1. Department of Biochemistry
Protein DNA Interactions From interactions to function prediction Sue Jones
Department of Biochemistry
University of Sussex
20th Sept 2004
EMBL Lecture Course

2. Outline Protein-DNA Interactions :importance Structural Data
Predicting DNA Binding Function
Alternative Method & New Perspectives

4. Protein-DNA Interactions : Importance
Gene expression
Transcription initiation (TATA binding protein)
RNA synthesis (RNA polymerase)
Transcription regulation (MAX protein)
DNA repair (DNA glycosylase : oxidative DNA damage)

5. Protein-DNA Interactions : Importance
DNA packaging (Histone H2A.e)
DNA replication (Polymerases, Ligases, single stranded binding proteins)

6. Outline Protein-DNA Interactions :importance Structural Data
Predicting DNA Binding Function
Alternative Method & New Perspectives

7. DNA B A Z DNA has structural flexibility
Structure described by Watson & Crick : B-form
Feature B A
Type of helix RH
Diameter
2.37
2.55
Rise per bp
0.34
0.29
# bp per turn 10 11
Major groove
Wide,
deep
Narrow,
Minor groove
shallow
Wide, shallow
B A Z

8. Structural Data
NDB : assemble and distribute structural information about nucleic acids
2490 structures (25/08/04)
Protein-DNA Complex
Number
Double Helix
593
Single Strand 57
Berman et al., Biophys J 63 p751

9. Protein-DNA Interactions : Structure

10. Protein-DNA Interactions : characteristics
Major and minor groove binding
DNA-binding motifs
Positively charged surface areas
Size ASA : 618Å Å2
Conformational changes
DNA bending
domain movements, quaternary changes
Nadassy et al., 1999 Biochemistry 38 p1999
Jones et al., 1999 J.Mol.Biol. 287 p877

11. Outline Protein-DNA Interactions :importance Structural Data
Predicting DNA Binding Function
New Perspectives

12. Predicting DNA Binding Function
Knowing a protein’s function is essential in understanding
cellular location
interactions
biochemical pathways
potential as drug targets
Prediction of protein DNA binding site given unbound protein structure
electrostatic patches
motifs

13. Predicting Function from Structure
Structural genomics : filling in the gaps of protein structure space
Structures solved that have low sequence identity (< 30% sequence identity)
Potentially little or no fold similarity to any currently in the PDB
Require algorithms to make fast & reliable function predictions

14. Predicting DNA Binding Function
Easy to make matches between globally homologous structures
Method aims to identify remote matches based on local homology of a specific motif
Helix-Turn-Helix (HTH)
C-terminal helix - major groove binding
1/3 DNA-binding protein families (16/54)

15. Catabolic Activator Protein
HTH Motif Proteins
Hin Recombinase
(1hcr)
Catabolic Activator Protein
(1j59)

16. HTH Motif Dataflow NDB PDB Literature PFAM SMART
PDB Chains
NDB
PDB
Literature
PFAM
SMART
26 Hidden Markov Models
PDB
SAM-T99
Literature
Rasmol
349 HTH Chains
227 HTH Proteins
28 HMMs
86 NI
Proteins
3D-Templates 7
HREPS 29
SREPS
84 NI
Proteins
232 HTH
Chains 30
SREPS
HTH Motif Dataflow

17. HTH Template Library 1ais 1hcr 1b9m 1eto 1jhg 1lmb 1orc 1hcrA160-181
1b9mA32-56
1etoA73-95
1aisB
1jhgA68-91
1lmb331-53
1orc016-36
1jhg
1lmb
1orc

18. Template Scanning Scanning template library against 3D structures
One template T (length n) scanned against protein P of length m, calculated optimal gapless superposition at each m-n+1 possible positions in P using RMSD
Based on Kabsch (1976) Acta Cryst A. 32 p922

19. RMSD Distributions 1.6Å Frequency
368/8266 = 3.5% false positives /84 = 1.4% false negatives

20. Improving Template Specificity
Extending templates
Assessing motif accessible surface area (ASA)
+2 templates 61/8264 = 0.7% false positives
ASA threshold (990Å2) 38/8264 = 0.5% false positives
3 ‘false’ positives were actually real HTH proteins not previously annotated

21. ‘New’ HTH Motif 1 DNA Methyltransferase (MGMT)
C-terminal domain
‘d’ and ‘e’ helices
Site directed mutagenesis
1mgtA

22. ‘New’ HTH Motif 2 1fy7A Histone acetyltransferase
C-terminal domain
zinc finger N-terminal domain
protein-protein interactions
SCOP : ‘winged helix’

23. ‘New’ HTH Motif 3 1taq 1tau Polymerase I 673-700 ‘fingers’ subdomain
DNA contacts ‘O’ helix
New HTH precedes ‘O’ helix

24. Generic Templates

25. Generic Templates Sequence Structure RMSD < 1.6
Full sequence HMMs (0.001)
Structure
RMSD < 1.6

26. Structural Genomics Targets
Scanned template library against 30 target structures from MCSG
21-49
1LMB331-53
1695
1.3
APS048
Location
Template
ASA
RMSD
MSGC Target
Isocitrate lyase regulator transcription
factor. (Zhang et al., J. Biol. Chem. 2002)

27. Summary
Method combined structural data from NDB and PDB with sequence data from PFAM and SMART
Structural template library of 7 HTH motifs
RMSD threshold from optimal superposition
Hit rate of 88% & false positive rate of 0.5%
Recognition across families
Template method independent of global fold similarity
Potential to identify new DNA binding HTH motifs

28. Online Function Prediction

29. Outline Protein-DNA Interactions :importance Structural Data
Predicting DNA Binding Function
Alternative Method & New Perspectives

30. Alternative Statistical Model
Statistical Models for discerning protein structures containing the
DNA-binding HTH motif. Mclaughlin and Berman,
J. Mol. Biol p43.
Decision tree model to identify key structural features
geometric measurements of recognition helix (RH) & helices & beta sheets preceding and following
Key features
High solvent accessibility of RH
Hydrophobic interaction between RH & 2nd helix preceding
Predicting HTH motifs within the PDB
98% accuracy & 0.7% false positive rate
Predicted new HTH motifs

31. Future Perspectives
Extend method to other DNA binding motifs : HLH, HhH, -ribbon
Using electrostatic potentials with motifs to improve method
Spatial templates for proteins that don’t use discrete motifs for DNA recognition

32. Acknowledgements Mario Garcia Carles Ferrer Department of Energy : USA
Jonathan
Barker
Janet
Thornton
Hugh
Shanahan
Helen
Berman
Mario Garcia Carles Ferrer
Department of Energy : USA
European Bioinformatics Institute
Rutgers The State University