Quantitative NMR analysis of the mechanism and kinetics of chaperone Hsp104 action on amyloid-ß42 aggregation and fibril formation.

The chaperone Hsp104, a member of the Hsp100/Clp family of translocases, prevents fibril formation of a variety of amyloidogenic peptides in a paradoxically substoichiometric manner. To understand the mechanism whereby Hsp104 inhibits fibril formation, we probed the interaction of Hsp104 with the Alzheimer's amyloid-ß42 (Aß42) peptide using a variety of biophysical techniques. Hsp104 is highly effective at suppressing the formation of Thioflavin T (ThT) reactive mature fibrils that are readily observed by atomic force (AFM) and electron (EM) microscopies. Quantitative kinetic analysis and global fitting was performed on serially recorded 1H-15N correlation spectra to monitor the disappearance of Aß42 monomers during the course of aggregation over a wide range of Hsp104 concentrations. Under the conditions employed (50 µM Aß42 at 20 °C), Aß42 aggregation occurs by a branching mechanism: an irreversible on-pathway leading to mature fibrils that entails primary and secondary nucleation and saturating elongation; and a reversible off-pathway to form nonfibrillar oligomers, unreactive to ThT and too large to be observed directly by NMR, but too small to be visualized by AFM or EM. Hsp104 binds reversibly with nanomolar affinity to sparsely populated Aß42 nuclei present in nanomolar concentrations, generated by primary and secondary nucleation, thereby completely inhibiting on-pathway fibril formation at substoichiometric ratios of Hsp104 to Aß42 monomers. Tight binding to sparsely populated nuclei likely constitutes a general mechanism for substoichiometric inhibition of fibrillization by a variety of chaperones. Hsp104 also impacts off-pathway oligomerization but to a much smaller degree initially reducing and then increasing the rate of off-pathway oligomerization.

Quantitative analysis of sterol-modulated monomer-dimer equilibrium of the ß1-adrenergic receptor by DEER spectroscopy.

G protein-coupled receptors (GPCR) activate numerous intracellular signaling pathways. The oligomerization properties of GPCRs, and hence their cellular functions, may be modulated by various components within the cell membrane (such as the presence of cholesterol). Modulation may occur directly via specific interaction with the GPCR or indirectly by affecting the physical properties of the membrane. Here, we use pulsed Q-band double electron-electron resonance (DEER) spectroscopy to probe distances between R1 nitroxide spin labels attached to Cys163 and Cys344 of the ß1-adrenergic receptor (ß1AR) in n-dodecyl-ß-D-maltoside micelles upon titration with two soluble cholesterol analogs, cholesteryl hemisuccinate (CHS) and sodium cholate. The former, like cholesterol, inserts itself into the lipid membrane, parallel to the phospholipid chains; the latter is aligned parallel to the surface of membranes. Global quantitative analysis of DEER echo curves upon titration of spin-labeled ß1AR with CHS and sodium cholate reveal the following: CHS binds specifically to the ß1AR monomer at a site close to the Cys163-R1 spin label with an equilibrium dissociation constant [Formula: see text] ~1.4 ± 0.4 mM. While no direct binding of sodium cholate to the ß1AR receptor was observed by DEER, sodium cholate induces specific ß1AR dimerization ([Formula: see text] ~35 ± 6 mM and a Hill coefficient n ~ 2.5 ± 0.4) with intersubunit contacts between transmembrane helices 1 and 2 and helix 8. Analysis of the DEER data obtained upon the addition of CHS to the ß1AR dimer in the presence of excess cholate results in dimer dissociation with species occupancies as predicted from the individual KD values.

Quantitative NMR analysis of the kinetics of prenucleation oligomerization and aggregation of pathogenic huntingtin exon-1 protein.

The N-terminal region of the huntingtin protein, encoded by exon-1 (httex1) and containing an expanded polyglutamine tract, forms fibrils that accumulate in neuronal inclusion bodies, resulting in Huntington's disease. We previously showed that reversible formation of a sparsely populated tetramer of the N-terminal amphiphilic domain, comprising a dimer of dimers in a four-helix bundle configuration, occurs on the microsecond timescale and is an essential prerequisite for subsequent nucleation and fibril formation that takes place orders of magnitude slower on a timescale of hours. For pathogenic httex1, such as httex1Q35 with 35 glutamines, NMR signals decay too rapidly to permit measurement of time-intensive exchange-based experiments. Here, we show that quantitative analysis of both the kinetics and mechanism of prenucleation tetramerization and aggregation can be obtained simultaneously from a series of 1H-15N band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC) correlation spectra. The equilibria and kinetics of tetramerization are derived from the time dependence of the 15N chemical shifts and 1H-15N cross-peak volume/intensity ratios, while the kinetics of irreversible fibril formation are afforded by the decay curves of 1H-15N cross-peak intensities and volumes. Analysis of data on httex1Q35 over a series of concentrations ranging from 200 to 750 µM and containing variable (7 to 20%) amounts of the Met7O sulfoxide species, which does not tetramerize, shows that aggregation of native httex1Q35 proceeds via fourth-order primary nucleation, consistent with the critical role of prenucleation tetramerization, coupled with first-order secondary nucleation. The Met7O sulfoxide species does not nucleate but is still incorporated into fibrils by elongation.

Time-resolved DEER EPR and solid-state NMR afford kinetic and structural elucidation of substrate binding to Ca2+-ligated calmodulin.

Recent advances in rapid mixing and freeze quenching have opened the path for time-resolved electron paramagnetic resonance (EPR)-based double electron-electron resonance (DEER) and solid-state NMR of protein-substrate interactions. DEER, in conjunction with phase memory time filtering to quantitatively extract species populations, permits monitoring time-dependent probability distance distributions between pairs of spin labels, while solid-state NMR provides quantitative residue-specific information on the appearance of structural order and the development of intermolecular contacts between substrate and protein. Here, we demonstrate the power of these combined approaches to unravel the kinetic and structural pathways in the binding of the intrinsically disordered peptide substrate (M13) derived from myosin light-chain kinase to the universal eukaryotic calcium regulator, calmodulin. Global kinetic analysis of the data reveals coupled folding and binding of the peptide associated with large spatial rearrangements of the two domains of calmodulin. The initial binding events involve a bifurcating pathway in which the M13 peptide associates via either its N- or C-terminal regions with the C- or N-terminal domains, respectively, of calmodulin/4Ca2+ to yield two extended "encounter" complexes, states A and A*, without conformational ordering of M13. State A is immediately converted to the final compact complex, state C, on a timescale τ ≤ 600 µs. State A*, however, only reaches the final complex via a collapsed intermediate B (τ ∼ 1.5 to 2.5 ms), in which the peptide is only partially ordered and not all intermolecular contacts are formed. State B then undergoes a relatively slow (τ ∼ 7 to 18 ms) conformational rearrangement to state C.

A Transient, Excited Species of the Autoinhibited α-State of the Bacterial Transcription Factor RfaH May Represent an Early Intermediate on the Fold-Switching Pathway.

RfaH is a two-domain transcription factor in which the C-terminal domain switches fold from an α-helical hairpin to a ß-roll upon binding the ops-paused RNA polymerase. To ascertain the presence of a sparsely populated excited state that may prime the autoinhibited resting state of RfaH for binding ops-paused RNA polymerase, we carried out a series of NMR-based exchange experiments to probe for conformational exchange on the millisecond time scale. Quantitative analysis of these data reveals exchange between major ground (∼95%) and sparsely populated excited (∼5%) states with an exchange lifetime of ∼3 ms involving residues at the interface between the N-terminal and C-terminal domains formed by the ß3/ß4 hairpin and helix α3 of the N-terminal domain and helices α4 and α5 of the C-terminal domain. The largest 15N backbone chemical shift differences are associated with the ß3/ß4 hairpin, leading us to suggest that the excited state may involve a rigid body lateral displacement/rotation away from the C-terminal domain to adopt a position similar to that seen in the active RNA polymerase-bound state. Such a rigid body reorientation would result in a reduction in the interface between the N- and C-terminal domains with the possible introduction of a cavity or cavities. This hypothesis is supported by the observation that the population of the excited species and the exchange rate of interconversion between ground and excited states are reduced at a high (2.5 kbar) pressure. Mechanistic implications for fold switching of the C-terminal domain in the context of RNA polymerase binding are discussed.

Interaction of the bacterial division regulator MinE with lipid bicelles studied by NMR spectroscopy.

The bacterial MinE and MinD division regulatory proteins form a standing wave enabling MinC, which binds MinD, to inhibit FtsZ polymerization everywhere except at the midcell, thereby assuring correct positioning of the cytokinetic septum and even distribution of contents to daughter cells. The MinE dimer undergoes major structural rearrangements between a resting six-stranded state present in the cytoplasm, a membrane-bound state, and a four-stranded active state bound to MinD on the membrane, but it is unclear which MinE motifs interact with the membrane in these different states. Using NMR, we probe the structure and global dynamics of MinE bound to disc-shaped lipid bicelles. In the bicelle-bound state, helix α1 no longer sits on top of the six-stranded ß-sheet, losing any contact with the protein core, but interacts directly with the bicelle surface; the structure of the protein core remains unperturbed and also interacts with the bicelle surface via helix α2. Binding may involve a previously identified excited state of free MinE in which helix α1 is disordered, thereby allowing it to target the membrane surface. Helix α1 and the protein core undergo nanosecond rigid body motions of differing amplitudes in the plane of the bicelle surface. Global dynamics on the sub-millisecond time scale between a ground state and a sparsely populated excited state are also observed and may represent a very early intermediate on the transition path between the resting six-stranded and active four-stranded conformations. In summary, our results provide insights into MinE structural rearrangements important during bacterial cell division.

Deconvoluting Monomer- and Dimer-Specific Distance Distributions between Spin Labels in a Monomer/Dimer Mixture Using T1-Edited DEER EPR Spectroscopy.

Double electron-electron resonance (DEER) EPR is a powerful tool in structural biology, providing distances between pairs of spin labels. When the sample consists of a mixture of oligomeric species (e.g., monomer and dimer), the question arises as to how to assign the peaks in the DEER-derived probability distance distribution to the individual species. Here, we propose incorporating an EPR longitudinal electron relaxation (T1) inversion recovery experiment within a DEER pulse sequence to resolve this problem. The apparent T1 between dipolar coupled electron spins measured from the inversion recovery time (τinv) dependence of the peak intensities in the T1-edited DEER-derived probability P(r) distance distribution will be affected by the number of nitroxide labels attached to the biomolecule of interest, for example, two for a monomer and four for a dimer. We show that global fitting of all the T1-edited DEER echo curves, recorded over a range of τinv values, permits the deconvolution of distances between spin labels originating from monomeric (longer T1) and dimeric (shorter T1) species. This is especially useful when the trapping of spin labels in different conformational states during freezing gives rise to complex P(r) distance distributions. The utility of this approach is demonstrated for two systems, the ß1 adrenergic receptor and a construct of the huntingtin exon-1 protein fused to the immunoglobulin domain of protein G, both of which exist in a monomer-dimer equilibrium.

NMR spectroscopy, excited states and relevance to problems in cell biology - transient pre-nucleation tetramerization of huntingtin and insights into Huntington's disease.

Solution nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for analyzing three-dimensional structure and dynamics of macromolecules at atomic resolution. Recent advances have exploited the unique properties of NMR in exchanging systems to detect, characterize and visualize excited sparsely populated states of biological macromolecules and their complexes, which are only transient. These states are invisible to conventional biophysical techniques, and play a key role in many processes, including molecular recognition, protein folding, enzyme catalysis, assembly and fibril formation. All the NMR techniques make use of exchange between sparsely populated NMR-invisible and highly populated NMR-visible states to transfer a magnetization property from the invisible state to the visible one where it can be easily detected and quantified. There are three classes of NMR experiments that rely on differences in distance, chemical shift or transverse relaxation (molecular mass) between the NMR-visible and -invisible species. Here, I illustrate the application of these methods to unravel the complex mechanism of sub-millisecond pre-nucleation oligomerization of the N-terminal region of huntingtin, encoded by exon-1 of the huntingtin gene, where CAG expansion leads to Huntington's disease, a fatal autosomal-dominant neurodegenerative condition. I also discuss how inhibition of tetramerization blocks the much slower (by many orders of magnitude) process of fibril formation.

Transverse relaxation optimized spectroscopy of NH2 groups in glutamine and asparagine side chains of proteins.

A transverse relaxation optimized spectroscopy (TROSY) approach is described for the optimal detection of NH2 groups in asparagine and glutamine side chains of proteins. Specifically, we have developed NMR experiments for isolating the slow-relaxing 15N and 1H components of NH2 multiplets. Although even modest sensitivity gains in 2D NH2-TROSY correlation maps compared to their decoupled NH2-HSQC counterparts can be achieved only occasionally, substantial improvements in resolution of the NMR spectra are demonstrated for asparagine and glutamine NH2 sites of a buried cavity mutant, L99A, of T4 lysozyme at 5 ºC. The NH2-TROSY approach is applied to CPMG relaxation dispersion measurements at the side chain NH2 positions of the L99A T4 lysozyme mutant - a model system for studies of the role of protein dynamics in ligand binding.

Atomic view of cosolute-induced protein denaturation probed by NMR solvent paramagnetic relaxation enhancement.

The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift the equilibrium toward the latter, while osmolytes stabilize the former. The molecular mechanism whereby cosolutes perturb protein stability is still the subject of considerable debate. Probing the molecular details of the cosolvent effect is experimentally challenging as the interactions are very weak and transient, rendering them invisible to most conventional biophysical techniques. Here, we probe cosolute-protein interactions by means of NMR solvent paramagnetic relaxation enhancement together with a formalism we recently developed to quantitatively describe, at atomic resolution, the energetics and dynamics of cosolute-protein interactions in terms of a concentration normalized equilibrium average of the interspin distance, [Formula: see text], and an effective correlation time, τc The system studied is the metastable drkN SH3 domain, which exists in dynamic equilibrium between native and unfolded states, thereby permitting us to probe the interactions of cosolutes with both states simultaneously under the same conditions. Two paramagnetic cosolute denaturants were investigated, one neutral and the other negatively charged, differing in the presence of a carboxyamide group versus a carboxylate. Our results demonstrate that attractive cosolute-protein backbone interactions occur largely in the unfolded state and some loop regions in the native state, electrostatic interactions reduce the [Formula: see text] values, and temperature predominantly impacts interactions with the unfolded state. Thus, destabilization of the native state in this instance arises predominantly as a consequence of interactions of the cosolutes with the unfolded state.

A methyl-TROSY based 13C relaxation dispersion NMR experiment for studies of chemical exchange in proteins.

A methyl Transverse Relaxation Optimized Spectroscopy (methyl-TROSY) based, multiple quantum (MQ) 13C Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiment is described. The experiment is derived from the previously developed MQ 13C-1H CPMG scheme (Korzhnev in J Am Chem Soc 126: 3964-73, 2004) supplemented with a CPMG train of refocusing 1H pulses applied with constant frequency and synchronized with the 13C CPMG pulse train. The optimal 1H 'decoupling' scheme that minimizes the amount of fast-relaxing methyl MQ magnetization present during CPMG intervals, makes use of an XY-4 phase cycling of the refocusing composite 1H pulses. For small-to-medium sized proteins, the MQ 13C CPMG experiment has the advantage over its single quantum (SQ) 13C counterpart of significantly reducing intrinsic, exchange-free relaxation rates of methyl coherences. For high molecular weight proteins, the MQ 13C CPMG experiment eliminates complications in the interpretation of MQ 13C-1H CPMG relaxation dispersion profiles arising from contributions to exchange from differences in methyl 1H chemical shifts between ground and excited states. The MQ 13C CPMG experiment is tested on two protein systems: (1) a triple mutant of the Fyn SH3 domain that interconverts slowly on the chemical shift time scale between the major folded state and an excited state folding intermediate; and (2) the 82-kDa enzyme Malate Synthase G (MSG), where chemical exchange at individual Ile δ1 methyl positions occurs on a much faster time-scale.

An S/T motif controls reversible oligomerization of the Hsp40 chaperone DNAJB6b through subtle reorganization of a ß sheet backbone.

Chaperone oligomerization is often a key aspect of their function. Irrespective of whether chaperone oligomers act as reservoirs for active monomers or exhibit a chaperoning function themselves, understanding the mechanism of oligomerization will further our understanding of how chaperones maintain the proteome. Here, we focus on the class-II Hsp40, human DNAJB6b, a highly efficient inhibitor of protein self-assembly in vivo and in vitro that forms functional oligomers. Using single-quantum methyl-based relaxation dispersion NMR methods we identify critical residues for DNAJB6b oligomerization in its C-terminal domain (CTD). Detailed solution NMR studies on the structure of the CTD showed that a serine/threonine-rich stretch causes a backbone twist in the N-terminal ß strand, stabilizing the monomeric form. Quantitative analysis of an array of NMR relaxation-based experiments (including Carr-Purcell-Meiboom-Gill relaxation dispersion, off-resonance R1ρ profiles, lifetime line broadening, and exchange-induced shifts) on the CTD of both wild type and a point mutant (T142A) within the S/T region of the first ß strand delineates the kinetics of the interconversion between the major twisted-monomeric conformation and a more regular ß strand configuration in an excited-state dimer, as well as exchange of both monomer and dimer species with high-molecular-weight oligomers. These data provide insights into the molecular origins of DNAJB6b oligomerization. Further, the results reported here have implications for the design of ß sheet proteins with tunable self-assembling properties and pave the way to an atomic-level understanding of amyloid inhibition.

Abrogation of prenucleation, transient oligomerization of the Huntingtin exon 1 protein by human profilin I.

Human profilin I reduces aggregation and concomitant toxicity of the polyglutamine-containing N-terminal region of the huntingtin protein encoded by exon 1 (httex1) and responsible for Huntington's disease. Here, we investigate the interaction of profilin with httex1 using NMR techniques designed to quantitatively analyze the kinetics and equilibria of chemical exchange at atomic resolution, including relaxation dispersion, exchange-induced shifts, and lifetime line broadening. We first show that the presence of two polyproline tracts in httex1, absent from a shorter huntingtin variant studied previously, modulates the kinetics of the transient branched oligomerization pathway that precedes nucleation, resulting in an increase in the populations of the on-pathway helical coiled-coil dimeric and tetrameric species (τex ≤ 50 to 70 µs), while leaving the population of the off-pathway (nonproductive) dimeric species largely unaffected (τex ∼750 µs). Next, we show that the affinity of a single molecule of profilin to the polyproline tracts is in the micromolar range (Kdiss ∼ 17 and ∼ 31 µM), but binding of a second molecule of profilin is negatively cooperative, with the affinity reduced ∼11-fold. The lifetime of a 1:1 complex of httex1 with profilin, determined using a shorter huntingtin variant containing only a single polyproline tract, is shown to be on the submillisecond timescale (τex ∼ 600 µs and Kdiss ∼ 50 µM). Finally, we demonstrate that, in stable profilin-httex1 complexes, the productive oligomerization pathway, leading to the formation of helical coiled-coil httex1 tetramers, is completely abolished, and only the pathway resulting in "nonproductive" dimers remains active, thereby providing a mechanistic basis for how profilin reduces aggregation and toxicity of httex1.

Theory and Applications of Nitroxide-based Paramagnetic Cosolutes for Probing Intermolecular and Electrostatic Interactions on Protein Surfaces.

Solvent paramagnetic relaxation enhancement (sPRE) arising from nitroxide-based cosolutes has recently been used to provide an atomic view of cosolute-induced protein denaturation and to characterize residue-specific effective near-surface electrostatic potentials (ϕENS). Here, we explore distinct properties of the sPRE arising from nitroxide-based cosolutes and provide new insights into the interpretation of the sPRE and sPRE-derived ϕENS. We show that: (a) the longitudinal sPRE rate Γ1 is heavily dependent on spectrometer field and viscosity, while the transverse sPRE rate Γ2 is much less so; (b) the spectral density J(0) is proportional to the inverse of the relative translational diffusion constant and is related to the quantity ⟨r-4⟩norm, a concentration-normalized equilibrium average of the electron-proton interspin separation; and (c) attractive intermolecular interactions result in a shortening of the residue-specific effective correlation time for the electron-proton vector. We discuss four different approaches for evaluating ϕENS based on Γ2, J(0), Γ1, or ⟨r-6⟩norm. The latter is evaluated from the magnetic field dependence of Γ1 in conjunction with Γ2. Long-range interactions dominate J(0) and Γ2, while, at high magnetic fields, the contribution of short-range interactions becomes significant for J(ω) and hence Γ1; the four ϕENS quantities enable one to probe both long- and short-range electrostatic interactions. The experimental ϕENS potentials were evaluated using three model protein systems, two folded (ubiquitin and native drkN SH3) and one intrinsically disordered (unfolded state of drkN SH3), in relation to theoretical ϕENS potentials calculated from atomic coordinates using the Poisson-Boltzmann theory with either a r-6 or r-4 dependence.

Quantitative Agreement between Conformational Substates of Holo Calcium-Loaded Calmodulin Detected by Double Electron-Electron Resonance EPR and Predicted by Molecular Dynamics Simulations.

Calcium-loaded calmodulin (CaM/4Ca2+) comprises two domains that undergo rigid body reorientation from a predominantly extended conformation to a compact one upon binding target peptides. A recent replica-exchange molecular dynamics (MD) simulation on holo CaM/4Ca2+ suggested the existence of distinct structural clusters (substates) along the path from extended to compact conformers in the absence of substrates. Here, we experimentally demonstrate the existence of CaM/4Ca2+ substates trapped in local minima by three freezing/annealing regimes (slow, 40 s; intermediate, 1.5 s; fast, 0.5 ms) using pulsed Q-band double electron-electron resonance (DEER) EPR spectroscopy to measure interdomain distances between nitroxide spin-labels positioned at A17C and A128C in the N- and C-terminal domains, respectively. The DEER echo curves were directly fit to population-optimized P(r) pairwise distance distributions calculated from the coordinates of the MD clusters and compact crystal structure. DEER data on fully deuterated CaM/4Ca2+ were acquired at multiple values of the second echo period (10-35 µs) and analyzed globally to eliminate instrumental and overfitting artifacts and ensure accurate populations, peak positions, and widths. The DEER data for all three freezing regimes are quantitatively accounted for within experimental error by 5-6 distinct conformers comprising a predominantly populated extended form (60-75%) and progressively more compact states whose populations decrease as the degree of compactness increases. The shortest interdomain separation is found in the compact crystal structure, which has an occupancy of 4-6%. Thus, CaM/4Ca2+ samples high energy local minima comprising a few discrete substates of increasing compactness in a rugged energy landscape.

Spatial domain organization in the HIV-1 reverse transcriptase p66 homodimer precursor probed by double electron-electron resonance EPR.

HIV type I (HIV-1) reverse transcriptase (RT) catalyzes the conversion of viral RNA into DNA, initiating the chain of events leading to integration of proviral DNA into the host genome. RT is expressed as a single polypeptide chain within the Gag-Pol polyprotein, and either prior to or following excision by HIV-1 protease forms a 66 kDa chain (p66) homodimer precursor. Further proteolytic attack by HIV-1 protease cleaves the ribonuclease H (RNase H) domain of a single subunit to yield the mature p66/p51 heterodimer. Here, we probe the spatial domain organization within the p66 homodimer using pulsed Q-band double electron-electron resonance (DEER) EPR spectroscopy to measure a large number of intra- and intersubunit distances between spin labels attached to surface-engineered cysteines. The DEER-derived distances are fully consistent with the structural subunit asymmetry found in the mature p66/p51 heterodimer in which catalytic activity resides in the p66 subunit, while the p51 subunit purely serves as a structural scaffold. Furthermore, the p66 homodimer precursor undergoes a conformational change involving the thumb, palm, and finger domains in one of the subunits (corresponding to the p66 subunit in the mature p66/p51 heterodimer) from a closed to a partially open state upon addition of a nonnucleoside inhibitor. The relative orientation of the domains was modeled by simulated annealing driven by the DEER-derived distances. Finally, the RNase H domain that is cleaved to generate p51 in the mature p66/p51 heterodimer is present in 2 major conformers. One conformer is fully solvent accessible thereby accounting for the observation that only a single subunit of the p66 homodimer precursor is susceptible to HIV-1 protease.

Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis.

J-domain chaperones are involved in the efficient handover of misfolded/partially folded proteins to Hsp70 but also function independently to protect against cell death. Due to their high flexibility, the mechanism by which they regulate the Hsp70 cycle and how specific substrate recognition is performed remains unknown. Here we focus on DNAJB6b, which has been implicated in various human diseases and represents a key player in protection against neurodegeneration and protein aggregation. Using a variant that exists mainly in a monomeric form, we report the solution structure of an Hsp40 containing not only the J and C-terminal substrate binding (CTD) domains but also the functionally important linkers. The structure reveals a highly dynamic protein in which part of the linker region masks the Hsp70 binding site. Transient interdomain interactions via regions crucial for Hsp70 binding create a closed, autoinhibited state and help retain the monomeric form of the protein. Detailed NMR analysis shows that the CTD (but not the J domain) self-associates to form an oligomer comprising ∼35 monomeric units, revealing an intricate balance between intramolecular and intermolecular interactions. The results shed light on the mechanism of autoregulation of the Hsp70 cycle via conserved parts of the linker region and reveal the mechanism of DNAJB6b oligomerization and potentially antiaggregation.

Probing transient excited states of the bacterial cell division regulator MinE by relaxation dispersion NMR spectroscopy.

Bacterial MinD and MinE form a standing oscillatory wave which positions the cell division inhibitor MinC, that binds MinD, everywhere on the membrane except at the midpoint of the cell, ensuring midcell positioning of the cytokinetic septum. During this process MinE undergoes fold switching as it interacts with different partners. We explore the exchange dynamics between major and excited states of the MinE dimer in 3 forms using 15N relaxation dispersion NMR: the full-length protein (6-stranded ß-sheet sandwiched between 4 helices) representing the resting state; a 10-residue N-terminal deletion (Δ10) mimicking the membrane-binding competent state where the N-terminal helix is detached to interact with membrane; and N-terminal deletions of either 30 (Δ30) or 10 residues with an I24N mutation (Δ10/I24N), in which the ß1-strands at the dimer interface are extruded and available to bind MinD, leaving behind a 4-stranded ß-sheet. Full-length MinE samples 2 "excited" states: The first is similar to a full-length/Δ10 heterodimer; the second, also sampled by Δ10, is either similar to or well along the pathway toward the 4-stranded ß-sheet form. Both Δ30 and Δ10/I24N sample 2 excited species: The first may involve destabilization of the ß3- and ß3'-strands at the dimer interface; changes in the second are more extensive, involving further disruption of secondary structure, possibly representing an ensemble of states on the pathway toward restoration of the resting state. The quantitative information on MinE conformational dynamics involving these excited states is crucial for understanding the oscillation pattern self-organization by MinD-MinE interaction dynamics on the membrane.

Probing initial transient oligomerization events facilitating Huntingtin fibril nucleation at atomic resolution by relaxation-based NMR.

The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic domain (httNT), a polyglutamine (Q n ) tract, and a proline-rich sequence. Polyglutamine expansion results in an aggregation-prone protein responsible for Huntington's disease. Here, we study the earliest events involved in oligomerization of a minimalistic construct, httNTQ7, which remains largely monomeric over a sufficiently long period of time to permit detailed quantitative NMR analysis of the kinetics and structure of sparsely populated [Formula: see text] oligomeric states, yet still eventually forms fibrils. Global fitting of concentration-dependent relaxation dispersion, transverse relaxation in the rotating frame, and exchange-induced chemical shift data reveals a bifurcated assembly mechanism in which the NMR observable monomeric species either self-associates to form a productive dimer (τex ∼ 30 µs, Kdiss ∼ 0.1 M) that goes on to form a tetramer ([Formula: see text] µs; Kdiss ∼ 22 µM), or exchanges with a "nonproductive" dimer that does not oligomerize further (τex ∼ 400 µs; Kdiss ∼ 0.3 M). The excited state backbone chemical shifts are indicative of a contiguous helix (residues 3-17) in the productive dimer/tetramer, with only partial helical character in the nonproductive dimer. A structural model of the productive dimer/tetramer was obtained by simulated annealing driven by intermolecular paramagnetic relaxation enhancement data. The tetramer comprises a D2 symmetric dimer of dimers with largely hydrophobic packing between the helical subunits. The structural model, validated by EPR distance measurements, illuminates the role of the httNT domain in the earliest stages of prenucleation and oligomerization, before fibril formation.

Microsecond Backbone Motions Modulate the Oligomerization of the DNAJB6 Chaperone.

DNAJB6 is a prime example of an anti-aggregation chaperone that functions as an oligomer. DNAJB6 oligomers are dynamic and subunit exchange is critical for inhibiting client protein aggregation. The T193A mutation in the C-terminal domain (CTD) of DNAJB6 reduces both chaperone self-oligomerization and anti-aggregation of client proteins, and has recently been linked to Parkinson's disease. Here, we show by NMR, including relaxation-based methods, that the T193A mutation has minimal effects on the structure of the ß-stranded CTD but increases the population and rate of formation of a partially folded state. The results can be rationalized in terms of ß-strand peptide plane flips that occur on a timescale of ≈100 µs and lead to global changes in the overall pleat/flatness of the CTD, thereby altering its ability to oligomerize. These findings help forge a link between chaperone dynamics, oligomerization and anti-aggregation activity which may possibly lead to new therapeutic avenues tuned to target specific substrates.