Despite over 75% of the eukaryotic proteome is composed by multi-domain proteins, much of our current knowledge on protein folding relies on studies on single domain proteins. In fact, protein domains are generally assumed to be able to fold independently, as proven by the possibility to express them in isolation. However, it cannot be excluded that the interaction between these structural subunits may play a critical role in the formation of the native structure, as well as in dictating misfolding events. Consequently, recent years have seen a considerable growing effort in developing methods to understand the folding of more complex multi-domain systems.
The present project is about the description of the folding mechanism of a multi-domain tandem construct, comprising two distinct covalently bound PDZ domains, belonging to a protein called Whirlin, a scaffolding protein of the hearing apparatus.
To exercise its biological function, Whirlin undergoes a mechanical process of unfolding/refolding, making this protein a suitable system for the studying of the role of multi-domain assembly in protein folding mechanisms.
The general knowledge on protein misfolding is intimately linked to dysfunction and disease. In fact, since decades it is well established how the aberrant incorrect structures are the recurrent trigger of protein aggregation leading to several diseases spanning from neurological disorders, such Alzheimer's or Parkinson's diseases, to systemic amyloidosis. Consequently, Nature has evolved several systems to solve incorrect protein folding or to degrade irreversibly misfolded polypeptides.
A peculiar protein misfolding event is represented by tandem domain swapping. In this cases, if the increasing complexity of protein architecture demands the presence of contiguous homologuous domains, the concurrent unfolding of two neighbouring subunits may induce intertwined interactions. This phenomenon causes two adjacent domains to form a central domain, equivalent to a circular permutation, formed from the central region of the two-domain sequence, and a terminal domain comprising the remaining parts of the sequence. Notably, by following this scenario, a misfolded intermediate may appear to contain more folded structure than that expected when studying a single domain.
Studies on titin and immunoglobulin domains have shown that evolution tends to minimize domain swapped misfolding by lowering the sequence identity of adjacent domains, which is almost universally less than 40% in tandem repeat proteins. In this context, it is of interest to notice that all the sequences of Whirlin in the UniProt database display unusually high sequence identities between their adjacent PDZ1 and PDZ2. Therefore, also by considering that PDZ domains are naturally prone to circular permutation, domain swapping and that previous protein engineering experiments suggested that PDZ domains may be successfully expressed as circularly permutated variants, it is somewhat surprising to find that the tendency to perform domain swapping might be evolutionary conserved in Whirlin. Can a possible misfolded intermediate play some physiological role in the function of Whirlin?
Mechanical proteins, as Whirlin, are exposed to tensile forces that might unfold them and lead to the accumulation of long-lived states displaying multiple unfolded domains. In these conditions, there could be a tendency to form aberrant structures leading to a loss of function. In the case of Whirlin, the anchoring to Sans protein via its PDZ1 domain, guarantees the mechanical communication from the surface of the stereocilia to the actin filaments, ensuring the hearing of sound waves. It is intriguing that the presence of two PDZ domains is almost universally conserved in Whirlin, and yet no physiological partners could be found for PDZ2. Thus, on the basis of our preliminary findings, we speculate that the second PDZ domain, could enhance the possibility to accumulate a functionally competent domain swapped state, which may represent a structural buffer to increase the repertoire of structures to conserve the anchoring to Sans, even in the presence of mechanical unfolding.