Paweł Sledź

Max Planck Institute of Biochemistry, München, Bavaria, Germany

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Publications (8)69.63 Total impact

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    ABSTRACT: The 26S proteasome is a 2.5 MDa molecular machine that executes the degradation of substrates of the ubiquitin-proteasome pathway. The molecular architecture of the 26S proteasome was recently established by cryo-EM approaches. For a detailed understanding of the sequence of events from the initial binding of polyubiquitylated substrates to the translocation into the proteolytic core complex, it is necessary to move beyond static structures and characterize the conformational landscape of the 26S proteasome. To this end we have subjected a large cryo-EM dataset acquired in the presence of ATP and ATP-γS to a deep classification procedure, which deconvolutes coexisting conformational states. Highly variable regions, such as the density assigned to the largest subunit, Rpn1, are now well resolved and rendered interpretable. Our analysis reveals the existence of three major conformations: in addition to the previously described ATP-hydrolyzing (ATPh) and ATP-γS conformations, an intermediate state has been found. Its AAA-ATPase module adopts essentially the same topology that is observed in the ATPh conformation, whereas the lid is more similar to the ATP-γS bound state. Based on the conformational ensemble of the 26S proteasome in solution, we propose a mechanistic model for substrate recognition, commitment, deubiquitylation, and translocation into the core particle.
    Proceedings of the National Academy of Sciences 03/2014; · 9.81 Impact Factor
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    ABSTRACT: This protocol describes the screening of a library of low-molecular-weight compounds (fragments) using a series of biophysical ligand-binding assays. Fragment-based drug discovery (FBDD) has emerged as a successful method to design high-affinity ligands for biomacromolecules of therapeutic interest. It involves detecting relatively weak interactions between the fragments and a target macromolecule using sensitive biophysical techniques. These weak binders provide a starting point for the development of inhibitors with submicromolar affinity. Here we describe an efficient fragment screening cascade that can identify binding fragments (hits) within weeks. It is divided into three stages: (i) preliminary screening using differential scanning fluorimetry (DSF), (ii) validation by NMR spectroscopy and (iii) characterization of binding fragments by isothermal titration calorimetry (ITC) and X-ray crystallography. Although this protocol is readily applicable in academic settings because of its emphasis on low cost and medium-throughput early-stage screening technologies, the core principle of orthogonal validation makes it robust enough to meet the quality standards of an industrial laboratory.
    Nature Protocol 11/2013; 8(11):2309-24. · 8.36 Impact Factor
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    ABSTRACT: The 26S proteasome is a 2.5 MDa molecular machine for the degradation of substrates of the ubiquitin-proteasome pathway with a key role in cellular proteostasis. Until recently, only the structure of its core particle, the 20S proteasome, could be studied in detail, whereas the 19S regulatory particle or the holocomplex remained elusive. Novel integrative approaches have now revealed the molecular architecture of the entire complex and provided the first insights into the conformational changes during its functional cycle. Here we review the problems in structural studies of the 26S proteasome, the methods that made possible its structure determination, the architectural principles of the holocomplex, and its conformational space. These advances provide valuable insights into the mechanism of substrate recruitment and processing preceding their destruction in the 20S core particle.
    Structure 09/2013; 21(9):1551-62. · 5.99 Impact Factor
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    ABSTRACT: The 26S proteasome is the executive arm of the ubiquitin-proteasome system. This 2.5-MDa complex comprising the 20S core particle (CP) and the 19S regulatory particle (RP) is able to effectively execute its function due to a tightly regulated network of allosteric interactions. From this perspective, we summarize the current state of knowledge on these regulatory interdependencies. We classify them into the three functional layers-within the CP, within the RP, and at the CP-RP interface. In the CP, allosteric effects are thought to couple the gate opening and substrate proteolysis. Gate opening depends on events occurring in the RP-ATP hydrolysis and substrate binding. Finally, a number of processes occurring solely in the RP, like ATP hydrolysis or substrate deubiquitylation, are also proposed to be allosterically regulated. Recent advances in structural studies of 26S proteasome open up new avenues for dissecting and rationalizing the molecular basis of these regulatory networks.
    Journal of Molecular Biology 02/2013; · 3.91 Impact Factor
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    ABSTRACT: An explicit solvent ligand-mapping approach was used to reveal an otherwise hidden hydrophobic pocket in polo-like kinase 1 (Plk1). It predicted a novel ligand binding mode that was used for the design of a new ligand with high affinity for Plk1. X-ray crystallography confirmed that the binding was specific to the intended pocket.
    Angewandte Chemie International Edition 09/2012; 51(40):10078-81. · 11.34 Impact Factor
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    ABSTRACT: Despite intensive effort, the majority of the annotated Mycobacterium tuberculosis genome consists of genes encoding proteins of unknown or poorly understood function. For example, there are seven conserved hypothetical proteins annotated as homologs of pyridoxine 5'-phosphate oxidase (PNPOx), an enzyme that oxidizes pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP) to form pyridoxal 5'-phosphate (PLP). We have characterized the function of Rv2607 from Mycobacterium tuberculosis H37Rv and shown that it encodes a PNPOx that oxidizes PNP to PLP. The k(cat) and K(M) for this reaction were 0.01 s(-1) and 360 µM, respectively. Unlike many PNPOx enzymes, Rv2607 does not recognize PMP as a substrate.
    PLoS ONE 01/2011; 6(11):e27643. · 3.53 Impact Factor
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    ABSTRACT: We report the experimental charge density of HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], which is a common buffering agent. The structure was refined using the Hansen-Coppens formalism. The ability of the HEPES molecule to form stable intermolecular interactions and intermolecular hydrogen bonds in the crystal structure is discussed in terms of its buffering properties. The protonation mode observed in the crystal structure is different from that expected in solution, suggesting that additional factors must be taken into consideration in order to explain the solution properties of the compound. As ordered HEPES molecules are found in the active sites of proteins in several protein crystal structures, our results will allow for quantitative analysis of the electrostatic potential of the interacting surfaces of those proteins.
    Acta crystallographica. Section B, Structural science 08/2010; 66(Pt 4):482-92. · 1.80 Impact Factor
  • Paweł Sledź, Marc Mauduit, Karol Grela
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    ABSTRACT: This tutorial review covers the use of ionic liquids as (co)solvents for the olefin metathesis reaction. Olefin metathesis is a simple and effective synthetic tool used to create many compounds which otherwise would require complex multistep syntheses. It is expected that the application of ionic liquids (IL) as well as the development of IL-enabled catalysts and separation techniques will create a green aspect to this important methodology.
    Chemical Society Reviews 12/2008; 37(11):2433-42. · 24.89 Impact Factor