Cooperativity and folding kinetics in a multi-domain protein with interwoven chain topology

Although a large percentage of eukaryotic proteomes consist of proteins with multiple domains, not much is known about their assembly mechanism, especially those with complicated native state architectures. Some have complex topology in which the structural elements along the sequence are interwoven in such a manner that the domains cannot be separated by cutting at any location along the sequence. We refer to such proteins as Multiply connected Multidomain Proteins (MMPs). The phoshotransferase enzyme Adenylate Kinase (ADK) with three domains (NMP, LID, and CORE), the subject of this study, is an example of MMP. We devised a coarse-grained model to simulate ADK folding initiated by changing either the temperature or guanidinium chloride (GdmCl) concentration. The simulations reproduce the experimentally measured melting temperatures that are associated with two equilibrium transitions, FRET efficiency as a function of GdmCl concentration, and the global folding times nearly quantitatively. Although the NMP domain orders independently, cooperative interactions between the LID and the CORE domains are required for complete assembly of the enzyme. The kinetic simulations show that on the collapse time scale, but less than the global folding time, multiple interconnected metastable states are populated, attesting to the folding heterogeneity. The network connectivity between distinct states shows that the CORE domain folds only after the NMP and LID domains are formed, reflecting the interwoven nature of the chain topology. We propose that the rules for MMP folding must also hold for the folding of RNA enzymes.