Mechanisms of activity of Non-Ribosomal Peptide Synthases.
Organic Molecular Chemistry - Synthesis and Characterisation
Final Report Abstract
We could answer all four questions we posed at the beginning of the project to completion. What confers specificity to the binding of the PCP domains to either the acceptor or donor sites of the condensation domain? Is specificity dictated by the substrate or is there a contribution from the PCP domains themselves? The donor and acceptor PCP domains are programmed to bind orthogonally to the respective faces of the BC. The APCP is recognised selectively by the N95 domain, which does not form an encounter complex with BPCP. In addition, there are some specific interactions between the APCP and the BC core domain (not discussed here). The APCP also does not bind to the acceptor binding surface of BC. Thus, we conclude that the specificity of APCP and BPCP recruitment is driven by the amino acids sequence of the PCP, while much of the affinity is given by the substrate and PPant arm. Which conformation of PCP domains bind to the condensation domain? All our PCP domains adopt the A/H conformation and there is no evidence of any other conformation. What drives substrate binding? What drives product release? Is a conformational change of the PCP domains implicated in these processes? No changes in the conformation of the PCP domains are observed in the process. APCP binding (before the condensation reaction) and unbinding (after the condensation reaction) is caused by the fact that a large portion of the binding affinity is provided by the substrate. Once the substrate is lost, the Ppant arm of APCP can easily exit the BC tunnel. Furthermore, we discovered that the BC protein is in at least two distinct conformations, a resting state and an APCP-bound, active state. These two states may be linked to the conformation of the side chain of the catalytic H227. We also discovered the existence of a new “docking” domain, N95, which, unlike all other docking domains known to date, has the same structure as PCP domains but has lost the capability to be linked to a Ppant arm. This domain is responsible for the specific recruitment of APCP.
