Bioinformatics Practical ���for���

Biochemists

Andrei Lupas, Birte Höcker, Steffen Schmidt WS 2013/14

03. Sequence Features

Günter Blobel, 1999, nobelprize.org

Targeting proteins

•  signal peptide •  targets proteins to the secretory pathway

•  N-terminal sequence recognized while peptide is still synthesized on the ribosome

Nielsen et al. (2007)

Signal Peptide Prediction

•  Sequence Logo of eukaryotic signal peptides

Signal Peptide Prediction - SignalP

http://www.cbs.dtu.dk/cgi-bin/webface2.fcgi?jobid=53329BB900004A2504297A29&wait=20

http://www.cbs.dtu.dk/services/SignalP/

PDB-id: 1c3w

Transmembrane Helices •  unusually long stretch of hydrophobic residues

•  >18 hydrophobic amino acids

•  hydrophobic interaction with lipids in membrane

•  orientation of helix / topology of the protein •  looking at the “loops”: R & K mainly found on cytoplasmic side “positive inside rule”

TGRPEWIWLALGTALMGLGTLYFLVKGMGVSDPDAKKFYAITTLVPAIAFTMYLSMLLGYGL

N

C

Sonnhammer et al. (1998)

Transmembrane Helices – TMHMM

•  http://www.cbs.dtu.dk/services/TMHMM/ •  Accuracy of predicting TM helices high > 90%

•  Accuracy of predicting the topology prediction > 75%

Transmembrane Helices – TMHMM

•  http://www.cbs.dtu.dk/services/TMHMM/

http://www.cbs.dtu.dk/cgi-bin/webface2.fcgi?jobid=5332A5E70000681F58EBAC3B&wait=20

William (1987) Biochim Biophys Acta

Secondary Structure – amino acid preferences

∝-helix" β-strand" β-turn "

Glu" 1.59" 0.52" 1.01"

Ala" 1.41" 0.72" 0.92"

Leu" 1.34" 1.22" 0.57"

Met" 1.3" 1.14" 0.52"

Gln" 1.27" 0.98" 0.84"

Lys" 1.23" 0.69" 1.07"

Arg" 1.21" 0.84" 0.9"

His" 1.05" 0.8" 0.81"

Val" 0.9" 1.87" 0.41"

Ile" 1.09" 1.67" 0.47"

Tyr" 0.74" 1.45" 0.76"

Cys" 0.66" 1.4" 0.54"

Trp" 1.02" 1.35" 0.65"

Phe" 1.16" 1.33" 0.59"

Thr" 0.76" 1.17" 0.9"

Gly" 0.43" 0.58" 1.77"

Asn" 0.76" 0.48" 1.34"

Pro" 0.34" 0.31" 1.32"

Ser" 0.57" 0.96" 1.22"

Asp" 0.99" 0.39" 1.24"

PDB: 1kgs, Buckler et al. (2002), Structure

Secondary Structure – buried ß-sheet

PDB: 1kgs, Buckler et al. (2002), Structure

Secondary Structure amphiphilic partially buried ∝-helix

PDB: 1jat, VanDenmark et al. (2001), Cell

Secondary Structure – amphiphilic ß-strand

Secondary Structure – collagen

Secondary Structure Prediction

•  Toolkit – Quick2D

Secondary Structure Prediction

•  Toolkit – Ali2D

http://www.nature.com/news/2011/110309/full/471151a/box/1.html

Disordered Regions

•  Today, programs predict that about 40% of all human proteins contain at least one intrinsically disordered segment of 30 amino acids or more, and that some 25% are likely to be disordered from beginning to end.

•  lack of hydrophobic residues

•  often with overrepresentation of a few amino acids

Secondary Structure Prediction

•  Toolkit - Ali2D

Disorder Prediction

•  IUPRED - http://iupred.enzim.hu/

Short Linear Motifs

•  Eukaryotic Linear Motifs (ELM) / Short Linear Motifs (SLiM)

•  Hunt T (1990) “These motifs are linear, in the sense that three- dimensional organization is not required to bring distant segments of the molecule together to make the recognizable unit. The conservation of these motifs varies: some are highly conserved while others allow substitutions that retain only a certain pattern of charge across the motif.”

Short Linear Motifs – Characteristics •  3-11 amino acids long

•  poorly conserved / evolve fast

•  1-3 amino acids in the motifs are “hot spots”

•  ~ 80% in disordered regions

•  relatively low affinity to interacting partner (1-150µM)

•  interaction via induced fit

Short Linear Motifs – Function •  protein-protein interactions

•  post-translational modifications

•  e.g. Phosphorylation

•  proteolytic cleavage/processing sites

•  KEN / D box in cell cycle - degradation signals

•  subcellular targeting sites

•  NES - nuclear export signal

➡  modulation of interactions - fine tuning

David Goodsell, http://www.rcsb.org/pdb/101/motm.do?momID=85

Short Linear Motifs – Nuclear Localization Signal (NLS) •  Impor'n-­‐beta  (1qgk;  blue)  recognizes  nuclear  pores  and  moves  through  them.  It  wraps  around  the  end  

of importin-alpha (1ee5; green), an adaptor molecule that connects importin-beta with the cargo, here nucleoplasmin(1k5j; yellow), a chaperone important in nucleosome assembly. All interactions are mediated by linear motifs in unstructured segments (bipartite nuclear localization signals).

ELM Resources

•  elm.eu.org

•  NUPL_XENLA

NLS in nucleoplasmin •  Quick 2D secondary structure prediction for nucleoplasmin, showing the unstructured

C-terminal tail and the bipartite nuclear localization motif

50 100 ! | | | | | | | | | | ! MASTVSNTSKLEKPVSLIWGCELNEQNKTFEFKVEDDEEKCEHQLALRTVCLGDKAKDEFHIVEIVTQEEGAEKSVPIATLKPSILPMATMVGIELTPPVTFRLKAGSG!SS PSIPRED EEEEEEEE EEEE EEEEEEEEE EEEEEE EEEEEEEE EEE EE EEEEEE !SS JNET EEEEEEE EEE HHHHHHHHHHHH EEEEEEEE EEEEEE EEEEEE !DO DISOPRED2 DDDDDDDDDDDDDDDDD !DO IUPRED DDD D DDDD DDD D D DDD DDDDDDD DDDD D DDDD DDDD !SO Prof (Rost) B B B BBBBB B B B B B BBB BBB BBBB BB B BB B BB BB BBBB B B B B !SO JNET B BBBBBBBB B B B B B BBBBBBBBBBB B BBBBBBB B BBBBBB B B BBBB B B B BBB B B!! 150 ! | | | | | | | | | |! PLYISGQHVAMEEDYSWAEEEDEGEAEGEEEEEEEEDQESPPKAVKRPAATKKAGQAKKKKLDKEDESSEEDSPTKKGKGAGRGRKPAAKK!SS PSIPRED EEEEEEEEEE HH !SS JNET EEE E !DO DISOPRED2 DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD!DO IUPRED DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD!SO Prof (Rost) BBB BBB B B B B B !SO JNET BBBBBBBBBB !!  SS = Alpha-Helix Beta-Sheet Secondary Structure!DO = Disorder!SO = Solvent accessibility (A burried residue has at most 25% of its surface exposed to the solvent.)!

DDX6 & Scd6 / EDC3 Interaction

DEAD-box helicase

FDF…FDK YjeF LSM EDC3

DDX6/Me31B

Scd6/Tral LSM FDF

DDX6 & Scd6 / EDC3 Interaction

PDB: 2wax, Tritschler et al. (2009), Mol Cell

DDX6 & Scd6 / EDC3 Interaction