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1.
Proc Natl Acad Sci U S A ; 118(37)2021 09 14.
Article in English | MEDLINE | ID: mdl-34504002

ABSTRACT

Intrinsically disordered proteins often form dynamic complexes with their ligands. Yet, the speed and amplitude of these motions are hidden in classical binding kinetics. Here, we directly measure the dynamics in an exceptionally mobile, high-affinity complex. We show that the disordered tail of the cell adhesion protein E-cadherin dynamically samples a large surface area of the protooncogene ß-catenin. Single-molecule experiments and molecular simulations resolve these motions with high resolution in space and time. Contacts break and form within hundreds of microseconds without a dissociation of the complex. The energy landscape of this complex is rugged with many small barriers (3 to 4 kBT) and reconciles specificity, high affinity, and extreme disorder. A few persistent contacts provide specificity, whereas unspecific interactions boost affinity.


Subject(s)
Antigens, CD/chemistry , Cadherins/chemistry , Intrinsically Disordered Proteins/chemistry , Protein Folding , beta Catenin/chemistry , Antigens, CD/metabolism , Cadherins/metabolism , Diffusion , Humans , Intrinsically Disordered Proteins/metabolism , Kinetics , Ligands , Molecular Dynamics Simulation , Protein Conformation , beta Catenin/metabolism
2.
Proc Natl Acad Sci U S A ; 118(28)2021 07 13.
Article in English | MEDLINE | ID: mdl-34244431

ABSTRACT

Dynamin oligomerizes into helical filaments on tubular membrane templates and, through constriction, cleaves them in a GTPase-driven way. Structural observations of GTP-dependent cross-bridges between neighboring filament turns have led to the suggestion that dynamin operates as a molecular ratchet motor. However, the proof of such mechanism remains absent. Particularly, it is not known whether a powerful enough stroke is produced and how the motor modules would cooperate in the constriction process. Here, we characterized the dynamin motor modules by single-molecule Förster resonance energy transfer (smFRET) and found strong nucleotide-dependent conformational preferences. Integrating smFRET with molecular dynamics simulations allowed us to estimate the forces generated in a power stroke. Subsequently, the quantitative force data and the measured kinetics of the GTPase cycle were incorporated into a model including both a dynamin filament, with explicit motor cross-bridges, and a realistic deformable membrane template. In our simulations, collective constriction of the membrane by dynamin motor modules, based on the ratchet mechanism, is directly reproduced and analyzed. Functional parallels between the dynamin system and actomyosin in the muscle are seen. Through concerted action of the motors, tight membrane constriction to the hemifission radius can be reached. Our experimental and computational study provides an example of how collective motor action in megadalton molecular assemblies can be approached and explicitly resolved.


Subject(s)
Dynamins/metabolism , Models, Biological , Biomechanical Phenomena , Dynamins/chemistry , Fluorescence Resonance Energy Transfer , Kinetics , Molecular Motor Proteins/chemistry , Molecular Motor Proteins/metabolism , Nucleotides/metabolism , Protein Domains , Protein Multimerization , Solutions
3.
Nat Commun ; 12(1): 2967, 2021 05 20.
Article in English | MEDLINE | ID: mdl-34016970

ABSTRACT

Allostery is a pervasive principle to regulate protein function. Growing evidence suggests that also DNA is capable of transmitting allosteric signals. Yet, whether and how DNA-mediated allostery plays a regulatory role in gene expression remained unclear. Here, we show that DNA indeed transmits allosteric signals over long distances to boost the binding cooperativity of transcription factors. Phenotype switching in Bacillus subtilis requires an all-or-none promoter binding of multiple ComK proteins. We use single-molecule FRET to demonstrate that ComK-binding at one promoter site increases affinity at a distant site. Cryo-EM structures of the complex between ComK and its promoter demonstrate that this coupling is due to mechanical forces that alter DNA curvature. Modifications of the spacer between sites tune cooperativity and show how to control allostery, which allows a fine-tuning of the dynamic properties of genetic circuits.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/genetics , DNA, Bacterial/chemistry , Gene Expression Regulation, Bacterial , Transcription Factors/genetics , Allosteric Regulation/genetics , Binding Sites/genetics , DNA, Bacterial/genetics , Gene Regulatory Networks , Nucleic Acid Conformation , Phenotype , Promoter Regions, Genetic/genetics
4.
Angew Chem Int Ed Engl ; 59(43): 19121-19128, 2020 10 19.
Article in English | MEDLINE | ID: mdl-32744783

ABSTRACT

Membrane proteins require lipid bilayers for function. While lipid compositions reach enormous complexities, high-resolution structures are usually obtained in artificial detergents. To understand whether and how lipids guide membrane protein function, we use single-molecule FRET to probe the dynamics of DtpA, a member of the proton-coupled oligopeptide transporter (POT) family, in various lipid environments. We show that detergents trap DtpA in a dynamic ensemble with cytoplasmic opening. Only reconstitutions in more native environments restore cooperativity, allowing an opening to the extracellular side and a sampling of all relevant states. Bilayer compositions tune the abundance of these states. A novel state with an extreme cytoplasmic opening is accessible in bilayers with anionic head groups. Hence, chemical diversity of membranes translates into structural diversity, with the current POT structures only sampling a portion of the full structural space.


Subject(s)
Membrane Transport Proteins/chemistry , Crystallography, X-Ray , Fluorescence Resonance Energy Transfer , Lipid Bilayers/chemistry , Membrane Transport Proteins/metabolism , Protein Conformation , Protein Transport
5.
Proc Natl Acad Sci U S A ; 115(3): 513-518, 2018 01 16.
Article in English | MEDLINE | ID: mdl-29298911

ABSTRACT

Protein dynamics are typically captured well by rate equations that predict exponential decays for two-state reactions. Here, we describe a remarkable exception. The electron-transfer enzyme quiescin sulfhydryl oxidase (QSOX), a natural fusion of two functionally distinct domains, switches between open- and closed-domain arrangements with apparent power-law kinetics. Using single-molecule FRET experiments on time scales from nanoseconds to milliseconds, we show that the unusual open-close kinetics results from slow sampling of an ensemble of disordered domain orientations. While substrate accelerates the kinetics, thus suggesting a substrate-induced switch to an alternative free energy landscape of the enzyme, the power-law behavior is also preserved upon electron load. Our results show that the slow sampling of open conformers is caused by a variety of interdomain interactions that imply a rugged free energy landscape, thus providing a generic mechanism for dynamic disorder in multidomain enzymes.


Subject(s)
Oxidoreductases/chemistry , Protozoan Proteins/chemistry , Trypanosoma brucei brucei/enzymology , Electron Transport , Kinetics , Oxidoreductases/metabolism , Protein Conformation , Protein Domains , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/chemistry
6.
FEBS Lett ; 588(2): 261-8, 2014 Jan 21.
Article in English | MEDLINE | ID: mdl-24161673

ABSTRACT

The translation machinery is the engine of life. Extracting the cytoplasmic milieu from a cell affords a lysate capable of producing proteins in concentrations reaching to tens of micromolar. Such lysates, derivable from a variety of cells, allow the facile addition and subtraction of components that are directly or indirectly related to the translation machinery and/or the over-expressed protein. The flexible nature of such cell-free expression systems, when coupled with high throughput monitoring, can be especially suitable for protein engineering studies, allowing one to bypass multiple steps typically required using conventional in vivo protein expression.


Subject(s)
Genetic Engineering/methods , Protein Biosynthesis , Animals , Cell-Free System/metabolism , Humans
7.
J Am Chem Soc ; 135(30): 11322-9, 2013 Jul 31.
Article in English | MEDLINE | ID: mdl-23822614

ABSTRACT

Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measured elongation rates and codon and isoacceptor tRNA usage, and provide a quantitative estimate of the effect on elongation rate of replacing a codon recognizing an abundant tRNA with a synonymous codon cognate to a rarer tRNA. Our results suggest that tRNA selection plays an important general role in modulating the rates and rhythms of protein synthesis, potentially influencing simultaneous co-translational processes such as folding and chemical modification.


Subject(s)
Fluorescence Resonance Energy Transfer , Peptide Chain Elongation, Translational , Amino Acid Sequence , Codon/genetics , Green Fluorescent Proteins/chemistry , Models, Molecular , Molecular Sequence Data , Mutation , Peptide Fragments/biosynthesis , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protein Conformation , RNA, Transfer/genetics , Ribosomes/genetics , Ribosomes/metabolism
8.
Bioconjug Chem ; 24(7): 1186-90, 2013 Jul 17.
Article in English | MEDLINE | ID: mdl-23734598

ABSTRACT

When suitably labeled bulk tRNAs are transfected into cells they give rise to FRET (fluorescence resonance energy transfer) signals via binding to ribosomes that provide a measure of total protein synthesis. Application of this approach to monitoring rates of specific protein synthesis requires achieving a very high signal-to-noise ratio. Such high ratios may be attainable using LRET (luminescence resonance energy transfer) in place of FRET. Lanthanide complexes containing an antenna chromophore are excellent LRET donors. Here we describe the synthesis of a Phe-tRNA(Phe) labeled with a Tb(3+) complex, denoted Tb(3+)-Phe-tRNA(Phe) that, notwithstanding the bulkiness of the Tb(3+) complex, is active in protein synthesis.


Subject(s)
Protein Biosynthesis , RNA, Transfer/chemistry , Terbium/chemistry , Energy Transfer , Magnetic Resonance Spectroscopy , Spectrometry, Mass, Electrospray Ionization
9.
Nucleic Acids Res ; 40(12): e88, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22422844

ABSTRACT

We present a flexible, real-time-coupled transcription-translation assay that involves the continuous monitoring of fluorescent Emerald GFP formation. Along with numerical simulation of a reaction kinetics model, the assay permits quantitative estimation of the effects on full-length protein synthesis of various additions, subtractions or substitutions to the protein synthesis machinery. Since the assay uses continuous fluorescence monitoring, it is much simpler and more rapid than other assays of protein synthesis and is compatible with high-throughput formats. Straightforward alterations of the assay permit determination of (i) the fraction of ribosomes in a cell-free protein synthesis kit that is active in full-length protein synthesis and (ii) the relative activities in supporting protein synthesis of modified (e.g. mutated, fluorescent-labeled) exogenous components (ribosomes, amino acid-specific tRNAs) that replace the corresponding endogenous components. Ribosomes containing fluorescent-labeled L11 and tRNAs labeled with fluorophores in the D-loop retain substantial activity. In the latter case, the extent of activity loss correlates with a combination of steric bulk and hydrophobicity of the fluorophore.


Subject(s)
Fluorometry/methods , Protein Biosynthesis , Cell-Free System , Green Fluorescent Proteins/biosynthesis , Luminescent Agents/analysis , Phenylalanine/metabolism , RNA, Transfer, Amino Acyl/metabolism , RNA, Transfer, Phe/metabolism , Ribosomes/metabolism , Transcription, Genetic
10.
Mol Cell ; 42(3): 367-77, 2011 May 06.
Article in English | MEDLINE | ID: mdl-21549313

ABSTRACT

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G⋅GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA⋅EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.


Subject(s)
Fluorescence Resonance Energy Transfer/methods , Molecular Dynamics Simulation , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Adenosine Triphosphate/metabolism , Carbocyanines/chemistry , Kinetics , Models, Chemical , Models, Genetic , Models, Molecular , Peptide Elongation Factor G/metabolism , Protein Biosynthesis/genetics , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Ribosomes/genetics
11.
PLoS One ; 5(6): e11043, 2010 Jun 16.
Article in English | MEDLINE | ID: mdl-20585385

ABSTRACT

Enzymatic processing of extracellular matrix (ECM) macromolecules by matrix metalloproteases (MMPs) is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen (3/4) fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease.


Subject(s)
Collagen/chemistry , Matrix Metalloproteinases/metabolism , Collagen/metabolism , Electrophoresis, Polyacrylamide Gel , Extracellular Matrix/metabolism , Microscopy, Atomic Force , Protein Conformation
12.
Biochim Biophys Acta ; 1803(1): 29-38, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19406173

ABSTRACT

The zinc-dependent matrix metalloproteinases (MMPs) belong to a large family of structurally homologous enzymes. These enzymes are involved in a wide variety of biological processes ranging from physiological cell proliferation and differentiation to pathological states associated with tumor metastasis, inflammation, tissue degeneration, and cell death. Controlling the enzymatic activity of specific individual MMPs by antagonist molecules is highly desirable, first, for studying their individual roles, and second as potential therapeutic agents. However, blocking the enzymatic activity with synthetic small inhibitors appears to be an extremely difficult task. Thus, this is an unmet need presumably due to the high structural homology between MMP catalytic domains. Recent reports have recognized a potential role for exosite or allosteric protein regions, distinct from the extended catalytic pocket, in mediating MMP activation and substrate hydrolysis. This raises the possibility that MMP enzymatic and non-enzymatic activities may be modified via antagonist molecules targeted to such allosteric sites or to alternative enzyme domains. In this review, we discuss the structural and functional bases for potential allosteric control of MMPs and highlight potential alternative enzyme domains as targets for designing highly selective MMP inhibitors.


Subject(s)
Matrix Metalloproteinase Inhibitors , Matrix Metalloproteinases/chemistry , Allosteric Regulation/drug effects , Animals , Antibodies, Blocking/pharmacology , Humans , Matrix Metalloproteinases/immunology , Matrix Metalloproteinases/metabolism , Models, Molecular , Protease Inhibitors/pharmacology
13.
Structure ; 15(10): 1227-36, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17937912

ABSTRACT

The multidomain zinc endopeptidase matrix metalloproteinase-9 (MMP-9) is a recognized therapeutic target in autoimmune diseases, vascular pathologies, and cancer. Despite its importance, structural characterization of full-length pro-MMP-9 is incomplete. Here, we report the structural model of full-length pro-MMP-9 and, in particular, the molecular character of its unique proline-rich and heavily O-glycosylated (OG) domain. Using a powerful combination of small-angle X-ray scattering and single-molecule imaging, we demonstrate that pro-MMP-9 possesses an elongated structure with two terminal globular domains connected by an unstructured OG domain. Image analysis highlights the flexibility of the OG domain, implicating its role in the varied enzyme conformations and in facilitating independent movements of the terminal domains. This may endorse recognition, binding, and processing of substrates, ligands, as well as receptors and marks this domain as an additional target for the design of selective regulators.


Subject(s)
Enzyme Precursors/chemistry , Matrix Metalloproteinase 9/chemistry , Enzyme Precursors/metabolism , Humans , Matrix Metalloproteinase 9/metabolism , Microscopy, Atomic Force , Protein Structure, Tertiary , Scattering, Small Angle , X-Ray Diffraction
14.
J Am Chem Soc ; 129(44): 13566-74, 2007 Nov 07.
Article in English | MEDLINE | ID: mdl-17929919

ABSTRACT

Activation of matrix metalloproteinase zymogen (pro-MMP) is a vital homeostatic process, yet its molecular basis remains unresolved. Using stopped-flow X-ray spectroscopy of the active site zinc ion, we determined the temporal sequence of pro-MMP-9 activation catalyzed by tissue kallikrein protease in milliseconds to several minutes. The identity of three intermediates seen by X-ray spectroscopy was corroborated by molecular dynamics simulations and quantum mechanics/molecular mechanics calculations. The cysteine-zinc interaction that maintains enzyme latency is disrupted via active-site proton transfers that mediate transient metal-protein coordination events and eventual binding of water. Unexpectedly, these events ensue as a direct result of complexation of pro-MMP-9 and kallikrein and occur before proteolysis and eventual dissociation of the pro-peptide from the catalytic site. Here we demonstrate the synergism among long-range protein conformational transitions, local structural rearrangements, and fine atomic events in the process of zymogen activation.


Subject(s)
Cysteine/chemistry , Enzyme Precursors/chemistry , Matrix Metalloproteinases/chemistry , Thermodynamics , Binding Sites , Enzyme Activation , Models, Molecular , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Spectrum Analysis/methods , Tissue Kallikreins/chemistry , X-Rays , Zinc/chemistry
15.
J Biol Chem ; 279(30): 31646-54, 2004 Jul 23.
Article in English | MEDLINE | ID: mdl-15102849

ABSTRACT

The metalloproteinase tumor necrosis factor-alpha-converting enzyme (TACE) is involved in the regulation of several key physiological and pathological processes. Therefore, potent and selective synthetic inhibitors are highly sought for the study of the physiological roles of TACE as well as for therapeutic purposes. Because of the high structural similarities between the active site of TACE and those of other related zinc endopeptidases such as disintegrin (ADAMs) and matrix metalloproteinases (MMPs), the design of such tailor-made inhibitors is not trivial. To obtain new insights into this problem, we have used a selective MMP inhibitor as a probe to examine the structural and kinetic effects occurring at the active site of TACE upon inhibition. Specifically, we used the selective MMP mechanism-based inhibitor SB-3CT to characterize the fine structural and electronic differences between the catalytic zinc ions within the active sites of TACE and MMP-2. We show that SB-3CT directly binds the metal ion of TACE as observed before with MMP-2. However, in contrast to MMP-2, the binding mode of SB-3CT to the catalytic zinc ion of TACE is different in the length of the Zn-S(SB-3CT) bond distance and the total effective charge of the catalytic zinc ion. In addition, SB-3CT inhibits TACE in a non-competitive fashion by inducing significant conformational changes in the structure. For MMP-2, SB-3CT behaved as a competitive inhibitor and no significant conformational changes were observed. An examination of the second shell amino acids surrounding the catalytic zinc ion of these enzymes indicated that the active site of TACE is more polar than that of MMP-2 and of other MMPs. On the basis of these results, we propose that although there is a seemingly high structural similarity between TACE and MMP-2, these enzymes are significantly diverse in the electronic and chemical properties within their active sites.


Subject(s)
Matrix Metalloproteinase 2/chemistry , Metalloendopeptidases/chemistry , ADAM Proteins , ADAM17 Protein , Catalytic Domain , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Heterocyclic Compounds, 1-Ring/chemistry , Heterocyclic Compounds, 1-Ring/pharmacology , Humans , In Vitro Techniques , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/genetics , Models, Molecular , Protein Structure, Tertiary , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Static Electricity , Sulfones/chemistry , Sulfones/pharmacology , Tumor Necrosis Factor-alpha/metabolism , Zinc/chemistry
16.
J Biol Chem ; 278(29): 27009-15, 2003 Jul 18.
Article in English | MEDLINE | ID: mdl-12679334

ABSTRACT

The zinc-dependent gelatinases belong to the family of matrix metalloproteinases (MMPs), enzymes that have been shown to play a key role in angiogenesis and tumor metastasis. These enzymes are capable of hydrolyzing extracellular matrix (ECM) components under physiological conditions. Specific and selective inhibitors aimed at blocking their activity are highly sought for use as potential therapeutic agents. We report herein on a novel mode of inhibition of gelatinase A (MMP-2) by the recently characterized inhibitors 4-(4-phenoxphenylsulfonyl)butane-1,2-dithiol (inhibitor 1) and 5-(4-phenoxphenylsulfonyl) pentane-1,2-dithiol (inhibitor 2). These synthetic inhibitors are selective for MMP-2 and MMP-9. We show that the dithiolate moiety of these inhibitors chelates the catalytic zinc ion of MMP-2 via two sulfur atoms. This mode of binding results in alternation of the coordination number of the metal ion and the induction of conformational changes at the microenvironment of the catalytic zinc ion; a set of events that is likely to be at the root of the potent slow binding inhibition behavior exhibited by these inhibitors. This study demonstrates a distinct approach for the understanding of the structural mechanism governing the molecular interactions between potent inhibitors and catalytic sites of MMPs, which may aid in the design of effective inhibitors.


Subject(s)
Matrix Metalloproteinase 2/chemistry , Matrix Metalloproteinase Inhibitors , Catalytic Domain , Circular Dichroism , Drug Design , Enzyme Precursors/chemistry , Enzyme Precursors/genetics , Enzyme Precursors/metabolism , Humans , In Vitro Techniques , Kinetics , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Models, Molecular , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrum Analysis , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/pharmacology , Sulfones/chemistry , Sulfones/pharmacology , X-Rays , Zinc/chemistry
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