Henry Jonker - Publicaties / Publications "The Human Cdc37-Hsp90 complex studied by heteronuclear NMR spectroscopy", Sreeramulu S, Jonker HRA, Langer T, Richter C, Lancaster CRD, Schwalbe H, JBC, 284 (2009), 3885-3896 Abstract: The cell division cycle protein 37 (Cdc37) and the 90 kDa heat shock protein (Hsp90) are molecular chaperones, which are crucial elements in the protein signalling pathway. The largest class of client proteins for Cdc37 and Hsp90 are protein kinases. The catalytic domains of these kinases are stabilized by Cdc37 and their proper folding and functioning is dependent on Hsp90. Here, we present the X-ray crystal structure of the 16 kDa middle domain of human Cdc37 at 1.88 Å resolution and the structure of this domain in complex with the 23 kDa N-terminal domain of human Hsp90 based on heteronuclear solution state NMR data and docking. Our results demonstrate that the middle domain of Cdc37 exists as a monomer. NMR and mutagenesis experiments reveal L205 in Cdc37 as a key residue enabling complex formation. These findings can be very useful in the development of small molecule inhibitors against cancer. (Copyright © 2008, American Society for Biochemistry and Molecular Biology) "The high resolution NMR structure of Parvulustat (Z-2685) from Streptomyces parvulus FH-1641: Comparison with Tendamistat from Streptomyces tendae 4158", Rehm S, Han S, Hassani I, Sokocevic A, Jonker HRA, Engels JW, Schwalbe H, ChemBioChem, 10 (2009), 119-127 Abstract: The protein parvulustat (Z-2685) from Streptomyces parvulus comprises 78 amino acids and functions as a highly efficient alpha-amylase inhibitor. Parvulustat shares 29.6% overall amino acid sequence identity to the well-known alpha-amylase inhibitor tendamistat. Among the conserved residues are the two disulfide bridges (C9–C25, C43–C70) and the active-site motif (W16, R17, Y18). Here, we report the high-resolution NMR structure of parvulustat based on NOEs, J couplings, chemical shifts and hydrogen-exchange data. In addition, we studied the dynamical properties of parvulustat by heteronuclear relaxation measurements. We compare the structure of parvulustat with the structure of tendamistat in terms of secondary structure elements, charges and hydrophobicity. The overall structural composition is very similar, but there are distinct differences including the active-site region. These structural and dynamical differences indicate that for parvulsutat an induced-fit mechanism for binding to alpha-amylase might take place, since the structure of tendamistat does not change upon binding to alpha-amylase. (Copyright © 2008, Wiley-VCH Verlag GmbH & Co KGaA, Weinheim) "Nuclear Magnetic Resonance (NMR) Spectroscopy: Overview of Applications in Chemical Biology", Nielsen G, Stadler M, Jonker H, Betz M, Schwalbe H, Wiley Encyclopedia of Chemical Biology, (2008), article online Abstract: Nuclear magnetic resonance (NMR) spectroscopy is a powerful method to determine the structure of biomacromolecules and their complexes in solution. It allows determination of the dynamics of proteins, RNA, DNA and their complexes at atomic resolution. NMR spectroscopy can therefore monitor the often transient weak interactions in the interactome of proteins and the interaction between proteins and small molecule ligands. In addition, intrinsically unstructured proteins can be investigated and first reports of structure determination of membrane proteins in the immobilized state (solid state) are emerging. This review will introduce the fundamental NMR observables, the methods to investigate structure and dynamics and will discuss a number of examples where NMR spectroscopy has provided valuable information in the context of Chemical Biology. (Copyright © 2008, John Wiley & Sons, Inc.) "L11 domain rearrangement upon binding to RNA and thiostrepton studied by NMR spectroscopy", Jonker HRA, Ilin S, Grimm SK, Wöhnert J & Schwalbe H, Nucleic Acids Research, 35 (2007), 441-454 Abstract: Ribosomal proteins are assumed to stabilize specific RNA structures and promote compact folding of the large rRNA. The conformational dynamics of the protein between the bound and unbound state play an important role in the binding process. We have studied those dynamical changes in detail for the highly conserved complex between the ribosomal protein L11 and the GTPase region of 23S rRNA. The RNA domain is compactly folded into a well defined tertiary structure, which is further stabilized by the association with the C-terminal domain of the L11 protein (L11ctd). In addition, the N-terminal domain of L11 (L11ntd) is implicated in the binding of the natural thiazole antibiotic thiostrepton, which disrupts the elongation factor function. We have studied the conformation of the ribosomal protein and its dynamics by NMR in the unbound state, the RNA bound state and in the ternary complex with the RNA and thiostrepton. Our data reveal a rearrangement of the L11ntd, placing it closer to the RNA after binding of thiostrepton, which may prevent binding of elongation factors. We propose a model for the ternary L11–RNA–thiostrepton complex that is additionally based on interaction data and conformational information of the L11 protein. The model is consistent with earlier findings and provides an explanation for the role of L11ntd in elongation factor binding. (Copyright © 2006 Oxford University Press) "Domain reorientation and induced fit upon RNA binding: Solution structure and dynamics of ribosomal protein L11 from Thermotoga maritima", Ilin S, Hoskins A, Ohlenschläger O, Jonker HRA, Schwalbe H and Wöhnert J, ChemBioChem, 6 (2005), 1611-1618 Abstract: L11, a protein of the large ribosomal subunit, binds to a highly conserved domain of 23S rRNA and mediates ribosomal GTPase activity. Its C-terminal domain is the main determinant for rRNA binding, whereas its N-terminal domain plays only a limited role in RNA binding. The N-terminal domain is thought to be involved in interactions with elongation and release factors as well as with the antibiotics thiostrepton and micrococcin. This report presents the NMR solution structure of the full-length L11 protein from the thermophilic eubacterium Thermotoga maritima in its free form. The structure is based on a large number of orientational restraints derived from residual dipolar couplings in addition to conventional NOE-based restraints. The solution structure of L11 demonstrates that, in contrast to many other multidomain RNA-binding proteins, the relative orientation of the two domains is well defined. This is shown both by heteronuclear 15N-relaxation and residual dipolar-coupling data. Comparison of this NMR structure with the X-ray structure of RNA-bound L11, reveals that binding not only induces a rigidification of a flexible loop in the C-terminal domain, but also a sizeable reorientation of the N-terminal domain. The domain orientation in free L11 shows limited similarity to that of ribosome-bound L11 in complex with elongation factor, EF-G. (Copyright © 2005, Wiley-VCH Verlag GmbH & Co KGaA, Weinheim) "The intrinsically unstructured domain of PC4 modulates the activity of the structured core through inter- and intramolecular interactions", Jonker HRA, Wechselberger RW, Boelens R, Kaptein R and Folkers GE, Biochemistry, 45 (2006), 5067-5081 Abstract: Proteins frequently contain unstructured regions apart from a functionally important and well-conserved structured domain. Functional and structural aspects for these regions are frequently less clear. The general human positive cofactor 4 (PC4), has such a domain organization and can interact with various DNA substrates, transcriptional activators, and basal transcription factors. While essential for the cofactor function, structural and functional knowledge about these interactions is limited. Using biochemical, NMR, and docking experiments, we show that the carboxy-terminal structured core domain (PC4ctd) is required and sufficient for binding to single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and the herpes simplex virion protein 16 (VP16) activation domain (VP16ad). We determined the interaction surfaces within PC4 and showed that VP16 and DNA binding are mutually exclusive. Although the amino-terminal domain of PC4 (PC4ntd) alone is devoid of any bioactivity, it increases the interaction with VP16ad. While it decreases the ssDNA-binding and DNA-unwinding activity, it does not influence dsDNA binding. Structural characterization of this domain showed that it is highly flexible and mostly unstructured both in the free form and in the complex. NMR titration experiments using various protein and DNA substrates of the individual domains and the full-length PC4 revealed local conformational or environmental changes in both the structured and unstructured subdomains, which are interpreted to be caused by inter- and intramolecular interactions. We propose that the unstructured PC4ntd regulates the PC4 cofactor function by specific interactions with the activator and through modulation and/or shielding of the interaction surface in the structured core of PC4ctd. (Copyright © 2006, American Chemical Society) "Gradual phosphorylation regulates PC4 coactivator function", Jonker HRA, Wechselberger RW, Pinkse M, Kaptein R and Folkers GE, FEBS, 273 (2006), 1430-1444 Abstract: The unstructured N-terminal domain of the transcriptional cofactor PC4 contains multiple phosphorylation sites that regulate activity. The phosphorylation status differentially influences the various biochemical functions performed by the structured core of PC4. Binding to ssDNA is slightly enhanced by phosphorylation of one serine residue, which is not augmented by further phosphorylation. The presence of at least two phosphoserines decreases DNA-unwinding activity and abrogates binding to the transcriptional activator VP16. Phosphorylation gradually decreases the binding affinity for dsDNA. These phosphorylation-dependent changes in PC4 activities correlate with the sequential functions PC4 fulfils throughout the transcription cycle. MS and NMR revealed that up to eight serines are progressively phosphorylated towards the N-terminus, resulting in gradual environmental changes in the C-terminal direction of the following lysine-rich region. Also within the structured core, primarily around the interaction surfaces, environmental changes are observed. We propose a model for co-ordinated changes in PC4 cofactor functions, mediated by phosphorylation status-dependent gradual masking of the lysine-rich region causing shielding or exposure of interaction surfaces. (Copyright © 2006, FEBS Journal, The Authors Journal compilation) "Structural properties of the promiscuous VP16 activation domain", Jonker HRA, Wechselberger RW, Boelens R, Folkers GE and Kaptein R, Biochemistry, 44 (2005), 827-839 Abstract: Herpes simplex virion protein 16 (VP16) contains two strong activation regions that can independently and cooperatively activate transcription in vivo. We have identified the regions and residues involved in the interaction with the human transcriptional coactivator positive cofactor 4 (PC4) and the general transcription factor TFIIB. NMR and biochemical experiments revealed that both VP16 activation regions are required for the interaction and undergo a conformational transition from random coil to alpha-helix upon binding to its target PC4. The interaction is strongly electrostatically driven and the binding to PC4 is enhanced by the presence of its amino-terminal domain. We propose models for binding of VP16 to the core domains of PC4 and TFIIB that are based on two independent docking approaches using NMR chemical shift changes observed in titration experiments. The models are consistent with results from site-directed mutagenesis and provide an explanation for the contribution of both acidic and hydrophobic residues for transcriptional activation by VP16. Both intrinsically unstructured activation domains are attracted to their interaction partner by electrostatic interactions, and adopt an alpha-helical conformation around the important hydrophobic residues. The models showed multiple distinct binding surfaces upon interaction with various partners, providing an explanation for the promiscuous properties, cooperativity, and the high activity of this activation domain. (Copyright © 2004, American Chemical Society) "Solid-like components in carbohydrate gels probed by NMR spectroscopy", van Duynhoven JPM, Kulik AS, Jonker HRA, Haverkamp J, Carbohydrate Polymers, 40 (1999), 211-219 Abstract: Solid-like components in maltodextrin and inulin model gels were probed with NMR spectroscopy, 1H NMR cross-relaxation experiments yielded quantitative information about the ageing process during which the immobile fraction of the polymer increased. The increase of immobile protons was correlated with crystallization in the gels. From the growth rate of the amount of solid-like component in inulin an maltodextrin gels different kinetics of ageing were deduced. In the case of inulin, the fraction of immobilized polymer was reduced at lower polymer concentration which was correlated with the observed decrease in the gel strength. Firming of the inulin gel took longer when starting from totally dissolved polysaccharide material, as was the case with solutions prepared at temperatures exceeding 82°C. However, neither the kinetics of solidification, nor the fraction of immobilized polymer at equilibrium appeared to be influenced by the temperature at which the starting solution had been prepared. Additionally, molecular mobility of maltodextrin chains and interaction with water in the gel was investigated by 13C 1D and 2D solid-state NMR. The molecular mobility of the polymer chains was studied as a function of storage time and storage temperature and turned out to be higher in gels stored at higher temperatures. Water binding was probed with WISE experiments. Lower molecular mobility of polysaccharide chains as well as stronger interaction with water in gels stored at lower temperature was attributed to the formation of a 3D polymer network accompanied by partial crystallization. (Copyright © 1999, Elsevier Science Ltd. All rights reserved)