Experimentally validated computational docking to characterize protein- protein interactions

Each biomolecule in a living organism needs to adopt a specific threedimensional conformation to function properly. Function itself is usually

achieved by specific interactions between biomolecular units. Structural

knowledge at atomic level of biomolecules and their interaction is important to

understand the mechanisms leading to biological response and to develop

strategies to interfere with them when necessary.

Antibodies are molecules of the immune system playing an ever more

prominent role in basic research as well as in the biotechnology and

pharmaceutical sectors. Characterizing their region of interaction with other

proteins (epitopes) is useful for purposes ranging from molecular biology

research to vaccine design.

During my PhD studies I used a combination of solution NMR mapping,

molecular biology and computational docking to provide a structural and

biophysical characterization of new neutralizing antibodies from Dengue

virus recovered subjects, comparing the binding of the same antibody to the

four Dengue serotypes and the binding of different antibodies to the same

serotype. We were able to rationally mutate an antibody to first alter its

selectivity for different viral strains and then increase its neutralization by

~40 folds. For the first time, this was achieved without the availability of an

x-ray structure.

In a second sub-project, I investigated the interaction of the chemokine

CXCL12 with the chromatin-associated protein HMGB1, confirming their

direct interaction (only proposed but never proved before) and providing a structural explanation for the HMGB1 dependent increase of CXCL12

cellular activity. High profile publications resulted from the two above

projects.

The above mentioned projects relied heavily on solution NMR spectroscopy,

which is ideally suited to the atomic level characterization of intermolecular

interfaces and, as a consequence, to antibody epitope discovery. Having

provided a residue-level description of a protein-protein interface by NMR, we

subsequently used this experimental information to guide and validate

computational docking experiments aimed at providing a three dimensional

structure of the protein-protein (or antibody-protein) complex of interest. In

collaboration with other members of my research group I validated the use of

NMR and computational simulations to study antibody-antigen interactions,

publishing two reviews in collaboration with other members of my research

group.