Strukturna biologija, bioinformatika, biologija sistema biologija 21-og veka

Strukturna biologija, bioinformatika, biologija sistema

biologija 21-og veka

Danasnja presentacija bice podeljena u tri dela:

1. Pozadina i opsti uvod- NMR- Rentgenska kristalografija2. Strukturna biologija – primeri iz moje

laboratorije3. Poseban osvrt na bioinformatiku i

biologiju sistema kao interdisciplinarne grane relvantne za kompiuterske nauke

Human & other genome sequences

Functionally cloned DNA moleculescoding for proteins of interest.

Amino acid sequences

Protein structure Protein function

Structural biology

Protein chemistry / yeast 2-hybrid screens / proteomics / enzymology / genetics / transgenes / knock-outs / knock-ins / chemical genetics / etc.

Bioinformatics

3D structures of molecules allow us to understand biological processes at the most basic level. We can ‘see’ which molecules interact, how they interact, how they function, how drugs act. They can help us understand disease at an atomic level. 3D structures can be exploited in development of new drugs.

structure-based drug design

Strukturna Biologija• Struktura moze ponekad da odkrije funkciju proteina

direktno • Struktura moza da racionalizuje eksperimentalnu

obzervaciju o selktivitetu i specificnosti enzimaticne reakcije

• Struktura moze da postane osnova za rational drug/inhibitor discovery.

• Struktura moze da razotkrije dinamicki aspekt proteinskog ponasanja.

• Trodimenzionalne topologije polipeptida obezbedjuju podatke za resavanje problema formiranja proteinske strukture -- ‘protein folding problem’.

• [Sometimes a 3D structure can be rather uninformative - the ‘structural genomics’ debate.]

Experimental modes of molecular structural biology

X-ray crystallography

Protein crystals

(3-50 mgs/ml; ca. 100 ml)

[Se-methionine labelling]

X-ray source/-Synchrotron

Applicable to proteins of any size (in principle).

NMR spectroscopy

Protein solutions

(> 0.5 mM; min. volume 0.3ml; 10-100 mg)

15N,13C-Isotope labelling

NMR Spectrometer

Applicable to small(ish) proteins (smaller than ca. 30,000 MW)

Cryo-electron microscopy

Macromolecular assemblies/particles frozen in vitreous ice

Electron microscope

Large particles, typically > 500,000 MW

High resolutionMedium resolution

‘Low’ resolution

x 10 x 10x 10x 10x 10x 10 0.2 nm2 nm20 nm200 nm2 m20 m200 m

CELLS MOLECULES

ORGANELLES ATOMS

Unaided eye

Light microscope

Electronmicroscope

1 m = 103 mm

= 106 m

= 109 nm

The sizes of cells and of their component parts

STA JE NMR?

Nuclear Magnetic Resonance (NMR) je mocna spektroskopska tehnika koja pruza informaciju o strukturnim i hemijskim osobinama molekula.

NMR je ne-destruktivna metoda za analizu strukture i dinamike molekula. NMR koristi osobine odredjenih atoma kada su izlozeni vrlo jakom magnetnom polju. For biochemists these are mainly 1H, 15N, 13C and 31P. 1H and 31P are highly abundant isotopes whilst 15N and 13C are present at only low levels < 1%. Studies using these nuclei generally require isotopic enrichment by production of the molecule from media that has been enriched in these isotopes.

Prof. Kurt WüthrichNobel Prize for Chemistry 2002

Typically the magnets used in NMR spectroscopy are 10,000-15,000 times stronger than the earth’s magnetic field. The NMR experiment generally consists of applying short bursts or pulses of energy in the radio frequency (RF) range, typically 40-800 MHz, to the sample. These pulses of RF cause the nuclei to rotate away from their equilibrium position and they start to precess (rotate) around the axis of the magnetic field. The exact frequency at which the nuclei precess is related to both the chemical and physical environment of the atom in the molecule. By using different combinations of RF pulses and delays it is possible to determine how each atom in the molecule interacts with other atoms in the molecule.

592 709

11 0

11 5

1 2 0

1 2 5

1 0 .0 9 .0 8 .0 7 .0

592 732

11 0

11 5

1 2 0

1 2 5

1 0 .0 9 .0 8 .0 7 .0

1H(ppm)

15N

(ppm

)

11 0

11 5

1 2 0

1 2 5

1 0 .0 9 .0 8 .0 7 .0

543 709

The NMR spectrum is exquisitely sensitive to the conformation of the polypeptide chain, and to the presence of interacting chemical ligands.

These and other features of the rich ‘spin physics’ that underlies the NMR phenomenon mean that NMR spectroscopy is a highly versatile tool for the characterisation of:

Structure Dynamics Molecular interactions

N

C

1

2

3

4

5

6

N

C

N

C

Harris et al. (2004) J. Mol. Biol.

NC

N

C E676E667

E664

E699

E700

E652

D709

4 2 4

3

1

5

6

Harris et al. (2004) J. Mol. Biol.

X-ray Crystallography

- An experimental technique involving diffraction of X-rays by crystalline material.

-X-ray wavelength ~ Å

-Based on the diffraction pattern, electron density of the molecule could be reconstructed. (Need intensities and phases)

-Model is built in the reconstructed electron-density

-Model – the molecular picture – molecular structure from global folds to atomic details

-Limited information about the molecule’s dynamic

-Depends on obtaining crystals

1. Why X-rays?

2. Why electron density?

3. Why crystals?

EYEPIECE LENSmagnification n

Scattered radiation

OBJECTIVE LENSmagnification m

OBJECT

VISIBLE LIGHT

Enlarged image of objectMagnification mn

X-RAYS

OBJECT(crystal)

Scattered radiation

DETECTOR

COMPUTER

PHASES

COMPUTEDELECTRON-DENSITYMAP

CRYSTALLOGRAPHER

Pregled procesa odredjivanja strukture proteina koriscenjem difrakcije X zraka

1. Proizvodnja izolovanog dovoljno velikog kristala kandidat proteina

2. Postavljanje kristala, prikupljanje i evaluacija preliminarnih difrakcionih podataka

3. Kompletno prikupljanje podataka i procena fazi

4. Izgradnja i rafiniranje proteinskih lanaca

5. Validacija strukture

European

Synchrotron

Radiation

Facility

(Grenoble, France)

Structural characterisation of drug-targets from M.tuberculosis

Snezana Djordjevic

Institute of Structural Molecular Biology

M. tuberculosis

• 2-3 million deaths from tuberculosis annually

• 1/3 of world population currently infected with the disease

• Drug resistance

-multidrug-resistant strains

-12.6 % M. tuberculosis isolates resistant to at least one drug

-2.2 % resistant to both isonazid and rifampin

New Drugs

-agents that exhibit activity against drug resistant strains

-completely sterilize infection

-shorten the duration of drug therapy and thus promote drug compliance

METRO – 06/03/2007

Mechanism of resistance to Isoniazid

-Isoniazid is a prodrug that is oxidized by KatG

-KatG is catalase-peroxidase

-Mutation of the KatG leads to resistance

KatG Prodrug activation ResistanceResistance

KatG activity is important for virulence !

-Physiological function of the KatG includes protection of the mycobacterium against H2O2 and other ROS produced by the microbe and its host.

?

KatG AhpC AhpD

AhpD

Alkylhydroperoxidase

From M. tuberculousisPaul Ortiz de Montellano

Dept. of Pharmaceutical Chemistry, UCSF

C2; a=186.38 Å, b=117.28 Å, c=88.99 Å, =113.97°

177 residues/monomer

Structure solution: SeMet/MAD

4 wavelengths data collected in Grenoble

2Fo-Fc map1.9 (1.7) Å resolution

N

CN

C

12

3

4

56

7

8

AhpD Monomer Topology

CXXC

Thioredoxins

-solvent exposed

-pKa ~ 7.1

Peroxiredoxins

From structure to function and From structure to function and the catalytic mechanismthe catalytic mechanism

Cys130

Cys133

His137

Glu118

Putative substrate binding site

Cys133

NADH

NAD+

Lpd(ox)

Lpd(red)

DlaT-LpH2

DlaT-Lp

AhpD(ox)

AhpD(red)

AhpC(red)

AhpC(ox)

ROOH

ROH

Novel redox pathway in M. tuberculosis

Lpd: Dihydrolipoamide dehydrogenase

SucB: Dihydrolipoamide acyltransferase

Components of pyruvate dehydrogenase complexes

E3 E2

Pyruvate Acetyl-CoA + CO2

NAD+ NADH

Molecular Surface

A Prototypical Two-Component Signal

Transduction System

Receptor / input / sensor domain

Kinase Core

ResponseRegulator (RR)

Periplasmic Space

P

ExternalStimulus Response

Histidine Kinase (HK) Sensory Protein

B

P

+CH3

A A

WW

P P+ATP

Y

-CH3

TarSAM

R

B

Chemotaxis

DosS

• Induced by exposure to hypoxia, NO and ethanol.

• Structural studies have been initiated with the aim of describing the signalling mechanism that leads to histidine kinase activation.

• Histidine kinase domain (HK) undergoes autophosphorylation and can carry out a Mg2+ dependant phosphotransfer reaction onto DosR.

• DosS : DosR are a cognate sensor-regulator pair.

Identification of domain boundaries

PDE2A_B 196 DVSVLLQEIITEARN-------LSNAEICSVFLLDQ------------NELVAKVFDGGVVDDe----sY

DosS GAF_A 3 DLEATLRAIVHSATS-------LVDARYGAMEVHDRQH---------RVLHFVYEGIDEETVR------R

cGMP PDE_1 154 DVTALCHKIFLHIHG-------LISADRYSLFLVCEdss-------ndKFLISRLFDVAEGSTleeasnN

cGMP PDE_2 336 SLEVILKKIAATIIS-------FMQVQKCTIFIVDEdcsdsf-ssvfhMECEELEKSSDTLTR------E

anfA 46 DLADALSIVLGVMQQ-------HLKMQRGIVTLYDMr----------aETIFIHDSFGLTEEEk-----K

cGMP PDE_3 228 DATSLQLKVLRYLQQ-------ETQATHCCLLLVSEd----------nLQLSCKVIGEKVLG-------E

ADEN_CYCL_1 79 GFENILQEMLQSITLkt---geLLGADRTTIFLLDEe----------kQELWSIVAAGEGDRS------L

ADEN_CYCL_2 271 DLEDTLKRVMDEAKE-------LMNADRSTLWLIDRd----------rHELWTKITQDNGST-------K

yebR 27 DLNRDFNALMAGETS-------FLATLANTSALLYErlt-------diNWAGFYLLEDDTLVLg----pF

Hypoth. Pro. 54 LIKATLQKTMEASIH-------QTGAQLGSLFLLDGd----------gRVTESILARGATDQSqk---kN

Nif-regul_1 68 RLEVTLANVVNVLSS-------MLQMRHGMICILDSe-----------GDPDMVATTGWTPEMa-----G

Nif-regul_2 46 RLEVTLANVLGLLQS-------FVQMRHGLVSLFNDd-----------GVPELTVGAGWSEG-------T

Nif-regul_3 35 NTARALAAILEVLHD-------HAFMQYGMVCLFDKe----------rNALFVESLHGIDGERkk--etR

Nif-regul_4 21 DLSKTLREVLNVLSA-------HLETKRVLLSLMQDs-----------GELQLVSAIGLSYEEf-----Q

consensus 1 DLEELLQTILEELRQ-------LLGADRVSIYLVDEDK---------

RGELVLVASDGLTLPE------L

PDE2A_B EIRIPADQ-----GIAGHVATTGQILNIP-DAYAHPl--fYRGVDDSTGFR-----TRNILCFPIKNEn- DosS GAF_A IGHLPKGL-----GVIGLLIEDPKPLRLD-DVSAHP----AS-IGFPPYHPP----MRTFLGVPVRVR-- cGMP PDE_1 CIRLEWNK-----GIVGHVAAFGEPLNIK-DAYEDPr--fNAEVDQITGYK-----TQSILCMPIKNHr- cGMP PDE_2 RDANRINY-----MYAQYVKNTMEPLNIP-DVSKDKr---FPWTNENMGNInq-qcIRSLLCTPIKNGk- anfA RGIYAVGE-----GITGKVVETGKAIVAR-RLQEHP-----DFLGRTRVSRng-kaKAAFFCVPIMRA-- cGMP PDE_3 EVSFPLTM-----GRLGQVVEDKQCIQLK-DLTSDD----VQQLQNMLGCE-----LRAMLCVPVISRa- ADEN_CYCL_1 EIRIPADK-----GIAGEVATFKQVVNIPfDFYHDPrsifAQKQEKITGYR-----TYTMLALPLLSEq- ADEN_CYCL_2 ELRVPIGK-----GFAGIVAASGQKLNIPfDLYDHPdsatAKQIDQQNGYR-----TCSLLCMPVFNGd- yebR QGKIACVRipvgrGVCGTAVARNQVQRIE-DVHVFD-------GHIACDAA-----SNSEIVLPLVVK-- Hypoth. Pro. IVGQVLDK-----GLAGWVRENKRTGLIN-DTTKDY----RWLKLPDEPYQ-----ALSALGVPIVWG-- Nif-regul_1 QIRAHVPQ-----KAIDQIVATQMPLVVQ-DVTADP-----LFAGHEDLFGppeeaTVSFIGVPIKAD-- Nif-regul_2 DERYRTCVp---qKAIHEIVATGRSLMVE-NVAAEt---aFSAADREVLGAsd-siPVAFIGVPIRVD-- Nif-regul_3 HVRYRMGE-----GVIGAVMSQRQALVLP-RISDDQ-----RFLDRLNIYDy----SLPLIGVPIPGAd- Nif-regul_4 SGRYRVGE-----GITGKIFQTETPIVVR-DLAQEP-----LFLARTSPRQsqdgeVISFVGVPIKAA--

consensus GVRFPLDE-----GLVGRVAETGRPLVIP-DVEADP----FFFLDLLQRYQL----IRSFLAVPLVAG--

12

3 44 5

2

3

Further structural investigation of GAF domains

Secondary Structure: 1MC0

PDE2A_B -QEVIGVAELVNK-------------------INGPWFSKFDEDLATAFSIYCGISIAHSLLYKKVN 345 DosS GAF_A -DESFGTLYLTDK-------------------TNGQPFSDDDEvlvqalaaaagiavanarlyqqak 150 cGMP PDE_1 -EEVVGVAQAINKk-----------------sGNGGTFTEKDEKDFAAYLAFCGIVLHNAQLYETSL 314 cGMP PDE_2 kNKVIGVCQLVNKmee--------------ttGKVKAFNRNDEQFLEAFVIFCGLGIQNTQMYEAVE 503 anfA -QKVLGTIAAERV-------------------YMNPRLLKQDVELLTMIATMIAPLVELYLIENIER 196 cGMP PDE_3 tDQVVALACAFNK-------------------LGGDFFTDEDERAIQHCFHYTGTVLTSTLAFQKEQ 375 ADEN_CYCL_1 -GRLVAVVQLLNKlkpyspp-----dallaerIDNQGFTSADEQLFQEFAPSIRLILESSRSFYIAT 249 ADEN_CYCL_2 -QELIGVTQLVNKkktgefppynpetwpiapeCFQASFDRNDEEFMEAFNIQAGVALQNAQLFATVK 441 yebR -NQIIGVLDIDST--------------------VFGRFTDEDEQGLRQLVAQLEKVLATTDYKKFFA 179 Hypoth. Pro. -DELLGILTLMHS--------------------QVNHFTPACATAMEKTAELIALVLNNARIQTKHK 202 Nif-regul_1 -HHVMGTLSIDRIw-----------------dGTARFRFDEDVRFLTMVANLVGQTVRLHKLVASDR 220 Nif-regul_2 -STVVGTLTIDRIp------------------EGSSSLLEYDARLLAMVANVIGQTIKLHRLFAGDR 198 Nif-regul_3 -NQPAGVLVAQPM-------------------ALHEDRLAASTRFLEMVANLISQPLRSATPPESLP 186 Nif-regul_4 -REMLGVLCVFRDg------------------QSPSRSVDHEVRLLTMVANLIGQTVRLYRSVAAER 180

consensus -GELLGVLALHRK-------------------DSPRPFTEEEEELLQALANQLAIALALAQLYEELR

56Secondary Structure|1MC0

SAMt99 : to detect remote structural homologues of this protein.

From the 11149 sequence homologies identified, 24 had a known structure but none of those identified produced significant global alignment. Local alignments covered either the C or N terminal regions. No alignment was found that covered both putative GAF domains.

1 structural homologue was identified for DosS GAF A domain : 1MC0

UV-Visible Characterisation of GAF A HaemAbsorption spectra of DosS 63-210

Wavelength (nm)

550 600 650

B. Ferric (solid line)A. Oxy-ferrous (dashed line)

C. Ferrous (dotted line)

D. Ferrous-CO (solid line)

E. Ferrous-NO (solid line)

A0.005

Absorption spectra of Haemoglobin

Wavelength (nm)

500 550 600 650 700

A0.1

A. Ferric haemoglobin (solid line)

B. Oxy-ferrous haemoglobin (dashed line)

C. Ferrous haemoglobin (dotted line)

D. Ferrous-CO haemoglobin (solid line)

E. Ferrous-NO haemoglobin (solid line)

Absorption spectra of Haemoglobin

Absorption spectra of DosS GAF A

Fe2+

His

CO / NO / O2

Visible/UV spectrum of the DosS GAF A (63-210) histidine to alanine mutants

The Model of Signalling

Fe2+

Fe2+

P

O2

PNO

OFF

ON

DosR

DosR

A

A

B

B

GAF B - NMR1H, 15N labeled DosS GAF B HSQC

NMR experiments: HNCO, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, HA(CA)NH and HA(CACO)NH were obtained at 1H frequency of 500MHz on a 0.6mM [1H, 13C, 15N]-labelled DosS 231-379, pH6, 20mM phosphate, 100mM NaCl.

GAF B - NMR

Predicted secondary structure for DosS GAF 2 using PSIPRED.

PROBLEMS:

- 48 residues are still to be assigned

- 21 expected cross-peaks are missing from the spectrum.Sekharan MR, Rajagopal et al. 2005. Backbone 1H, 13C, and 15N resonance assignment of the 46 kDa dimeric GAF A domain of phosphodiesterase 5 J Biomol NMR. 33(1):75

- Some of the cross-peaks do not form one peak but multiple peaks.

- High content of Val, Leu and Ala residues in the sequence.

Signalling mechanism

N

C

STRUCTURAL GENOMICS CENTRES IN

NORTH AMERICA, UK, FRANCE, JAPAN • OXFORD STRUCTURAL GENOMICS• Announced in 2003, with operations commencing in July

2004 for an initial three-year period, this initiative received funding from Canadian, Swedish and British sponsors from both the public and private sectors. For the second phase, July 2007, over £49 million is being made from public funding agencies in Canada, Sweden and Ontario, charitable foundations in the UK and Sweden, GlaxoSmithKline plc, Novartis and Merck. Laboratories at the University of Oxford , University of Toronto and Karolinska Institutet, Stockholm.

BIOINFORMATIKA

U toku poslednjih nekoliko dekada, napredak u molekularnoj biologiji, zajedno sa progresom u genetskoj tehnologiji doveo je do eksplozije u kolicini informacija stvorenih u naucnoj zajednici. Pojava te mase informacija proizvela je potrebu i zahtev za kompiuterizovanim bankama podataka (databases) da bi se cuvali, organizovali i katalogovali podaci. Pritom neophodno je bilo razviti sredstva (tools) za pregled, vizualizaciju i analizu tih podataka.   

Computational biology(sam proces analize i interpretacije podataka)

• Razvoj i primena alatki (tools) koji omogucavaju pristup, upotrebu i organizaciju raznih informacija

• Razvoj novih algoritma i statistike sa kojima se mogu proceniti relazije medju komponentama u velikoj grupi podataka. Na primer metode za lociranje gene u okviru sekvence, predvidjanje strukture proteina/funkcije, i grupisanje proteinskih sekvenci u familije povezanih (related) slicnih sekvenci.

“Organizmi funkcionisu kao integrisani sistemi – nasa cula, nasi misici, nas metabolizam i nas um rade zajedno u povezanoj celini. Biolozi su tradicionalno proucavali organizme deo po deo i uzivali u modernoj moci da proucavaju molekul po molekul, gen po gen. ISM je posvecen novoj nauci, kriticnoj nauci buducnosti kojoj je za cilj da razume integraciju delova koji sacinjavaju bioloski system.”

David Baltimore (Nobel Laureate) President, Cal. Institute of Tech., Pasadena

Systems biology requires:

-Integration of biology, technology, computation medicine

-a strong cross-disciplinary team of researchers.

-Institutes include scientists trained in biology, physics, chemistry, engineering, computing, mathematics, medicine, immunology, biochemistry, and genetics.

-They all speak the language of biology assembled into a multiplicity of teams that are attacking focused and important problems of systems biology.

Health Care in the 21st Century:• Predictive

(genetic makeup, protein markers) • Preventive

(probability of disease and response to treatment)• Personalized

(customized therapeutic drugs)

http://csbi.mit.edu:8080/infoglueDeliverWorking/

The MIT CSBI links biologists, computer scientists and engineers in a multi-disciplinary approach to the systematic analysis of complex biological phenomena.

http://www.sbml.org/Main_Page

The Systems Biology Markup Language (SBML) is a computer-readable format for representing models of biochemical reaction networks in software. It's applicable to models of metabolism, cell-signaling, and many others. SBML has been evolving since mid-2000 thanks to an international community of software developers and users. This website is the portal for the global SBML development effort; here you can find information about all aspects of SBML.

Manchester Centre for Integrative Systems Biology (MCISB)

• Molecular Biology / Biochemistry / Biophysics), mathematical and computational (Modelling / Data Integration / Text Mining

• Development and exploitation of methods for the quantitative measurement of kinetic and binding constants on a genome-wide scale

• Combined approaches will lead to computer models of parts of living cells. Some of these 'silicon cells' are already available for in silico experimentation, through the Biomodels and JWS databases.

VIRTUELNA CELIJA

My Group: NMR:Sunita Sardiwal Paul DriscollSyeed Hussain Richard HarrisShreenal PatelMark Jeeves ITC/CD:Christine Nunn John Ladbury

Paul Leonard

Collaborators at RVC: UV-VIS SpectraNeil Stoker Peter RichSharon Kendall Doug MarshallFarahnaz MoahedzadehStuart Rison

PHOSPHORYLATION Studies: EM:Irina Tsaneva Helen Saibil

Nadav Elad

Acknowledgements: