RESUMEN
The unfoldase ClpC1 is one of the most exciting drug targets against tuberculosis. This AAA+ unfoldase works in cooperation with the ClpP1P2 protease and is the target of at least four natural product antibiotics: cyclomarin, ecumicin, lassomycin, and rufomycin. Although these molecules are promising starting points for drug development, their mechanisms of action remain largely unknown. Taking advantage of a middle domain mutant, we determined the first structure of Mycobacterium tuberculosis ClpC1 in its apo, cyclomarin-, and ecumicin-bound states via cryo-EM. The obtained structure displays features observed in other members of the AAA+ family and provides a map for further drug development. While the apo and cyclomarin-bound structures are indistinguishable and have N-terminal domains that are invisible in their respective EM maps, around half of the ecumicin-bound ClpC1 particles display three of their six N-terminal domains in an extended conformation. Our structural observations suggest a mechanism where ecumicin functions by mimicking substrate binding, leading to ATPase activation and changes in protein degradation profile.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Antibacterianos/farmacología , Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Chaperonas Moleculares/metabolismoRESUMEN
Tim chaperones transport membrane proteins to the two mitochondrial membranes. TIM9·10, a 70 kDa protein complex formed by 3 copies of Tim9 and Tim10, guides its clients across the aqueous compartment. The TIM9·10·12 complex is the anchor point at the inner-membrane insertase TIM22. The subunit composition of TIM9·10·12 remains debated. Joint NMR, small-angle X-ray scattering, and MD simulation data allow us to derive a structural model of the TIM9·10·12 assembly, with a 2:3:1 stoichiometry (Tim9:Tim10:Tim12). Both TIM9·10 and TIM9·10·12 hexamers are in a dynamic equilibrium with their constituent subunits, exchanging on a minutes timescale. NMR data establish that the subunits exhibit large conformational dynamics: when the conserved cysteines of the CX3C-Xn-CX3C motifs are formed, short α helices are formed, and these are fully stabilized only upon formation of the mature hexameric chaperone. We propose that the continuous subunit exchange allows mitochondria to control their level of inter-membrane space chaperones.
Asunto(s)
Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/química , Multimerización de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Chaperones are essential for assisting protein folding and for transferring poorly soluble proteins to their functional locations within cells. Hydrophobic interactions drive promiscuous chaperone-client binding, but our understanding of how additional interactions enable client specificity is sparse. Here, we decipher what determines binding of two chaperones (TIM8·13 and TIM9·10) to different integral membrane proteins, the all-transmembrane mitochondrial carrier Ggc1 and Tim23, which has an additional disordered hydrophilic domain. Combining NMR, SAXS, and molecular dynamics simulations, we determine the structures of Tim23/TIM8·13 and Tim23/TIM9·10 complexes. TIM8·13 uses transient salt bridges to interact with the hydrophilic part of its client, but its interactions to the transmembrane part are weaker than in TIM9·10. Consequently, TIM9·10 outcompetes TIM8·13 in binding hydrophobic clients, while TIM8·13 is tuned to few clients with both hydrophilic and hydrophobic parts. Our study exemplifies how chaperones fine-tune the balance of promiscuity versus specificity.
Asunto(s)
Membranas Mitocondriales , Chaperonas Moleculares , Humanos , Proteínas de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Chaperonas Moleculares/química , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation.
Asunto(s)
Proteínas Bacterianas , Endopeptidasa Clp , Inhibidores de Proteasas/química , Thermus thermophilus/enzimología , Regulación Alostérica , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Dominio Catalítico , Cristalografía por Rayos X , Endopeptidasa Clp/antagonistas & inhibidores , Endopeptidasa Clp/químicaRESUMEN
The exchange of metabolites between the mitochondrial matrix and the cytosol depends on ß-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and ß-barrel protein biogenesis. Our work reveals how a single mitochondrial "transfer-chaperone" system is able to guide α-helical and ß-barrel membrane proteins in a "nascent chain-like" conformation through a ribosome-free compartment.
Asunto(s)
Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Membranas Intracelulares/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Dominios Proteicos , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
Mycobacterium tuberculosis can remain dormant in the host, an ability that explains the failure of many current tuberculosis treatments. Recently, the natural products cyclomarin, ecumicin, and lassomycin have been shown to efficiently kill Mycobacterium tuberculosis persisters. Their target is the N-terminal domain of the hexameric AAA+ ATPase ClpC1, which recognizes, unfolds, and translocates protein substrates, such as proteins containing phosphorylated arginine residues, to the ClpP1P2 protease for degradation. Surprisingly, these antibiotics do not inhibit ClpC1 ATPase activity, and how they cause cell death is still unclear. Here, using NMR and small-angle X-ray scattering, we demonstrate that arginine-phosphate binding to the ClpC1 N-terminal domain induces millisecond dynamics. We show that these dynamics are caused by conformational changes and do not result from unfolding or oligomerization of this domain. Cyclomarin binding to this domain specifically blocked these N-terminal dynamics. On the basis of these results, we propose a mechanism of action involving cyclomarin-induced restriction of ClpC1 dynamics, which modulates the chaperone enzymatic activity leading eventually to cell death.
Asunto(s)
Antibacterianos/farmacología , Arginina/análogos & derivados , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Oligopéptidos/farmacología , Tuberculosis/tratamiento farmacológico , Arginina/farmacología , Muerte Celular , Cristalografía por Rayos X , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Transporte Iónico , Compuestos Organofosforados/farmacología , Fosforilación , Conformación Proteica , Dominios Proteicos , Tuberculosis/metabolismo , Tuberculosis/microbiologíaRESUMEN
Characterizing the structure of membrane proteins (MPs) generally requires extraction from their native environment, most commonly with detergents. Yet, the physicochemical properties of detergent micelles and lipid bilayers differ markedly and could alter the structural organization of MPs, albeit without general rules. Dodecylphosphocholine (DPC) is the most widely used detergent for MP structure determination by NMR, but the physiological relevance of several prominent structures has been questioned, though indirectly, by other biophysical techniques, e.g., functional/thermostability assay (TSA) and molecular dynamics (MD) simulations. Here, we resolve unambiguously this controversy by probing the functional relevance of three different mitochondrial carriers (MCs) in DPC at the atomic level, using an exhaustive set of solution-NMR experiments, complemented by functional/TSA and MD data. Our results provide atomic-level insight into the structure, substrate interaction and dynamics of the detergent-membrane protein complexes and demonstrates cogently that, while high-resolution NMR signals can be obtained for MCs in DPC, they systematically correspond to nonfunctional states.
Asunto(s)
Detergentes/química , Micelas , Proteínas de Transporte de Membrana Mitocondrial/química , Fosforilcolina/análogos & derivados , Translocasas Mitocondriales de ADP y ATP/química , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Fosforilcolina/química , Conformación Proteica , Estabilidad Proteica , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
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.
Asunto(s)
Proteínas y Péptidos de Choque por Frío/química , Proteínas y Péptidos de Choque por Frío/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Pliegue del ARN , ARN/química , Aminoácidos Aromáticos/química , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , ARN/metabolismoRESUMEN
The neutrophil gelatinase-associated lipocalin (NGAL, also known as LCN2) and its cellular receptor (LCN2-R, SLC22A17) are involved in many physiological and pathological processes such as cell differentiation, apoptosis, and inflammation. These pleiotropic functions mainly rely on NGAL's siderophore-mediated iron transport properties. However, the molecular determinants underlying the interaction between NGAL and its cellular receptor remain largely unknown. Here, using solution-state biomolecular NMR in conjunction with other biophysical methods, we show that the N-terminal domain of LCN2-R is a soluble extracellular domain that is intrinsically disordered and interacts with NGAL preferentially in its apo state to form a fuzzy complex. The relatively weak affinity (≈10 µm) between human LCN2-R-NTD and apoNGAL suggests that the N terminus on its own cannot account for the internalization of NGAL by LCN2-R. However, human LCN2-R-NTD could be involved in the fine-tuning of the interaction between NGAL and its cellular receptor or in a biochemical mechanism allowing the receptor to discriminate between apo- and holo-NGAL.
Asunto(s)
Proteínas de Fase Aguda/química , Lipocalinas/química , Proteínas de Transporte de Catión Orgánico/química , Proteínas Proto-Oncogénicas/química , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Lipocalina 2 , Lipocalinas/genética , Lipocalinas/metabolismo , Ratones , Resonancia Magnética Nuclear Biomolecular , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
AIMS: The small molecule indirubin-3'-monoxime (I3MO) has been shown to inhibit vascular smooth muscle cell (VSMC) proliferation and neointima formation in vivo. The influence of I3MO on VSMC migration and vascular inflammation, two additional key players during the onset of atherosclerosis and restenosis, should be investigated. METHODS AND RESULTS: We examined the influence of I3MO on VSMC migration, with focus on monocyte-derived leukotrienes (LTs) and platelet-derived growth factors (PDGFs) as elicitors. Exogenous LTB4 and cysteinyl leukotrienes as well as LT-enriched conditioned medium of activated primary human monocytes induced VSMC migration, which was inhibited by I3MO. I3MO also blunted migration of VSMC stimulated with the PDGF, the strongest motogen tested in this study. Induction of haem oxygenase 1 accounted for this anti-migratory activity of I3MO in VSMC. Notably, I3MO not only interfered with the migratory response in VSMC, but also suppressed the production of pro-migratory LT in monocytes. Conditioned media from monocytes that were activated in the presence of I3MO failed to induce VSMC migration. In cell-based and cell-free assays, I3MO selectively inhibited 5-lipoxygenase (5-LO), the key enzyme in LT biosynthesis, with an IC50 in the low micromolar range. CONCLUSION: Our study reveals a novel dual inhibitory mode of I3MO on LT-mediated VSMC migration: (i) I3MO interferes with pro-migratory signalling in VSMC and (ii) I3MO suppresses LT biosynthesis in monocytes by direct inhibition of 5-LO. These inhibitory actions on both migratory stimulus and response complement the previously demonstrated anti-proliferative properties of I3MO and may further promote I3MO as promising vasoprotective compound.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Cisteína/metabolismo , Indoles/farmacología , Leucotrienos/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Oximas/farmacología , Transducción de Señal/efectos de los fármacos , Araquidonato 5-Lipooxigenasa/metabolismo , Movimiento Celular/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Neointima/tratamiento farmacológico , Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
(13)C-α-ketoacid metabolic precursors of phenylalanine and tyrosine effectively enter the metabolism of a protein overexpressing E. coli strain to label Phe- and Tyr-residues devoid of any cross-labelling. The methodology gives access to highly selective labelling patterns as valuable tools in protein NMR spectroscopy without the need of (15)N-chiral amino acid synthesis using organic chemistry.
Asunto(s)
Cetoácidos/química , Cetoácidos/metabolismo , Fenilalanina/metabolismo , Proteínas Recombinantes/metabolismo , Tirosina/metabolismo , Escherichia coli/metabolismo , Marcaje Isotópico , Isótopos/análisis , Isótopos/química , Isótopos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Fenilalanina/química , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Estereoisomerismo , Tirosina/químicaRESUMEN
The addition of labeled α-ketoisovalerate to the growth medium of a protein-expressing host organism has evolved into a versatile tool to achieve concomitant incorporation of specific isotopes into valine- and leucine- residues. The resulting target proteins represent excellent probes for protein NMR analysis. However, as the sidechain resonances of these residues emerge in a narrow spectral range, signal overlap represents a severe limitation in the case of high-molecular-weight NMR probes. We present a protocol to eliminate leucine labeling by supplying the medium with unlabeled α-ketoisocaproate. The resulting spectra of a model protein exclusively feature valine signals of increased intensity, confirming the method to be a first example of independent valine and leucine labeling employing α-ketoacid precursor compounds.