.

Huntington's disease arises from polyQ expansion within the exon-1 region of huntingtin (httex1 ), resulting in an aggregation-prone protein that accumulates in neuronal inclusion bodies. We investigate the interaction of various httex1 constructs with the bacterial analog (GroEL) of the human chaperonin Hsp60. Using fluorescence spectroscopy and electron and atomic force microscopy, we show that GroEL inhibits fibril formation. The binding kinetics of httex1 constructs with intact GroEL and a mini-chaperone comprising the apical domain is characterized by relaxation-based NMR measurements. The lifetimes of the complexes range from 100 to 400 µs with equilibrium dissociation constants (KD ) of ∼1-2 mM. The binding interface is formed by the N-terminal amphiphilic region of httex1 (which adopts a partially helical conformation) and the H and I helices of the GroEL apical domain. Sequestration of monomeric httex1 by GroEL likely increases the critical concentration required for fibrillization.

Probing the mechanism of inhibition of amyloid-ß(1-42)-induced neurotoxicity by the chaperonin GroEL.

The human chaperonin Hsp60 is thought to play a role in the progression of Alzheimer's disease by mitigating against intracellular ß-amyloid stress. Here, we show that the bacterial homolog GroEL (51% sequence identity) reduces the neurotoxic effects of amyloid-ß(1-42) (Aß42) on human neural stem cell-derived neuronal cultures. To understand the mechanism of GroEL-mediated abrogation of neurotoxicity, we studied the interaction of Aß42 with GroEL using a variety of biophysical techniques. Aß42 binds to GroEL as a monomer with a lifetime of ∼1 ms, as determined from global analysis of multiple relaxation-based NMR experiments. Dynamic light scattering demonstrates that GroEL dissolves small amounts of high-molecular-weight polydisperse aggregates present in fresh soluble Aß42 preparations. The residue-specific transverse relaxation rate profile for GroEL-bound Aß42 reveals the presence of three anchor-binding regions (residues 16-21, 31-34, and 40-41) located within the hydrophobic GroEL-consensus binding sequences. Single-molecule FRET analysis of Aß42 binding to GroEL results in no significant change in the FRET efficiency of a doubly labeled Aß42 construct, indicating that Aß42 samples a random coil ensemble when bound to GroEL. Finally, GroEL substantially slows down the disappearance of NMR visible Aß42 species and the appearance of Aß42 protofibrils and fibrils as monitored by electron and atomic force microscopies. The latter observations correlate with the effect of GroEL on the time course of Aß42-induced neurotoxicity. These data provide a physical basis for understanding how Hsp60 may serve to slow down the progression of Alzheimer's disease.

Extensive Sampling of the Cavity of the GroEL Nanomachine by Protein Substrates Probed by Paramagnetic Relaxation Enhancement.

The chaperonin GroEL is a 800 kDa nanomachine comprising two heptameric rings, each of which encloses a large cavity or folding chamber. The GroEL cycle involves ATP-dependent capping of the cavity by the cochaperone GroES to create a nanocage in which a single protein molecule can fold. We investigate how protein substrates sample the cavity prior to encapsulation by GroES using paramagnetic relaxation enhancement to detect transient, sparsely populated interactions between apo GroEL, paramagnetically labeled at several sites within the cavity, and three variants of an SH3 protein domain (the fully native wild type, a triple mutant that exchanges between a folded state and an excited folding intermediate, and a stable folding intermediate mimetic). We show that the substrate not only interacts with the hydrophobic inner rim of GroEL at the mouth of the cavity but also penetrates deep within the cavity, transiently contacting the disordered C-terminal tail, and, in the case of the folding intermediate mimetic, the base as well. Transient interactions with the C-terminal tail may facilitate substrate capture and retention prior to encapsulation.

Disassembly/reassembly strategy for the production of highly pure GroEL, a tetradecameric supramolecular machine, suitable for quantitative NMR, EPR and mutational studies.

GroEL, a prototypical member of the chaperonin class of chaperones, is a large supramocular machine that assists protein folding and plays an important role in proteostasis. GroEL comprises two heptameric rings, each of which encloses a large cavity that provides a folding chamber for protein substrates. Many questions remain regarding the mechanistic details of GroEL facilitated protein folding. Thus, data at atomic resolution of the type provided by NMR and EPR are invaluable. Such studies often require complete deuteration of GroEL, uniform or residue specific 13C and 15N isotope labeling, and the introduction of selective cysteine mutations for site-specific spin labeling. In addition, high purity GroEL is essential for detailed studies of substrate-GroEL interactions as quantitative interpretation is impossible if the cavities are already occupied and blocked by other protein substrates present in the bacterial expression system. Here we present a new purification protocol designed to provide highly pure GroEL devoid of non-specific protein substrate contamination.

Binding of Polythiophenes to Amyloids: Structural Mapping of the Pharmacophore.

Luminescent conjugated polythiophenes bind to amyloid proteins with high affinity. Their fluorescence properties, which are modulated by the detailed conformation in the bound state, are highly sensitive to structural features of the amyloid. Polythiophenes therefore represent diagnostic markers for the detection and differentiation of pathological amyloid aggregates. We clarify the binding site and mode of two different polythiophenes to fibrils of the prion domain of the HET-s protein by solid-state NMR and correlate these findings with their fluorescence properties. We demonstrate how amyloid dyes recognize distinct binding sites with specific topological features. Regularly spaced surface charge patterns and well-accessible grooves on the fibril surface define the pharmacophore of the amyloid, which in turn determines the binding mode and fluorescence wavelength of the polythiophene.

Chaperonin GroEL accelerates protofibril formation and decorates fibrils of the Het-s prion protein.

We have studied the interaction of the prototypical chaperonin GroEL with the prion domain of the Het-s protein using solution and solid-state NMR, electron and atomic force microscopies, and EPR. While GroEL accelerates Het-s protofibril formation by several orders of magnitude, the rate of appearance of fibrils is reduced. GroEL remains bound to Het-s throughout the aggregation process and densely decorates the fibrils at a regular spacing of ∼200 Å. GroEL binds to the Het-s fibrils via its apical domain located at the top of the large open ring. Thus, apo GroEL and bullet-shaped GroEL/GroES complexes in which only a single ring is capped by GroES interact with the Het-s fibrils; no evidence is seen for any interaction with football-shaped GroEL/GroES complexes in which both rings are capped by GroES. EPR spectroscopy shows that rotational motion of a nitroxide spin label, placed at the N-terminal end of the first ß-strand of Het-s fibrils, is significantly reduced in both Het-s/GroEL aggregates and Het-s fibrils, but virtually completely eliminated in Het-s/GroEL fibrils, suggesting that in the latter, GroEL may come into close proximity to the nitroxide label. Solid-state NMR measurements indicate that GroEL binds to the mobile regions of the Het-s fibril comprising the N-terminal tail and a loop connecting ß-strands 4 and 5, consistent with interactions involving GroEL binding consensus sequences located therein.

Fast NMR-Based Determination of the 3D Structure of the Binding Site of Protein-Ligand Complexes with Weak Affinity Binders.

In early drug discovery approaches, screening hits are often weak affinity binders that are difficult to characterize in structural detail, particularly towards obtaining the 3D structure of protein-ligand complexes at atomic resolution. NMR is the outstanding technique to tackle such problems, yet suffers from a tedious structure calculation process. NMR2 was recently developed to alleviate the laborious element of routine NMR structure calculation procedures and provides the structural information at protein-ligand interaction sites orders of magnitude faster than standard procedures. The NMR2 method was extended to weak binders and applied to the oncoproteins HDM2 and MDMX. The structure of the MDMX-SJ212 complex is reported with a Kd of approximately 0.7 µm; the complex structure of HDM2 with the mm affinity ligand #845 exhibits a new scaffold.

Long Distance Measurements up to 160†Šin the GroEL Tetradecamer Using Q-Band DEER EPR Spectroscopy.

Current distance measurements between spin-labels on multimeric protonated proteins using double electron-electron resonance (DEER) EPR spectroscopy are generally limited to the 15-60 Šrange. Here we show how DEER experiments can be extended to dipolar evolution times of ca. 80 µs, permitting distances up to 170 Što be accessed in multimeric proteins. The method relies on sparse spin-labeling, supplemented by deuteration of protein and solvent, to minimize the deleterious impact of multispin effects and substantially increase the apparent spin-label phase memory relaxation time, complemented by high sensitivity afforded by measurements at Q-band. We demonstrate the approach using the tetradecameric molecular machine GroEL as an example. Two engineered surface-exposed mutants, R268C and E315C, are used to measure pairwise distance distributions with mean values ranging from 20 to 100 Šand from 30 to 160 Å, respectively, both within and between the two heptameric rings of GroEL. The measured distance distributions are consistent with the known crystal structure of apo GroEL. The methodology presented here should significantly expand the use of DEER for the structural characterization of conformational changes in higher order oligomers.