The beta3-beta4 loop is able to effectively block the active site from antibiotics, but it also prevents peptidoglycan from entering. Yet previous research had shown that PBP2a was functional even in the presence of beta-lactam antibiotics. Researchers then began to investigate PBP2a for a potential allosteric binding site that could cause the necessary conformational change required to reveal the active site. 60Å away in what was called the non-penicillin binding domain, researchers discovered an allosteric binding site between lobes 1, 2, and 3. Nascent peptidoglycan and peptidoglycan mimics, such as muric acid which is structurally similar to peptidoglycan, were able to non-covalently bind to the allosteric site and induce significant conformational change. Upon allosteric binding, the ligand repulses an asparagine residue at position 146 which disrupts surrounding salt bridges and other intermolecular forces. More specifically, this shift breaks the salt bridge between the lysine located at residue 148 and the glutamic acid at residue 150. This glutamic acid is now capable of forming new salt bridge interactions with amino acids outside of the allosteric site. While in other proteins this might result in a small conformational change, PBP2a is a highly charged enzyme with 125 charged residues on its surface and 79 hypothesized salt bridges in the apo-structure. As a result, this new glutamic acid salt bridge interaction perpetuates the cycle and sets off a salt bridge cascade of bond breaking and forming along the length of the protein. A series of conformational changes follows this cascade which culminates in the opening of the active site.

In order to support the salt bridge cascade hypothesis, researchers explored the effects of various point mutations throughout the protein. A positive histidine located at position 351, outside of both the allosteric and active sites, was mutated to a neutral asparagine. This H351N mutation reduced catalytic activity to the point that the enzyme was essentially inactive. Within the active site, a E447K mutation resulted in a similar reduction in catalytic activity. This mutation is thought to form a new salt bridge with E460, diverting the signal away from the active site and failing to culminate in the necessary conformational change required. Comparing both the allosteric holo and apo-structure of PBP2a revealed that each conformation has a substantial number of unique salt bridges. In both the closed and open active site, the salt bridges unique to each confirmation were centered around the active and allosteric sites. These findings ultimately support the salt bridge cascade hypothesis as it stresses the importance of a connected chain of interactions reaching from the allosteric site to the active site. If this chain is severed either in the middle, as in the case of H351N mutation, or at the end, as with E447K mutation, the signal stops and so does any conformational change. When the salt bridge cascade is successfully completed and the beta3-beta4 loop subsequently shifts to expose the PBP2a active site, it culminates in the unique salt bridges seen in prior data.

With the discovery of an allosteric binding site, researchers began to investigate the potential of antibiotics that target the allosteric site in addition to the active site. A drug called ceftaroline successfully bound to both the active and allosteric site and became the prescribed treatment for MRSA infections. Within two years of ceftaroline being on the market, hospitals had already began reporting resistant strains. Researchers began to investigate the extent of PBP2a conformational change for a variety of different allosterically bound molecules. Ultimately it was determined that the size and identity of the allosteric modulator affects both the salt bridge cascade as well as the conformational change. Ceftaroline was able to create a salt bridge cascade that transmits a signal from the allosteric site to the active site, yet there were less salt bridges formed in comparison to when nascent peptidoglycan was bound. This finding indicates that there isn't one single defined salt bridge cascade. Rather, due to the sheer amount of charged residues on the surface of the protein, a salt bridge cascade can take a myriad of different pathways from the allosteric to the active site depending on the size of the initial allosteric modulator.