Structural Biology Michael Sattler Institute of Structural Biology - PDF document

Structural Biology Michael Sattler Institute of Structural Biology (STB) http://www.nmr.ch.tum.de http://www.helmholtz-muenchen.de/stb http://www.helmholtz-muenchen.de/PEPF (Protein Expression & Purification Facility) http://www.bnmrz.org (Bayerisches NMR Zentrum) Jan 2012 Outline • What is structural biology? Aims, methods and challenges • Institute of Structural Biology at HMGU Research, methods, resources • Biomolecular NMR spectroscopy • Liquid state NMR & solid state NMR • Structure-based drug design • Application

Structural Biology What is it? A branch of biochemistry and biophysics which provides three-dimensional structures of proteins, RNA, DNA using X-ray crystallography, NMR and electron microscopy (EM) techniques Why? • To obtain atomic resolution and molecular details about the molecular functions of biomacromolecules • To understand their molecular functions and as a starting point for structure-based drug design K. Scheffzek

1962 – Nobel Year of Structural Biology We need an EMBL J. Watson F. Crick M. Wilkins J. Kendrew M. Perutz K. Scheffzek Structural Biology Milestones DNA t-RNA Myoglobin Photoreaction center Ion channels Ribosome ‘Pol II’ F1-ATPase Nucleosome K. Scheffzek

Protein Data Bank • First crystal structure determined in 1958, first NMR structure in 1985 • Coordinates are stored in the Protein Data Bank mirror sites in the USA, Europe and Japan however, the different tools and analyses are provided at these sites • NMR data are stored at BioMagResBank (BMRB) Total #: 78500 (15.1.2012) 87.5% X-ray 11.7% NMR 0.5% EM http://www.brmb.wisc.edu http://www.pdbe.org Structural Biology - from atoms to cells Dave Stuart, U Oxford, INSTRUCT

From tissue to molecules Dave Stuart, U Oxford, INSTRUCT Structure/imaging from molecules to animals – Integrated Structural Biology Animal MRI Size, spatial resolution Chemical Light Static picture, Cell microscopy Biology snapshots Small molecules EM tomography Protein complexes Cryo EM Molecular machines SAXS, SANS Proteins, X-ray NMR domains Dynamics: regulation 10 -9 1 10 3 [s] Dynamics, timescales Light NMR MRI microscopy

I nstitute of Structural Biology 12 Institute of Structural Biology (STB) Structural biology of cellular pathways and disease mechanism • Prof. Dr. Michael Sattler – STB and TUM (since 2007) • Solution state NMR; Molecular recognition in gene regulation and signaling • Dr. Dierk Niessing – STB and LMU (since 2005) • X-ray crystallography; RNA localization and intracellular transport • Prof. Dr. Bernd Reif – TUM and STB (since 2010) • Solid-state NMR; amyloids and membrane proteins • Dr. Arie Geerlof – Protein Expression & Purification Facility (PEPF) • Since 2010; sample production for structural/chemical biology, biophysics I nstitute of Structural Biology 13 Mission of STB • Structural and molecular basis of cellular pathways • Gene regulation and signalling pathways • Molecular basis of disease mechanisms • Quantitative biochemical and biophysical characterization • Knowledge and resources in structural biology, protein biochemistry and biophysical analysis • Chemical biology & structure-based drug discovery • Protein production (PEPF) • NMR spectroscopy and X-ray crystallography

I nstitute of Structural Biology 15 Methods Structural Biology Biophysical techniques • Liquid state NMR • Isothermal titration calorimetry (ITC) • Solid state NMR • Surface plasmon resonance (SPR, Biacore) • X-ray crystallography • Light Scattering (SLS, DLS) • Integrated approaches: • Fluorescence techniques, FRET, … NMR, X-ray, SAXS/ SANS, EM SANS SAXS RDCs I nstitute of Structural Biology Sattler Group 16 DNA / GENE Spliceosome assembly, Non-coding RNAs SF1/ intron RNA Liu, Luyten et al. Sattler Science (2001) Xist A-repeat RNA Duszczyk et al Sattler Spliceosome assembly, RNA (2011) U2AF65/ SF1; Selenko et al pre-mRNA Sattler, Mol. Cell. (2003) Xist RNA U2AF/ DEK, Soares et al Sattler, Splicing Valcarcel Science (2006) on X i mRNA RNA interference SPF45 UHM miRNAs Corsini et al Sattler Nat Struct Mol Biol (2007) C'  2 FHV B2 suppressor of RNAi N' Splicing regulation: U2AF/ Py tract, K62' Lingel et al Sattler EMBO Rep Mackereth et al Sattler, Nature (2011) R36 PROTEIN R54' (2005)  1' K47 10 A K47' R54 N  2' K62 R36' C Argonaute PAZ domain/ siRNA Lingel et al Sattler Nature (2003) Dimethyl-arginine recognition by Tudor Sam68 Qua1 Lingel et al Sattler Nat Struct Mol Biol (2004) domains; ripsianes et al Sattler, Meyer et al Sattler Nat Struct Mol Biol (2011) J Biol Chem (2010)

I nstitute of Structural Biology Sattler Group 17 Cellular pathw ays and diseases Peroxisomal biogenesis LRRK2 Construct Expression 1000 2528 Kin No expression • Peroxiosomal biogenesis disorders: Kin long1 Exp, weak “Zellweger syndrome” Kin long2 Exp. Weak • Understand basic mechanisms LRR Roc COR MAP3K WD40 KinD Expr. Weak KinD long1 Overexp. W ith M. Ueffing ( PROT) KinD long2 Overexpr. Funding: Michael J. Fox Foundation; Roc-Cor-Kin Expression HGF Allianz HelMA ( applied) Cor-Kin long1 Expression • Structural biology of LRRK2 and its Pex5 Kin long1-WD Expression W xxxF LRR Overexpr. R25 molecular interactions • Understand molecular mechanisms Pex5 ROC-COR Overexpr. K56 • Drug design K55 W118 WD40 Overexpr. K34 Delta/Notch: Dll1/Magi2 PDZ F122 Pex14 R40 90° W ith G. Przem eck, Pex1 4 ( N) M. Hrabe de Angelis ( I EG) R25 15 N PDZ4 pH 6.9 Pex19 • Dll1 peptide/PDZ4: Kd ~ nM K55 F75 K56 • Mutant 100-fold reduced • Next steps: K34 • 3D structure F71 Pex14 • Small molecules R40 F70 Pex1 9 FFxxxF 10 9 8 Neufeld et al EMBO J 2009 7 F2 [ ppm] I nstitute of Structural Biology Niessing Group 18 Research topics in Niessing lab EpCAM signal mRNA & Mitotic spindle transduction vesicle transport orientation Motor complexes for mitotic spindle orientation Associated diseases: Aneuploidy, Neurodegeneration, Dementia Epithelial cancer multiple diseases Griselli syndrome Microvillus inclusion disease Key Publications: Müller et al. & Niessing PLoS Biol 2011 Graebsch et al. & Niessing PNAS 2009 Heuck et al. & Niessing J. Cell Biol . 2010 Heuck et al. & Niessing PNAS 2007 Graebsch et al. & Niessing PLoS One 2010 Niessing et al. Burley Cell 2004

I nstitute of Structural Biology Reif Group 19 Research topics in Reif lab Mem brane Proteins Soluble Protein Protein Misfolding Com plexes Maltose ABC ß-Amyloid Fibril Small heat Transporter Structure shock proteins MalEFG ‧ K 2 Proteasome Interactions with Small Molecules Small Multidrug And Molecular Chaperones Transporter (EmrE) Alzheimer’s disease, Protein degradation Multidrug resistance Diabetes type II, AL-Amyloidosis Key Publications: Linser et al. Angewandte Chemie Int. Edt. (2011) Richter et al. PNAS (2010) Bieschke et al., Nature Chem. Biol. (2011) Mainz et al., JACS (2009) Bertini et al., PNAS (2011) Narayanan et al. PNAS (2003) I nstitute of Structural Biology 20 Structure-based drug design Protein Expression & Purification Facility (Dr. Arie Geerlof) • Support for protein production and biophysical characterization X-ray, NMR-based ligand binding/ screening (Dr. Ana Messias) • Fragment-based drug design (N.N.) X-ray crystallography core facility (Dr. Dierk Niessing) • Structures of ligand complexes • Access to HGF DESY/ Petra III beamline

I nstitute of Structural Biology 21 Sam ple production for structural biology Purification Crystallization Gene 2-30 mg Amplification >95% purity Isotope Culture Fermentor labeling Expression NMR Gene X-ray Cloning Structure determination Tag Gene Expression screening antibiotic E. coli Transformation SF9/baculo virus http://www.helmholtz-muenchen.de/PEPF I nstitute of Structural Biology 22 Protein Expression and Purification Facility http: / / www.helmholtz-muenchen.de/ PEPF Group leader: Arie Geerlof • Established October 2009 • Support and Training - help with sample production for structural, functional and chemical biology - providing materials and facilities for the cultivation of bacteria and insect cells - help with biochemical and biophysical characterization • Development and Implementation of new methods and protocols • Protein Production and characterization - production of proteins for general use ( e.g. proteases and polymerases) - sample preparation for chemical biology/assay development - biochemical and biophysical characterization of proteins - collaboration with individual researchers

Why solution state NMR? Nature 2007 Nature 2007 Nature 2011 Nature 2009 HeLa In vitro Biomolecular NMR • Structure determination of biomacromolecules  no crystal needed, native-like conditions: solution,  macromolecular crowding, “in cell” NMR (Xenopus oocyctes)  transient regulatory interactions, flexible linkers • Ligand binding and molecular interactions in solution  “NMR fingerprint: macromolecular and small molecule interactions • Dynamics and mobility (ps  days)  conformational dynamics  enzyme turnover, kinetics, folding • Multidisciplinary approaches  combine NMR and X-ray with SAXS/SANS, EPR, FRET, …  free  bound 250 250 200 200 T2 [ms] 150 150 SANS 100 100 RDCs SAXS 50 50 0 0 140 160 180 200 220 240 260 280 300 320 340 residue