Molecular mechanism of domain swapping in proteins: an analysis of slower motions

Domain swapping is a structural phenomenon that plays an important role in the mechanism of oligomerization of some proteins. The monomer units in the oligomeric structure become entangled with each other. Here we investigate the mechanism of domain swapping in diphtheria toxin and the structural criteria required for it to occur by analyzing the slower modes of motion with elastic network models, Gaussian network model and anisotropic network model. We take diphtheria toxin as a representative of this class of domain-swapped proteins and show that the domain, which is being swapped in the dimeric state, rotates and twists, in going from the "open" to the "closed" state, about a hinge axis that passes through the middle of the loop extending between two domains. A combination of the intra- and intermolecular contacts of the dimer is almost equivalent to that of the monomer, which shows that the relative orientations of the residues in both forms are almost identical. This is also reflected in the calculated B-factors when compared with the experimentally determined B-factors in x-ray crystal structures. The slowest modes of both the monomer and dimer show a common hinge centered on residue 387. The differences in distances between the monomer and the dimer also shows the hinge at nearly the same location (residue 381). Finally, the first three dominant modes of anisotropic network model together shows a twisting motion about the hinge centered on residue 387. We further identify the location of hinges for a set of another 12 domain swapped proteins and give the quantitative measures of the motions of the swapped domains toward their "closed" state, i.e., the overlap and correlation between vectors.     

3D domain swapping: as domains continue to swap

Three-dimensional (3D) domain swapping creates a bond between two or more protein molecules as they exchange their identical domains. Since the term '3D domain swapping' was first used to describe the dimeric structure of diphtheria toxin, the database of domain-swapped proteins has greatly expanded. Analyses of the now about 40 structurally characterized cases of domain-swapped proteins reveal that most swapped domains are at either the N or C terminus and that the swapped domains are diverse in their primary and secondary structures. In addition to tabulating domain-swapped proteins, we describe in detail several examples of 3D domain swapping which show the swapping of more than one domain in a protein, the structural evidence for 3D domain swapping in amyloid proteins, and the flexibility of hinge loops. We also discuss the physiological relevance of 3D domain swapping and a possible mechanism for 3D domain swapping. The present state of knowledge leads us to suggest that 3D domain swapping can occur under appropriate conditions in any protein with an unconstrained terminus. As domains continue to swap, this review attempts not only a summary of the known domain-swapped proteins, but also a framework for understanding future findings of 3D domain swapping.     

Three-dimensional domain swapping in the protein structure space

Since the proposal of three-dimensional (3D) domain swapping, many 3D domain-swapped structures have been reported. However, when compared with the vast protein structure space, it is still unclear whether 3D domain swapping is a general mechanism for protein assembly. Here, we investigated this possibility by constructing a dataset consisting of more than 500 domain-swapped structures. The domain-swapped structures were mapped into the protein structure space. We found that about 10% of protein folds and 5% of protein families contain domain-swapped structures. When comparing the domain-swapped structures in a family/superfamily, we found that proteins within a family/superfamily can swap in different ways. Interface analysis revealed that the hinge loops contributed more than half of the open interface in 70% of bona fide domain-swapped dimers, indicating that the hinge loops play an important role in stabilizing the domain-swapped conformations. Our study supports the suggestion that domain swapping is a general property of all proteins and will facilitate further understanding the mechanism of 3D domain swapping.     

Protein oligomerization through domain swapping: role of inter-molecular interactions and protein concentration

Domain swapping has been shown to be an important mechanism controlling multiprotein assembly and has been suggested recently as a possible mechanism underlying protein aggregation. Understanding oligomerization via domain swapping is therefore of theoretical and practical importance. By using a symmetrized structure-based (Gō) model, we demonstrate that in the free-energy landscape of domain swapping, a large free-energy barrier separates monomeric and domain-swapped dimeric configurations. We investigate the effect of finite monomer concentration, by implementing a new semi-analytical method, which involves computing the second virial coefficient, a thermodynamic indicator of inter-molecular interactions. This method, together with the symmetrized structure-based (Gō) model, minimizes the need for expensive many-protein simulations, providing a convenient framework to investigate concentration effect. Finally, we perform direct simulations of domain-swapped trimer formation, showing that this modeling approach can be used for higher-order oligomers.     

Amyloid-like fibrils from a domain-swapping protein feature a parallel, in-register conformation without native-like interactions

The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular β-sheet contacts present in the monomeric state could constitute intermolecular β-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded β-sheet and one α-helix. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register β-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular β-strand interactions seen in the dimeric state, may be preserved in "domain-swapping" fibrils. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.     

Mutation of interfaces in domain-swapped human betaB2-crystallin

The superfamily of eye lens betagamma-crystallins is highly modularized, with Greek key motifs being used to form symmetric domains. Sequences of monomeric gamma-crystallins and oligomeric beta-crystallins fold into two domains that pair about a further conserved symmetric interface. Conservation of this assembly interface by domain swapping is the device adopted by family member betaB2-crystallin to form a solution dimer. However, the betaB1-crystallin solution dimer is formed from an interface used by the domain-swapped dimer to form a tetramer in the crystal lattice. Comparison of these two structures indicated an intriguing relationship between linker conformation, interface ion pair networks, and higher assembly. Here the X-ray structure of recombinant human betaB2-crystallin showed that domain swapping was determined by the sequence and not assembly conditions. The solution characteristics of mutants that were designed to alter an ion pair network at a higher assembly interface and a mutant that changed a proline showed they remained dimeric. X-ray crystallography showed that the dimeric mutants did not reverse domain swapping. Thus, the sequence of betaB2-crystallin appears well optimized for domain swapping. However, a charge-reversal mutation to the conserved domain-pairing interface showed drastic changes to solution behavior. It appears that the higher assembly of the betagamma-crystallin domains has exploited symmetry to create diversity while avoiding aggregation. These are desirable attributes for proteins that have to exist at very high concentration for a very long time.     

Topological determinants of protein domain swapping

Protein domain swapping has been repeatedly observed in a variety of proteins and is believed to result from destabilization due to mutations or changes in environment. Based on results from our studies and others, we propose that structures of the domain-swapped proteins are mainly determined by their native topologies. We performed molecular dynamics simulations of seven different proteins, known to undergo domain swapping experimentally, under mildly denaturing conditions and found in all cases that the domain-swapped structures can be recapitulated by using protein topology in a simple protein model. Our studies further indicated that, in many cases, domain swapping occurs at positions around which the protein tends to unfold prior to complete unfolding. This, in turn, enabled prediction of protein structural elements that are responsible for domain swapping. In particular, two distinct domain-swapped dimer conformations of the focal adhesion targeting domain of focal adhesion kinase were predicted computationally and were supported experimentally by data obtained from NMR analyses.     

Propensity for C-terminal domain swapping correlates with increased regional flexibility in the C-terminus of RNase A

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.     

Method for identification of rigid domains and hinge residues in proteins based on exhaustive enumeration

Many proteins undergo large‐scale motions where relatively rigid domains move against each other. The identification of rigid domains, as well as the hinge residues important for their relative movements, is important for various applications including flexible docking simulations. In this work, we develop a method for protein rigid domain identification based on an exhaustive enumeration of maximal rigid domains, the rigid domains not fully contained within other domains. The computation is performed by mapping the problem to that of finding maximal cliques in a graph. A minimal set of rigid domains are then selected, which cover most of the protein with minimal overlap. In contrast to the results of existing methods that partition a protein into non‐overlapping domains using approximate algorithms, the rigid domains obtained from exact enumeration naturally contain overlapping regions, which correspond to the hinges of the inter‐domain bending motion. The performance of the algorithm is demonstrated on several proteins. Proteins 2015; 83:1054–1067. © 2015 Wiley Periodicals, Inc.     

A proposed structural model for amyloid fibril elongation: domain swapping forms an interdigitating beta-structure polymer

We propose a model illustrating how proteins, which differ in their overall sequences and structures, can form the propagating, twisted beta-sheet conformations, characteristic of amyloids. Some cases of amyloid formation can be explained through a "domain swapping" event, where the swapped segment is either a beta-hairpin or an unstable conformation which can partially unfold and assume a beta-hairpin structure. As in domain swapping, here the swapped beta-hairpin is at the edge of the structure, has few (if any) salt bridges and hydrogen bonds connecting it to the remainder of the structure and variable extents of buried non-polar surface areas. Additionally, in both cases the swapped piece constitutes a transient "building block" of the structure, with a high population time. Whereas in domain swapping the swapped fragment has been shown to be an alpha-helix, loop, strand or an entire domain, but so far not a beta-hairpin, despite the large number of cases in which it was already detected, here swapping may involve such a structural motif. We show how the swapping of beta-hairpins would form an interdigitated, twisted beta-sheet conformation, explaining the remarkable high stability of the protofibril in vitro. Such a swapping mechanism is attractive as it involves a universal mechanism in proteins, critical for their function, namely hinge-bending motions. Our proposal is consistent with structural superpositioning of mutational variants. While the overall r.m.s.d.s of the wild-type and mutants are small, the proposed hinge-bending region consistently shows larger deviations. These larger deviations illustrate that this region is more prone to respond to the mutational changes, regardless of their location in the sequence or in the structure. Nevertheless, above all, we stress that this proposition is hypothetical, since it is based on assumptions lacking definitive experimental support.     

The structure of an engineered domain-swapped ribonuclease dimer and its implications for the evolution of proteins toward oligomerization

BACKGROUND: Domain swapping has been proposed as a mechanism that explains the evolution from monomeric to oligomeric proteins. Bovine and human pancreatic ribonucleases are monomers with no biological properties other than their RNA cleavage ability. In contrast, the closely related bovine seminal ribonuclease is a natural domain-swapped dimer that has special biological properties, such as cytotoxicity to tumour cells. Several recombinant ribonuclease variants are domain-swapped dimers, but a structure of this kind has not yet been reported for the human enzyme. RESULTS: The crystal structure at 2 A resolution of an engineered ribonuclease variant called PM8 reveals a new kind of domain-swapped dimer, based on the change of N-terminal domains between the two subunits. The swapping is fastened at both hinge peptides by the newly introduced Gln101, involved in two intermolecular hydrogen bonds and in a stacking interaction between residues of different chains. Two antiparallel salt bridges and water-mediated hydrogen bonds complete a new interface between subunits, while the hinge loop becomes organized in a 3(10) helix structure. CONCLUSIONS: Proteins capable of domain swapping may quickly evolve toward an oligomeric form. As shown in the present structure, a single residue substitution reinforces the quaternary structure by forming an open interface. An evolutionary advantage derived from the new oligomeric state will fix the mutation and favour others, leading to a more extended complementary dimerization surface, until domain swapping is no longer necessary for dimer formation. The newly engineered swapped dimer reported here follows this hypothetical pathway for the rapid evolution of proteins.     

Dimer formation through domain swapping in the crystal structure of the Grb2-SH2-Ac-pYVNV complex

Src homology 2 (SH2) domains are key modules in intracellular signal transduction. They link activated cell surface receptors to downstream targets by binding to phosphotyrosine-containing sequence motifs. The crystal structure of a Grb2-SH2 domain-phosphopeptide complex was determined at 2.4 A resolution. The asymmetric unit contains four polypeptide chains. There is an unexpected domain swap so that individual chains do not adopt a closed SH2 fold. Instead, reorganization of the EF loop leads to an open, nonglobular fold, which associates with an equivalent partner to generate an intertwined dimer. As in previously reported crystal structures of canonical Grb2-SH2 domain-peptide complexes, each of the four hybrid SH2 domains in the two domain-swapped dimers binds the phosphopeptide in a type I beta-turn conformation. This report is the first to describe domain swapping for an SH2 domain. While in vivo evidence of dimerization of Grb2 exists, our SH2 dimer is metastable and a physiological role of this new form of dimer formation remains to be demonstrated.     

A Hinge Region cis-Proline in Ribonuclease A Acts as a Conformational Gatekeeper for C-Terminal Domain Swapping

Domain swapping, the process in which a structural unit is exchanged between monomers to create a dimer containing two subunits of the monomeric fold, is believed to be an important mechanism for oligomerization and the formation of amyloid fibrils. Structural studies have implicated proline as an important residue for domain swapping due to its increased frequency in hinge regions preceding swapped arms. We hypothesized that proline's unique ability to populate both cis and trans peptide bond conformations may allow proline to act as a conformational gatekeeper, regulating interconversion between monomer and domain-swapped dimer forms. The hinge region of RNase A contains a proline at residue 114 that adopts a cis conformation in the monomer and extends to a trans conformation in the C-terminal domain-swapped dimer. Substitution of P114 with residues that strongly prefer a trans peptide bond (Ala, Gly) results in significant population of the C-terminal domain-swapped dimer under near-physiological conditions (pH 8.0, 37 °C). This is in stark contrast to dimerization of wild-type RNase A, which requires incubation under extreme conditions such as lyophilization from acetic acid or elevated temperature. In addition, we observe similar results when cis-P114 is mutated to glycine in a homologous RNase, human pancreatic RNase 1. Our results suggest that isomerization at P114 may facilitate population of a partially unfolded intermediate or alternative structure competent for domain swapping and provide support for a more general role for proline isomerization as a conformational gatekeeper in domain swapping and oligomerization.     

Global Transitions of Proteins Explored by a Multiscale Hybrid Methodology: Application to Adenylate Kinase

Efficient and accurate mapping of transition pathways is a challenging problem in allosteric proteins. We propose here a to our knowledge new methodology called collective molecular dynamics (coMD). coMD takes advantage of the collective modes of motions encoded by the fold, simultaneously evaluating the interactions and energetics via a full-atomic MD simulation protocol. The basic approach is to deform the structure collectively along the modes predicted by the anisotropic network model, upon selecting them via a Monte Carlo/Metropolis algorithm from among the complete pool of all accessible modes. Application to adenylate kinase, an allosteric enzyme composed of three domains, CORE, LID, and NMP, shows that both open-to-closed and closed-to-open transitions are readily sampled by coMD, with large-scale motions of the LID dominating. An energy-barrier crossing occurs during the NMP movements. The energy barrier originates from a switch between the salt bridges K136-D118 at the LID-CORE interface and K57-E170 and D33-R156 at the CORE-NMP and LID-NMP interfaces, respectively. Despite its simplicity and computing efficiency, coMD yields ensembles of transition pathways in close accord with detailed full atomic simulations, lending support to its utility as a multiscale hybrid method for efficiently exploring the allosteric transitions of multidomain or multimeric proteins.     

Three-dimensional domain swapping in the folded and molten-globule states of cystatins, an amyloid-forming structural superfamily

Cystatins, an amyloid-forming structural superfamily, form highly stable, domain-swapped dimers at physiological protein concentrations. In chicken cystatin, the active monomer is a kinetic trap en route to dimerization, and any changes in solution conditions or mutations that destabilize the folded state shorten the lifetime of the monomeric form. In such circumstances, amyloidogenesis will start from conditions where a domain-swapped dimer is the most prevalent species. Domain swapping occurs by a rearrangement of loop I, generating the new intermonomer interface between strands 2 and 3. The transition state for dimerization has a high level of hydrophobic group exposure, indicating that gross conformational perturbation is required for domain swapping to occur. Dimerization also occurs when chicken cystatin is in its reduced, molten-globule state, implying that the organization of secondary structure in this state mirrors that in the folded state and that domain swapping is not limited to the folded states of proteins. Although the interface between cystatin-fold units is poorly defined for cystatin A, the dimers are the appropriate size to account for the electron-dense regions in amyloid protofilaments.     

Global Transitions of Proteins Explored by a Multiscale Hybrid Methodology: Application to Adenylate Kinase

Efficient and accurate mapping of transition pathways is a challenging problem in allosteric proteins. We propose here a to our knowledge new methodology called collective molecular dynamics (coMD). coMD takes advantage of the collective modes of motions encoded by the fold, simultaneously evaluating the interactions and energetics via a full-atomic MD simulation protocol. The basic approach is to deform the structure collectively along the modes predicted by the anisotropic network model, upon selecting them via a Monte Carlo/Metropolis algorithm from among the complete pool of all accessible modes. Application to adenylate kinase, an allosteric enzyme composed of three domains, CORE, LID, and NMP, shows that both open-to-closed and closed-to-open transitions are readily sampled by coMD, with large-scale motions of the LID dominating. An energy-barrier crossing occurs during the NMP movements. The energy barrier originates from a switch between the salt bridges K136-D118 at the LID-CORE interface and K57-E170 and D33-R156 at the CORE-NMP and LID-NMP interfaces, respectively. Despite its simplicity and computing efficiency, coMD yields ensembles of transition pathways in close accord with detailed full atomic simulations, lending support to its utility as a multiscale hybrid method for efficiently exploring the allosteric transitions of multidomain or multimeric proteins.     

Swapping of three-dimensional domains as a molecular mechanism of dimerization of aminoacyl-tRNA synthetases

It has been shown recently that many proteins undergo oligomerization through exchange of structural elements. That process, termed a 3D domain swapping, is the replacement of a portion of the tertiary structure of a protein with an identical piece from a second polypeptide chain. When the exchange is reciprocated, domain-swapped dimers embrace with the exchange of elements of secondary structure or domains; however, if the exchange is not reciprocated but propagated along multiple polynucleotide chains, higher-order assemblies may form. In this paper we discuss swapping as a general mechanism of aminoacyl-tRNA synthetases dimerization, specifically plant methionyl-tRNA synthetase.     

Dynamic properties of the N-terminal swapped dimer of ribonuclease A

Bovine pancreatic ribonuclease (RNase A) forms two 3-dimensional domain-swapped dimers with different quaternary structures. One dimer is characterized by the swapping of the C-terminal region (C-Dimer) and presents a rather loose structure. The other dimer (N-Dimer) exhibits a very compact structure with exchange of the N-terminal helix. Here we report the results of a molecular dynamics/essential dynamics (MD/ED) study carried out on the N-Dimer. This investigation, which represents the first MD/ED analysis on a three-dimensional domain-swapped enzyme, provides information on the dynamic properties of the active site residues as well as on the global motions of the dimer subunits. In particular, the analysis of the flexibility of the active site residues agrees well with recent crystallographic and site-directed mutagenesis studies on monomeric RNase A, thus indicating that domain swapping does not affect the dynamics of the active sites. A slight but significant rearrangement of N-Dimer quaternary structure, favored by the formation of additional hydrogen bonds at subunit interface, has been observed during the MD simulation. The analysis of collective movements reveals that each subunit of the dimer retains the functional breathing motion observed for RNase A. Interestingly, the breathing motion of the two subunits is dynamically coupled, as they open and close in phase. These correlated motions indicate the presence of active site intercommunications in this dimer. On these bases, we propose a speculative mechanism that may explain negative cooperativity in systems preserving structural symmetry during the allosteric transitions.     

The GB1 amyloid fibril: recruitment of the peripheral beta-strands of the domain swapped dimer into the polymeric interface

Three-dimensional domain swapping has been evoked as a mechanism for oligomerization of proteins. Here, we show for the immunoglobulin-binding domain B1 of streptococcal protein G (GB1) that fibril formation is observed readily for variants that exist as domain-swapped dimers. No fibril was formed by a revertant that exhibits the stable wild-type GB1 fold or a mutant comprising a highly destabilized, fluctuating ensemble of conformers. Structural features of the GB1 amyloid fibril were characterized by cysteine disulfide cross-linking. Residues in the outer edge beta-strands of the domain-swapped dimer readily form intermolecular disulfide bonds prior to and during fibril formation. On the basis of these data, a structural model for the assembly of domain-swapped dimers into a polymeric structure of the GB1 fibril is proposed.