Transient interactions correspond to intermolecular binding events that do not yield a stable, long-lived complex. The dissociation rate of the complex is fast enough to yield frequent dissociation (and reassociation) events over the duration of most experimental observations. The equilibrium state of the system is characterized by a mixture of individual and complex species, whose relative population depend on the total concentration and the underlying equilibrium constant(s). Transient interactions are at the heart of many enzymatic mechanisms, where a fast dissociation rate of the enzyme substrate/product complex supports cycling through the catalytic process.
NMR is virtually the only structural biology technique able to determine the structure of a transient complex without requiring any stabilizing modifications. NMR uniquely yields information on both the structure of the complex and the process of association/dissociation, providing insights into the structural changes necessary for catalysis. Nevertheless, NMR structural studies of (large) transient complexes remain challenging.
In our laboratory, we study non-ribosomal peptidyl synthetases (NRPSs), which accomplish the synthesis of unnatural peptides in microorganisms. NRPSs are a biological exemplar of the concept of ‘division of labour’, both structurally and functionally. Each NRPS is organized as separate modules, which may be located on the same or different polypeptide chains. Every module is composed of a set of independently folded domains that catalyse a particular reaction step in the pathway. The modules are classified as initiation, elongation and termination modules, which respectively start the peptide synthesis, extend the peptide chain and release the final product. The interactions between the catalytic domains and the peptidyl carrier domains (PCP), which carry the growing peptide chain, are transient, as required by the reaction cycle. We are studying the elongation step of one such system, with particular focus on how the donor PCP interacts with the domain catalysing formation of the peptide bond, as well as on the final termination step. These studies are highly relevant for ongoing efforts towards engineering NRPSs for synthesis of artificial and specifically tailored non-ribosomal peptides.