1. Atomic Structure of protein-protein
interactions

2. Overview

8. The peptide bond
(----ALA-HIS-GLY-ILE-LEU-PHE-TYR-LYSGLY---)n

14. CATH: Class Class (C), Architecture (A), Topology (T) and
Homologous superfamily (H).
SCOP: Structural Classification of Proteins

16. Quaternary Structure
Homodimers Tetramers
Icosahedral virus capsids Heterocomplex

18. Atomic resolution structures of biomolecules are
stored at the Protein Data Bank
Contains 60000 structures (mostly determined by X-ray crystallography and NMR
About 3-5 new structures per day
year

21. Application of Computational tools to solve specific
biological problems
Specific protein-protein interactions is a rule rather than exception and non-
specific interactions can lead to several disease
Protein-protein interactions in binary complexes:
 What makes the specificity?
 Discriminate specific and non-specific interfaces

22. Specific and non-spacific interactions
Homodimers Heterocomplex
Crystal-packing interfaces of monomeric protein
Is that pair a
biological
dimer?
The non-covalent interactions that hold crystals together:
are the same as in protein-protein complexes and oligomeric proteins
BUT
they are not subject to natural selection, thus, biologically non-
specific.

23. The dataset and methodology
Non-redundant dataset (taken from Protein Data Bank)
• 70 Protein-protein complexes (Chakrabarti and Janin, 2002)
• 120 Homodimers (Bahadur et al., 2003)
• 183 Crystal packing interfaces of 145 monomeric proteins
2fold related interface =105
Non-2fold related interface=83 (Bahadur et al., 2004)
Tools for the analysis
• Structural features
Solvent accessible surface are, Buried atoms, core-rim, atomic density
• Physico-chemical properties
Polar-non polar interactions, residue composition, hydration

24. Interface area definition
Molecule A Molecule B Complex AB
w
w
Interface area (B) = ASA(A) + ASA(B) – ASA(AB)
(Lee & Richards, 1971)
(‘Naccess’ by Hubbard SJ. 1992)
Interface atoms and residues are all atoms and residues that lose ASA in the complex and
contribute to B

25. Protein-protein interfaces
Dimeric
k-bungarotoxin
(1kba)
BSA = 1000 Å2
(Bahadur et al., 2004)
Crystal dimer
Pokeweed
antiviral protein
(1qci)
BSA = 1000 Å2
(Bahadur et al., 2004)

26. Size of the protein-protein interfaces
70
No two-fold Interface Interface Ref.
Crystal dimers
area B
60 Homodimers
Complexes (s.d.)
50
Homodimer 3880 Bahadur et
Interfaces
40 al., (2003)
(±2200)
30
20 Chakrabarti
Complex 1910
& Janin,
10
(±760) (2002)
0
800 1000 1600 2400 3200 >3600
2
Interface area B (Å ) Crystal- 1510 Bahadur et.
al., (2004)
packing
3% Homo +Complex 16% Crystal (±520)
30% Crystal
1200 Å2 > 2000 Å2
Standard size interface (Lo Conte et al, 1999)
Interfaces formed in protein-protein complex are of ‘Standard size’
Homodimer interfaces are very large compared to crystal-packing interfaces

27. Non-polar interface area
No two-fold
60
fnp*B = Interface area contributed by the C- Crystal dimers
Homodimers
Complexes
containing groups only / Total interface area
40
Interfaces
fnp* B (%)
20
Homodimer 65
Complex 58
Crystal-packing 58 0
>80
40
50
60
70
80
Non-polar fraction of the interface area (%)
0 Homo + 6% Crystal
0 Complex
< 50 > 70
Homodimers have hydrophobic interfaces compared to protein-protein complexes and crystal-
packing interfaces.

28. Clustering of Interface atoms: single or multiple patch?
…we cluster interface atoms by the average
linkage method on a purely geometric basis.
1 3
A threshold distance dM must be used in clustering.
4
It is set to half the diameter of the interface; 5
2
dM= 15 Å in a typical protein-protein complex, 22
Å in homodimers.
d13+d14+d15+d23+d24+d25
dM =
6
(Chakrabarti & Janin, 2002)
(Bahadur et. al., 2003)

29. Standard size single patch and multi-patch interfaces
Cytochrome c’
(2ccy) B (Å2) #res #atoms
Homodimers (70) 2740 74 280
Complex (46) 1560 47 170
95% of the crystal-packing interafces are of ‘single patch’
Thrombin-
ornithodorin
(1toc)
B (Å2) #res #atoms
Homodimers (35) 4760 (0.67, 0.33) 126 486
Complex (18) 2510 (0.63, 0.37) 73 217
Multi-patch interfaces contains at least one large patch with ‘standard size’

30. Evaluating packing density of the interface
Homodimer interface Crystal-packing interface
1qci, Antiviral
1kba, Kappa- protien complex-
bungarotoxin ed with adenine
Ai
Local density index Global density index
count the number ni of interface atoms Calculate the principal moments of inertia Na2,
within D = 12 Å of interface atom Ai Nb2, Nc2 of the set of interface atoms; the
inertia ellipsoid has half-axes a>b> c
average ni over all interface atoms
the area at the equator is A=πab
LD = Σ (ni) / N GD = N/A
B(Å2) LD GD
1kba 998 34 0.95
1qci 994 14 0.31
Specific interfaces are well packed compared to non-specific interfaces

31. Buried atoms at the interface
No two-fold
Partially buried 50 Crystal dimers
Homodimers
w interface atoms Complexes
40
Interfaces
30
20
10
Fully buried
0
interface atoms
>50
10
20
30
40
50
Fraction of fully buried interface atoms (%)
87% Crystal- 71% Homodimer +
Number of fully buried interface atoms
fbu= Total number of interface
packing Protein-protein complex
fbu<30% fbu>30%
atoms
Specific interfaces: 34-36% of the interface atoms are fully buried
Non-specific interfaces: this fraction is only 21%

32. Dissecting the interface: Core and Rim
A
Core residue: with at least one fully buried atom
Rim residue: Contain accessible atoms only d all
CI2 inhibitor bound to B
subtilisin (2sni) Enolase (1ebh) d
72% (B) in core 74% (B) in B
core B
A
Core (B%)
Homodimers 77
Complexes
72
‘Core’ region is absent in crystal-packing interfaces

33. Amino acid composition of the specific interfaces
The amino acid composition
of the interface core and rim D E K
S
Numberwise
fi = number of core (rim) residues of type i / T
FYWM
total number of core (rim) residues IL
G
Areawise
f0i = interface area contributed by core (rim)
residues of type i / total core (rim) interface
area
Core: aromatic and hydrophobic residues are abundant at the
Rim: polar and charged residues

34. Composition of interface relative to surface
Euclidean distance (Δf) between amino
acid compositions of the interface and
rest of the protein surface:
(∆f)2 = 1/19 ∑ i=1 to 20 (ki – k0i)2
ki = composition of amino acid residues at interface
k0i = composition of amino acid residues at surface
1.6
Monomers Interface Surface
2.1 0.5
Homodimers Interface Surface
3.4
Homodimer: interface differs from the protein surface
Crystal-packing: Difference is negligible

35. Residue Propensity (RP) score
RP = Σ ni * Pi , ni number of residues in the interface
No two-fold
Propensity of a residue to be 50
Crystal dimers
at homodimer interface 40
Homodimers
Complexes
Pi = ln (ki/k0i)
Interfaces
30
ki = composition of amino acid 20
residues at interface
10
k0i = composition of amino acid
residues at surface 0
<-6 -4.5 -1.5 1.5 4.5 >6
Residue Propensity Score (RP)
Residue propensity
Only 5% 5% Crystal
score (RP)
Homo+Complex
Homodimer 4.3 (±4.9)
Complex 0.9 (±2.3)
Crystal-packing -1.1 (±2.7) < -3 >3
Specific interfaces have +ve RP and non-specific interfaces have –ve RP score

36. Identifying homodimers and crystal dimers
50
Use a combination of parameters
40
Fraction of fully buried atoms (fbu)
Fraction of buried atoms (%)
Non-polar interface area (fnp*B)
30
U Residue Propensity score (RP)
20
Classification fnp*B (Å2) fbu (%) RP
10 Monomer ≤ 800 or
≤ 2000 and ≤ 20
M D
Homodimer ≥ 2000 or
0
0 1000 2000 3000 4000 ≥ 800 and ≥ 30
Non-polar interface area ( Å2 ) Undecided 800-2000 and 20-30 < 1.5 (M)
≥1.5 (D)
Monomer 95%
Homodimer 93%

37. Structural rule of specificity
 Minimum size: specific protein-protein interfaces have B ≥ 800 Å2
Most crystal packing interfaces are below that size, which may be the minimum for
a biologically relevant macromolecular interaction.
 Standard-size (1200-2000 Å2) interfaces make stable, specific and fully functional
interactions
 . Specificity is expressed in the following features:
• close-packed interface atoms
• a high fraction of buried atoms
A combination of parameters, non-polar interface area, fraction of fully
buried interface atoms and residue propensity score discriminates the
specific interfaces from the non-specific ones with a 95% success rate.

38. ProFace: http://www.boseinst.ernet.in/resources/bioinfo/stag.html