Marcel J. J. Blommers

Crystal structure of human estrogen-related receptor alpha in complex with a synthetic inverse agonist reveals its novel molecular mechanism.

Inverse agonists of the constitutively active human estrogen-related receptorα (ERRα, NR3B1) are of potential interest for several disease indications (e.g. breast cancer, metabolic diseases, or osteoporosis). ERRα is constitutively active, because its ligand binding pocket (LBP) is practically filled with side chains (in particular with Phe328, which is replaced by Ala in ERRβ and ERRγ). We present here the crystal structure of the ligand binding domain of ERRα (containing the mutation C325S) in complex with the inverse agonist cyclohexylmethyl-(1-p-tolyl-1H-indol-3-ylmethyl)-amine (compound 1a), to a resolution of 2.3Å. The structure reveals the dramatic multiple conformational changes in the LBP, which create the necessary space for the ligand. As a consequence of the new side chain conformation of Phe328 (on helix H3), Phe510(H12) has to move away, and thus the activation helix H12 is displaced from its agonist position. This is a novel mechanism of H12 inactivation, different from ERRγ, estrogen receptor (ER) α, and ERβ. H12 binds (with a surprising binding mode) in the coactivator groove of its ligand binding domain, at a similar place as a coactivator peptide. This is in contrast to ERRγ but resembles the situation for ERα (raloxifene or 4-hydroxytamoxifen complexes). Our results explain the novel molecular mechanism of an inverse agonist for ERRα and provide the basis for rational drug design to obtain isotype-specific inverse agonists of this potential new drug target. Despite a practically filled LBP, the finding that a suitable ligand can induce an opening of the cavity also has broad implications for other orphan nuclear hormone receptors (e.g. the NGFI-B subfamily).

NMR structural and dynamical investigation of the isolated voltage-sensing domain of the potassium channel KvAP: implications for voltage gating.

The structure and dynamics of the isolated voltage-sensing domain (VSD) of the archaeal potassium channel KvAP was studied by high-resolution NMR. The almost complete backbone resonance assignment and partial side-chain assignment of the (2)H,(13)C,(15)N-labeled VSD were obtained for the protein domain solubilized in DPC/LDAO (2:1) mixed micelles. Secondary and tertiary structures of the VSD were characterized using secondary chemical shifts and NOE contacts. These data indicate that the spatial structure of the VSD solubilized in micelles corresponds to the structure of the domain in an open state of the channel. NOE contacts and secondary chemical shifts of amide protons indicate the presence of tightly bound water molecule as well as hydrogen bond formation involving an interhelical salt bridge (Asp62-R133) that stabilizes the overall structure of the domain. The backbone dynamics of the VSD was studied using (15)N relaxation measurements. The loop regions S1-S2 and S2-S3 were found mobile, while the S3-S4 loop (voltage-sensor paddle) was found stable at the ps-ns time scale. The moieties of S1, S2, S3, and S4 helices sharing interhelical contacts (at the level of the Asp62-R133 salt bridge) were observed in conformational exchange on the micros-ms time scale. Similar exchange-induced broadening of characteristic resonances was observed for the VSD solubilized in the membrane of lipid-protein nanodiscs composed of DMPC, DMPG, and POPC/DOPG lipids. Apparently, the observed interhelical motions represent an inherent property of the VSD of the KvAP channel and can play an important role in the voltage gating.

Lipid-protein nanoscale bilayers: a versatile medium for NMR investigations of membrane proteins and membrane-active peptides.

In the present Communication we demonstrate the possibility to use high-resolution NMR for the investigation of membrane proteins in reconstituted high-density lipoprotein (rHDL) particles. The rHDL particles are nanoscale phospholipid bilayers wrapped around by a dimer of apolipoprotein A-1 (Bayburt, T. H.; Grinkova, Y. V.; Sligar, S. G. Nano Lett. 2002, 2, 853−856). In contrast to the commonly used spherical micelles, the rHDL particles incorporate a lipid bilayer like in biological membranes. These particles still undergo isotropic motion on the NMR time scale, providing the application of high-resolution NMR spectroscopy of the peptides and proteins embedded into their bilayer. As an example, the topology of the membrane-active peptide Antiamoebin-I in the bilayer of the rHDL particles was determined by using the lipid-soluble relaxation probe technique.

Time efficient detection of protein–ligand interactions with the polarization optimized PO-WaterLOGSY NMR experiment

The identification of compounds that bind to a protein of interest is of central importance in contemporary drug research. For screening of compound libraries, NMR techniques are widely used, in particular the Water-Ligand Observed via Gradient SpectroscopY (WaterLOGSY) experiment. Here we present an optimized experiment, the polarization optimized WaterLOGSY (PO-WaterLOGSY). Based on a water flip-back strategy in conjunction with model calculations and numerical simulations, the PO-WaterLOGSY is optimized for water polarization recovery. Compared to a standard setup with the conventional WaterLOGSY, time consuming relaxation delays have been considerably shortened and can even be omitted through this approach. Furthermore, the robustness of the pulse sequence in an industrial setup was increased by the use of hard pulse trains for selective water excitation and water suppression. The PO-WaterLOGSY thus yields increased time efficiency by factor of 3–5 when compared with previously published schemes. These time savings have a substantial impact in drug discovery, since significantly larger compound libraries can be tested in screening campaigns.

Interaction of Epothilone B (Patupilone) with Microtubules as Detected by Two‐Dimensional Solid‐State NMR Spectroscopy

Solid evidence: Induction of the polymerization of β-tubulin dimers into microtubules by epothilones, such as patupilone, by an as yet unknown mechanism leads to the apoptosis of cancer cells. Solid-state NMR spectroscopy of patupilone bound to microtubules has now enabled the identification of atomic positions of the drug that undergo clear chemical-shift changes upon binding (see correlation spectra of free (black) and complexed patupilone (red)).

Expression of G-protein coupled receptors in Escherichia coli for structural studies

To elaborate a high-performance system for expression of genes of G-protein coupled receptors (GPCR), methods of direct and hybrid expression of 17 GPCR genes in Escherichia coli and selection of strains and bacteria cultivation conditions were investigated. It was established that expression of most of the target GPCR fused with the N-terminal fragment of OmpF or Mistic using media for autoinduction provides high output (up to 50 mg/liter).

Determination of the backbone torsion angle ɛ in nucleic acids

SummaryThe multiplet structure of cross peaks in double-quantum-filtered COSY NMR spectra is analysed for those resonances that include passive heteronuclear couplings. Interestingly, the cross peak involving the sugar-ring protons H2′ and H3′ in nucleic acids display an E.COSY-type appearance exclusively when the backbone torsion angle ɛ (C4′-C3′-O3′-P) adopts a gauche(-) conformation. This observation allows an unambiguous analysis of the conformation around ɛ, without the knowledge of 3Jcp coupling constants.

Strategies for the NMR‐Based Identification and Optimization of Allosteric Protein Kinase Inhibitors

Protein kinases are important drug targets, but kinase inhibitors ought to be selective and specific in order to avoid side effects in the clinic. Kinase inhibitors that do not target the highly conserved ATP-binding site, but that target an allosteric site, are generally expected to be more selective for the target kinase and thus have a better clinical profile. Here we propose an NMR-based strategy to discover and optimize allosteric kinase inhibitors. The approach uses a spin-labeled adenine analogue to detect allosteric kinase ligands by paramagnetic relaxation enhancement. Protein kinases comprise a large family of enzymes that catalyze the transfer of the terminal phosphate from ATP (adenosine triphosphate) to protein substrates, specifically to the hydroxyl group of serine or threonine (Ser/Thr kinases) or tyrosine (Tyr kinases). Protein kinases play a crucial role in signal transduction and thereby regulate central cellular processes such as cell-cycle control, growth control, apoptosis, and transcriptional activation. Kinase activity is generally tightly regulated, but can get out of control with overactive or constitutionally activated kinases. Several pathological states or diseases, such as cancer, can be a consequence of kinase overactivation. Small molecules that can modulate kinase activity in vivo are therefore of high therapeutic interest, and those kinases with a central and specific role in a particular disease are pharmaceutically highly relevant drug targets. 3] A recent example of successful target selection and inhibitor design is the clinical success of Gleevec;, a low-molecular-weight inhibitor of the constitutionally activated tyrosine kinase, Bcr-Abl. Protein kinases generally consist of a catalytic (SH1) domain and one or several regulatory (e.g. SH2 or SH3) domains. The catalytic domains have a conserved three-dimensional fold with a bilobed structure: an N-terminal lobe consisting mainly of b sheets and a C-terminal helical lobe (Figure 1). The catalytic site is located near a hinge region that connects these two domains. Kinases can adopt multiple conformational states that are associated with the degree of catalytic activity : fully active kinases are generally phosphorylated in their activation loop, which adopts a conformation that allows for optimal binding of ATP/Mg and substrate protein, and for efficient transfer of the phosphate group of ATP. There are several regulatory mechanisms by which a kinase becomes down-regulated or “inactive”. The conformational consequence of kinase downregulation can be movement of the activation loop or other components so that the substrate cannot be efficiently bound to the kinase catalytic domain. Besides the ATP-binding site and the substrate binding site, allosteric binding sites occur in kinases, often at sites with regulatory control function. More than 500 kinases are estimated to be encoded in the human genome. All of them bind ATP/Mg , and the ATP-binding site is highly conserved both in amino acid sequence and in three-dimensional structure. Kinase inhibitors that target the ATP site in an active kinase conformation (type I inhibitors) might therefore have a higher risk of clinical liabilities due to lack of selectivity against other kinases. The ATP-binding site changes shape and becomes structurally less conserved when it is in a down-regulated conformation. Kinase inhibitors that target the ATP-binding site in a down-regulated conformation (type II inhibitors) might therefore have better selectivity and specificity, and hopefully a better clinical profile. Glivec/Gleevec is such a type II kinase inhibitor. While not being perfectly selective, it targets the ATP site of Bcr-Abl in its down-regulated conformation. The best selectivity profile might be possible for inhibitors that bind outside the ATP site, at the substrate site or an allosteric binding site. These sites are not generally conserved, and high selectivity against other kinases can hopefully be achieved. Most known kinase inhibitors are type I or II inhibitors. 14] This is probably due to the fact that most kinase inhibitor screens are performed by using biochemical functional assays with purified and activated recombinant kinase. Allosteric kinase inhibitors are not identified by these assay types if they do not inhibit kinase catalysis per se, although they might inhibit kinase activation or signal transduction. Allosteric kinase Figure 1. Principle of the experiment. Spin-labeled adenine analogue 3 is bound to the ATP-binding site. Any ligand that binds simultaneously but at a different binding site feels the paramagnetic relaxation enhancement mediated by 3. The structure of MEK2 in complex with PD334581 and ATP is shown here solely to illustrate the technique.

Optimisation of metric matrix embedding by genetic algorithms

SummaryTo improve the convergence properties of ‘embedding’ distance geometry, a new approach was developed by combining the distance-geometry methodology with a genetic algorithm. This new approach is called DG-OMEGA (DGΩ, optimised metric matrix embedding by genetic algorithms). The genetic algorithm was used to combine well-defined parts of individual structures generated by the distance-geometry program, and to identify new lower and upper distance bounds within the original experimental restraints in order to restrict the sampling of the metrisation algorithm to promising regions of the conformational space. The algorithm was tested on cyclosporin A, which is notorious for its intrinsic difficult sampling properties. A set of 58 distance restraints was employed. It was shown that DGΩ resulted in an improvement of convergence behaviour as well as sampling properties with respect to the standard distance-geometry protocol.

Direct Measurement of Dihedral Angles with High‐Resolution NMR Spectroscopy

Help in determining biomolecular structure by NMR spectroscopy is found in a new method recently proposed by Reif, Hennig, and Griesinger, which enables the direct measurement of angles between bond vectors (see picture; X, Y=13 C, 15 N). This work may be another milestone in the progress of NMR spectroscopy toward simpler and more generally applicable structure determination of biomolecules.