Uwe Mueller

Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin

The three macromolecular crystallography beamlines BL14.1, BL14.2 and BL14.3 at the BESSY II storage ring at the Helmholtz-Zentrum Berlin are described.

Two exposed amino acid residues confer thermostability on a cold shock protein

Thermophilic organisms produce proteins of exceptional stability. To understand protein thermostability at the molecular level we studied a pair of cold shock proteins, one of mesophilic and one of thermophilic origin, by systematic mutagenesis. Although the two proteins differ in sequence at 12 positions, two surface-exposed residues are responsible for the increase in stability of the thermophilic protein (by 15.8 kJ mol−1 at 70 °C). 11.5 kJ mol−1 originate from a predominantly electrostatic contribution of Arg 3 and 5.2 kJ mol−1 from hydrophobic interactions of Leu 66 at the carboxy terminus. The mesophilic protein could be converted to a highly thermostable form by changing the Glu residues at positions 3 and 66 to Arg and Leu, respectively. The variation of surface residues may thus provide a simple and powerful approach for increasing the thermostability of a protein.

A Mesoporous Metal–Organic Framework

A new class of porous materials namely metal–organic frameworks (MOFs) has set records in recent years regarding specific surface area and pore volume. Nevertheless, the search for compounds with very large pores and higher specific surface area remains a key challenge in the rapidly expanding field of MOFs, especially for applications in catalysis, drug delivery, and high-pressure gas storage. Compounds containing small windows or pores that are inaccessible for anchoring molecular catalysts, impregnation with catalyst precursors, or larger drug molecules pose limitations for MOFs in fine chemical transformation, nanoparticle formation, or drug delivery. For energy-storage applications at 200 bar, larger pores (2-3 nm) are essential to achieve a shift of the excess adsorption maximum towards higher pressure. Despite a somewhat reduced heat of adsorption, in practice such large pore MOFs outperform small pore MOFs as a result of the higher pore volume. A common concept to enhance the pore size in MOFs uses a linear extension of the linker in a given network topology. In such MOFs the pore diameter achievable is limited by interpenetration. A prominent example is the IRMOF series (isoreticular MOFs). Other examples of increasing pore size through linker extensions are [Cu3(btc)2] [11] (btc = benzene-1,3,5-tricarboxylate; tbo-topology), PCN-6 ([Cu3(tatb)2], tatb = 4,4’,4’’-s-triazine-2,4,6-triyltribenzoate; tbo), or MOF-14 ([Cu3(btb)2], btb = benzene1,3,5-tribenzoate; pto), built from paddle wheel clusters and tritopic linkers. However, in PCN-6 and MOF-14, which have the btb linker (a longer version of the btc linker), the porosity is reduced because of the presence of two interwoven 3D nets in the structure. The non-interpenetrated analogue of PCN-6 (PCN-6’) is obtained using a templating strategy, while a non-interpenetrated analogue of MOF-14 is unknown. Herein, we report an approach that avoids interpenetration by using a secondary linker to stabilize a highly open framework structure by crosslinking an extended Pt3O4topology. The resulting new mesoporous MOF material, DUT-6 (DUT= Dresden University of Technology), shows no interpenetration and has an extremely high gas adsorption capacity for n-butane, hydrogen, and methane. Single crystals of [Zn4O(2,6-ndc)(btb)4/3(def)16(H2O)9/2] (DUT-6; def = N,N-diethylformamide, 2,6-ndc = 2,6-naphthalenedicarboxylate) suitable for X-ray diffraction analysis were obtained from the reaction of H3(btb), H2(2,6-ndc), and zinc nitrate in a ratio of 3:2:14. The compound crystallizes in the cubic space group Pm3̄n. Dodecahedral mesoporous cages 2.5–3 nm in diameter are formed by twelve Zn4O 6+

XDSAPP: a graphical user interface for the convenient processing of diffraction data using XDS

XDSAPP is a Tcl/Tk-based graphical user interface for the easy and convenient processing of diffraction data sets using XDS. It provides easy access to all XDS functionalities, automates the data processing and generates graphical plots of various data set statistics provided by XDS. By incorporating additional software, further information on certain features of the data set, such as radiation decay during data collection or the presence of pseudo-translational symmetry and/or twinning, can be obtained. Intensity files suitable for CCP4, CNS and SHELX are generated.

The 1.8-A Crystal Structure of α1-Acid Glycoprotein (Orosomucoid) Solved by UV RIP Reveals the Broad Drug-Binding Activity of This Human Plasma Lipocalin

Alpha(1)-acid glycoprotein (AGP) is an important drug-binding protein in human plasma and, as an acute-phase protein, it has a strong influence on pharmacokinetics and pharmacodynamics of many pharmaceuticals. We report the crystal structure of the recombinant unglycosylated human AGP at 1.8 A resolution, which was solved using the new method of UV-radiation-damage-induced phasing (UV RIP). AGP reveals a typical lipocalin fold comprising an eight-stranded beta-barrel. Of the four loops that form the entrance to the ligand-binding site, loop 1, which connects beta-strands A and B, is among the longest observed so far and exhibits two full turns of an alpha-helix. Furthermore, it carries one of the five N-linked glycosylation sites, while a second one occurs underneath the tip of loop 2. The branched, partly hydrophobic, and partly acidic cavity, together with the presumably flexible loop 1 and the two sugar side chains at its entrance, explains the diverse ligand spectrum of AGP, which is known to vary with changes in glycosylation pattern.

Dye Encapsulation Inside a New Mesoporous Metal–Organic Framework for Multifunctional Solvatochromic‐Response Function

Metal–organic frameworks (MOFs), hybrid materials built up from metal clusters and organic linkers, have shown a huge potential for a wide range of applications. In recent years, MOFs have set new records in terms of specific surface areas and pore volumes and therefore are highly suitable as storage materials for small and large molecules. The development of new materials is crucial for the improvement of storage devices, but MOFs are also ideal candidates for functionalization. One functionalization strategy is the integration of complex organic linker molecules containing secondary functional groups. However, this approach is synthetically demanding and not general, because functional donor atoms may affect the linker connectivity resulting in unexpected network topologies. A second powerful strategy is postsynthetic modification of the framework. In this case, the range of functions is restricted due to the limited stability of MOFs against aggressive chemicals. A modular and more versatile approach may be the encapsulation of functional guest molecules into the MOF material. However, for these systems leaching is critical. A crucial requirement in all cases is also the accessibility of MOF functionalities for guest molecules. In this context, a large pore size is highly beneficial. However, the development of such mesoporous frameworks is challenging, because expanded frameworks are often more fragile leading to a collapse of the framework during removal of included guests molecules. A very effective concept to achieve robust frameworks lies in the creation of hierarchical pore structures by combination of two different linker molecules. The most prominent examples of such copolymerization approach are UMCM-1/ 2/ 3, DUT-6 and DUT-23 or MOF210. In the case of DUT-23, auxiliary linker was used successfully to avoid interpenetration and to enhance the robustness, which resulted in highly porous structures. To date, this concept was restricted only to the combination of triand ditopic linkers. On the other hand, DUT-10(M) (M= Zn, Cu, Co) compounds based on the tetratopic N,N,N’,N’benzidinetetrabenzoate (benztb) ligand and the paddlewheel secondary building unit (SBU) undergo structural change upon solvent removal. By enhancing the connectivity of the framework by using a six-connecting [Zn4O] 6+

Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells CHARACTERIZATION OF A SELECTIVE ALLOSTERIC KINASE INHIBITOR

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1–5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.

Structural fine-tuning of a multifunctional cytochrome p450 monooxygenase.

AurH is a unique cytochrome P450 monooxygenase catalyzing the stepwise formation of a homochiral oxygen heterocycle, a key structural and pharmacophoric component of the antibiotic aureothin. The exceptional enzymatic reaction involves a tandem oxygenation process including a regio- and stereospecific hydroxylation, followed by heterocyclization. For the structural and biochemical basis of this unparalleled sequence, four crystal structures of AurH variants in different conformational states and in complex with the P450 inhibitor ancymidol were solved, which represent the first structures of the CYP151A group. Structural data in conjunction with computational docking, site-directed mutagenesis, and chemical analyses unveiled a switch function when recognizing the two substrates, deoxyaureothin and the hydroxylated intermediate, thus allowing the second oxygenation-heterocyclization step. Furthermore, we were able to modify the chemo- and regioselectivity of AurH, yielding mutants that catalyze the regioselective six-electron transfer of a nonactivated methyl group to a carboxylic acid via hydroxyl and aldehyde intermediates.

Crystal structure of Homo sapiens protein hp14.5

Babu A. Manjasetty, Heinrich Delbrück, Dinh-Trung Pham, Uwe Mueller, Martin Fieber-Erdmann, Christoph Scheich, Volker Sievert, Konrad Büssow, Frank H. Neisen, Wilhelm Weihofen, Bernhard Loll, Wolfram Saenger, and Udo Heinemann* Protein Structure Factory, c/o BESSY GmbH, Berlin, Germany Forschungsgruppe Kristallographie, Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Institut für Chemie/Kristallographie, Freie Universität Berlin, Germany Protein Structure Factory, Berlin, Germany Alpha-Bioverfahrenstechnik GmbH, Kleinmachnow, Germany Max-Planck-Institut für Molekulare Genetik, Berlin, Germany Universitätsklinikum Charité, Institut für Medizinische Physik & Biophysik Berlin, Germany

Six Biophysical Screening Methods Miss a Large Proportion of Crystallographically Discovered Fragment Hits: A Case Study

Fragment-based lead discovery (FBLD) has become a pillar in drug development. Typical applications of this method comprise at least two biophysical screens as prefilter and a follow-up crystallographic experiment on a subset of fragments. Clearly, structural information is pivotal in FBLD, but a key question is whether such a screening cascade strategy will retrieve the majority of fragment-bound structures. We therefore set out to screen 361 fragments for binding to endothiapepsin, a representative of the challenging group of aspartic proteases, employing six screening techniques and crystallography in parallel. Crystallography resulted in the very high number of 71 structures. Yet alarmingly, 44% of these hits were not detected by any biophysical screening approach. Moreover, any screening cascade, building on the results from two or more screening methods, would have failed to predict at least 73% of these hits. We thus conclude that, at least in the present case, the frequently applied biophysical prescreening filters deteriorate the number of possible X-ray hits while only the immediate use of crystallography enables exhaustive retrieval of a maximum of fragment structures, which represent a rich source guiding hit-to-lead-to-drug evolution.