Design of amyloidogenic peptide traps.

Segments of proteins with high ß-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in ß-strand and ß-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein-peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid ß1-42 (Aß42). The Aß binders block the assembly of Aß fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aß42 species.

Activation of caspase-9 on the apoptosome as studied by methyl-TROSY NMR.

Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins. Failure to activate caspase-9 enables the evasion of programmed cell death, which occurs in various forms of cancer. Despite the critical apoptotic function of caspase-9, the structural mechanism by which it is activated on the apoptosome has remained elusive. Here, we used a combination of methyl-transverse relaxation-optimized NMR spectroscopy, protein engineering, and biochemical assays to study the activation of caspase-9 bound to the apoptosome. In the absence of peptide substrate, we observed that both caspase-9 and its isolated protease domain (PD) only very weakly dimerize with dissociation constants in the millimolar range. Methyl-NMR spectra of isotope-labeled caspase-9, within the 1.3-MDa native apoptosome complex or an engineered 480-kDa apoptosome mimic, reveal that the caspase-9 PD remains monomeric after recruitment to the scaffold. Binding to the apoptosome, therefore, organizes caspase-9 PDs so that they can rapidly and extensively dimerize only when substrate is present, providing an important layer in the regulation of caspase-9 activation. Our work highlights the unique role of NMR spectroscopy to structurally characterize protein domains that are flexibly tethered to large scaffolds, even in cases where the molecular targets are in excess of 1 MDa, as in the present example.

Disrupting the α-synuclein-ESCRT interaction with a peptide inhibitor mitigates neurodegeneration in preclinical models of Parkinson's disease.

Accumulation of α-synuclein into toxic oligomers or fibrils is implicated in dopaminergic neurodegeneration in Parkinson's disease. Here we performed a high-throughput, proteome-wide peptide screen to identify protein-protein interaction inhibitors that reduce α-synuclein oligomer levels and their associated cytotoxicity. We find that the most potent peptide inhibitor disrupts the direct interaction between the C-terminal region of α-synuclein and CHarged Multivesicular body Protein 2B (CHMP2B), a component of the Endosomal Sorting Complex Required for Transport-III (ESCRT-III). We show that α-synuclein impedes endolysosomal activity via this interaction, thereby inhibiting its own degradation. Conversely, the peptide inhibitor restores endolysosomal function and thereby decreases α-synuclein levels in multiple models, including female and male human cells harboring disease-causing α-synuclein mutations. Furthermore, the peptide inhibitor protects dopaminergic neurons from α-synuclein-mediated degeneration in hermaphroditic C. elegans and preclinical Parkinson's disease models using female rats. Thus, the α-synuclein-CHMP2B interaction is a potential therapeutic target for neurodegenerative disorders.

Exploiting conformational dynamics to modulate the function of designed proteins.

With the recent success in calculating protein structures from amino acid sequences using artificial intelligence-based algorithms, an important next step is to decipher how dynamics is encoded by the primary protein sequence so as to better predict function. Such dynamics information is critical for protein design, where strategies could then focus not only on sequences that fold into particular structures that perform a given task, but would also include low-lying excited protein states that could influence the function of the designed protein. Herein, we illustrate the importance of dynamics in modulating the function of C34, a designed α/ß protein that captures ß-strands of target ligands and is a member of a family of proteins designed to sequester ß-strands and ß hairpins of aggregation-prone molecules that lead to a variety of pathologies. Using a strategy to "see" regions of apo C34 that are invisible to NMR spectroscopy as a result of pervasive conformational exchange, as well as a mutagenesis approach whereby C34 molecules are stabilized into a single conformer, we determine the structures of the predominant conformations that are sampled by C34 and show that these attenuate the affinity for cognate peptide. Subsequently, the observed motion is exploited to develop an allosterically regulated peptide binder whose binding affinity can be controlled through the addition of a second molecule. Our study emphasizes the unique role that NMR can play in directing the design process and in the construction of new molecules with more complex functionality.

Opening of a cryptic pocket in ß-lactamase increases penicillinase activity.

Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in ß-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol-labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across ß-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM ß-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR chemical exchange saturation transfer experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket's functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.

Structural basis for the stabilization of amyloidogenic immunoglobulin light chains by hydantoins.

Misfolding and aggregation of immunoglobulin light chains (LCs) leads to the degeneration of post-mitotic tissue in the disease immunoglobulin LC amyloidosis (AL). We previously reported the discovery of small molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which slow or stop the LC aggregation cascade at the outset. A predominant structural category of kinetic stabilizers emerging from the high-throughput screen are coumarins substituted at the 7-position, which bind at the interface between the two variable domains of the light chain dimer. Here, we report the binding mode of another, more polar, LC kinetic stabilizer chemotype, 3,5-substituted hydantoins. Computational docking, solution nuclear magnetic resonance experiments, and x-ray crystallography show that the aromatic substructure emerging from the hydantoin 3-position occupies the same LC binding site as the coumarin ring. Notably, the hydantoin ring extends beyond the binding site mapped out by the coumarin hits. The hydantoin ring makes hydrogen bonds with both LC monomers simultaneously. The alkyl substructure at the hydantoin 5-position partially occupies a novel binding pocket proximal to the pocket occupied by the coumarin substructure. Overall, the hydantoin structural data suggest that a larger area of the LC variable-domain-variable-domain dimer interface is amenable to small molecule binding than previously demonstrated, which should facilitate development of more potent full-length LC kinetic stabilizers.

A methyl-TROSY approach for NMR studies of high-molecular-weight DNA with application to the nucleosome core particle.

The development of methyl-transverse relaxation-optimized spectroscopy (methyl-TROSY)-based NMR methods, in concert with robust strategies for incorporation of methyl-group probes of structure and dynamics into the protein of interest, has facilitated quantitative studies of high-molecular-weight protein complexes. Here we develop a one-pot in vitro reaction for producing NMR quantities of methyl-labeled DNA at the C5 and N6 positions of cytosine (5mC) and adenine (6mA) nucleobases, respectively, enabling the study of high-molecular-weight DNA molecules using TROSY approaches originally developed for protein applications. Our biosynthetic strategy exploits the large number of naturally available methyltransferases to specifically methylate DNA at a desired number of sites that serve as probes of structure and dynamics. We illustrate the methodology with studies of the 153-base pair Widom DNA molecule that is simultaneously methyl-labeled at five sites, showing that high-quality 13C-1H spectra can be recorded on 100 µM samples in a few minutes. NMR spin relaxation studies of labeled methyl groups in both DNA and the H2B histone protein component of the 200-kDa nucleosome core particle (NCP) establish that methyl groups at 5mC and 6mA positions are, in general, more rigid than Ile, Leu, and Val methyl probes in protein side chains. Studies focusing on histone H2B of NCPs wrapped with either wild-type DNA or DNA methylated at all 26 CpG sites highlight the utility of NMR in investigating the structural dynamics of the NCP and how its histone core is affected through DNA methylation, an important regulator of transcription.

Confronting the Invisible: Assignment of Protein 1HN Chemical Shifts in Cases of Extreme Broadening.

NMR studies of intrinsically disordered proteins (IDPs) at neutral pH values are hampered by the rapid exchange of backbone amide protons with solvent. Although exchange rates can be modulated by changes in pH, interactions between IDPs that lead to phase separation sometimes only occur at neutral pH values or higher, where backbone amide-based experiments fail. Here we describe a simple NMR experiment for measuring amide proton chemical shifts in cases where 1HN spectra cannot be obtained. The approach uses a weak 1H B1 field, searching for elusive 1HN resonance frequencies that become encoded in the intensities of cross-peaks in three-dimensional 1Hα-detect spectra. Applications to the CAPRIN1 protein in both dilute- and phase-separated states highlight the utility of the method, establishing that accurate 1HN chemical shifts can be obtained even in cases where solvent hydrogen exchange rates are on the order of 1500 s-1.

An allosteric switch regulates Mycobacterium tuberculosis ClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR.

The 300-kDa ClpP1P2 protease from Mycobacterium tuberculosis collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all of the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of antituberculosis (anti-TB) therapeutics that target the Clp system. Here, we present a combined biophysical and biochemical study to explore the structure-dynamics-function relationship in MtClpP1P2. Electron cryomicroscopy (cryo-EM) structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key ß-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that, on binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod-Wyman-Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function and shows extensive allosteric interactions involving both intra- and interring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target M. tuberculosis.

Exploring long-range cooperativity in the 20S proteasome core particle from Thermoplasma acidophilum using methyl-TROSY-based NMR.

The 20S core particle (CP) proteasome is a molecular assembly catalyzing the degradation of misfolded proteins or proteins no longer required for function. It is composed of four stacked heptameric rings that form a barrel-like structure, sequestering proteolytic sites inside its lumen. Proteasome function is regulated by gates derived from the termini of α-rings and through binding of regulatory particles (RPs) to one or both ends of the barrel. The CP is dynamic, with an extensive allosteric pathway extending from one end of the molecule to catalytic sites in its center. Here, using methyl-transverse relaxation optimized spectroscopy (TROSY)-based NMR optimized for studies of high-molecular-weight complexes, we evaluate whether the pathway extends over the entire 150-Å length of the molecule. By exploiting a number of different labeling schemes, the two halves of the molecule can be distinguished, so that the effects of 11S RP binding, or the introduction of gate or allosteric pathway mutations at one end of the barrel can be evaluated at the distal end. Our results establish that while 11S binding and the introduction of key mutations affect each half of the CP allosterically, they do not further couple opposite ends of the molecule. This may have implications for the function of so-called "hybrid" proteasomes where each end of the CP is bound with a different regulator, allowing the CP to be responsive to both RPs simultaneously. The methodology presented introduces a general NMR strategy for dissecting pathways of communication in homo-oligomeric molecular machines.

The Role of Protein Thermodynamics and Primary Structure in Fibrillogenesis of Variable Domains from Immunoglobulin Light Chains.

Immunoglobulin light-chain amyloidosis is a protein aggregation disease that leads to proteinaceous deposits in a variety of organs in the body and, if untreated, ultimately results in death. The mechanisms by which light-chain aggregation occurs are not well understood. Here we have used solution NMR spectroscopy and biophysical studies to probe immunoglobulin variable domain λV6-57 VL aggregation, a process that appears to drive the degenerative phenotypes in amyloidosis patients. Our results establish that aggregation proceeds via the unfolded state. We identify, through NMR relaxation experiments recorded on the unfolded domain ensemble, a series of hotspots that could be involved in the initial phases of aggregate formation. Mutational analysis of these hotspots reveals that the region that includes K16-R24 is particularly aggregation prone. Notably, this region includes the site of the R24G substitution, a mutation that is found in variable domains of λ light-chain deposits in 25% of patients. The R24G λV6-57 VL domain aggregates more rapidly than would be expected on the basis of thermodynamic stability alone, while substitutions in many of the aggregation-prone regions significantly slow down fibril formation.

Stabilization of amyloidogenic immunoglobulin light chains by small molecules.

In Ig light-chain (LC) amyloidosis (AL), the unique antibody LC protein that is secreted by monoclonal plasma cells in each patient misfolds and/or aggregates, a process leading to organ degeneration. As a step toward developing treatments for AL patients with substantial cardiac involvement who have difficulty tolerating existing chemotherapy regimens, we introduce small-molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which can slow or stop the amyloidogenicity cascade at its origin. A protease-coupled fluorescence polarization-based high-throughput screen was employed to identify small molecules that kinetically stabilize LCs. NMR and X-ray crystallographic data demonstrate that at least one structural family of hits bind at the LC-LC dimerization interface within full-length LCs, utilizing variable-domain residues that are highly conserved in most AL patients. Stopping the amyloidogenesis cascade at the beginning is a proven strategy to ameliorate postmitotic tissue degeneration.

Role of domain interactions in the aggregation of full-length immunoglobulin light chains.

Amyloid light-chain (LC) amyloidosis is a protein misfolding disease in which the aggregation of an overexpressed antibody LC from a clonal plasma cell leads to organ toxicity and patient death if left untreated. While the overall dimeric architecture of LC molecules is established, with each LC composed of variable (VL) and constant (CL) domains, the relative contributions of LC domain-domain interfaces and intrinsic domain stabilities to protection against LC aggregation are not well understood. To address these topics we have engineered a number of domain-destabilized LC mutants and used solution NMR spectroscopy to characterize their structural properties and intrinsic stabilities. Moreover, we used fluorescence spectroscopy to assay their aggregation propensities. Our results point to the importance of both dimerization strength and intrinsic monomer stability in stabilizing VL domains against aggregation. Notably, in all cases considered VL domains aggregate at least 10-fold faster than full-length LCs, establishing the important protective role of CL domains. A strong protective coupling is found between VL-VL and CL-CL dimer interfaces, with destabilization of one interface adversely affecting the stability of the other. Fibril formation is observed when either the VL or CL domain in the full-length protein is severely destabilized (i.e., where domain unfolding free energies are less than 2 kcal/mol). The important role of CL domains in preventing aggregation highlights the potential of the CL-CL interface as a target for the development of drugs to stabilize the dimeric LC structure and hence prevent LC amyloidosis.

Exploiting conformational plasticity in the AAA+ protein VCP/p97 to modify function.

p97/VCP, a member of the AAA+ (ATPases associated with diverse cellular activities) family of proteins, is implicated in the etiology of a group of degenerative diseases affecting bone and muscle tissue as well as the central nervous system. Methyl-TROSY-based NMR studies have previously revealed how disease-causing mutations deregulate a subtle dynamic conformational equilibrium involving the N-terminal domain (NTD) with implications for the binding of certain adaptors, providing insight into how disease mutations lead to abnormal function. Herein the conformational plasticity of the p97 system is explored in an attempt to identify hotspots that can serve as targets for restoring function in disease mutants by shifting the position of the NTD back to its wild-type location. Although p97 is overall robust with respect to extensive mutagenesis throughout the protein involving conservative substitutions of hydrophobic residues, key positions have been identified that alter the NTD equilibrium; these lie in specific regions that localize to the interface between the NTD and the D1 nucleotide-binding domain of the complex. Notably, for a severe disease mutant involving an R155C substitution the NTD equilibrium can be shifted back to its wild-type position by mutation at a secondary site with restoration of wild-type two-pronged binding of the UBXD1 adaptor protein that is impaired in disease; this underlies the potential for recovering function by targeting p97 disease mutants with drug molecules.

RNA binding and chaperone activity of the E. coli cold-shock protein CspA.

Ensuring the correct folding of RNA molecules in the cell is of major importance for a large variety of biological functions. Therefore, chaperone proteins that assist RNA in adopting their functionally active states are abundant in all living organisms. An important feature of RNA chaperone proteins is that they do not require an external energy source to perform their activity, and that they interact transiently and non-specifically with their RNA targets. So far, little is known about the mechanistic details of the RNA chaperone activity of these proteins. Prominent examples of RNA chaperones are bacterial cold shock proteins (Csp) that have been reported to bind single-stranded RNA and DNA. Here, we have used advanced NMR spectroscopy techniques to investigate at atomic resolution the RNA-melting activity of CspA, the major cold shock protein of Escherichia coli, upon binding to different RNA hairpins. Real-time NMR provides detailed information on the folding kinetics and folding pathways. Finally, comparison of wild-type CspA with single-point mutants and small peptides yields insights into the complementary roles of aromatic and positively charged amino-acid side chains for the RNA chaperone activity of the protein.

Self-Assembly of Human Profilin-1 Detected by Carr-Purcell-Meiboom-Gill Nuclear Magnetic Resonance (CPMG NMR) Spectroscopy.

Protein oligomerization in the cell has important implications for both health and disease, and an understanding of the mechanisms by which proteins can self-associate is, therefore, of critical interest. Initial stages of the oligomerization process can be hard to detect, as they often involve the formation of sparsely populated and transient states that are difficult to characterize by standard biophysical approaches. Using relaxation dispersion nuclear magnetic resonance spectroscopy, we study the oligomerization of human profilin-1, a protein that regulates the polymerization of actin. We show that in solution and at millimolar concentrations profilin-1 is predominantly monomeric. However, fits of concentration-dependent relaxation data are consistent with the formation of a higher-order oligomer that is generated via a multistep process. Together with crystallographic data for profilin-2, a homologue of the protein studied here, our results suggest that profilin-1 forms a sparsely populated tetrameric conformer in solution.

Quantitative measurement of exchange dynamics in proteins via (13)C relaxation dispersion of (13)CHD2-labeled samples.

Methyl groups have emerged as powerful probes of protein dynamics with timescales from picoseconds to seconds. Typically, studies involving high molecular weight complexes exploit (13)CH3- or (13)CHD2-labeling in otherwise highly deuterated proteins. The (13)CHD2 label offers the unique advantage of providing (13)C, (1)H and (2)H spin probes, however a disadvantage has been the lack of an experiment to record (13)C Carr-Purcell-Meiboom-Gill relaxation dispersion that monitors millisecond time-scale dynamics, implicated in a wide range of biological processes. Herein we develop an experiment that eliminates artifacts that would normally result from the scalar coupling between (13)C and (2)H spins that has limited applications in the past. The utility of the approach is established with a number of applications, including measurement of ms dynamics of a disease mutant of a 320 kDa p97 complex.

ClpB N-terminal domain plays a regulatory role in protein disaggregation.

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.

(13)CHD2-CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins.

An NMR experiment for quantifying slow (millisecond) time-scale exchange processes involving the interconversion between visible ground state and invisible, conformationally excited state conformers is presented. The approach exploits chemical exchange saturation transfer (CEST) and makes use of (13)CHD2 methyl group probes that can be readily incorporated into otherwise highly deuterated proteins. The methodology is validated with an application to a G48A Fyn SH3 domain that exchanges between a folded conformation and a sparsely populated and transiently formed unfolded ensemble. Experiments on a number of different protein systems, including a 360 kDa half-proteasome, establish that the sensitivity of this (13)CHD2 (13)C-CEST technique can be upwards of a factor of 5 times higher than for a previously published (13)CH3 (13)C-CEST approach (Bouvignies and Kay in J Biomol NMR 53:303-310, 2012), suggesting that the methodology will be powerful for studies of conformational exchange in high molecular weight proteins.

Measuring hydrogen exchange in proteins by selective water saturation in (1)H- (15)N SOFAST/BEST-type experiments: advantages and limitations.

HET(ex)-SOFAST NMR (Schanda et al. in J Biomol NMR 33:199-211, 2006) has been proposed some years ago as a fast and sensitive method for semi-quantitative measurement of site-specific amide-water hydrogen exchange effects along the backbone of proteins. Here we extend this concept to BEST readout sequences that provide a better resolution at the expense of some loss in sensitivity. We discuss the theoretical background and implementation of the experiment, and demonstrate its performance for an intrinsically disordered protein, 2 well folded globular proteins, and a transiently populated folding intermediate state. We also provide a critical evaluation of the level of accuracy that can be obtained when extracting quantitative exchange rates from HET(ex) NMR measurements.