Tail-Mediated Collapse of HMGB1 Is Dynamic and Occurs via Differential Binding of the Acidic Tail to the A and B Domains

The architectural DNA-binding protein HMGB1 consists of two tandem HMG-box domains joined by a basic linker to a C-terminal acidic tail, which negatively regulates HMGB1-DNA interactions by binding intramolecularly to the DNA-binding faces of both basic HMG boxes. Here we demonstrate, using NMR chemical-shift mapping at different salt concentrations, that the tail has a higher affinity for the B box and that A box-tail interactions are preferentially disrupted. Previously, we proposed a model in which the boxes are brought together in a collapsed, tail-mediated assembly, which is in dynamic equilibrium with a more extended form. Small-angle X-ray scattering data are consistent with such a dynamic equilibrium between collapsed and extended structures and are best represented by an ensemble. The ensembles contain a significantly higher proportion of collapsed structures when the tail is present. ¹⁵N NMR relaxation measurements show that full-length HMGB1 has a significantly lower rate of rotational diffusion than the tail-less protein, consistent with the loss of independent domain motions in an assembled complex. Mapping studies using the paramagnetic spin label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidin-3-yl)methyl methanethiosulfonate] placed at three locations in the tail confirm our previous findings that the tail binds to both boxes with some degree of specificity. The end of the tail lies further from the body of the protein and is therefore potentially free to interact with other proteins. MTSL labelling at a single site in the A domain (C44) causes detectable relaxation enhancements of B domain residues, suggesting the existence of a “sandwich”-like collapsed structure in which the tail enables the close approach of the basic domains. These intramolecular interactions are presumably important for the dynamic association of HMGB1 with chromatin and provide a mechanism by which protein-protein interactions or posttranslational modifications might regulate the function of the protein at particular sites, or at particular stages in the cell cycle.     

Quaternary dynamics of αB-crystallin as a direct consequence of localised tertiary fluctuations in the C-terminus

The majority of proteins exist in vivo within macromolecular assemblies whose functions are dependent on dynamical processes spanning a wide range of time scales. One such assembly is formed by the molecular chaperone αB-crystallin that exists in a variety of exchanging oligomeric states, centred on a mass of approximately 560 kDa. For many macromolecular assemblies, including αB-crystallin, the inherent dynamics, heterogeneity and high mass contribute to difficulties in quantitative studies. Here, we demonstrate a strategy based on correlating solution-state nuclear magnetic resonance spectroscopy and mass spectrometry data to characterize simultaneously the organization and dynamics of the polydisperse αB-crystallin ensemble. We show that protomeric dimers assemble into oligomers via the binding of extended C-termini, with each monomer donating and receiving one terminus. Moreover, we establish that the C-termini undergo millisecond fluctuations that regulate the interconversion of oligomeric forms. The combined biophysical approach allows construction of an energy profile for a single monomer that completely describes the equilibrium dynamics of the ensemble. It also facilitates an analysis of dynamics spanning the millisecond to hour time scales and secondary to quaternary structural levels, and provides an approach for, obtaining simultaneously detailed structural, thermodynamic and kinetic information on a heterogeneous protein assembly.     

The denatured state of N-PGK is compact and predominantly disordered

The organisation of the structure present in the chemically denatured N-terminal domain of phosphoglycerate kinase (N-PGK) has been determined by paramagnetic relaxation enhancements (PREs) to define the conformational landscape accessible to the domain. Below 2.0 M guanidine hydrochloride (GuHCl), a species of N-PGK (denoted I_b) is detected, distinct from those previously characterised by kinetic experiments [folded (F), kinetic intermediate (I_k) and denatured (D)]. The transition to I_b is never completed at equilibrium, because F predominates below 1.0 M GuHCl. Therefore, the ability of PREs to report on transient or low population species has been exploited to characterise I_b. Five single cysteine variants of N-PGK were labelled with the nitroxide electron spin-label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate] and the denaturant dependences of the relaxation properties of the amide NMR signals between 1.2 and 3.6 M GuHCl were determined. Significant PREs for I_b were obtained, but these were distributed almost uniformly throughout the sequence. Furthermore, the PREs indicate that no specific short tertiary contacts persist. The data indicate a collapsed state with no coherent three-dimensional structure, but with a restricted radius beyond which the protein chain rarely reaches. The NMR characteristics of I_b indicate that it forms from the fully denatured state within 100 μs, and therefore a rapid collapse is the initial stage of folding of N-PGK from its chemically denatured state. By extrapolation, I_b is the predominant form of the denatured state under native conditions, and the non-specifically collapsed structure implies that many non-native contacts and chain reversals form early in protein folding and must be broken prior to attaining the native state topology.     

Distances between the b-subunits in the tether domain of F0F1-ATP synthase from E. coli

The arrangement of the b-subunits in the holo-enzyme F₀F₁-ATP synthase from E. coli is investigated by site-directed mutagenesis spin-label EPR. F₀F₁-ATP synthases couple proton translocation with the synthesis of ATP from ADP and phosphate. The hydrophilic F₁-part and the hydrophobic membrane-integrated F₀-part are connected by a central and a peripheral stalk. The peripheral stalk consists of two b-subunits. Cysteine mutations are introduced in the tether domain of the b-subunit at b-40, b-51, b-53, b-62 or b-64 and labeled with a nitroxide spin label. Conventional (9 GHz), high-field (95 GHz) and pulsed EPR spectroscopy reveal: All residues are in a relatively polar environment, with mobilities consistent with helix sites. The distance between the spin labels at each b-subunit is 2.9 nm in each mutant, revealing a parallel arrangement of the two helices. They can be in-register but separated by a large distance (1.9 nm), or at close contact and displaced along the helix axes by maximally 2.7 nm, which excludes an in-register coiled-coil model suggested previously for the b-subunit. Binding of the non-hydrolysable nucleotide AMPPNP to the spin-labeled enzyme had no significant influence on the distances compared to that in the absence of nucleotides.     

A single residue mutation in Hha preserving structure and binding to H-NS results in loss of H-NS mediated gene repression properties

In this study, we report that a single mutation of cysteine 18 to isoleucine (C18I) in Escherichia coli Hha abolishes the repression of the hemolysin operon observed in the wild-type protein. The phenotype also includes a significant decrease in the growth rate of E. coli cells at low ionic strength. Other substitutions at this position (C18A, C18S) have no observable effects in E. coli growth or hemolysin repression. All mutants are stable and well folded and bind H-NS in vitro with similar affinities suggesting that Cys 18 is not directly involved in H-NS binding but this position is essential for the activity of the H-NS/Hha heterocomplexes in the regulation of gene expression.     

NMR characterization of the interactions between lyso-GM1 aqueous micelles and amyloid β

Gangliosides are targets for a variety of pathologically relevant proteins, including amyloid β (Aβ), an important component implicated in Alzheimer’s disease (AD). To provide a structural basis for this pathogenic interaction associated with AD, we conducted NMR analyses of the Aβ interactions with gangliosides using lyso-GM1 micelles as a model system. Our NMR data revealed that the sugar-lipid interface is primarily perturbed upon binding of Aβ to the micelles, underscoring the importance of the inner part of the ganglioside cluster for accommodating Aβ in comparison with the outer carbohydrate branches that provide microbial toxin- and virus-binding sites.     

Structural characterization of the intrinsically unfolded protein beta-synuclein, a natural negative regulator of alpha-synuclein aggregation

The synuclein family of intrinsically unfolded proteins is composed of three highly homologous members, alpha-synuclein (alphaS), beta-synuclein (betaS) and gamma-synuclein (gammaS), which are linked to neurodegenerative disorders and cancer. alphaS has been studied intensively after its identification as the major protein component of amyloid-like deposits in Parkinson's disease and dementia with Lewy bodies. betaS, on the other hand, was found to act as a potent inhibitor of alphaS amyloid formation, and it is proposed as a natural regulator of its neurotoxicity. It is then of particular interest to elucidate the structural and dynamic features of the soluble state of betaS as a first step to understand the molecular basis of its anti-amyloidogenic effect on alphaS. We present here the characterization of natively unstructured betaS by high resolution heteronuclear NMR techniques. A combination of pulse-field gradient, three-dimensional heteronuclear correlation, residual dipolar couplings, paramagnetic relaxation enhancement and backbone relaxation experiments were employed to characterize the ensemble of conformations populated by the protein. The results indicate that betaS adopts extended conformations in its native state, characterized by the lack of the long-range contacts as previously reported for alphaS. Despite the lack of defined secondary structure, we found evidence for transient polyproline II conformations clustered at the C-terminal region. The structuring of the backbone at the C terminus is locally encoded, stabilized by the presence of eight proline residues embedded in a polypeptide stretch rich in hydrophilic and negatively charged amino acids. The structural and functional implications of these findings are analyzed via a thorough comparison with its neurotoxic homolog alphaS.     

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b₆f with the electron transfer proteins plastocyanin and cytochrome c₆. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c₆ from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c₆ were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge–charge interactions pre-orient cytochrome c₆ with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.     

Backbone Dynamics and Global Effects of an Activating Mutation in Minimized Mtu RecA Inteins

Inteins mediate protein splicing, which has found many applications in biotechnology and protein engineering. A single valine-to-leucine mutation (V67L) can globally enhance splicing and related cleavage reactions in minimized Mycobacterium tuberculosis RecA inteins. However, V67L mutation causes little change in crystal structures. To test whether protein dynamics contribute to activity enhancement in the V67L mutation, we have studied the conformations and dynamics of the minimized and engineered intein ΔΔIhh-V67CM and a single V67L mutant, ΔΔIhh-L67CM, by solution NMR. Chemical shift perturbations established that the V67L mutation causes global changes, including changes at the N-terminus and C-terminus of the intein, which are active sites for protein splicing. The single V67L mutation significantly slows hydrogen-exchange rates globally, indicating a shift to more stable conformations and reduction in ensemble distribution. Whereas the V67L mutation causes little change for motions on the picosecond-to-nanosecond timescale, motions on the microsecond-to-millisecond timescale affect a region involving the conserved F-block histidine and C-terminal asparagine, which are residues important for C-terminal cleavage. The V67L mutation is proposed to activate splicing by reducing the ensemble distribution of the intein structure and by modifying the active sites.     

The Role of Prefibrillar Structures in the Assembly of a Peptide Amyloid

The self-assembly of proteins into stable, fibrillar aggregates is a general property of polypeptides most notably associated with degenerative diseases termed amyloidoses. These nano- to micrometer scale structures are formed predominantly of β-sheets that self-assemble by a nucleation-dependent mechanism. The rate-limiting step of assembly involves stabilization of high-energy intermediates in a kinetic step termed nucleation. Determination of the structural characteristics of these high-energy intermediates has been elusive, as its members are the least populated states on the assembly pathway. Using a peptide derived from diabetes-related amyloid, we use electron paramagnetic resonance (EPR) spectroscopy and disulfide crosslinking to show that fibers are composed of parallel, in-register β-sheets. Kinetic studies are then used to infer the structural elements of the pre-nucleation intermediates. Notably, stabilization of this ensemble is shown to depend on the number but not the position of amide side chains within the primary sequence. Additionally, fiber formation is accelerated by constructs that mimic the intra-sheet structure of the fiber. Our data suggest that pre-nucleation intermediates sample intra- β-sheet structure and place bounds on the possible nucleation mechanisms for fiber assembly. Understanding the nucleation of fibrillogenesis is critical so that this process can be prevented in disease and productively controlled by design.     

Structural dynamics of the actomyosin complex probed by a bifunctional spin label that cross-links SH1 and SH2

We have used a bifunctional spin label (BSL) to cross-link Cys⁷⁰⁷ (SH1) and Cys⁶⁹⁷ (SH2) in the catalytic domain of myosin subfragment 1 (S1). BSL induces the same weakened ATPase activity and actin-binding affinity that is observed when SH1 and SH2 are cross-linked with pPDM, which traps an analog of the post-hydrolysis state A·M·ADP·P. Electron paramagnetic resonance showed that BSL reports the global orientation and dynamics of S1. When bound to actin in oriented muscle fibers in the absence of ATP, BSL-S1 showed almost complete orientational disorder, as reported previously for the weakly bound A·M·ADP. In contrast, helical order is observed for the strongly bound state A·M. Saturation transfer electron paramagnetic resonance showed that the disorder of cross-linked S1 on actin is nearly static on the microsecond timescale, at least 30 times slower than that of A·M·ADP. We conclude that cross-linked S1 exhibits rotational disorder comparable to that of A·M·ADP, slow rotational mobility comparable to that of A·M, and intermediate actin affinity. These results support the hypothesis that the catalytic domain of myosin is orientationally disordered on actin in a post-hydrolysis state in the early stages of force generation.     

Solution NMR structure of putidaredoxin-cytochrome P450cam complex via a combined residual dipolar coupling-spin labeling approach suggests a role for Trp106 of putidaredoxin in complex formation

The 58-kDa complex formed between the [2Fe-2S] ferredoxin, putidaredoxin (Pdx), and cytochrome P450cam (CYP101) from the bacterium Pseudomonas putida has been investigated by high-resolution solution NMR spectroscopy. Pdx serves as both the physiological reductant and effector for CYP101 in the enzymatic reaction involving conversion of substrate camphor to 5-exo-hydroxycamphor. In order to obtain an experimental structure for the oxidized Pdx-CYP101 complex, a combined approach using orientational data on the two proteins derived from residual dipolar couplings and distance restraints from site-specific spin labeling of Pdx has been applied. Spectral changes for residues in and near the paramagnetic metal cluster region of Pdx in complex with CYP101 have also been mapped for the first time using ¹⁵N and ¹³C NMR spectroscopy, leading to direct identification of the residues strongly affected by CYP101 binding. The new NMR structure of the Pdx-CYP101 complex agrees well with results from previous mutagenesis and biophysical studies involving residues at the binding interface such as formation of a salt bridge between Asp38 of Pdx and Arg112 of CYP101, while at the same time identifying key features different from those of earlier modeling studies. Analysis of the binding interface of the complex reveals that the side chain of Trp106, the C-terminal residue of Pdx and critical for binding to CYP101, is located across from the heme-binding loop of CYP101 and forms non-polar contacts with several residues in the vicinity of the heme group on CYP101, pointing to a potentially important role in complex formation.     

Structural Reorganization of α-Synuclein at Low pH Observed by NMR and REMD Simulations

α-Synuclein is an intrinsically disordered protein that appears in aggregated forms in the brains of patients with Parkinson's disease. The conversion from monomer to aggregate is complex, and aggregation rates are sensitive to changes in amino acid sequence and environmental conditions. It has previously been observed that α-synuclein aggregates faster at low pH than at neutral pH. Here, we combine NMR spectroscopy and molecular simulations to characterize α-synuclein conformational ensembles at both neutral and low pH in order to understand how the altered charge distribution at low pH changes the structural properties of these ensembles and leads to an increase in aggregation rate. The N-terminus, which has a small positive charge at neutral pH due to a balance of positively and negatively charged amino acid residues, is very positively charged at low pH. Conversely, the acidic C-terminus is highly negatively charged at neutral pH and becomes essentially neutral and hydrophobic at low pH. Our NMR experiments and replica exchange molecular dynamics simulations indicate that there is a significant structural reorganization within the low-pH ensemble relative to that at neutral pH in terms of long-range contacts, hydrodynamic radius, and the amount of heterogeneity within the conformational ensembles. At neutral pH, there is a very heterogeneous ensemble with transient contacts between the N-terminus and the non-amyloid β component (NAC); however, at low pH, there is a more homogeneous ensemble that exhibits strong contacts between the NAC and the C-terminus. At both pH values, transient contacts between the N- and C-termini are observed, the NAC region shows similar exposure to solvent, and the entire protein shows similar propensities to secondary structure. Based on the comparison of the neutral- and low-pH conformational ensembles, we propose that exposure of the NAC region to solvent and the secondary-structure propensity are not factors that account for differences in propensity to aggregate in this context. Instead, the comparison of the neutral- and low-pH ensembles suggests that the change in long-range interactions between the low- and neutral-pH ensembles, the compaction of the C-terminal region at low pH, and the uneven distribution of charges across the sequence are key to faster aggregation.     

Solution conformation, backbone dynamics and lipid interactions of the intrinsically unstructured malaria surface protein MSP2

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of the merozoite stage of Plasmodium falciparum, is a potential component of a malaria vaccine, having shown some efficacy in a clinical trial in Papua New Guinea. MSP2 is a GPI-anchored protein consisting of conserved N- and C-terminal domains and a variable central region. Previous studies have shown that it is an intrinsically unstructured protein with a high propensity for fibril formation, in which the conserved N-terminal domain has a key role. Secondary structure predictions suggest that MSP2 contains long stretches of random coil with very little α-helix or β-strand. Circular dichroism spectroscopy confirms this prediction under physiological conditions (pH 7.4) and in more acidic solutions (pH 6.2 and 3.4). Pulsed field gradient NMR diffusion measurements showed that MSP2 under physiological conditions has a large effective hydrodynamic radius consistent with an intrinsic pre-molten globule state, as defined by Uversky. This was supported by sedimentation velocity studies in the analytical ultracentrifuge. NMR resonance assignments have been obtained for FC27 MSP2, allowing the residual secondary structure and backbone dynamics to be defined. There is some motional restriction in the conserved C-terminal region in the vicinity of an intramolecular disulfide bond. Two other regions show motional restrictions, both of which display helical structure propensities. One of these helical regions is within the conserved N-terminal domain, which adopts essentially the same conformation in full-length MSP2 as in corresponding peptide fragments. We see no evidence of long-range interactions in the full-length protein. MSP2 associates with lipid micelles, but predominantly through the N-terminal region rather than the C terminus, which is GPI-anchored to the membrane in the parasite.     

Probing the structure of the Ff bacteriophage major coat protein transmembrane helix dimer by solution NMR

The transmembrane (TM) segment of the major coat protein from Ff bacteriophage has been extensively studied as an example of dimerization in detergent and lipid bilayer systems. However, almost all the information regarding this interaction has been gained through mutagenesis studies, with little direct structural information being available. To this end solution NMR has the potential to provide new insights into structure of the dimer. In order to evaluate the utility of this approach we have studied a selectively ¹⁵N-labeled peptide containing the TM segment of MCP (MCP_TM) by solution NMR. This peptide was found to give rise to detergent concentration-dependent spectra that were assigned to monomeric and dimeric forms. The standard free energy of this interaction in SDS was estimated from these spectra and found to be consistent with weak but specific dimerization. In addition, similar spectra could be obtained in β-octyl glucoside with intermolecular paramagnetic relaxation experiments demonstrating a parallel arrangement of TM helices in the dimer. In both detergents backbone chemical shift differences between monomeric and dimeric forms of MCP_TM showed that the largest changes occur around its GXXXG motif. The resulting structural model is consistent with observations made for MCP mutants previously characterized in biological membranes, opening the door to detailed structural characterization of this form of MCP. These results also have general implications for the study of weakly interacting TM segments by solution NMR since the use of similar sample conditions should allow structural data to be accessed for oligomeric states from a wide range systems that undergo biologically relevant but weak associations in the membrane.     

Characterization of conformational and dynamic properties of natively unfolded human and mouse alpha-synuclein ensembles by NMR: implication for aggregation

Conversion of human α-synuclein (aS) from the free soluble state to the insoluble fibrillar state has been implicated in the etiology of Parkinson's disease. Human aS is highly homologous in amino acid sequence to mouse aS, which contains seven substitutions including the A53T that has been linked to familial Parkinson's disease, and including five substitutions in the C-terminal region. It has been shown that the rate of fibrillation is highly dependent on the exact sequence of the protein, and mouse aS is reported to aggregate more rapidly than human aS in vitro. Nuclear magnetic resonance experiments of mouse and human aS at supercooled temperatures (263 K) are used to understand the effect of sequence on conformational fluctuations in the disordered ensembles and to relate these to differences in propensities to aggregate. We show that both aS are natively unfolded at low temperature with different propensities to secondary structure, backbone dynamics and long-range contacts across the protein. Mouse aS exhibits a higher propensity to helical conformation around the C-terminal substitutions as well as the loss of transient long-range contacts from the C- to the N-terminal end and hydrophobic central regions of the protein relative to human aS. Lack of back-folding from the C-terminal end of mouse aS exposes the N-terminal region, which is shown, by ¹⁵N relaxation experiments, to be very restricted in mobility relative to human aS. We propose that the restricted mobility in the N-terminal region may arise from transient interchain interactions, suggesting that the N-terminal KTK(E/Q)GV repeats may serve as initiation sites for aggregation in mouse aS. These transient interchain interactions coupled with a non-Aβ amyloid component (NAC) region that is both more exposed and has a higher propensity to β structure may accelerate the rate of fibril formation of aS.