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Amyloids are fibrillar protein superstructures that are commonly associated with diseases in humans and with physiological functions in various organisms. The precise mechanisms of amyloid formation remain to be elucidated. Surprisingly, we discovered that a bacterial Escherichia coli chaperone-like ATPase, regulatory ATPase variant A (RavA), and specifically the LARA domain in RavA, forms amyloids under acidic conditions at elevated temperatures. RavA is involved in modulating the proper assembly of membrane respiratory complexes. LARA contains an N-terminal loop region followed by a β-sandwich-like folded core. Several approaches, including nuclear magnetic resonance spectroscopy and molecular dynamics simulations, were used to determine the mechanism by which LARA switches to an amyloid state. These studies revealed that the folded core of LARA is amyloidogenic and is protected by its N-terminal loop. At low pH and high temperatures, the interaction of the N-terminal loop with the folded core is disrupted, leading to amyloid formation.

Membrane encapsulation is frequently used by the cell to sequester biomolecules and compartmentalize their function. Cells also concentrate molecules into phase-separated protein or protein/nucleic acid "membraneless organelles" that regulate a host of biochemical processes. Here, we use solution NMR spectroscopy to study phase-separated droplets formed from the intrinsically disordered N-terminal 236 residues of the germ-granule protein Ddx4. We show that the protein within the concentrated phase of phase-separated Ddx4, [Formula: see text], diffuses as a particle of 600-nm hydrodynamic radius dissolved in water. However, NMR spectra reveal sharp resonances with chemical shifts showing [Formula: see text] to be intrinsically disordered. Spin relaxation measurements indicate that the backbone amides of [Formula: see text] have significant mobility, explaining why high-resolution spectra are observed, but motion is reduced compared with an equivalently concentrated nonphase-separating control. Observation of a network of interchain interactions, as established by NOE spectroscopy, shows the importance of Phe and Arg interactions in driving the phase separation of Ddx4, while the salt dependence of both low- and high-concentration regions of phase diagrams establishes an important role for electrostatic interactions. The diffusion of a series of small probes and the compact but disordered 4E binding protein 2 (4E-BP2) protein in [Formula: see text] are explained by an excluded volume effect, similar to that found for globular protein solvents. No changes in structural propensities of 4E-BP2 dissolved in [Formula: see text] are observed, while changes to DNA and RNA molecules have been reported, highlighting the diverse roles that proteinaceous solvents play in dictating the properties of dissolved solutes.

ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB-substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD-substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD-substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD-substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.

Allosteric transmission of information between distant sites in biological macromolecules often involves collective transitions between active and inactive conformations. Nuclear magnetic resonance (NMR) spectroscopy can yield detailed information on these dynamics. In particular, relaxation dispersion techniques provide structural, dynamic, and mechanistic information on conformational transitions occurring on the millisecond to microsecond timescales. In this review, we provide an overview of the theory and analysis of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments and briefly describe their application to the study of allosteric dynamics in the homeodomain from the PBX transcription factor (PBX-HD). CPMG NMR data show that local folding (helix/coil) transitions in one part of PBX-HD help to communicate information between two distant binding sites. Furthermore, the combination of CPMG and other spin relaxation data show that this region can also undergo local misfolding, reminiscent of conformational ensemble models of allostery.

Cells chemically isolate molecules in compartments to both facilitate and regulate their interactions. In addition to membrane-encapsulated compartments, cells can form proteinaceous and membraneless organelles, including nucleoli, Cajal and PML bodies, and stress granules. The principles that determine when and why these structures form have remained elusive. Here, we demonstrate that the disordered tails of Ddx4, a primary constituent of nuage or germ granules, form phase-separated organelles both in live cells and in vitro. These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing. Sequence determinants are used to identify proteins found in both membraneless organelles and cell adhesion. Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA. We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles.

In yeast, the cyclin-dependent kinase inhibitor Sic1 is a disordered protein which interacts with a single site on its acceptor Cdc4 only upon multi-phosphorylation of its dispersed sites. To gain insight into the multi-phosphorylation dependence in Sic1-Cdc4 interaction, the conformational properties of the disordered Sic1 N-terminal targeting region were studied using single-molecule fluorescence spectroscopy.At least two Sic1 conformational populations with different sensitivities to charge screening by non-denaturing salts and ionic denaturants were identified. As described by the polyelectrolyte theory, the chain dimensions decrease monotonically with salt concentration and roll over at high denaturant, although a scaling factor of 1.2 indicates that Sic1 is not accurately described as a random chain. Fluorescence correlation analysis shows that Sic1 structure fluctuations occur on fast (10-100 ns) and slow (10-100 ms) ranges, with the fast phase being absent at low salt. Our data provides direct evidence that intrachain charge repulsions are significant for the conformational landscape of Sic1, and support the role of electrostatics in determining the size and shape of intrinsically-disordered proteins.View Large Image | View Hi-Res Image | Download PowerPoint Slide

For intrinsically disordered proteins (IDPs) containing a high density of charged residues, a Gaussian chain is an inadequate description of the protein's shape. One such IDP is Sic1, a highly positively charged IDP in the budding yeast Saccharomyces Cerevisiae which prevents the cell cycle from entering the S-phase from the G1-phase. Sic1's binding affinity for Cdc4 is highly phosphorylation state dependent, although the corresponding physical basis is not fully understood. NMR data supports the presence of a dynamic complex of Sic1:Cdc4 and a poly-electrostatic model has been proposed by Forman-Kay and coworkers.We studied the Sic1 N-terminal targeting region (1-90) to better understand the role of intrachain electrostatics, and to compare with the structural ensembles calculated from NMR and SAXS data. Sic1 is characterized using time-resolved fluorescence anisotropy (TRFA), which is sensitive to rotational and conformational dynamics. Sic1 exists in a dynamic ensemble of conformations and ensemble-averaged experiments have limitations in identifying and characterizing static and/or dynamic inhomogeneity in the motional dynamics. Therefore, we also performed burst spectroscopy and single-molecule TRFA on freely diffusing Sic1. Shape and flexibility were probed by varying the degree of intrachain repulsion screening through adjusting salt concentrations and described within a polymer physics framework, the polyelectrolyte model. To investigate the relative contributions of “global rotation” and “conformational flexibility”, Sic1 was labelled at three different sites. Sic1 is found to be well modelled as a prolate ellipsoid with internal flexibility.The single-molecule derived TRFA distribution data may be valuable as a constraint incorporated in future conformational ensemble calculations, complementary to SAXS and NMR data. Additionally, these measurements raise questions about the accuracy of highly charged IDP's radii of gyration when calculated from sm-FRET derived end-to-end distances, which often assume a Gaussian chain model for the end-to-end distance probability distribution.

Intrinsically disordered proteins and regions (IDPs/IDRs), which do not have stable secondary and tertiary structure, are capable of adopting different structural states. Many IDPs/IDRs populate conformationally heterogeneous monomeric states or engage in discrete interactions with other proteins, leading to folding upon binding or retaining significant disorder in the bound state. Others are involved in large-scale association having different degrees of order, from more defined fibers, to variably networked gels and to disordered liquid demixed states or droplets. These latter have been suggested to provide the physical basis for cellular membrane-less organelles such as the nucleolus.We have studied the N-terminal disordered region of Ddx4, an RNA DEAD-box helicase that is essential for formation of a class of membrane-less organelles termed nuage or germ granules functioning in spermatogenesis. When expressed in HeLa cells, the protein forms spherical, micron-sized, liquid-like cellular organelles. In vitro, it phase separates to form droplets with similar morphological and dynamic properties to the organelles observed in cells. Phase separation is sensitive to salt, pointing to the importance of electrostatic interactions. The sequence features of the disordered N-terminus of Ddx4 that underlie phase separation include clustering of charged residues into blocks of net positive and negative charge, with over-representation of FG/GF pairs and RG/GR pairs within the positive blocks. Perturbations of these properties disrupt phase separation, pointing to multi-valent cation-pi interactions playing an important role. The transient sampling of multi-valent interactions in self-association of Ddx4 extends previous observations of dynamic multi-valent interactions in discrete complexes of IDPs/IDRs, such as for Sic1:Cdc4 binding. The insights obtained from these and ongoing biophysical studies of Ddx4 will be valuable for developing a general understanding of the biogenesis and disassembly of membrane-less cellular organelles.

Cystic fibrosis-causing mutations in CFTR can lead to protein misprocessing or channel gating defects, perturbing the ion and fluid flow across epithelial surfaces. CFTR transmembrane helices extend into the cytoplasm and interface with two nucleotide-binding domains, NBD1 and NBD2, via short 'coupling helices' (CLs). Channel gating is regulated by ATP binding and hydrolysis in the NBD1/NBD2 dimer and by phosphorylation of the NBD1 regulatory insert (RI) and R region immediately C-terminal of NBD1. We have used NMR spectroscopy to probe changes in conformational populations caused by ligand binding or mutation, revealing an allosteric network linking the CL4 binding site to regions near the RI and the R region. Conformational exchange occurs on multiple timescales in the NBD1 αRI construct, including μs-ms exchange and elevated flexibility in loops and C-terminal helices H8 and H9. Q637R, a mutation between H8 and H9 near to where the R region begins, changes dynamics in the RI deletion site and the CL4 binding site. VX-809, a drug that partially rescues both folding and gating defects, binds to the β-strands beneath H8/H9 of full-length NBD1, destabilizing these helices and perturbing some chemical shifts in the CL4 binding site. Titration of a CL4 peptide results in remote changes to H8/H9 and correlated chemical shifts observed at the RI. An allosteric network coupling the CL4 binding site to regions near both of the phospho-regulatory sites may contribute to the folding and gating defects that arise from mutations at the CL4:NBD1 interface, including those of the most common CF-causing mutation, F508del. Coupling of remote NBD1 sites by the observed allosteric network suggests that distinct inputs are integrated in CFTR processing and function and implies that correlation of NBD-ICL interactions might be achieved through targeting of these allosterically-linked sites.

We have characterized the backbone dynamics of NADH oxidase from Thermus thermophilus (NOX) using a recently-developed suite of NMR experiments designed to isolate exchange broadening, together with (15)N R (1), R (1ρ ), and {(1)H}-(15)N steady-state NOE relaxation measurements performed at 11.7 and 18.8 T. NOX is a 54 kDa homodimeric enzyme that belongs to a family of structurally homologous flavin reductases and nitroreductases with many potential biotechnology applications. Prior studies have suggested that flexibility is involved in the catalytic mechanism of the enzyme. The active site residue W47 was previously identified as being particularly important, as its level of solvent exposure correlates with enzyme activity, and it was observed to undergo "gating" motions in computer simulations. The NMR data are consistent with these findings. Signals from W47 are dynamically broadened beyond detection and several other residues in the active site have significant R ( ex ) contributions to transverse relaxation rates. In addition, the backbone of S193, whose side chain hydroxyl proton hydrogen bonds directly with the FMN cofactor, exhibits extensive mobility on the ns-ps timescale. We hypothesize that these motions may facilitate structural rearrangements of the active site that allow NOX to accept both FMN and FAD as cofactors.

The PBX1 homeodomain (PBX-HD) cooperatively binds DNA with Hox transcription factors and helps to regulate gene expression during vertebrate development. Allostery plays an important role in these interactions. DNA binding on one surface of PBX-HD enhances interactions with Hox proteins at a different interface. In addition, DNA binding causes a 15-residue extension at the C-terminus of PBX-HD to undergo a disorder-to-helix transition, although this region does not directly contact the DNA. Deletion of the C-terminal extension reduces both the DNA affinity of PBX-HD and the cooperativity of forming the DNA/Hox/PBX-HD ternary complex. To better understand the mechanism underlying these allosteric interactions, we used NMR relaxation dispersion dynamics experiments to characterize millisecond-timescale motions in PBX-HD over a range of temperatures. The data show that the C-terminal extension folds to form a fourth α-helix to a level of 5–10%, even in the absence of binding partners. This suggests that PBX-HD transiently preorganizes prior to binding DNA, reminiscent of the “conformational selection” model of molecular recognition. Folding of the C-terminal extension in the unbound protein is accompanied by structural rearrangements in both the DNA binding site and the Hox binding site, suggesting a possible role for these dynamics in the allosteric mechanism of PBX-HD.

Some marginally stable proteins undergo microsecond time scale folding reactions that involve significant populations of partly ordered forms, making it difficult to discern individual steps in their folding pathways. It has been suggested that many of these proteins fold non-cooperatively, with no significant barriers to separate the energy landscape into distinct thermodynamic states. Here we present an approach for studying the cooperativity of rapid protein folding with a combination of differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) relaxation dispersion experiments, and an analysis of the temperature dependence of amide (1)H and (15)N chemical shifts. We applied this method to the PBX homeodomain (PBX-HD), which folds on the microsecond time scale and produces a broad DSC thermogram with an elevated and steeply sloping native-state heat capacity baseline, making it a candidate for barrierless folding. However, by globally fitting the NMR thermal melt and DSC data, and by comparing these results to those obtained from the NMR relaxation dispersion experiments, we show that the native form of the protein undergoes two-state exchange with a small population of the thermally denatured form, well below the melting temperature. This result directly demonstrates the coexistence of distinct folded and unfolded forms and firmly establishes that folding of PBX-HD is cooperative. Further, we see evidence of large-scale structural and dynamical changes within the native state by NMR, which helps to explain the broad and shallow DSC profile. This study illustrates the potential of combining calorimetry with NMR dynamics experiments to dissect mechanisms of protein folding.

Cystic fibrosis is caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator), leading to folding and processing defects and to chloride channel gating misfunction. CFTR is regulated by ATP binding to its cytoplasmic nucleotide-binding domains, NBD1 and NBD2, and by phosphorylation of the NBD1 regulatory insert (RI) and the regulatory extension (RE)/R region. These regulatory effects are transmitted to the rest of the channel via NBD interactions with intracellular domain coupling helices (CL), particularly CL4. Using a sensitive method for detecting inter-residue correlations between chemical shift changes in NMR spectra, an allosteric network was revealed within NBD1, with a construct lacking RI. The CL4-binding site couples to the RI-deletion site and the C-terminal residues of NBD1 that precede the R region in full-length CFTR. Titration of CL4 peptide into NBD1 perturbs the conformational ensemble in these sites with similar titration patterns observed in F508del, the major CF-causing mutant, and in suppressor mutants F494N, V510D and Q637R NBD1, as well as in a CL4-NBD1 fusion construct. Reciprocally, the C-terminal mutation, Q637R, perturbs dynamics in these three sites. This allosteric network suggests a mechanism synthesizing diverse regulatory NBD1 interactions and provides biophysical evidence for the allosteric coupling required for CFTR function.