MSOE Sandbox HA

From Proteopedia

Hemagglutinin Bound to Antibody CR6261 and Bound to de novo Protein HB36

Abstract:

The threat of an influenza pandemic looms eminent in the future of public health around the globe, similar to the previous 1918 H1N1 Spanish flu, 1957 H2N2 Asian flu, and 1968 H3N2 Hong Kong flu epidemics. Synthesizing a broad-spectrum influenza vaccine remains elusive due to the highly mutable nature of the head region of the viral protein hemagglutinin (HA). However, it is known that the stem portion of this molecule is highly stable and that the rare human antibody CR6261 Fab can dock into a stem region binding site in the presence of low pH to inactivate the membrane fusion process. It is this process that allows the virus to infect the cell. The development of a de novo protein molecule that mimics the docking action of CR6261 into the binding site was done computationally, and 88 designs were tested for effectiveness using the yeast strain EBY 100. Following affinity maturation, the 3R2X protein was shown to effectively bind at the conserved surface patch and inhibit the HA conformational change at low pH. This protocol of producing de novo designed proteins may alleviate the problem of producing sufficient quantities of CR6261 in the event of a pandemic.

Hemagglutinin

Hemagglutin exists a functional trimer that begins influenza infection with a complex of other proteins. The each of the three HA proteins are constructed: HA1 and HA2 . The Hemagglutinin (HA) protein undergoes a major conformational shift to enable infection which involves several major helices of the HA2 chain. This region along with an adjacent region of the HA1 from the stem region of HA. Because this region is so important to the conformational change for functionality of this protein, this region is highly conserved across a variety of species of influenza (needs citation)and thus an ideal target to create a vaccine that will be effective against many strains of influenza. HA is divided into two regions . The Head region is highly mutable and thus poor target for drug design in vaccinations. The stem is highly conserved across multiple species of flu.

CR6261: An Antibody that May Provide Immunity to Diverse Influenza Strains

HB36.3: Making a De Novo Protein

Sarel J. Fleishman et al have created a denovo protein called HB36.3 based upon a new computational design strategy. To initiate this process, they first began by looking at the epitope (or binding area) of the CR6261 on the Hemagglutinin stem (discussed above). This antibody was found by Damian Ekiert et al to bind to the stem region of the 1918 Influenza Hemagglutinin (HA) and to be able to bind to many influenza hemagglutinin molecules. Fleishman's group identified three "hot spot" residues called HS1, HS2, and HS3 located within the groove on the HA stem region. This groove, located between two alpha helices, is lined with multiple hydrophobic residues making it an excellent potential binding-site for the new protein. The team identified the amino acid side chains that formed an energetically favorable interaction with each of the hot spots. Next they searched for protein scaffolds that could be used to support the various identified amino acid side chains.These scaffolds were further tested and residues compatible to the identified hot spots were added and analyzed using new, complex computer software programs.

There were 51 potential proteins containing 2 HS residues and 37 with 3 of the HS residues selected for testing. To determine the HA binding success, the genes encoding the protein designs were cloned into a yeast-display vector and transformed into a yeast strain. The designed proteins then displayed on the yeast cell surface. Of the 73 proteins displayed on the yeast surface, two showed reproducible HA stem binding. HB36.3 is one of these proteins. HB36.3 features a row of hydrophic residues (F61, W57, M53, and F49) on a helix that fits into the HA groove. In the refining HB36.3, a library of molecules was generated using site mutation error-prone polymerase chain reacttion and then binding to HA using yeast surface display. This process generated two changes in structure: A60V which produced a 25% increase in binding and D47S which produced a 40% increase in binding.