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

• 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

Outline

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

• M. Wilkins
• F. Crick
• J. Watson
• M. Perutz
• J. Kendrew
• K. Scheffzek

Structural Biology Milestones

F1-ATPase DNA

Photoreaction center Myoglobin

t-RNA Nucleosome Ribosome Ion channels ‘Pol II’

• 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)

http://www.pdbe.org http://www.brmb.wisc.edu

87.5% X-ray 11.7% NMR 0.5% EM Total #: 78500 (15.1.2012)

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

Dynamics, timescales Size, spatial resolution Proteins, domains Protein complexes Molecular machines

Cryo EM EM tomography Light microscopy Light microscopy NMR

10-9 1

X-ray NMR

103 [s]

MRI

Animal

MRI SAXS, SANS

Static picture, snapshots Dynamics: regulation

Chemical Biology

Small molecules

Cell

I nstitute of Structural Biology 12

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

Institute of Structural Biology (STB)

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

• Liquid state NMR
• Solid state NMR
• X-ray crystallography
• Integrated approaches:

NMR, X-ray, SAXS/ SANS, EM

RDCs SANS SAXS

Biophysical techniques

• Isothermal titration calorimetry (ITC)
• Surface plasmon resonance (SPR, Biacore)
• Light Scattering (SLS, DLS)
• Fluorescence techniques, FRET, …

I nstitute of Structural Biology 16

pre-mRNA DNA / GENE mRNA PROTEIN

RNA interference miRNAs

Splicing

FHV B2 suppressor of RNAi Lingel et al Sattler EMBO Rep (2005) Spliceosome assembly, SF1/ intron RNA Liu, Luyten et al. Sattler Science (2001) Spliceosome assembly, U2AF65/ SF1; Selenko et al Sattler, Mol. Cell. (2003) SPF45 UHM Corsini et al Sattler Nat Struct Mol Biol (2007) Argonaute PAZ domain/ siRNA Lingel et al Sattler Nature (2003) Lingel et al Sattler Nat Struct Mol Biol (2004) U2AF/ DEK, Soares et al Sattler, Valcarcel Science (2006) Sam68 Qua1 Meyer et al Sattler J Biol Chem (2010)

Non-coding RNAs

Xist A-repeat RNA Duszczyk et al Sattler RNA (2011) Xist RNA

• n Xi

Sattler Group

Dimethyl-arginine recognition by Tudor domains; ripsianes et al Sattler, Nat Struct Mol Biol (2011) Splicing regulation: U2AF/ Py tract, Mackereth et al Sattler, Nature (2011)

I nstitute of Structural Biology 17

Cellular pathw ays and diseases LRRK2 Delta/Notch: Dll1/Magi2 PDZ Peroxisomal biogenesis

90°

W118 R25 R25 K55 K55 K56 K56 R40 R40 K34 K34

Pex5 Pex14 Pex14 Pex19

F75 F122 F70 F71

Pex1 4 ( N) Pex5 W xxxF Pex1 9 FFxxxF

Neufeld et al EMBO J 2009 Roc LRR WD40 MAP3K COR 1000 2528

Construct Expression Kin No expression Kin long1 Exp, weak Kin long2

• Exp. Weak

KinD

• Expr. Weak

KinD long1 Overexp. KinD long2 Overexpr. Roc-Cor-Kin Expression Cor-Kin long1 Expression Kin long1-WD Expression LRR Overexpr. ROC-COR Overexpr. WD40 Overexpr.

• Structural biology of LRRK2 and its

molecular interactions

• Understand molecular mechanisms
• Drug design

pH 6.9 10 9 8 7 F2 [ ppm]

• Dll1 peptide/PDZ4: Kd ~ nM
• Mutant 100-fold reduced
• Next steps:
• 3D structure
• Small molecules

W ith M. Ueffing ( PROT) Funding: Michael J. Fox Foundation; HGF Allianz HelMA ( applied) W ith G. Przem eck,

• M. Hrabe de Angelis ( I EG)
• Peroxiosomal biogenesis disorders:

“Zellweger syndrome”

• Understand basic mechanisms

Sattler Group

I nstitute of Structural Biology 18

Research topics in Niessing lab

Epithelial cancer

Mitotic spindle

• rientation

mRNA & vesicle transport EpCAM signal transduction

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

Niessing Group

Associated diseases: Aneuploidy, multiple diseases Neurodegeneration, Dementia Griselli syndrome Microvillus inclusion disease

Motor complexes for mitotic spindle orientation

I nstitute of Structural Biology 19

Research topics in Reif lab

Protein degradation Multidrug resistance Alzheimer’s disease, 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)

Protein Misfolding

Interactions with Small Molecules And Molecular Chaperones ß-Amyloid Fibril Structure

Soluble Protein Com plexes

Proteasome Small heat shock proteins

Mem brane Proteins

Maltose ABC Transporter MalEFG‧K2 Small Multidrug Transporter (EmrE)

Reif Group

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 Transformation

Crystallization

Purification

Culture

Expression screening Structure determination Amplification

• E. coli

SF9/baculo virus

Isotope labeling Fermentor

X-ray NMR Expression

Gene Gene

Cloning

Tag

antibiotic

Gene

2-30 mg >95% purity

http://www.helmholtz-muenchen.de/PEPF I nstitute of Structural Biology 22 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

Protein Expression and Purification Facility http: / / www.helmholtz-muenchen.de/ PEPF

Why solution state NMR?

Nature 2007 Nature 2007 Nature 2011 Nature 2009 In vitro HeLa

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, …

bound free

T2 [ms] residue

RDCs SANS SAXS

Nuclear spins, Magnetic moments & Resonance

precession frequency B0 nuclear magnetic dipole

Magnetic field

Apply radio frequency at resonance to measure the nuclear precession frequencies frequency 

NMR spectrum Nuclear spin Resonance

= B0

B0

NMR Magnet

time t

Fourier transformation

Richard Ernst, Nobel Prize Chemistry 1991

60 MHz 250 MHz 900 MHz

• 1

11 10 9 8 7 6 5 4 3 2 1 ppm

Ribonuklease 40 MHz Lysozym 900 MHz

München 2002

• M. Saunders et al.

J.Amer.Chem.Soc. 1957, 79, 3289

I nstitute of Structural Biology 34

Instruments

• 900 MHz cryo
• 800 MHz cryo (HMGU)
• 750 MHz
• 600 MHz cryo
• 600 MHz
• 500 MHz wide bore
• 400 MHz wide bore

Advanced NMR Technology Platform

• Protein expression & isotope labeling  PEPF
• Molecular interactions
• Wirkstoffzentrum (hit validation/optimization)

Workshops/training

• Jul 27- Aug 3, 2009, 2011: EMBO Practical Course, BNMRZ, Munich
• Oct 16-18, 2008: NMR-Life Workshop, Murnau
• Sep 8-15, 2008: EMBO World Practical Course, Beijing, China
• Jul 24/25: Protein expression and isotope labeling for structural biology, BNMRZ
• Jan 16, 2008: Symposium "Perspectives of Biomolecular NMR“, BNMRZ

Personnel

• PD Dr. Gerd Gemmecker
• Dr. Rainer Haessner

Bayerisches NMR Zentrum (BNMRZ)

800 MHz @ HMGU 900 MHz @ TUM

Structure determination by NMR

Molecular dynamics

A 1D NMR spectrum of a protein

H H H

Is my protein folded?

Unfolded 20 kDa protein

• 1

11 10 9 8 7 6 5 4 3 2 1 ppm

Trp H backbone HN side chain NH2 aromatic CH CH CH2 CH3

• 0.5

0.0 ppm

• 1

11 10 9 8 7 6 5 4 3 2 1 ppm

backbone HN side chain NH2 aromatic CH aliphatic

• 0.5

0.0 ppm

CH3

Folded 20 kDa protein

NMR fingerprint spectra

Side view Contour plot

15N 1H 15N 1H

2D 1H,15N correlation (HSQC, TROSY)

N H C O C

NMR structure determination: distance restraints

18 kDa protein/RNA complex

protein/protein NOEs intermolecular protein/RNA NOEs Proton “density” in a 15 kDa protein

Structure calculation

• The NOE intensities measured in a NOESY spectrum are calibrated and used

to derive proton/proton distance restraints (NOE ~ 1/r6)

• These are applied in a restrained molecular dynamics calculation.
• Different and randomized starting structures are used.

The result is an ensemble of structures

An ensemble of NMR structures

• btained from a

restrained MD/SA calculation

Kurt Wüthrich Nobel Prize Chemistry 2002

Dynamics – exchange

Fast exchange k >> Ω Coalescence k ≈ Ω Slow exchange k << Ω

k1

A  B

k-1 kex = k1+ k-1

223 K 243 K 253 K 263 K 273 K

Eyring equation

Bain A.D. Prog NMR Spectroscopy (2003) 43, 63-103.; Kessler H. Angew Chem (1970) 9, 219-235.

∆Hǂ conformational coordinate energy

Transition state theory

Temperature dependence of kex

Ligand binding in NMR titrations - fast exchange

Fraction bound [PL] ~ obs- free = f([Ltot])

Binding in fast exchange

• n the NMR chemical shift time scale

[P] < KD

Dissociation constant KD from binding isotherm koff

P + L  PL

kon

Kd= [P][L] / [PL] = kB/kA kA = kon [L]; kB = koff B = protein-ligand complex PL A = free protein P

bound free

Selenko et al (2001) Nature Struct. Biol. 8, 27-31.

The NMR „band shift“ and binding site mapping

Protein Protein-RNA

Chemical shift perturbation upon ligand binding Mapping of the ligand binding site onto the structure

Lingel et al (2003) Nature 426, 465-9 11. Lingel et al (2004) Nat Struct Mol Biol 11, 576-7.

Folding upon ligand binding seen by NMR

NMR spectrum of a novel RNA binding domain

Protein (misfolded) Protein Protein+RNA

when bound to an RNA oligonucleotide

Mourao et al. RNA. (2010) in press.

Structure based drug discovery

Hits from activity screening Compound library Positive hits Optimization of ligand affinity and selectivity NMR-based screening 3D structure determination protein-ligand complex

Protein expression (PEPF) Assays & Screening (ADSF) NMR X-ray NMR

NMR in drug research: SAR by NMR

Structure-Activity Relationships (SAR) by NMR

Science (1996) 274, 1531

SAR by NMR ... ... is a nuclear magnetic resonance (NMR)-based method in which small organic molecules that bind to proximal subsites of a protein are identified, optimized, and linked together to produce high- affinity ligands. The approach is called "SAR by NMR" because structure-activity relationships (SAR) are obtained from NMR. With this technique, compounds with nanomolar affinities for a target protein can be rapidly discovered by tethering two ligands with micromolar affinities. The method reduces the amount of chemical synthesis and time required for the discovery of high- affinity ligands and is particularly useful in target-directed drug research.

SAR by NMR

Peptide binding Bcl-2 inhibitor binding Nature (2005) Science (1997)

Regulation of pre-mRNA splicing

Constitutive splice signals intronic cis elements

Pre-mRNA exon

ESE: exonic splicing enhancer ESS: exonic splicing silencer

exon exon intron

Constitutive splicing Alternative splicing ISE: intronic splicing enhancer ISS: intronic splicing silencer

3’ ss 5’ ss

RRM2 RRM1

Py tract RNA recognition involves a dynamic equilibrium between

• pen and closed conformations of U2AF65



RRM2 RRM1

Mackereth et al Nature (2011)

• pen

closed

U2AF65

UACUAAC UUUUUUU AG

SF1 U2AF35

Human U2 introns

Py tract 3’ ss

Solution conformation differs from crystal structure

RRM1 RRM2

NMR

RRM1 RRM2’ RRM1’ RRM2

RNA RRM1+2 ∆linker

RRM2 RRM1

Importance of using solution methods for studying multidomain proteins

X-ray

Sickmier et al Mol. Cell (2006)

RDCexp [Hz] RDCexp [Hz] RDCcalc [Hz] RDCcalc [Hz]

Multi-domain conformational selection

Literature – Structural Biology

Structural biology of proteins and nucleic acids

• Protein Structure and Function . Gregory A. Petsko, Dagmar
• Ringe. New Science Press NY 2004. Paperback.
• Introduction to Protein Structure. Carl Branden, John Tooze.

Garland Science 1998, 2nd Edition. Paperback.

• Principles of nucleic acid structure. Wolfram Sänger.

Springer 1988. Paperback

Literature - Biomolecular NMR

Modern/biomolecular NMR:

• Structural Biology – Practical NMR Applications.

Quincy Teng. Springer (2005)

• Protein NMR spectroscopy – Principles and Practice.

Cavanagh, Fairbrother, PalmerIII, Skelton. Academic Press 2nd ed (2007) Basic reading, practical approaches:

• NMR of Proteins and Nucleic Acids. Kurt Wüthrich. Wiley (1986)
• NMR of Macromolecules: A Practical Approach. Oxford (1993). Gordon Roberts

(Ed.) Introduction to NMR (chemists):

• Understanding NMR spectroscopy – James Keeler (free on-line!)

Literature – X-ray crystallography

An almost perfect combination of modern approaches with moderate theoretical load. Some Math-textbook may be necessary (Klaus Scheffzek). Macromolecular crystallography

• Outline of crystallography for biologists.

David Blow. Oxford (2003)