Protein Interfaces and Computational Alanine Scanning

Macromolecular complexes

• Functionality of protein is usually of macromolecular assemblies

• There are many examples of proteins fulfilling their biological role only after the formation of a macromolecular complex

Protein complex example

• Quaternary structure of haemoglobin (4-mer)

• Different chain (e.g., alpha, beta etc.) from hetero-oligomers

• Important in the transportation of respiratory gases

• Involved in regulation of iron metabolism

Protein-DNA interaction example

• Catabolite gene activator protein (cAMP receptor protein) involved in DNA bonding to regulate gene expression

• In a homodimer but associates with DNA

• Helix-turn-helix structural motif allows major groove DNA binding

• Required for transcription of lac operon

Protein Interfaces, Surfaces and Assemblies (PISA-PDBe)

Investigates the stability of formation of macromolecular complexes based on theoretical physiochemical calculations

• Protein

• DNA/RNA

• Ligand

The following physiochemical properties govern the stability of any macromolecular complex in a biological system

• Free energy of formation

• Solvation energy gain

• Number of hydrogen bonding interactions

• Cross-interface salt bridge

• Hydrophobic specificity

Nat

Indicates the total number of atoms in the corresponding structure

Nres

Indicates the total number of residues in the corresponding structure

SNat

Indicates the total number of surface atoms in the corresponding structure

SNres

Indicates the total number of surface-exposed residues in the corresponding structure

Area

Indicates the solvent-accessible surface of the corresponding structure, in A²

Delta G

Indicates the solvation free energy of folding for the folding for the corresponding structure, in kcal/M

Results and terminology - interfaces

1. Dimer interface

2. C-terminal trimer interface

3. N-terminal trimer interface

Overview of structures

• Red and Green = Dimer interface

• Red, olive and slate blue = N-terminal trimer interface

• Red, gold and cyan = C-terminal trimer interface

Delta iG

Indicates the solvation free energy gain upon formation of the interface. Negative corresponds to hydrophobic interfaces, or positive protein affinity. This value does not include the effect of satisfied hydrogen bonds and salt bridges across the interface

The P-value

Measures the probability of getting a lower than observed delta iG, when the interface atoms are picked randomly from the protein surface. P>0.5 means that the interface is less hydrophobic than it could be. P>0.5 indicates interfaces with surprising, higher than would-be-average features, interface can be interaction-specific (driven by hydrophobicity for example)

N_HB

Potential number of cross interface hydrogen bonding interactions (worth 0.5 kcal/mol)

N_SB

Potential number of cross interface salt bridges (worth 0.3 kcal/mol)

N_DS

Potential number of cross interface disulphide bridges (worth 4.0 kcal/mol)

CSS

Stands for the Complexation Significance Score, which indicates how significant for assembly formation the interface is. The score is defined as a maximal fraction of the total free energy of binding that belongs to the interface in stable assemblies

Alanine Scanning

A technique to ‘walk’ the length of a protein, mutating each residue, one at a time, to Alanine, and monitoring the phenotypic effects the mutation causes…

• Assembly of proteins

• Stability

• Catalytic/Enzyme activity

Computational Alanine Scanning

• Uses free energy functions to calculate the effects of alanine mutations on the binding free energy of a protein-protein complex with each interfacing chain chosen

• Output is a list of ‘hotspots’ or amino acid side chains that are predicted to significantly destabilise the interface when mutated to alanine, analogous to the results of experimental alanine-scanning mutagenesis

An interface residue is regarded as:

• A residue that has at least one atom within a sphere with a 4A radius of an atom belonging to the other partner in the protein complex OR

• A residue that becomes significantly buried upon complex formation, as measured by an increase in the number of C beta atoms of the residue of interest

Int_ID

Measure of whether a residue side chain atom is within 4 A of an atom on the other partner (1) or not contacting directly, but buried upon binding (0)

res#

residue numbering

aa

numerical amino acid type

DDG (complex)

contains the predicted changes in binding free energy (delta delta Gbind) upon alanine mutation. Positive values mean that replacement by alanine is predicted to destabilise the complex; negative values predict a stabilising effect

DG(partner)

predicted change in protein stability of the mutated complex partner upon alanine mutation

What are hotspots?

Defined as those for which alanine mutations have destabilising effects on delta delta Gbind of more than 1 kcal/mol

Combining results with PISA

Highest DDG (complex) for DpsC interfaces) residue Arg-118 with a value of 4.82

PISA analysis shows this residue has two hydrogen binding interactions

Alanine Scanning Drawbacks

• Coupling effects between different mutations cannot be taken into account, and multiple mutations are always assumed to be addictive - homo-oligomers would have multiplication of the effect of a single residue change (although this will ignore the environment-dependent effects)

• Indirect effects on the binding energy exerted by residues not making direct interactions in the interface are generally not captured:

• Side chain steric hindrances

• Conformational changes upon ligand binding

• Effects on intramolecular interactions

• Residues not directly participating in contacts across the interface can affect the binding free energy by changing the environment of hydrogen-bonding interactions