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Bioinformatics Master Course Sequence Alignment

Lecture 9aPattern matching

part I

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Sequence Patterns vs. Protein Structure

I. Protein-Protein interaction1. enzyme (protein) substrate : serine protease trypsin2. receptor (protein) ligand : growth hormone receptor3. antibody (protein) antigen : immunoglobulin (Ig)

II. Protein-Ion and small molecule interaction1. protein ion (Ca2+, Mg2+, Na+, K+, Cl–, HCO3

–, SO42–) :

calmodulin2. pump ion, coupled to enzymatic function : ATPase3. channel water : aquaporin

III. Protein-DNA/RNA interaction1. enzyme DNA : Eco-RI ribozyme2. binder DNA groove : leucine zipper, zinc finger3. regulator RNA : KH domain

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Reactions and Interactions

• What is the difference between a reaction and an interaction? change in chemical bonding

• Which one of these is a chemical bond?1. H3C-CH2-O-H2. Na+ Cl–

3. H-O-H···OH2

4. H-O-CH2-CH3···H3C-CH2-O-H

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Bond Strength

• Bond strength and lifetime are a function of temperature vibration (bond stretching), thermal background

• Non-covalent interactions depend very much on the medium compare salt crystal with salt solution

• Interaction strength has a strong distance dependence ion-ion ~ r–2, dipole-dipole ~ r–4

quadrupole-quadrupole ~ r–6

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Binding: Complementary Interfaces

Binding requires complementary interfaces:

Interfaces have characteristic and conserved residues patterns or motifs

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Sequence Patterns and Profiles• Comparison between sequence pattern matching and

similarity scoring

PATTERN SCORE

exact word identity

regular expression weight matrix

Hidden Markov Model profile

generalized profilegeneral Hidden Markov Model

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Resources• PROSITE: biologically significant sites, patterns and profiles

– www.ebi.ac.uk/ppsearch/

• PFAM: large collection of multiple sequence alignments– www.sanger.ac.uk/Software/Pfam/

• DIP: interacting proteins– dip.doe-mbi.ucla.edu/

• Specialized Databases– Immunoglobins: imgt.cines.fr/– Ca2+-binding proteins structbio.vanderbilt.edu/cabp_database/

• Molecular visualisation packages– VMD: www.ks.uiuc.edu/Research/vmd/– MOLMOL: www.mol.biol.ethz.ch/wuthrich/software/molmol/– Rasmol: www.umass.edu/microbio/rasmol/

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Protein-Protein Interactions

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Protein Interaction NetworksMost proteins are functionally linked to other proteins

H Jeong, SP Mason, A-L Barabási & ZN Oltvai "Lethality and centrality in protein networks" Nature 2001;411(6833):41

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I.1 Enzyme: Serine Protease Trypsin

• Specific class of hydrolases– cleave peptide bonds at specific residue positions.

• aspartate proteases, cysteine proteases, serine proteases

• Trypsin is a serine protease– cleaves C-terminal of the basic residues Lys and Arg– one of the three principal digestive proteases

• other two are pepsin and chymotrypsin

– produced in an inactive form by the pancreas

• Pattern: His57, Asp102 and Ser195 (H-D-S)

NC

CN

'R'

OH

H

HN

CC

'R'

OHH

O

H2O

NC

C

'R'

OHH

OH

CH2

Trypsin

HOCH2

Trypsin

HOCH2

Trypsin

N

H

HN

H

H

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Serine Protease: Trypsin

• Pattern: His57, Asp102 and Ser195 (H-D-S)

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Principle of Catalysis

http://www.chemguide.co.uk/physical/basicrates/catalyst.html

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Trypsin Complex with Inhibitor

1btc.pdb

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I.2 Receptor: Growth Hormone Receptor

• Membrane-borne receptors:– extra-cellular domain

• ligand-binding site

– transmembrane domain• anchoring in the cell membrane

– intracellular domain• kinase or another signalling module (typically)

• Receptor for growth hormone – member of the cytokine receptor superfamily– dimerizes upon binding growth hormone as ligand– activates intracellular kinase, triggers cellular signalling cascade.

• Most structures only contain extra/intracellular domain– transmembrane domain is difficult to crystallize

• Patterns:– YGEFS (growth hormone receptor)

– WSxWS (cytokine receptor family)

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Growth Hormone Receptor Complex with Growth Hormone

1a22.pdb

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I.3 Immune System: Antibody• Antibodies (immunoglobulins, or Ig)

– immune system: bind ’foreign’ (non-self) characteristic structures

• e.g. protein surfaces

• Heavy Chain and Light Chain• Constant part (Fc) and Variable part (Fv).

– Fv specific recognition of target molecule (‘antigen’)

• structure called ‘Ig fold’:– Two -sheets face-to-face, with ‘Greek-key’ motif– binding site between two Ig folds– hypervariable loops participate in binding:

• H1, H2, H3 and L1, L2, L3• composition characteristic for antigen

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Pfam Ig Family Alignment

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Patterns of Hypervariable Loops

Loop Before After Length

CDR-L1 always Cys always Trp 10 to 17

CDR-L2 generally Ile-Tyr, also Val-Tyr, Ile-Lys, Ile-Phe

- always 7

CDR-L3 always Cys always Phe-Gly-xxx-Gly 7 to 11

CDR-H1 always Cys-xxx-xxx-xxx always Trp 10 to 12

CDR-H2 typically Leu-Glu-Trp-Ile-Gly Lys, Arg-Leu, Ile, Val, Phe, Thr, Ala-Thr, Ser, Ile, Ala

16 to 19

CDR-H3 always Cys-xxx-xxxx always Trp-Gly-xxx-Gly 3 to 25

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Antibody Structure

Kontou et al. Eur J Biochem 2000 267 23891F3R.pdb

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Antibody Diversity• Gene translocation

• heavy chain – multiple VH genes join with one DH and one JH

• light chain – multiple VL genes join with one JL gene

www.cat.cc.md.us/courses/bio141/lecguide/unit3/humoral/antibodies/abydiversity/abydiversity.html

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Protein-Ion and Protein-’small molecule’

Interactions

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II.1 Ion Binding: Calmodulin

• Two domains, each two ‘EF-hands’: – helix-loop-helix structure– loop contains Ca2+-binding motif.

• Ca2+-ion: 6-fold coordinated: – Oxygens from residues 1, 3, 5, 7, 9, and 12 in EF loop:

D-K-D-G-D-G-T-I-T-T-K-Q– one water molecule– three are negatively charged

• Ca2+-binding changes conformation of entire protein from closed to open– open conformation exposes hydrophobic surface area– binding site for calmodulin target proteins

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Calmodulin Complex with Calcium Ions

1exr.pdb

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II.2 Ion Pump: 2. Calcium ATPase (ATP synthase)• protein complex

– links electrical potential to ATP hydrolysis/synthesis– interconversion between mechanical and electrochemical energy in

molecular motors.

• F1F0 ATPase: reversible proton pump/motor• P-type ATPases: transport ions across membrane against a

concentration gradient.– Pattern: D-K-T-G-T-[LIVM]-[TIS]– Next to aspartate which is phosphorylated during reaction cycle

• Na+/K+-ATPase: ubiquitous membrane transport protein in mammalian cells– maintains high K+ and low Na+ in cytoplasm for normal membrane potentials

and cellular activities

• Ca-ATPases: Ca2+ from cytoplasm to organels (mammalian)– e.g. sarcoplasmic reticulum, endoplasmic reticulum

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ATPases

F1Fo-ATPase Ca2+-ATPasewww.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/f1fo.htm

www.utoronto.ca/maclennan/rint1.htm

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ATPase: Calcium Ions in Active Site

1eul.pdb

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II.3 Membrane Channel: Aquaporin

Conserved NPA motifs: Asn, Pro and Ala stabilise loops through multiple hydrogen bonds

Bert de Groot: www.mpibpc.mpg.de/groups/de_groot/bgroot.html

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Aquaporin: Motifs

•NPA: stabilizes loops B and E

• G(a)xxxG(a)xxG(a):– Crossing of

right-handhelicalbundles

Andreas Engel and Henning Stahlberg, in: Current Topics in Membranes (2001), Hohmann, Agre & Nielsen (Eds.) Academic Press

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Aqu

apor

in S

ubun

it

Ber

t de

Gro

ot: w

ww

.mpi

bpc.

mpg

.de/

grou

ps/d

e_gr

oot/b

groo

t.htm

l

1j4n.pdb

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Protein-DNA/RNA Interactions

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III.1 Enzyme: Eco-RI• Restriction enzyme:

– cut palindrome sequences – complex of one

DNA molecule with two Eco-RI molecules with inversion symmetry

www.accessexcellence.org/RC/VL/GG/restriction.html

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Eco-RI

1qrh.pdb

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III.2a DNA recognition: Leucine Zipper

• Dimer – Leu interactions– binds DNA by a fork-shaped structure

• ‘coiled-coil’ structure:– leucines on one side of helix– 7-residue repeat; one helix turn is 3.6 residues

a b c d e f g (position)

256 KV E E L L S KN Y H L E N EV A R L K K LV G 279

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Leucine Zipper: Complex with DNA

1an2.pdb

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Leucine Zipper: 7-Residue Repeat

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III.2b DNA Recognition: Zinc Finger Proteins

• zinc coordinates several side chains– pulls them together to form ‘finger’ loops

• Pattern: C-x2-4-C-x12-15-H-x3-5-H or C-x2-4-C-x12-15-C

– recognize nucleic acids (DNA or RNA) • modulate genes (also proteins can be targeted)

• modulate important functions:– gene expression– reverse transcription and virus assembly

• drug discovery targets: – pathogen-specific 3D structures – different from endogeneous (cellular) zinc finger proteins

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Zinc Finger Complex with DNA

1a1h.pdb

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III.3 RNA Regulation: KH Domain

• bind to specific DNA/RNA locations– regulation of RNA synthesis and metabolism– combination with other domains– Pattern: G-x-x-G

• ribonucleoprotein (RNP) domain• double stranded RNA binding domain (dsRBD)• K Homology (KH) domain

– recognize tetranucleotide motifs – high affinity/specificity:

• RNA secondary structure• repeated sequence elements

• alpha/beta fold similar to ribosomal proteins

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KH Domain Complex with RNA

1k1g.pdb

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Copyright ©2005 American Society of Plant BiologistsPrzybilski, R., et al. Plant Cell 2005;17:1877-1885

The HHRzHammerhead Motif of Ribozyme

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Hammerhead Motif of Ribozyme

• three base-paired helices (I-III) • core of 11 highly conserved, non-complementary

nucleotides – necessary for the catalysis.

• catalytic motif discovered by sequence comparison of plant viroids– site-specific,

self-catalyzed cleavage

(Birikh, 1997)academic.brooklyn.cuny.edu/chem/zhuang/QD/toppage1.htm

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Hammerhead Ribozyme Action

488d.pdb

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Copyright ©2005 American Society of Plant Biologists

Przybilski, R., et al. Plant Cell 2005;17:1877-1885

Modeling of the Arabidopsis HHRz Ara2

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