Biology Reference
In-Depth Information
FIGURE 6.1
'Hotspot-design' method to design novel protein-protein interactions.
backbone atoms of the hotspot residues are superimposable onto the backbone atoms of
any one residue of the candidate scaffold, without the scaffold backbone overlapping with
the target protein. To achieve this, protein
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protein docking algorithms are used, where the
target protein is held constant and the candidate scaffold is docked. To guide sampling,
coordinate restraints between the candidate scaffold and the hotspot residues are imposed
during the docking calculation. After the scaffold has been properly placed, all residues in
the vicinity of the target protein (except the hotspot residues) are redesigned with side-chain
placement algorithms to obtain the final designed sequence. In practice, after the hotspot
residues have been placed, the docking and design procedure is carried out millions of
times, with the candidate scaffolds picked from a library of several hundred small,
monomeric, globular proteins for which high-resolution crystal structures are available, the
resulting designs ranked by energy, and a few dozen of the highest-ranking designs are
considered for expression.
The most impressive example of a successful execution of the hotspot design strategy is
represented by Fleishman et al.
s designed protein that binds to an evolutionary conserved
surface region of influenza hemagglutinin, referenced above. Hemagglutinin is a protein on
the flu surface that plays a vital role in the virus
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capacity to infect cells, undergoing a
conformational change when the virus binds to a host cell and becomes endocytosed. After
inspecting a co-crystal structure of hemagglutinin with a broadly neutralizing antibody, the
authors placed hotspot residues in the vicinity of the antibody binding site, and were
subsequently able to design two proteins that bind hemagglutinin, one with a K
D
of
200 nM and one weaker. After affinity maturation, the binding affinity was increased to a
K
D
of 22 nM and 38 nM for the two designs, and the designed proteins inhibited the
conformational change that hemagglutinin undergoes during infection. X-ray
crystallographic analysis confirmed that the designed proteins bound in the intended
location and orientation. It is well conceivable that such designed proteins can play a role as
therapeutics in the future.
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