Quantitative Characterization of Chain-Flipping of Acyl Carrier Protein of Escherichia coli Using Chemical Exchange NMR.

The acyl carrier protein of Escherichia coli, termed AcpP, is a prototypical example of type II fatty acid synthase systems found in many bacteria. It serves as a central hub by accepting diverse acyl moieties (4-18 carbons) and shuttling them between its multiple enzymatic partners to generate fatty acids. Prior structures of acyl-AcpPs established that thioester-linked acyl cargos are sequestered within AcpP's hydrophobic lumen. In contrast, structures of enzyme-bound acyl-AcpPs showed translocation of AcpP-tethered acyl chains into the active sites of enzymes. The mechanistic underpinnings of this conformational interplay, termed chain-flipping, are unclear. Here, using heteronuclear NMR spectroscopy, we reveal that AcpP-tethered acyl chains (6-10 carbons) spontaneously adopt lowly populated solvent-exposed conformations. To this end, we devised a new strategy to replace AcpP's thioester linkages with 15N-labeled amide bonds, which facilitated direct "visualization" of these excited states using NMR chemical exchange saturation transfer and relaxation dispersion measurements. Global fitting of the corresponding data yielded kinetic rate constants of the underlying equilibrium and populations and lifetimes of solvent-exposed states. The latter were influenced by acyl chain composition and ranged from milliseconds to submilliseconds for chains containing six, eight, and ten carbons, owing to their variable interactions with AcpP's hydrophobic core. Although transient, the exposure of AcpP-tethered acyl chains to the solvent may allow relevant enzymes to gain access to its active thioester, and the enzyme-induced selection of this conformation will culminate in the production of fatty acids.

Phase Separation and Fibrillization of Human Annexin A7 Are Mediated by Its Proline-Rich Domain.

Human annexin A7, a calcium- and phospholipid-binding protein, governs calcium homeostasis, plasma membrane repair, apoptosis, and tumor progression. A7 contains an N-terminal proline-rich domain (PRD; 180 residues, ∼24% prolines) that determines its functional specificity. Using microscopy and dye-binding assays, we show that recombinant A7 and its isolated PRD spontaneously phase separate into spherical condensates, which subsequently transform into ß-sheet-rich fibrils. We demonstrate that fibrillization of A7-PRD proceeds via primary nucleation and fibril-catalyzed secondary nucleation processes, as determined by chemical kinetics, providing a mechanistic basis for its amyloid assembly. This study confirms and highlights a subclass of eukaryotic PRDs prone to forming aggregates with important physiological and pathological implications.

Avidity-Based Method for the Efficient Generation of Monoubiquitinated Recombinant Proteins.

Monoubiquitination of proteins governs diverse physiological processes, and its dysregulation is implicated in multiple pathologies. The difficulty of preparing sufficient material often complicates the biophysical studies of monoubiquitinated recombinant proteins. Here we describe a robust avidity-based method that overcomes this problem. As a proof-of-concept, we produced milligram quantities of two monoubiquitinated targets, Parkinson's protein α-synuclein and ESCRT-protein ALIX, using NEDD4-family E3 ligases. Monoubiquitination hotspots were identified by quantitative chemical proteomics. Using FRAP and dye-binding assays, we uncovered strikingly opposite effects of monoubiquitination on the phase separation and fibrillization properties of these two amyloidogenic proteins, reflecting differences in their intermolecular interactions, thereby providing unique insights into the impact of monoubiquitination on protein aggregation.

Proline-rich domain of human ALIX contains multiple TSG101-UEV interaction sites and forms phosphorylation-mediated reversible amyloids.

Proline-rich domains (PRDs) are among the most prevalent signaling modules of eukaryotes but often unexplored by biophysical techniques as their heterologous recombinant expression poses significant difficulties. Using a "divide-and-conquer" approach, we present a detailed investigation of a PRD (166 residues; ∼30% prolines) belonging to a human protein ALIX, a versatile adaptor protein involved in essential cellular processes including ESCRT-mediated membrane remodeling, cell adhesion, and apoptosis. In solution, the N-terminal fragment of ALIX-PRD is dynamically disordered. It contains three tandem sequentially similar proline-rich motifs that compete for a single binding site on its signaling partner, TSG101-UEV, as evidenced by heteronuclear NMR spectroscopy. Global fitting of relaxation dispersion data, measured as a function of TSG101-UEV concentration, allowed precise quantitation of these interactions. In contrast to the soluble N-terminal portion, the C-terminal tyrosine-rich fragment of ALIX-PRD forms amyloid fibrils and viscous gels validated using dye-binding assays with amyloid-specific probes, congo red and thioflavin T (ThT), and visualized by transmission electron microscopy. Remarkably, fibrils dissolve at low temperatures (2 to 6 °C) or upon hyperphosphorylation with Src kinase. Aggregation kinetics monitored by ThT fluorescence shows that charge repulsion dictates phosphorylation-mediated fibril dissolution and that the hydrophobic effect drives fibril formation. These data illuminate the mechanistic interplay between interactions of ALIX-PRD with TSG101-UEV and polymerization of ALIX-PRD and its central role in regulating ALIX function. This study also demonstrates the broad functional repertoires of PRDs and uncovers the impact of posttranslational modifications in the modulation of reversible amyloids.

Mechanistic roles of tyrosine phosphorylation in reversible amyloids, autoinhibition, and endosomal membrane association of ALIX.

Human apoptosis-linked gene-2 interacting protein X (ALIX), a versatile adapter protein, regulates essential cellular processes by shuttling between late endosomal membranes and the cytosol, determined by its interactions with Src kinase. Here, we investigate the molecular basis of these transitions and the effects of tyrosine phosphorylation on the interplay between structure, assembly, and intramolecular and intermolecular interactions of ALIX. As evidenced by transmission electron microscopy, fluorescence and circular dichroism spectroscopy, the proline-rich domain of ALIX, which encodes binding epitopes of multiple cellular partners, formed rope-like ß-sheet-rich reversible amyloid fibrils that dissolved upon Src-mediated phosphorylation and were restored on protein-tyrosine phosphatase 1B-mediated dephosphorylation of its conserved tyrosine residues. Analyses of the Bro1 domain of ALIX by solution NMR spectroscopy elucidated the conformational changes originating from its phosphorylation by Src and established that Bro1 binds to hyperphosphorylated proline-rich domain and to analogs of late endosomal membranes via its highly basic surface. These results uncover the autoinhibition mechanism that relocates ALIX to the cytosol and the diverse roles played by tyrosine phosphorylation in cellular and membrane functions of ALIX.

Quantitative NMR Study of Insulin-Degrading Enzyme Using Amyloid-ß and HIV-1 p6 Elucidates Its Chaperone Activity.

Insulin-degrading enzyme (IDE) hydrolyzes monomeric polypeptides, including amyloid-ß (Aß) and HIV-1 p6. It also acts as a nonproteolytic chaperone to prevent Aß polymerization. Here we compare interactions of Aß and non-amyloidogenic p6 with IDE. Although both exhibited similar proteolysis rates, the binding kinetics to an inactive IDE characterized using relaxation-based NMR were remarkably different. IDE and Aß formed a sparsely populated complex with a lifetime of milliseconds in which a short hydrophobic cleavage segment of Aß was anchored to IDE. Strikingly, a second and more stable complex was significantly populated with a subsecond lifetime owing to multiple intermolecular contacts between Aß and IDE. By selectively sequestering Aß in this nonproductive complex, IDE likely increases the critical concentration required for fibrillization. In contrast, IDE and p6 formed a transient, submillisecond complex involving a single anchoring p6 motif. Modulation of intermolecular interactions, thus, allows IDE to differentiate between non-amyloidogenic and amyloidogenic substrates.

Binding kinetics and substrate selectivity in HIV-1 protease-Gag interactions probed at atomic resolution by chemical exchange NMR.

The conversion of immature noninfectious HIV-1 particles to infectious virions is dependent upon the sequential cleavage of the precursor group-specific antigen (Gag) polyprotein by HIV-1 protease. The precise mechanism whereby protease recognizes distinct Gag cleavage sites, located in the intrinsically disordered linkers connecting the globular domains of Gag, remains unclear. Here, we probe the dynamics of the interaction of large fragments of Gag and various variants of protease (including a drug resistant construct) using Carr-Purcell-Meiboom-Gill relaxation dispersion and chemical exchange saturation transfer NMR experiments. We show that the conformational dynamics within the flaps of HIV-1 protease that form the lid over the catalytic cleft play a significant role in substrate specificity and ordered Gag processing. Rapid interconversion between closed and open protease flap conformations facilitates the formation of a transient, sparsely populated productive complex between protease and Gag substrates. Flap closure traps the Gag cleavage sites within the catalytic cleft of protease. Modulation of flap opening through protease-Gag interactions fine-tunes the lifetime of the productive complex and hence the likelihood of Gag proteolysis. A productive complex can also be formed in the presence of a noncognate substrate but is short-lived owing to lack of optimal complementarity between the active site cleft of protease and the substrate, resulting in rapid flap opening and substrate release, thereby allowing protease to differentiate between cognate and noncognate substrates.

Inhibition of HIV Maturation via Selective Unfolding and Cross-Linking of Gag Polyprotein by a Mercaptobenzamide Acetylator.

For HIV to become infectious, any new virion produced from an infected cell must undergo a maturation process that involves the assembly of viral polyproteins Gag and Gag-Pol at the membrane surface. The self-assembly of these viral proteins drives formation of a new viral particle as well as the activation of HIV protease, which is needed to cleave the polyproteins so that the final core structure of the virus will properly form. Molecules that interfere with HIV maturation will prevent any new virions from infecting additional cells. In this manuscript, we characterize the unique mechanism by which a mercaptobenzamide thioester small molecule (SAMT-247) interferes with HIV maturation via a series of selective acetylations at highly conserved cysteine and lysine residues in Gag and Gag-Pol polyproteins. The results provide the first insights into how acetylation can be utilized to perturb the process of HIV maturation and reveal a new strategy to limit the infectivity of HIV.

Transient HIV-1 Gag-protease interactions revealed by paramagnetic NMR suggest origins of compensatory drug resistance mutations.

Cleavage of the group-specific antigen (Gag) polyprotein by HIV-1 protease represents the critical first step in the conversion of immature noninfectious viral particles to mature infectious virions. Selective pressure exerted by HIV-1 protease inhibitors, a mainstay of current anti-HIV-1 therapies, results in the accumulation of drug resistance mutations in both protease and Gag. Surprisingly, a large number of these mutations (known as secondary or compensatory mutations) occur outside the active site of protease or the cleavage sites of Gag (located within intrinsically disordered linkers connecting the globular domains of Gag to one another), suggesting that transient encounter complexes involving the globular domains of Gag may play a role in guiding and facilitating access of the protease to the Gag cleavage sites. Here, using large fragments of Gag, as well as catalytically inactive and active variants of protease, we probe the nature of such rare encounter complexes using intermolecular paramagnetic relaxation enhancement, a highly sensitive technique for detecting sparsely populated states. We show that Gag-protease encounter complexes are primarily mediated by interactions between protease and the globular domains of Gag and that the sites of transient interactions are correlated with surface exposed regions that exhibit a high propensity to mutate in the presence of HIV-1 protease inhibitors.

Conformation and dynamics of the Gag polyprotein of the human immunodeficiency virus 1 studied by NMR spectroscopy.

Assembly and maturation of the human immunodeficiency virus type 1 (HIV-1) are governed by the Gag polyprotein. Here we study the conformation and dynamics of a large HIV-1 Gag fragment comprising the matrix, capsid, spacer peptide 1 and nucleocapsid domains (referred to as ΔGag) by heteronuclear multidimensional NMR spectroscopy. In solution, ΔGag exists in a dynamic equilibrium between monomeric and dimeric states. In the presence of nucleic acids and at low ionic strength ΔGag assembles into immature virus-like particles. The structured domains of ΔGag (matrix, the N- and C-terminal domains of capsid, and the N- and C-terminal zinc knuckles of nucleocapsid) retain their fold and reorient semi-independently of one another; the linkers connecting the structural domains, including spacer peptide 1 that connects capsid to nucleocapsid, are intrinsically disordered. Structural changes in ΔGag upon proteolytic processing by HIV-1 protease, monitored by NMR in real-time, demonstrate that the conformational transition of the N-terminal 13 residues of capsid from an intrinsically disordered coil to a ß-hairpin upon cleavage at the matrix|capsid junction occurs five times faster than cleavage at the capsid|spacer peptide 1 junction. Finally, nucleic acids interact with both nucleocapsid and matrix domains, and proteolytic processing at the spacer peptide 1|nucleocapsid junction by HIV-1 protease is accelerated in the presence of single-stranded DNA.

Targeting a Dark Excited State of HIV-1 Nucleocapsid by Antiretroviral Thioesters Revealed by NMR Spectroscopy.

HIV-1 nucleocapsid (NCp7) is a two Cys2 HisCys zinc knuckle (N-Zn and C-Zn) protein that plays a key role in viral replication. NCp7 conformational dynamics is characterized by NMR relaxation dispersion and chemical exchange saturation transfer measurements. While the N-Zn knuckle is conformationally stable, the C-Zn knuckle interconverts on the millisecond timescale between the major state, in which the zinc is coordinated by three cysteines and a histidine, and two folded minor species (with populations around 1 %) in which one of the coordination bonds (Cys413-Sγ-Zn or His421-Nϵ2-Zn) is hydrolyzed. These findings explain why antiretroviral thioesters specifically disrupt the C-Zn knuckle by initial acylation of Cys413, and show that transient, sparsely-populated ("dark"), excited states of proteins can present effective targets for rational drug design.

Quantitative Characterization of Configurational Space Sampled by HIV-1 Nucleocapsid Using Solution NMR, X-ray Scattering and Protein Engineering.

Nucleic-acid-related events in the HIV-1 replication cycle are mediated by nucleocapsid, a small protein comprising two zinc knuckles connected by a short flexible linker and flanked by disordered termini. Combining experimental NMR residual dipolar couplings, solution X-ray scattering and protein engineering with ensemble simulated annealing, we obtain a quantitative description of the configurational space sampled by the two zinc knuckles, the linker and disordered termini in the absence of nucleic acids. We first compute the conformational ensemble (with an optimal size of three members) of an engineered nucleocapsid construct lacking the N- and C-termini that satisfies the experimental restraints, and then validate this ensemble, as well as characterize the disordered termini, using the experimental data from the full-length nucleocapsid construct. The experimental and computational strategy is generally applicable to multidomain proteins. Differential flexibility within the linker results in asymmetric motion of the zinc knuckles which may explain their functionally distinct roles despite high sequence identity. One of the configurations (populated at a level of ≈40 %) closely resembles that observed in various ligand-bound forms, providing evidence for conformational selection and a mechanistic link between protein dynamics and function.

Mutations Proximal to Sites of Autoproteolysis and the α-Helix That Co-evolve under Drug Pressure Modulate the Autoprocessing and Vitality of HIV-1 Protease.

N-Terminal self-cleavage (autoprocessing) of the HIV-1 protease precursor is crucial for liberating the active dimer. Under drug pressure, evolving mutations are predicted to modulate autoprocessing, and the reduced catalytic activity of the mature protease (PR) is likely compensated by enhanced conformational/dimer stability and reduced susceptibility to self-degradation (autoproteolysis). One such highly evolved, multidrug resistant protease, PR20, bears 19 mutations contiguous to sites of autoproteolysis in retroviral proteases, namely clusters 1-3 comprising residues 30-37, 60-67, and 88-95, respectively, accounting for 11 of the 19 mutations. By systematically replacing corresponding clusters in PR with those of PR20, and vice versa, we assess their influence on the properties mentioned above and observe no strict correlation. A 10-35-fold decrease in the cleavage efficiency of peptide substrates by PR20, relative to PR, is reflected by an only ∼4-fold decrease in the rate of Gag processing with no change in cleavage order. Importantly, optimal N-terminal autoprocessing requires all 19 PR20 mutations as evaluated in vitro using the model precursor TFR-PR20 in which PR is flanked by the transframe region. Substituting PR20 cluster 3 into TFR-PR (TFR-PR(PR20-3)) requires the presence of PR20 cluster 1 and/or 2 for autoprocessing. In accordance, substituting PR clusters 1 and 2 into TFR-PR20 affects the rate of autoprocessing more drastically (>300-fold) compared to that of TFR-PR(PR20-3) because of the cumulative effect of eight noncluster mutations present in TFR-PR20(PR-12). Overall, these studies imply that drug resistance involves a complex synchronized selection of mutations modulating all of the properties mentioned above governing PR regulation and function.

Skelemin association with αIIbß3 integrin: a structural model.

Over the last two decades, our knowledge concerning intracellular events that regulate integrin's affinity to their soluble ligands has significantly improved. However, the mechanism of adhesion-induced integrin clustering and development of focal complexes, which could further mature to form focal adhesions, still remains under-investigated. Here we present a structural model of tandem IgC2 domains of skelemin in complex with the cytoplasmic tails of integrin αIIbß3. The model of tertiary assembly is generated based upon NMR data and illuminates a potential link between the essential cell adhesion receptors and myosin filaments. This connection may serve as a basis for generating the mechanical forces necessary for cell migration and remodeling.

Investigation of the structure and dynamics of the capsid-spacer peptide 1-nucleocapsid fragment of the HIV-1 gag polyprotein by solution NMR spectroscopy.

Structural studies of HIV-1 Gag, the primary structural polyprotein involved in retroviral assembly, have been challenging, owing to its flexibility and conformational heterogeneity. Using residual dipolar couplings, we show that the four structural units of the capsid (CA)-spacer peptide 1 (SP1)-nucleocapsid (NC) fragment of HIV-1 Gag (namely, the N- and C-terminal domains of capsid, and the N- and C-terminal Zn knuckles of nucleocapsid) have the same structures as their individually isolated counterparts, and tumble semi-independently of one another in the absence of nucleic acids. Nucleic acids bind exclusively to the nucleocapsid domain and fix the orientation of the two Zn knuckles relative to one another so that the nucleocapsid domain/nucleic acid complex behaves as a single structural unit. The low (15) N-{(1) H} heteronuclear NOE values (≤0.4), the close to zero values for the residual dipolar couplings of the backbone amides, and minimal deviations from random-coil chemical shifts for the C-terminal tail of capsid and SP1, both in the absence and presence of nucleic acids, indicate that these regions are intrinsically disordered in the context of CA-SP1-NC.

Structure and dynamics of full-length HIV-1 capsid protein in solution.

The HIV-1 capsid protein plays a crucial role in viral infectivity, assembling into a cone that encloses the viral RNA. In the mature virion, the N-terminal domain of the capsid protein forms hexameric and pentameric rings, while C-terminal domain homodimers connect adjacent N-terminal domain rings to one another. Structures of disulfide-linked hexamer and pentamer assemblies, as well as structures of the isolated domains, have been solved previously. The dimer configuration in C-terminal domain constructs differs in solution (residues 144-231) and crystal (residues 146-231) structures by ∼30°, and it has been postulated that the former connects the hexamers while the latter links pentamers to hexamers. Here we study the structure and dynamics of full-length capsid protein in solution, comprising a mixture of monomeric and dimeric forms in dynamic equilibrium, using ensemble simulated annealing driven by experimental NMR residual dipolar couplings and X-ray scattering data. The complexity of the system necessitated the development of a novel computational framework that should be generally applicable to many other challenging systems that currently escape structural characterization by standard application of mainstream techniques of structural biology. We show that the orientation of the C-terminal domains in dimeric full-length capsid and isolated C-terminal domain constructs is the same in solution, and we obtain a quantitative description of the conformational space sampled by the N-terminal domain relative to the C-terminal domain on the nano- to millisecond time scale. The positional distribution of the N-terminal domain relative to the C-terminal domain is large and modulated by the oligomerization state of the C-terminal domain. We also show that a model of the hexamer/pentamer assembly can be readily generated with a single configuration of the C-terminal domain dimer, and that capsid assembly likely proceeds via conformational selection of sparsely populated configurations of the N-terminal domain within the capsid protein dimer.

ALS Variants of Annexin A11's Proline-Rich Domain Impair Its S100A6-Mediated Fibril Dissolution.

Mutations in the proline-rich domain (PRD) of annexin A11 are linked to amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, and generate abundant neuronal A11 inclusions by an unknown mechanism. Here, we demonstrate that recombinant A11-PRD and its ALS-associated variants form liquidlike condensates that transform into ß-sheet-rich amyloid fibrils. Surprisingly, these fibrils dissolved in the presence of S100A6, an A11 binding partner overexpressed in ALS. The ALS variants of A11-PRD showed longer fibrillization half-times and slower dissolution, even though their binding affinities for S100A6 were not significantly affected. These findings indicate a slower fibril-to-monomer exchange for these ALS variants, resulting in a decreased level of S100A6-mediated fibril dissolution. These ALS-A11 variants are thus more likely to remain aggregated despite their slower fibrillization.

Reversible phase separation of ESCRT protein ALIX through tyrosine phosphorylation.

Cytokinetic abscission, the last step of cell division, is regulated by the ESCRT machinery. In response to mitotic errors, ESCRT proteins, namely, ALIX, CHMP4B, and CHMP4C, accumulate in the cytosolic compartments termed "abscission checkpoint bodies" (ACBs) to delay abscission and prevent tumorigenesis. ALIX contributes to the biogenesis and stability of ACBs via an unknown mechanism. We show that ALIX phase separates into nondynamic condensates in vitro and in vivo, mediated by the amyloidogenic portion of its proline-rich domain. ALIX condensates confined CHMP4 paralogs in vitro. These condensates dissolved and reformed upon reversible tyrosine phosphorylation of ALIX, mediated by Src kinase and PTP1B, and sequestration of CHMP4C altered their Src-mediated dissolution. NMR analysis revealed how ALIX triggers the activation of CHMP4 proteins, which is required for successful abscission. These results implicate ALIX's phase separation in the modulation of ACBs. This study also highlights how posttranslational modifications can control protein phase separation.

Tyrosine phosphorylation as a conformational switch: a case study of integrin ß3 cytoplasmic tail.

Reversible protein phosphorylation is vital for many fundamental cellular processes. The actual impact of adding and removing phosphate group(s) is 3-fold: changes in the local/global geometry, alterations in the electrostatic potential and, as the result of both, modified protein-target interactions. Here we present a comprehensive structural investigation of the effects of phosphorylation on the conformational as well as functional states of a crucial cell surface receptor, α(IIb)ß(3) integrin. We have analyzed phosphorylated (Tyr(747) and Tyr(759)) ß(3) integrin cytoplasmic tail (CT) primarily by NMR, and our data demonstrate that under both aqueous and membrane-mimetic conditions, phosphorylation causes substantial conformational rearrangements. These changes originate from novel ionic interactions and revised phospholipid binding. Under aqueous conditions, the critical Tyr(747) phosphorylation prevents ß(3)CT from binding to its heterodimer partner α(IIb)CT, thus likely maintaining an activated state of the receptor. This conclusion was tested in vivo and confirmed by integrin-dependent endothelial cells adhesion assay. Under membrane-mimetic conditions, phosphorylation results in a modified membrane embedding characterized by significant changes in the secondary structure pattern and the overall fold of ß(3)CT. Collectively these data provide unique molecular insights into multiple regulatory roles of phosphorylation.

Structural studies of a signal peptide in complex with signal peptidase I cytoplasmic domain: the stabilizing effect of membrane-mimetics on the acquired fold.

A protein destined for export from the cell cytoplasm is synthesized as a preprotein with an amino-terminal signal peptide. In Escherichia coli, typically signal peptides that guide preproteins into the SecYEG protein conduction channel are subsequently removed by signal peptidase I. To understand the mechanism of this critical step, we have assessed the conformation of the signal peptide when bound to signal peptidase by solution nuclear magnetic resonance. We employed a soluble form of signal peptidase, which laks the two transmembrane domains (SPase I Δ2-75), and the E. coli alkaline phosphatase signal peptide. Using a transferred NOE approach, we found clear evidence of a weak peptide-enzyme complex formation. The peptide adopts a U-turn shape originating from the proline residues within the primary sequence that is stabilized by its interaction with the peptidase and leaves key residues of the cleavage region exposed for proteolysis. In dodecylphosphocholine (DPC) micelles the signal peptide also adopts a U-turn shape comparable with that observed in association with the enzyme. In both environments this conformation is stabilized by the signal peptide phenylalanine side chain-interaction with enzyme or lipid mimetic. Moreover, in the presence of DPC, the N-terminal core region residues of the peptide adopt a helical motif and based on PRE (paramagnetic relaxation enhancement) experiments are shown to be buried within the membrane. Taken together, this is consistent with proteolysis of the preprotein occurring while the signal peptide remains in the bilayer and the enzyme active site functioning at the membrane surface.