The document discusses function prediction for unknown proteins. It begins with an overview of common methods for function prediction, including sequence and structure similarity, domains and motifs, gene expression, and interactions. It then uses a protein called Msa as a case study, analyzing it with various tools and finding evidence it may function as a signal transducer in bacterial response to environment. Finally, it briefly discusses another protein M46 and challenges in evaluating prediction accuracy.

1. July 17th 2014
Vijayaraj Nagarajan PhD
Computational Molecular Biology Specialist
BCBB/OCICB/NIAID/NIH

3. 3

4. § The function prediction problem
§ Methods and approaches
§ MSA case study
§ M46 : a different direction
§ How good are the predictions…?
Outline

5. http://www.clker.com/clipart-24887.html
“Unknown function”
3951259

6. 6
7754904
“Unknown function”

7. Databases
Tool
Boxes
Which tool to use..?

8. Problem or opportunity….?
Tool
Boxes
Databases

9. ü The function prediction problem
§ Methods and approaches
§ MSA case study
§ M46 : a different direction
§ How good are the predictions…?

10. § Based on…..
• Physicochemical properties
• MW, pI, Amino acid composition etc.
• Sequence similarity
• Primary sequence, Patterns, Domains, Motifs and Profiles
• Structure similarity
• CATH, SCOP, PDB, fold libraries
• Functional properties
• Catalytic activity, Post-translational modifications, Binding
activity
Function Prediction

11. • Gene expression data
• GEO, ArrayExpress, Microarray Meta-Miner (MMM)
• Biomolecular interaction information
• Interaction partners
• Epigenetics (methylation, histone modifications etc.)
• Associations from genome scale data – NGS
• Phylogeny
• Sequence and structure similarity
• Gene Ontology
• Semantic distance
• Text mining
• Associations from literature

12. Function prediction workflow

13. ü The function prediction problem
ü Methods and approaches
§ MSA case study
§ M46 : a different direction
§ How good are the predictions…?

14. Environment…?
agr
cap
hlgA
etc.,
sarA
nuc
aur
ssp
etc.,
SA1233
Sambanthamoorthy et al., Microbiology (152) 2006
Nagarajan et al., BMC Bioinformatics. 8 (Suppl 7):S5 2007
msa
Msa – a case study

15. http://blast.ncbi.nlm.nih.gov/Blast.cgi
BLASTp

16. BLASTn

17. PSI BLAST Iteration I PSI BLAST Iteration 2 PSI BLAST Iteration 3
PSI-BLAST Hits

18.
List of Observations
First 40 – pattern..?
Transporter/efflux/membrane..?
PSI-BLAST Hits

19. PSI BLAST Iteration 3 with PAM70
BLAST against PDB Database

20. http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
NCBI Conserved Domain Database (CDD)

21.
List of Observations
First 40 – pattern.. (DUF?)
Transporter/efflux/membrane

22. § Physicochemical properties
§ Amino acid scale representation
• GRAVY (Grand Average of Hydropathy)
— Msa - 1.021 (Hydrophobic)
Lasergene
List of Observations
First 40 – pattern.. (DUF?)
Transporter/efflux/membrane..?
Hydrophobic..?
The next step…?

23. § Solubility and localization
– ProtFun – envelope, non-enzyme
– SVMProt – transmembrane
– CELLO – membrane
– PSORT – membrane
– ProtCompB – membrane
– PSLPred – membrane
§ Msa – insoluble, non-enzyme, membrane
List of Observations
First 40 – pattern.. (DUF?)
Transporter/efflux/membrane..?
Hydrophobic/insoluble/non-enzyme

24. Signals/Domains/Patterns/Motifs
SP – Signal Peptide
CP – Cleavage Position
If membrane, what next…?

25. SP – Signal Peptide
CP – Cleavage Position
More about membrane

26. TMHMM
DAS
Membrane topology

27. +1
0
+5
-1
Von Heijne, J Mol Biol. 225(2): 487-494. 1992
List of Observations
First 40 – pattern.. (DUF?)
Transporter/efflux/membrane..?
Hydrophobic/insoluble/non-enzyme
Signal peptide 1-20
TM1, TM2, TM3 – N-inside ✔
N
N
N
N
C
C
C
C
Positive-Inside rule and
Charge bias

28. http://pbil.ibcp.fr/htm/index.php
List of Observations
First 40 – pattern.. (DUF?)
Transporter/efflux/membrane..?
Hydrophobic/insoluble/non-enzyme
Signal peptide 1-20
TM1, TM2, TM3 – N-inside ✔
Three Transmembrane HELIX
Network Protein Sequence
Analysis

29. http://smart.embl-heidelberg.de/

30. PreATP-­‐grasp
domain
in
Msa.
§ Structural
Classifica:on
of
Proteins
(SCOP)
entry:
d1gsa
1
§ Usually
precedes
the
ATP-­‐grasp
domain
and
could
contain
a
substrate-­‐binding
func:on.
§ Located
between
the
85th
and
116th
residue.
§ Interes:ngly
this
loca:on
is
predicted
to
be
in
a
cytoplasmic
loop
region
of
Msa
List of Observations
First 40 – pattern.. (DUF?)
Transporter/efflux/membrane..?
Hydrophobic/insoluble/non-enzyme
Signal peptide 1-20
TM1, TM2, TM3 – N-inside ✔
Three Transmembrane HELIX
PreATP-grasp domain
Functional sites

31. http://www.ebi.ac.uk/intact/site/index.jsf
Any interactions…?

32. http://www.ebi.ac.uk/intact/site/index.jsf
Any interactions…?

33. http://www.ebi.ac.uk/intact/site/index.jsf
Any interactions…?
List of Observations
First 40 – pattern.. (DUF?)
Transporter/efflux/membrane..?
Hydrophobic/insoluble/non-enzyme
Signal peptide 1-20
TM1, TM2, TM3 – N-inside ✔
Three Transmembrane HELIX
PreATP-grasp domain
Responds to environment…?

34. http://www.bioinformatics.org/sammd/
List of Observations
First 40 – pattern.. (DUF?)
Transporter/efflux/membrane..?
Hydrophobic/insoluble/non-enzyme
Signal peptide 1-20
TM1, TM2, TM3 – N-inside ✔
Three Transmembrane HELIX
PreATP-grasp domain
Responds to environment ✔
Gene expression

35. http://www.ebi.ac.uk/interpro/
InterProScan

36.
List of Observations
First 40 – pattern.. (DUF?)
Transporter/efflux/membrane..?
Hydrophobic/insoluble/non-enzyme
Signal peptide 1-20
TM1, TM2, TM3 – N-inside ✔
Three Transmembrane HELIX
PreATP-grasp domain
Responds to environment ✔
Phosphorylation sites 48, 49, 99
**
*
Patterns

37. http://www.ihop-net.org/UniPub/iHOP/
Literature mining

38. • Homology Modeling
• No homologous structures in PDB
• FOLD recognition
• Phyre
Structure based predictions

39. § Swiss-Pdb Viewer
– Energy minimization
– PHI/PSI angle
– Loop
§ Structure validation
– Verify3D
Model of Msa

40. Ramachandran plot for the predicted tertiary structure of the
Msa protein pre (A) and post (B) refinement
Quality of the model

41.
List of Observations
First 40 – pattern.. (DUF?)
Transporter/efflux/membrane..?
Hydrophobic/insoluble/non-enzyme
Signal peptide 1-20
TM1, TM2, TM3 – N-inside ✔
Three Transmembrane HELIX
PreATP-grasp domain
Responds to environment ✔
Phosphorylation sites 48, 49, 99
Predicted 3D structure of Msa

42. Binding site predictions for the Msa protein. (A) ProFunc
predicted binding site (red); (B) PINUP predicted binding
site (interface in green); (C) Q-SiteFinder predicted binding
site and binding residues (pink)
List of Observations
First 40 – pattern.. (DUF?)
Transporter/efflux/membrane..?
Hydrophobic/insoluble/non-enzyme
Signal peptide 1-20
TM1, TM2, TM3 – N-inside ✔
Three Transmembrane HELIX
PreATP-grasp domain
Responds to environment ✔
Phosphorylation sites 48, 49, 99
Binding sites - cytoplasmic region?
Binding sites

43. ProFunc
– “nest” near the putative phosphorylation site (47-50)
– 47-50; predicted outside membrane
– All residues conserved at the “nest”
– “nest” shows features of anion-binding site
– “nest” characteristic functional motifs in ATP or GTP
binding proteins
List of Observations
First 40 – pattern.. (DUF?)
Transporter/efflux/membrane..?
Hydrophobic/insoluble/non-enzyme
Signal peptide 1-20
TM1, TM2, TM3 – N-inside ✔
Three Transmembrane HELIX
PreATP-grasp domain
Responds to environment ✔
Phosphorylation sites 48, 49, 99
Binding sites - cytoplasmic region?
Function site (“nest”) – outside..?
Predicted “nest”

44. § Multiple sequence alignment (ClustalW) of Msa protein
sequence from 11 different strains
– 12 variations in strain RF122
§ 1 replacement, 11 substitutions
• 1 substitution in pre-ATP grasp domain
– 7 variations in strain MRSA252
§ 2 replacements, 5 substitutions
• 1 replacement in pre-ATP grasp domain
• Replacement
• Hydrophilic -> Hydrophobic
• Ser -> Gly
• Substitution
• Hydrophilic -> Hydrophilic
• Ser -> Glu
Function motifs: conserved…?

45. – Variation at aa positions 111, 131, 133 common
– None in Phosphorylation sites, signal peptide, “nest”
List of Observations
First 40 – pattern.. (DUF?)
Transporter/efflux/membrane..?
Hydrophobic/insoluble/non-enzyme
Signal peptide 1-20
TM1, TM2, TM3 – N-inside ✔
Three Transmembrane HELIX
PreATP-grasp domain
Responds to environment ✔
Phosphorylation sites 48, 49, 99
Binding sites - cytoplasmic region?
Function site (“nest”) – outside..?
Functional sites highly conserved..?
Function motifs: conserved…?

46.
List of Observations
First 40 – pattern.. (DUF?)
Transporter/efflux/membrane..?
Hydrophobic/insoluble/non-enzyme
Signal peptide 1-20
TM1, TM2, TM3 – N-inside ✔
Three Transmembrane HELIX
PreATP-grasp domain
Responds to environment ✔
Phosphorylation sites 48, 49, 99
Binding sites - cytoplasmic region?
Function site (“nest”) – outside..?
Functional sites highly conserved..?
Msa – a putative
signal transducer

47. Msa – a putative signal transducer

48. msa and sarA transcriptome

49. msa transcriptome - sammd

50. Msa – regulatory network

51. ü The function prediction problem
ü Methods and approaches
ü MSA case study
§ M46 : a different direction
§ How good are the predictions…?
Any questions…?

52. § Mold specific
• Histoplasma capsulatum
• Only in mold not in yeast
M46 – a different direction

53. M46 – PSI-BLAST

54. § Nucleotide binding
– DNA/RNA
– S-S bonds
§ Look for motifs
– Predict motifs, build HMM, search for similar
§ Localization
– Secreted, ER signal, ER modifications
M46 – the clues

55. ü The function prediction problem
ü Methods and approaches
ü MSA case study
ü M46 : a different direction
§ How good are the
predictions…?

57. • Only
good
if
it
would
make
any
biological
sense
• Only
good
if
it
could
be
supported
by
follow
up
experimental
evidence
• No
hits
• Reduce
threshold
-­‐
expect
worst
evalues-­‐pvalues
• Cut-­‐off
value
paradox
(if
<0.05
is
significant,
what
about
0.051…?)
• It
is
OK
to
look
at
hits
with
poor
evalue-­‐pvalue
• Cannot
assign
homology,
can
pick
clues
(your
only
hope)
How good are the predictions…?

58. nagarajanv@mail.nih.gov
ScienceApps@niaid.nih.gov
hYp://bioinforma:cs.niaid.nih.gov
Questions…?