3D domain swapping, protein oligomerization, and amyloid formation

In 3D domain swapping, first described by Eisenberg, a structural element of a monomeric protein is replaced by the same element from another subunit. This process requires partial unfolding of the closed monomers that is then followed by adhesion and reconstruction of the original fold but from elements contributed by different subunits. If the interactions are reciprocal, a closed-ended dimer will be formed, but the same phenomenon has been suggested as a mechanism for the formation of open-ended polymers as well, such as those believed to exist in amyloid fibrils. There has been a rapid progress in the study of 3D domain swapping. Oligomers higher than dimers have been found, the monomer-dimer equilibrium could be controlled by mutations in the hinge element of the chain, a single protein has been shown to form more than one domain-swapped structure, and recently, the possibility of simultaneous exchange of two structural domains by a single molecule has been demonstrated. This last discovery has an important bearing on the possibility that 3D domain swapping might be indeed an amyloidogenic mechanism. Along the same lines is the discovery that a protein of proven amyloidogenic properties, human cystatin C, is capable of 3D domain swapping that leads to oligomerization. The structure of domain-swapped human cystatin C dimers explains why a naturally occurring mutant of this protein has a much higher propensity for aggregation, and also suggests how this same mechanism of 3D domain swapping could lead to an open-ended polymer that would be consistent with the cross-beta structure, which is believed to be at the heart of the molecular architecture of amyloid fibrils.     

Sequence-structure signals of 3D domain swapping in proteins

Three-dimensional domain swapping occurs when two or more identical proteins exchange identical parts of their structure to generate an oligomeric unit. It affects proteins with diverse sequences and structures, and is expected to play important roles in evolution, functional regulation and even conformational diseases. Here, we search for traces of domain swapping in the protein sequence, by means of algorithms that predict the structure and stability of proteins using database-derived potentials. Regions whose sequences are not optimal with regard to the stability of the native structure, or showing marked intrinsic preferences for non-native conformations in absence of tertiary interactions are detected in most domain-swapping proteins. These regions are often located in areas crucial in the swapping process and are likely to influence it on a kinetic or thermodynamic level. In addition, cation-pi interactions are frequently observed to zip up the edges of the interface between intertwined chains or to involve hinge loop residues, thereby modulating stability. We end by proposing a set of mutations altering the swapping propensities, whose experimental characterization would contribute to refine our in silico derived hypotheses.     

3D domain swapping modulates the stability of members of an icosahedral virus group

BACKGROUND: Rice yellow mottle virus (RYMV) is a major pathogen that dramatically reduces rice production in many African countries. RYMV belongs to the genus sobemovirus, one group of plant viruses with icosahedral capsids and single-stranded, positive-sense RNA genomes. RESULTS: The structure of RYMV was determined and refined to 2.8 A resolution by X-ray crystallography. The capsid contains 180 copies of the coat protein subunit arranged with T = 3 icosahedral symmetry. Each subunit adopts a jelly-roll beta sandwich fold. The RYMV capsid structure is similar to those of other sobemoviruses. When compared with these viruses, however, the betaA arm of the RYMV C subunit, which is a molecular switch that regulates quasi-equivalent subunit interactions, is swapped with the 2-fold-related betaA arm to a similar, noncovalent bonding environment. This exchange of identical structural elements across a symmetry axis is categorized as 3D domain swapping and produces long-range interactions throughout the icosahedral surface lattice. Biochemical analysis supports the notion that 3D domain swapping increases the stability of RYMV. CONCLUSIONS: The quasi-equivalent interactions between the RYMV proteins are regulated by the N-terminal ordered residues of the betaA arm, which functions as a molecular switch. Comparative analysis suggests that this molecular switch can also modulate the stability of the viral capsids.     

Role of the hinge peptide and the intersubunit interface in the swapping of N-termini in dimeric bovine seminal RNase.

Bovine seminal ribonuclease (BS-RNase) is the only known dimeric enzyme characterized by an equilibrium between two different 3D structures: MxM, with exchange (or swapping) of the N-terminal 1-20 residues, and M=M, without exchange. As a consequence, the hinge region 16-22 has a different tertiary structure in the two forms. In the native protein, the equilibrium ratio between MxM and M=M is about 7 : 3. Kinetic analysis of the swapping process for a recombinant sample shows that it folds mainly in the M=M form, then undergoes interconversion into the MxM form, reaching the same 7 : 3 equilibrium ratio. To investigate the role of the regions that are most affected structurally by the swapping, we expressed variant proteins by replacing two crucial residues with the corresponding ones from RNase A: Pro19, within the hinge peptide, and Leu28, located at the interface between subunits. We compared the structural properties of the monomeric forms of P19A-BS-RNase, L28Q-BS-RNase and P19A/L28Q-BS-RNase variants with those of the parent protein, and investigated the exchange kinetics of the corresponding dimers. The P19A mutation slightly increases the thermal stability of the monomer, but it does not alter the swapping tendency of the dimer. In contrast, the L28Q mutation significantly affects both the dimerization and swapping processes but not the thermal stability of the monomer. Overall, these results suggest that the structural determinants that control the exchange of N-terminal arms in BS-RNase may not be located within the hinge peptide, and point to a crucial role of the interface residues.     

The swapping of terminal arms in ribonucleases: comparison of the solution structure of monomeric bovine seminal and pancreatic ribonucleases

Bovine seminal ribonuclease (BS-RNase), the only dimeric protein among the pancreatic-like ribonucleases, is endowed with special structural features and with biological functions beyond enzymatic activity. In solution, the protein exists as an equilibrium mixture of two forms, with or without exchange (or swapping) of the N-terminal arms. After selective reduction and alkylation of the two intrachain disulfide bridges, the dimeric protein can be transformed into a monomeric derivative that has a ribonuclease activity higher than that of the parent dimeric protein but is devoid of the special biological functions. A detailed investigation of the structural features of this protein in solution, in comparison with those of other monomeric ribonucleases, may help unveil the structural details which induce swapping of the N-terminal arms of BS-RNase. The solution structure of the recombinant monomeric form of BS-RNase, as determined by 3D heteronuclear NMR, shows close similarity with that of bovine pancreatic ribonuclease (RNase A) in all regions characterized by regular elements of secondary structure. However, significant differences are present in the flexible regions, which could account for the different behavior of the two proteins. To characterize in detail these regions, we have measured H/D exchange rate constants, temperature coefficients and heteronuclear NOEs of backbone amides for both RNase A and monomeric BS-RNase. The results indicate a large difference in the backbone flexibility of the hinge peptide segment 16-22 of the two proteins, which could provide the molecular basis to explain the ability of BS-RNase subunits to swap their N-terminal arms.     

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.     

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.     

Molecular dynamics simulation of the SH3 domain aggregation suggests a generic amyloidogenesis mechanism

We use molecular dynamics simulation to study the aggregation of Src SH3 domain proteins. For the case of two proteins, we observe two possible aggregation conformations: the closed form dimer and the open aggregation state. The closed dimer is formed by "domain swapping"-the two proteins exchange their RT-loops. All the hydrophobic residues are buried inside the dimer so proteins cannot further aggregate into elongated amyloid fibrils. We find that the open structure-stabilized by backbone hydrogen bond interactions-packs the RT-loops together by swapping the two strands of the RT-loop. The packed RT-loops form a beta-sheet structure and expose the backbone to promote further aggregation. We also simulate more than two proteins, and find that the aggregate adopts a fibrillar double beta-sheet structure, which is formed by packing the RT-loops from different proteins. Our simulations are consistent with a possible generic amyloidogenesis scenario.     

Domain swapping of CD4 upon dimerization

It has recently been shown that disulfide bond Cys130-Cys159 in domain 2 of monomeric CD4 is involved in the formation of CD4 disulfide-bonded dimers on cell surfaces and that it can influence the permissiveness of cells to HIV infection. Because this disulfide bond is buried in the monomer, a large conformational change must take place in order to allow for such disulfide exchange. Using standard optimization techniques, whose efficiency was first checked in the well-documented CD2 case, we have shown that 3D domain swapping is a likely candidate for the conformational change, the hinge loop, or linker, being loop E-F. Indeed, as a consequence of domain swapping, because Cys130 and Cys159 belong to beta-strands C and F, respectively, two disulfide bonds become established between Cys130 in one monomer and Cys159 in the other one. Such a disulfide exchange has already been observed when the nuclear magnetic resonance (NMR) structure of the prion protein was compared to the crystallographic, dimeric one. In both cases, domain swapping implies disulfide exchange because the linker is located in the sequence between two disulfide-bonded cysteines. As in the CD2 case, the proposed configuration of the CD4 dimer is found as a pair of neighboring monomers in the crystallographic unit cell. Moreover, because in this configuration the epitope of monoclonal antibody MT151, which does not compete with Gp120 for CD4 binding, is in the cleft between the pair of CD4 monomers, it is suggested that MT151 achieves its HIV-blocking activity by interfering with the formation of CD4 domain-swapped dimers on cell surface.     

Chain termini cross-talk in the swapping process of bovine pancreatic ribonuclease

3D domain swapping is the process by which two or more protein molecules exchange part of their structure to form intertwined dimers or higher oligomers. Bovine pancreatic ribonuclease (RNase A) is able to swap the N-terminal α-helix (residues 1-13) and/or the C-terminal β-strand (residues 116-124), thus forming a variety of oligomers, including two different dimers. Cis-trans isomerization of the Asn113-Pro114 peptide group was observed when the protein formed the C-terminal swapped dimer. To study the effect of the substitution of Pro114 on the swapping process of RNase A, we have prepared and characterized the P114A monomeric and dimeric variants of the enzyme. In contrast with previous reports, the crystal structure and NMR data on the monomer reveals a mixed cis-trans conformation for the Asn113-Ala114 peptide group, whereas the X-ray structure of the C-terminal swapped dimer of the variant is very close to that of the corresponding dimer of RNase A. The mutation at the C-terminus affects the capability of the N-terminal α-helix to swap and the stability of both dimeric forms. The present results underscore the importance of the hydration shell in determining the cross-talk between the chain termini in the swapping process of RNase A.     

Unique biogenesis of high-molecular mass multimeric metalloenzyme nitrile hydratase: intermediates and a proposed mechanism for self-subunit swapping maturation

Rhodococcus rhodochrous J1 produces high- and low-molecular mass nitrile hydratases (H-NHase and L-NHase, respectively), depending on the inducer. The incorporation of cobalt into L-NHase has been found to depend on the α-subunit exchange between cobalt-free L-NHase (apo-L-NHase) and its cobalt-containing mediator, NhlAE (holo-NhlAE), this novel mode of post-translational maturation having been named self-subunit swapping and NhlE having been recognized as a self-subunit swapping chaperone. We discovered an H-NHase maturation mediator, NhhAG, consisting of NhhG and the α-subunit of H-NHase. The incorporation of cobalt into H-NHase was confirmed to be dependent on self-subunit swapping. For the first time, particles larger than apo-H-NHase were observed during the swapping process via dynamic light scattering measurements, suggesting the formation of intermediate complexes. On the basis of these findings, we initially proposed a possible mechanism for self-subunit swapping. Electron paramagnetic resonance analysis demonstrated that the coordination environment of a cobalt ion in holo-NhhAG is subtly different from that in H-NHase. Cobalt is inserted into cobalt-free NhhAG (apo-NhhAG) but not into apo-H-NHase, suggesting that NhhG functions not only as a self-subunit swapping chaperone but also as a metallochaperone. In addition, α-subunit swapping did not occur between apo-L-NHase and holo-NhhAG or between apo-H-NHase and holo-NhlAE in vitro. These findings revealed that self-subunit swapping is a subunit-specific reaction.     

The Molecular Mechanism of Domain Swapping of the C-Terminal Domain of the SARS-Coronavirus Main Protease

In three-dimensional domain swapping, two protein monomers exchange a part of their structures to form an intertwined homodimer, whose subunits resemble the monomer. Several viral proteins domain swap to increase their structural complexity or functional avidity. The main protease (M^pro) of the severe acute respiratory syndrome (SARS) coronavirus proteolyzes viral polyproteins and has been a target for anti-SARS drug design. Domain swapping in the α-helical C-terminal domain of M^pro (M^proC) locks M^pro into a hyperactive octameric form that is hypothesized to promote the early stages of viral replication. However, in the absence of a complete molecular understanding of the mechanism of domain swapping, investigations into the biological relevance of this octameric M^pro have stalled. Isolated M^proC can exist as a monomer or a domain-swapped dimer. Here, we investigate the mechanism of domain swapping of M^proC using coarse-grained structure-based models and molecular dynamics simulations. Our simulations recapitulate several experimental features of M^proC folding. Further, we find that a contact between a tryptophan in the M^proC domain-swapping hinge and an arginine elsewhere forms early during folding, modulates the folding route, and promotes domain swapping to the native structure. An examination of the sequence and the structure of the tryptophan containing hinge loop shows that it has a propensity to form multiple secondary structures and contacts, indicating that it could be stabilized into either the monomer- or dimer-promoting conformations by mutations or ligand binding. Finally, because all residues in the tryptophan loop are identical in SARS-CoV and SARS-CoV-2, mutations that modulate domain swapping may provide insights into the role of octameric M^pro in the early-stage viral replication of both viruses.     

NMR studies on structure and dynamics of the monomeric derivative of BS-RNase: new insights for 3D domain swapping

Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges.     

Using the Cre/lox system for targeted integration into the human genome: loxFAS-loxP pairing and delayed introduction of Cre DNA improve gene swapping efficiency

Cre recombinase is a commonly-used genome editing tool suitable for site-specific integrations in mammalian genomes; however, the efficiency of transgenic swapping events compared to excision remains limited. Here we sought to identify important parameters and limiting factors that influence swapping propensity in this system, especially when using one wild-type loxP site. To modulate and increase the occurrence of swapping events, we identified two novel parameters. First, we identified the loxFAS-loxP pairing, a sequence never before used in mammalian systems, as the best choice for increasing swapping events in human cell lines. Second, for the first time we implicate the importance of delayed introduction of Cre DNA for optimal swapping efficiency. This same modification could potentially be of use to other systems catalyzing trimolecular reactions such as ΦC31 integrase and FLP recombinase where we hypothesize that transport of the exchange cassette is likewise initially rate limiting. The total number of recombination events, but not the ratio of swapping to excision, was found to be influenced by the quantity of Cre DNA transfected. Through this study, we were able to obtain Cre-mediated swapping frequencies of 8-12% without antibiotic enrichment, which represents nearly an order of magnitude increase over prior reports in the literature.     

3D domain-swapped human cystatin C with amyloidlike intermolecular beta-sheets

Oligomerization of human cystatin C (HCC) leads to amyloid deposits in brain arteries, and this process is greatly accelerated with a naturally occurring L68Q variant. The crystal structures of N-truncated and full-length HCC (cubic form) showed dimer formation via three-dimensional (3D) domain swapping, and this observation has led to the suggestion that an analogous domain-swapping mechanism, but propagated in an open-ended fashion, could be the basis of HCC fibril formation. Here we report that full-length HCC, when crystallized in a new, tetragonal form, dimerizes by swapping the same secondary structure elements but with a very different overall structure generated by the flexibility of the hinge linking the moveable elements. The beta-strands of the beta-cores of the two folding units of the present dimer are roughly parallel, while they formed an angle of about 100 degrees in the previous two structures. The dimers pack around a crystallographic dyad by extending their molecular beta-sheets in an intermolecular context. At the other edge of the molecular beta-sheet, side-chain-side-chain hydrogen bonds propagate the beta-structure in the same direction. In consequence, a supramolecular crystal structure is generated, with all the beta-strands of the domain-swapped dimers being perpendicular to one crystallographic direction. This observation is relevant to amyloid aggregation of HCC, as X-ray diffraction studies of amyloid fibrils show them to have ordered, repeating structure, consistent with the so-called cross-beta structure, in which extended polypeptide chains are perpendicular to the fiber axis and form infinite beta-sheets that are parallel to this axis.     

The unfolding story of three-dimensional domain swapping

Three-dimensional domain swapping is the event by which a monomer exchanges part of its structure with identical monomers to form an oligomer where each subunit has a similar structure to the monomer. The accumulating number of observations of this phenomenon in crystal structures has prompted speculation as to its biological relevance. Domain swapping was originally proposed to be a mechanism for the emergence of oligomeric proteins and as a means for functional regulation, but also to be a potentially harmful process leading to misfolding and aggregation. We highlight experimental studies carried out within the last few years that have led to a much greater understanding of the mechanism of domain swapping and of the residue- and structure-specific features that facilitate the process. We discuss the potential biological implications of domain swapping in light of these findings.     

Homology-dependent interactions determine the order of strand exchange by IntDOT recombinase

The Bacteroides conjugative transposon CTnDOT encodes an integrase, IntDOT, which is a member of the tyrosine recombinase family. Other members of this group share a strict requirement for sequence identity within the region of strand exchange, called the overlap region. Tyrosine recombinases catalyze recombination by making an initial cleavage, strand exchange and ligation, followed by strand swapping isomerization requiring sequence identity in the overlap region, followed by the second cleavage, strand exchange and ligation. IntDOT is of particular interest because it has been shown to utilize a three-step mechanism: a sequence identity-dependent initial strand exchange that requires two base pairs of complementary DNA at the site of cleavage; a sequence identity-independent strand swapping isomerization, followed by a sequence identity-independent cleavage, strand exchange and ligation. In addition to the sequence identity requirement in the overlap region, Lambda Int interactions with arm-type sites dictate the order of strand exchange regardless of the orientation of the overlap region. Although IntDOT has an arm-binding domain, we show here that the location of sequence identity within the overlap region dictates where the initial cleavage takes place and that IntDOT can recombine substrates containing mismatches in the overlap region so long as a single base of sequence identity exists at the site of initial cleavage.