Exploring Host-Guest Interactions within a 600 kDa DegP Protease Cage Complex Using Hydrodynamics Measurements and Methyl-TROSY NMR.

The DegP protease-chaperone operates within the periplasm of Gram-negative bacteria, where it assists in the regulation of protein homeostasis, promotes virulence, and is essential to survival under stress. To carry out these tasks, DegP forms a network of preorganized apo oligomers that facilitate the capture of substrates within distributions of cage-like complexes which expand to encapsulate clients of various sizes. Although the architectures of DegP cage complexes are well understood, little is known about the structures, dynamics, and interactions of client proteins within DegP cages and the relationship between client structural dynamics and function. Here, we probe host-guest interactions within a 600 kDa DegP cage complex throughout the DegP activation cycle using a model α-helical client protein through a combination of hydrodynamics measurements, methyl-transverse relaxation optimized spectroscopy-based solution nuclear magnetic resonance studies, and proteolytic activity assays. We find that in the presence of the client, DegP cages assemble cooperatively with few intermediates. Our data further show that the N-terminal half of the bound client, which projects into the interior of the cages, is predominantly unfolded and flexible, and exchanges between multiple conformational states over a wide range of time scales. Finally, we show that a concerted structural transition of the protease domains of DegP occurs upon client engagement, leading to activation. Together, our findings support a model of DegP as a highly cooperative and dynamic molecular machine that stabilizes unfolded states of clients, primarily via interactions with their C-termini, giving rise to efficient cleavage.

Poly(ADP-ribosyl)ation enhances nucleosome dynamics and organizes DNA damage repair components within biomolecular condensates.

Nucleosomes, the basic structural units of chromatin, hinder recruitment and activity of various DNA repair proteins, necessitating modifications that enhance DNA accessibility. Poly(ADP-ribosyl)ation (PARylation) of proteins near damage sites is an essential initiation step in several DNA-repair pathways; however, its effects on nucleosome structural dynamics and organization are unclear. Using NMR, cryoelectron microscopy (cryo-EM), and biochemical assays, we show that PARylation enhances motions of the histone H3 tail and DNA, leaving the configuration of the core intact while also stimulating nuclease digestion and ligation of nicked nucleosomal DNA by LIG3. PARylation disrupted interactions between nucleosomes, preventing self-association. Addition of LIG3 and XRCC1 to PARylated nucleosomes generated condensates that selectively partition DNA repair-associated proteins in a PAR- and phosphorylation-dependent manner in vitro. Our results establish that PARylation influences nucleosomes across different length scales, extending from the atom-level motions of histone tails to the mesoscale formation of condensates with selective compositions.

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.

Flexible Client-Dependent Cages in the Assembly Landscape of the Periplasmic Protease-Chaperone DegP.

The periplasmic protein DegP, which is implicated in virulence factor transport leading to pathogenicity, is a bi-functional protease and chaperone that helps to maintain protein homeostasis in Gram-negative bacteria and is essential to bacterial survival under stress conditions. To perform these functions, DegP captures clients inside cage-like structures, which we have recently shown to form through the reorganization of high-order preformed apo oligomers, consisting of trimeric building blocks, that are structurally distinct from client-bound cages. Our previous studies suggested that these apo oligomers may allow DegP to encapsulate clients of various sizes under protein folding stresses by forming ensembles that can include extremely large cage particles, but how this occurs remains an open question. To explore the relation between cage and substrate sizes, we engineered a series of DegP clients of increasing hydrodynamic radii and analyzed their influence on DegP cage formation. We used dynamic light scattering and cryogenic electron microscopy to characterize the hydrodynamic properties and structures of the DegP cages that are adopted in response to each client. We present a series of density maps and structural models that include those for novel particles of approximately 30 and 60 monomers. Key interactions between DegP trimers and the bound clients that stabilize the cage assemblies and prime the clients for catalysis are revealed. We also provide evidence that DegP can form cages which approach subcellular organelles in terms of size.

Correlating histone acetylation with nucleosome core particle dynamics and function.

Epigenetic modifications of chromatin play a critical role in regulating the fidelity of the genetic code and in controlling the translation of genetic information into the protein components of the cell. One key posttranslational modification is acetylation of histone lysine residues. Molecular dynamics simulations, and to a smaller extent experiment, have established that lysine acetylation increases the dynamics of histone tails. However, a systematic, atomic resolution experimental investigation of how this epigenetic mark, focusing on one histone at a time, influences the structural dynamics of the nucleosome beyond the tails, and how this translates into accessibility of protein factors such as ligases and nucleases, has yet to be performed. Herein, using NMR spectroscopy of nucleosome core particles (NCPs), we evaluate the effects of acetylation of each histone on tail and core dynamics. We show that for histones H2B, H3, and H4, the histone core particle dynamics are little changed, even though the tails have increased amplitude motions. In contrast, significant increases to H2A dynamics are observed upon acetylation of this histone, with the docking domain and L1 loop particularly affected, correlating with increased susceptibility of NCPs to nuclease digestion and more robust ligation of nicked DNA. Dynamic light scattering experiments establish that acetylation decreases inter-NCP interactions in a histone-dependent manner and facilitates the development of a thermodynamic model for NCP stacking. Our data show that different acetylation patterns result in nuanced changes to NCP dynamics, modulating interactions with other protein factors, and ultimately controlling biological output.

Less is more: A simple methyl-TROSY based pulse scheme offers improved sensitivity in applications to high molecular weight complexes.

The HMQC pulse sequence and variants thereof have been exploited in studies of high molecular weight protein complexes, taking advantage of the fact that fast and slow relaxing magnetization components are sequestered along two distinct magnetization transfer pathways. Despite the simplicity of the HMQC scheme an even shorter version can be designed, based on elimination of the terminal refocusing period, as a further means of increasing signal. Here we present such an experiment, and show that significant sensitivity gains, in some cases by factors of two or more, are realized in studies of proteins varying in molecular masses from 100 kDa to 1 MDa.

Structural basis of protein substrate processing by human mitochondrial high-temperature requirement A2 protease.

The human high-temperature requirement A2 (HtrA2) protein is a trimeric protease that cleaves misfolded proteins to protect cells from stresses caused by toxic, proteinaceous aggregates, and the aberrant function of HtrA2 is closely related to the onset of neurodegenerative disorders. Our methyl-transverse relaxation optimized spectroscopy (TROSY)­based NMR studies using small-peptide ligands have previously revealed a stepwise activation mechanism involving multiple distinct conformational states. However, very little is known about how HtrA2 binds to protein substrates and if the distinct conformational states observed in previous peptide studies might be involved in the processing of protein clients. Herein, we use solution-based NMR spectroscopy to investigate the interaction between the N-terminal Src homology 3 domain from downstream of receptor kinase (drk) with an added C-terminal HtrA2-binding motif (drkN SH3-PDZbm) that exhibits marginal folding stability and serves as a mimic of a physiological protein substrate. We show that drkN SH3-PDZbm binds to HtrA2 via a two-pronged interaction, involving both its C-terminal PDZ-domain binding motif and a central hydrophobic region, with binding occurring preferentially via an unfolded ensemble of substrate molecules. Multivalent interactions between several clients and a single HtrA2 trimer significantly stimulate the catalytic activity of HtrA2, suggesting that binding avidity plays an important role in regulating substrate processing. Our results provide a thermodynamic, kinetic, and structural description of the interaction of HtrA2 with protein substrates and highlight the importance of a trimeric architecture for function as a stress-protective protease that mitigates aggregation.

Competing stress-dependent oligomerization pathways regulate self-assembly of the periplasmic protease-chaperone DegP.

DegP is an oligomeric protein with dual protease and chaperone activity that regulates protein homeostasis and virulence factor trafficking in the periplasm of gram-negative bacteria. A number of oligomeric architectures adopted by DegP are thought to facilitate its function. For example, DegP can form a "resting" hexamer when not engaged to substrates, mitigating undesired proteolysis of cellular proteins. When bound to substrate proteins or lipid membranes, DegP has been shown to populate a variety of cage- or bowl-like oligomeric states that have increased proteolytic activity. Though a number of DegP's substrate-engaged structures have been robustly characterized, detailed mechanistic information underpinning its remarkable oligomeric plasticity and the corresponding interplay between these dynamics and biological function has remained elusive. Here, we have used a combination of hydrodynamics and NMR spectroscopy methodologies in combination with cryogenic electron microscopy to shed light on the apo-DegP self-assembly mechanism. We find that, in the absence of bound substrates, DegP populates an ensemble of oligomeric states, mediated by self-assembly of trimers, that are distinct from those observed in the presence of substrate. The oligomeric distribution is sensitive to solution ionic strength and temperature and is shifted toward larger oligomeric assemblies under physiological conditions. Substrate proteins may guide DegP toward canonical cage-like structures by binding to these preorganized oligomers, leading to changes in conformation. The properties of DegP self-assembly identified here suggest that apo-DegP can rapidly shift its oligomeric distribution in order to respond to a variety of biological insults.

Dissecting the role of interprotomer cooperativity in the activation of oligomeric high-temperature requirement A2 protein.

The human high-temperature requirement A2 (HtrA2) mitochondrial protease is critical for cellular proteostasis, with mutations in this enzyme closely associated with the onset of neurodegenerative disorders. HtrA2 forms a homotrimeric structure, with each subunit composed of protease and PDZ (PSD-95, DLG, ZO-1) domains. Although we had previously shown that successive ligand binding occurs with increasing affinity, and it has been suggested that allostery plays a role in regulating catalysis, the molecular details of how this occurs have not been established. Here, we use cysteine-based chemistry to generate subunits in different conformational states along with a protomer mixing strategy, biochemical assays, and methyl-transverse relaxation optimized spectroscopy-based NMR studies to understand the role of interprotomer allostery in regulating HtrA2 function. We show that substrate binding to a PDZ domain of one protomer increases millisecond-to-microsecond timescale dynamics in neighboring subunits that prime them for binding substrate molecules. Only when all three PDZ-binding sites are substrate bound can the enzyme transition into an active conformation that involves significant structural rearrangements of the protease domains. Our results thus explain why when one (or more) of the protomers is fixed in a ligand-binding-incompetent conformation or contains the inactivating S276C mutation that is causative for a neurodegenerative phenotype in mouse models of Parkinson's disease, transition to an active state cannot be formed. In this manner, wild-type HtrA2 is only active when substrate concentrations are high and therefore toxic and unregulated proteolysis of nonsubstrate proteins can be suppressed.

Oligomeric assembly regulating mitochondrial HtrA2 function as examined by methyl-TROSY NMR.

Human High temperature requirement A2 (HtrA2) is a mitochondrial protease chaperone that plays an important role in cellular proteostasis and in regulating cell-signaling events, with aberrant HtrA2 function leading to neurodegeneration and parkinsonian phenotypes. Structural studies of the enzyme have established a trimeric architecture, comprising three identical protomers in which the active sites of each protease domain are sequestered to form a catalytically inactive complex. The mechanism by which enzyme function is regulated is not well understood. Using methyl transverse relaxation optimized spectroscopy (TROSY)-based solution NMR in concert with biochemical assays, a functional HtrA2 oligomerization/binding cycle has been established. In the absence of substrates, HtrA2 exchanges between a heretofore unobserved hexameric conformation and the canonical trimeric structure, with the hexamer showing much weaker affinity toward substrates. Both structures are substrate inaccessible, explaining their low basal activity in the absence of the binding of activator peptide. The binding of the activator peptide to each of the protomers of the trimer occurs with positive cooperativity and induces intrasubunit domain reorientations to expose the catalytic center, leading to increased proteolytic activity. Our data paint a picture of HtrA2 as a finely tuned, stress-protective enzyme whose activity can be modulated both by oligomerization and domain reorientation, with basal levels of catalysis kept low to avoid proteolysis of nontarget proteins.

Global multi-method analysis of interaction parameters for reversibly self-associating macromolecules at high concentrations.

Weak macromolecular interactions assume a dominant role in the behavior of highly concentrated solutions, and are at the center of a variety of fields ranging from colloidal chemistry to cell biology, neurodegenerative diseases, and manufacturing of protein drugs. They are frequently measured in different biophysical techniques in the form of second virial coefficients, and nonideality coefficients of sedimentation and diffusion, which may be related mechanistically to macromolecular distance distributions in solution and interparticle potentials. A problem arises for proteins where reversible self-association often complicates the concentration-dependent behavior, such that grossly inconsistent coefficients are measured in experiments based on different techniques, confounding quantitative conclusions. Here we present a global multi-method analysis that synergistically bridges gaps in resolution and sensitivity of orthogonal techniques. We demonstrate the method with a panel of monoclonal antibodies exhibiting different degrees of self-association. We show how their concentration-dependent behavior, examined by static and dynamic light scattering and sedimentation velocity, can be jointly described in a self-consistent framework that separates nonideality coefficients from self-association properties, and thereby extends the quantitative interpretation of nonideality coefficients to probe dynamics in highly concentrated protein solutions.

Parallel reaction pathways accelerate folding of a guanine quadruplex.

G-quadruplexes (G4s) are four-stranded, guanine-rich nucleic acid structures that can influence a variety of biological processes such as the transcription and translation of genes and DNA replication. In many cases, a single G4-forming nucleic acid sequence can adopt multiple different folded conformations that interconvert on biologically relevant timescales, entropically stabilizing the folded state. The coexistence of different folded conformations also suggests that there are multiple pathways leading from the unfolded to the folded state ensembles, potentially modulating the folding rate and biological activity. We have developed an experimental method for quantifying the contributions of individual pathways to the folding of conformationally heterogeneous G4s that is based on mutagenesis, thermal hysteresis kinetic experiments and global analysis, and validated our results using photocaged kinetic NMR experiments. We studied the regulatory Pu22 G4 from the c-myc oncogene promoter, which adopts at least four distinct folded isomers. We found that the presence of four parallel pathways leads to a 2.5-fold acceleration in folding; that is, the effective folding rate from the unfolded to folded ensembles is 2.5 times as large as the rate constant for the fastest individual pathway. Since many G4 sequences can adopt many more than four isomers, folding accelerations of more than an order of magnitude are possible via this mechanism.

Analyzing multi-step ligand binding reactions for oligomeric proteins by NMR: Theoretical and computational considerations.

Solution NMR spectroscopy is widely used to investigate the thermodynamics and kinetics of the binding of ligands to their biological receptors, as it provides detailed, atomistic information, potentially leading to microscopic affinities for each binding event, and, to the development of allosteric pathways describing how the binding at one site affects distal sites in the molecule. Importantly, weak interactions that are often invisible to other biophysical methods can also be probed. Methodological advancements in NMR have enabled the investigation of high molecular weight, homo-oligomeric complexes that bind multiple ligand molecules, with increasing numbers of studies of the structural dynamics and binding properties of these systems. It therefore becomes of interest to consider how binding and kinetics parameters can be extracted from experiments on these more complicated molecules. Here we present the theoretical framework for analyzing binding reactions of homo-oligomeric complexes by NMR, taking into account all of the chemical species in solution and their corresponding NMR observables. A number of simulations are presented to illustrate the utility of the derived expressions.

Conformational Dynamics of Strand Register Shifts in DNA G-Quadruplexes.

The complex folding energy landscape of DNA G-quadruplexes leads to numerous conformations for this functionally important class of noncanonical DNA structures. A new layer of conformational heterogeneity comes from sequences with different numbers of G-nucleotides in each of the DNA G-strands that form the four-stranded G-quartet core. The mechanisms by which G-quadruplexes transition from one folded conformation to another are currently unknown. To address this question, we studied two different G-quadruplexes, selecting a single conformation by blocking hydrogen bonding with photolabile protection groups. Upon irradiation, the block can be released and the kinetics of re-equilibration to the native conformational equilibrium can be determined by time-resolved NMR. We compared the NMR-derived refolding kinetics with data derived from thermal hysteresis folding kinetic experiments and found excellent agreement. The outlined methodological approach allows separation of K+-induced G-quadruplex formation and subsequent refolding and provides key insight into rate-limiting steps of G-quadruplex conformational dynamics.

G-quadruplex dynamics.

G-quadruplexes (GQs) are four-stranded nucleic acid secondary structures formed by guanosine (G)-rich DNA and RNA sequences. It is becoming increasingly clear that cellular processes including gene expression and mRNA translation are regulated by GQs. GQ structures have been extensively characterized, however little attention to date has been paid to their conformational dynamics, despite the fact that many biological GQ sequences populate multiple structures of similar free energies, leading to an ensemble of exchanging conformations. The impact of these dynamics on biological function is currently not well understood. Recently, structural dynamics have been demonstrated to entropically stabilize GQ ensembles, potentially modulating gene expression. Transient, low-populated states in GQ ensembles may additionally regulate nucleic acid interactions and function. This review will underscore the interplay of GQ dynamics and biological function, focusing on several dynamic processes for biological GQs and the characterization of GQ dynamics by nuclear magnetic resonance (NMR) spectroscopy in conjunction with other biophysical techniques. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.

Stabilization of i-motif structures by 2'-ß-fluorination of DNA.

i-Motifs are four-stranded DNA structures consisting of two parallel DNA duplexes held together by hemi-protonated and intercalated cytosine base pairs (C:CH(+)). They have attracted considerable research interest for their potential role in gene regulation and their use as pH responsive switches and building blocks in macromolecular assemblies. At neutral and basic pH values, the cytosine bases deprotonate and the structure unfolds into single strands. To avoid this limitation and expand the range of environmental conditions supporting i-motif folding, we replaced the sugar in DNA by 2-deoxy-2-fluoroarabinose. We demonstrate that such a modification significantly stabilizes i-motif formation over a wide pH range, including pH 7. Nuclear magnetic resonance experiments reveal that 2-deoxy-2-fluoroarabinose adopts a C2'-endo conformation, instead of the C3'-endo conformation usually found in unmodified i-motifs. Nevertheless, this substitution does not alter the overall i-motif structure. This conformational change, together with the changes in charge distribution in the sugar caused by the electronegative fluorine atoms, leads to a number of favorable sequential and inter-strand electrostatic interactions. The availability of folded i-motifs at neutral pH will aid investigations into the biological function of i-motifs in vitro, and will expand i-motif applications in nanotechnology.

G-register exchange dynamics in guanine quadruplexes.

G-quadruplexes (GQs) are 4-stranded DNA structures formed by tracts of stacked, Hoogsteen-hydrogen bonded guanosines. GQs are found in gene promoters and telomeres where they regulate gene transcription and telomere elongation. Though GQ structures are well-characterized, many aspects of their conformational dynamics are poorly understood. For example, when there are surplus guanosines in some of the tracts, they can slide with respect to one another, a process we term G-register (GR) exchange. These motions could in principle entropically stabilize the folded state, crucially benefitting GQs as their stabilities are closely tied to biological function. We have developed a method for characterizing GR exchange where each isomer in the wild-type conformational ensemble is trapped by mutation and thermal denaturation data for the set of trapped mutants and wild-type are analyzed simultaneously. This yields GR isomer populations as a function of temperature, quantifies conformational entropy and sheds light on correlated sliding motions of the G-tracts. We measured entropic stabilizations from GR exchange up to 14.3 ± 1.6 J mol(-1) K(-1), with melting temperature increases up to 7.3 ± 1.6°C. Furthermore, bioinformatic analysis suggests a majority of putative human GQ sequences are capable of GR exchange, pointing to the generality of this phenomenon.