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Domain swapping is a type of oligomerization in which monomeric proteins exchange a structural element, resulting in oligomers whose subunits recapitulate the native, monomeric fold. It has been implicated as a potential mechanism for protein aggregation, which provides a strong impetus to understand the structural determinants and folding mechanisms that trigger domain swapping. Bovine pancreatic ribonuclease A (RNase A) is a well-studied protein known to domain swap under extreme conditions, such as lyophilization from acetic acid. The major domain-swapped dimer form of RNase A exchanges a β-strand at its C-terminus to form a C-terminal domain-swapped dimer. To study the mechanism by which C-terminal swapping occurs, we used a variant of RNase A containing a P114G mutation that readily domain swaps under physiological conditions. Using NMR and hydrogen–deuterium exchange, we find that the P114G variant has decreased protection from hydrogen exchange compared to the wild-type protein near the C-terminal hinge region. Our results suggest that domain swapping occurs via a local high-energy fluctuation at the C-terminus.
- Top of page
- Materials and Methods
- Supporting Information
Domain swapping is a unique mechanism of protein dimerization and higher order oligomerization that results in the formation of subunits that recapitulate native, monomeric structure. It has been implicated as a means for evolution of quaternary structure and modification of enzymatic activity. It is most often studied in terms of its potential as a mechanism for protein aggregation and amyloid formation as both processes are self-complementing and often involve polar zipper-like interactions.1 For proteins that populate the dimer at equilibrium, there must be favorable enthalpic interactions stabilizing the dimer, most likely in the open interface.
In spite of its implications for protein evolution and aggregation, very little is known about the driving forces that cause proteins to domain swap. Although there are over 50 domain-swapped structures in the PDB,2 there are only a handful of investigations into the folding mechanisms that lead to domain swapping.3–9 Among these studies, there is debate as to whether domain swapping occurs via the unfolded state or some high energy, partially unfolded state. In the first case, domain swapping serves as an alternative folding pathway9; in the latter, the monomer may undergo a fluctuation to some non-native, partially unfolded form, M*, that is capable of swapping and forming a domain-swapped dimer (Fig. 1).
Figure 1. Structural models for C-terminal domain swapping in RNase A. Monomeric RNase A (PDB ID 2AAS) may dimerize (PDB ID 1F0V) either via complete unfolding or some partial unfolding event that produces a hypothetical exchange-competent intermediate, M*.
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Bovine pancreatic ribonuclease A (RNase A) is an example of a protein that exists primarily in its monomeric form but is capable of domain swapping. RNase A can swap at either its N- or C-termini to form dimers or higher order oligomers. To form domain-swapped structures, the protein is subjected to lyophilization from 50% acetic acid. Under these conditions, 80% of the dimers are C-terminal domain-swapped dimers, while 20% are N-terminal domain-swapped dimers. Since this process requires lyophilization, in which the protein goes through the solid state, it has been difficult to study the process in solution.3, 8 Thus, the mechanism of domain swapping in RNase A under more physiological conditions, particularly at the C-terminus, remains poorly understood.
Previous experiments have examined the role of the N-terminal hinge region on domain swapping in RNase A and related RNases, bovine seminal RNase (BS-RNase) and human pancreatic RNase (HP-RNase). While WT-RNase A does not domain swap under physiological conditions, BS-RNase readily forms N-terminal domain-swapped dimers in solution,10, 11 most likely due to differences in the hinge regions. The exact role of these sequence differences in affecting the swapping propensity remains unclear, as RNase A still does not readily domain swap even when its N-terminal hinge region residues are replaced with those of BS-RNase.10 Deletion of this hinge, however, can drive HP-RNase, a monomeric protein, to form N-terminal domain-swapped dimers.11 It is worth noting that the exchanged N-terminal region in these N-terminal-swapped dimers is the same helix that is cleaved by subtilisin in RNase A to form RNase S, demonstrating that, as seen in domain swapping, the two regions can associate noncovalently.12
The structural determinants that lead to the C-terminal dimer formation are both less obvious and potentially more interesting than those of the N-terminal dimer. There are no existing data to suggest that wild-type RNase A populates some partially unfolded intermediate in which the C-terminal β-strand is exposed and prone to exchange. The C-terminal β-sheet is one of the first elements of RNase A to fold.13 It is highly protected from hydrogen exchange and is protected from carboxypeptidase A cleavage in its thermally unfolded state.14–16 In fact, Laurents and coworkers3 have suggested, based on their experimental studies on the folding of RNase A, that the protein domain swaps via a previously identified folding intermediate, IN, and therefore swapping involves accessing the unfolded state. Computational studies have also suggested that significant unfolding is required for swapping from the C-terminus.6 Our recent results,17 however, demonstrate that under physiological conditions, an increase in the population of the unfolded state does not increase the propensity for domain swapping.
Resolving the mechanism of C-terminal dimer formation in RNase A is of particular interest as it applies to the link between domain swapping and amyloid formation. The open interface in the C-terminal dimer contains two stacked β-strands, reminiscent of the polar–zipper interactions present in amyloid-β spines, suggesting that the mechanism that leads to C-terminal dimer formation may be more similar to events that result in aggregation.18 In fact, an engineered version of RNase A with an amyloidogenic polyglutamine sequence in the C-terminal hinge region was the first well-defined example implicating domain swapping in amyloid formation.19
Recently, we showed that a cis-proline in the C-terminal hinge region (P114) acts as a conformational gatekeeper for domain swapping in RNase A.17 This result strongly suggests that isomerization at P114 is a key event in the mechanism of RNase A C-terminal domain swapping. Mutation of P114 to an amino acid that strongly prefers a trans conformation, such as glycine or alanine, resulted in variants of RNase A that readily domain swap under physiological conditions. These RNase A variants provide an excellent system for studying the mechanism of domain swapping under experimentally tractable conditions.
Our hypothesis was that differences in the dynamics of P114G and WT-RNase A might correlate with the change in swapping propensity of the P114G variant and elucidate some partially unfolded, exchange-competent intermediate state. Rather than directly comparing the P114G variant to WT, we used another variant, P93A, that has the same global stability as P114G but does not readily domain swap under physiological conditions.17 To probe for differences between these variants, we implemented NMR hydrogen–deuterium exchange (HX) techniques.
Increased HX rates in specific regions of a protein are interpreted as reflecting either an increased population or easier access to some “open” or exposed state for that amide site via the following standard model as described by Linderstrom-Lang20:
The more flexible a site in the protein is, either due to local fluctuations, partial or global unfolding events, the faster it will exchange. By comparing the HX behavior of the two RNase A variants, P114G and P93A, under identical conditions, we can make inferences about how local changes in conformational flexibility affect domain swapping propensity.