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.     

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.     

Insight into ribonuclease A domain swapping by molecular dynamics unfolding simulations

Bovine pancreatic ribonuclease (RNase A) deserves a special place among the numerous proteins that form oligomers by three-dimensional domain swapping. In fact, under destabilizing conditions and at high protein concentrations, it can swap two different domains, the N-terminal alpha-helix or the C-terminal beta-strand, leading to dimers with different quaternary structures. With the change in the unfolding conditions, the relative abundance of the two dimers varies, and the prevalence of one dimer over the other is inverted. To investigate the dynamic behavior of the termini, four independent 10 ns high-temperature molecular dynamics simulations of RNase A were carried out at two different pH values in an attempt to reproduce the experimental conditions of neutral and very low pH that favor the formation of the N- and C-terminal domain-swapped dimers, respectively. In agreement with experimental data, under mild unfolding conditions, a partial or complete opening of the N-terminal arm is observed, whereas the dislocation of the C-terminus away from the core of the structure occurs only during the low-pH simulations. Furthermore, the picture emerging from this study indicates that the same protein can have different pathways for domain swapping. Indeed, in RNase A the C-terminal swapping requires a substantial unfolding of the monomers, whereas the N-terminal swapping can occur through only partial unfolding.     

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.     

3D domain swapping provides a minor alternative refolding pathway for ribonuclease A

The C-terminal β-hairpin of RNase A contains a turn with a cis Asn113-Pro114 peptide bond. Pioneering pulsed HX experiments have shown that the C-terminal β-hairpin forms early during refolding. This is puzzling since the Asn113-Pro114 bond is predominately trans at this stage and this conformation destabilizes the native monomer. RNase A, when refolded at high concentration, forms a series of 3D domain-swapped oligomers. In the oligomers formed by C-terminal β-strand swapping, Asn113-Pro114 is trans and permits the formation of a new intersubunit β-sheet. We hypothesize that oligomeric species with trans Asn113-Pro114 may form during refolding. Such species could account for the HX results while comfortably accommodating Asn113-Pro114 in the trans conformation. Here, we test this hypothesis by employing chromatographic methods to detect oligomers forming in refolding conditions and find significant amounts of dimer. We propose that a 3D domain-swapped dimeric intermediate provides a minor alternative pathway for RNase A refolding.     

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.     

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.     

Folding and association of the human cell cycle regulatory proteins ckshs1 and ckshs2.

The two human proteins ckshs1 and ckshs2 are each 79 amino acids in length and consist of a four-stranded beta-sheet capped at one end by two alpha-helices. They are members of the cks family of essential cell cycle regulatory proteins that can adopt two native states, a monomer and a domain-swapped dimer formed by exchange of a C-terminal beta-strand. ckshs1 and ckshs2 both have marginal thermodynamic stability (the free energies of unfolding at 25 degrees C are 3.0 and 2.5 kcal/mol, respectively) and low kinetic stability (the rates of unfolding in water are approximately 1 s(-1)). Refolding of their denatured states to the monomeric forms of the proteins is slowed by transient oligomerization that is likely to occur via domain swapping. The folding behavior of ckshs1 and ckshs2 is markedly different from that of suc1, the cks protein from Schizosaccharomyces pombe, but the domain swapping propensities are similar. The greater thermodynamic and kinetic stability of suc1 and the population of a folding intermediate are most likely a consequence of its larger size (113 residues). The similarity in the domain swapping propensities, despite the contrast in other biophysical properties, may be attributable to the common double-proline motif in the hinge loop that connects the swapped domain to the rest of the protein. The motif was shown previously for suc1 to control the equilibrium between the monomer and the domain-swapped dimer. Finally, according to our model, the kinetic barrier separating the monomer and the domain-swapped dimer arises because the protein must unfold for beta-strand exchange to occur. Consistent with this, interconversion between the two states is much faster in the human proteins than it is for suc1, reflecting the faster unfolding rates of the former.     

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.     

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.     

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.     

Effect of pH and salt bridges on structural assembly: molecular structures of the monomer and intertwined dimer of the Eps8 SH3 domain

The SH3 domain of Eps8 was previously found to form an intertwined, domain-swapped dimer. We report here a monomeric structure of the EPS8 SH3 domain obtained from crystals grown at low pH, as well as an improved domain-swapped dimer structure at 1.8 A resolution. In the domain-swapped dimer the asymmetric unit contains two "hybrid-monomers." In the low pH form there are two independently folded SH3 molecules per asymmetric unit. The formation of intermolecular salt bridges is thought to be the reason for the formation of the dimer. On the basis of the monomer SH3 structure, it is argued that Eps8 SH3 should, in principle, bind to peptides containing a PxxP motif. Recently it was reported that Eps8 SH3 binds to a peptide with a PxxDY motif. Because the "SH3 fold" is conserved, alternate binding sites may be possible for the PxxDY motif to bind. The strand exchange or domain swap occurs at the n-src loops because the n-src loops are flexible. The thermal b-factors also indicate the flexible nature of n-src loops and a possible handle for domain swap initiation. Despite the loop swapping, the typical SH3 fold in both forms is conserved structurally. The interface of the acidic form of SH3 is stabilized by a tetragonal network of water molecules above hydrophobic residues. The intertwined dimer interface is stabilized by hydrophobic and aromatic stacking interactions in the core and by hydrophilic interactions on the surface.     

A domain-swapped RNase A dimer with implications for amyloid formation

Bovine pancreatic ribonuclease (RNase A) forms two types of dimers (a major and a minor component) upon concentration in mild acid. These two dimers exhibit different biophysical and biochemical properties. Earlier we reported that the minor dimer forms by swapping its N-terminal alpha-helix with that of an identical molecule. Here we find that the major dimer forms by swapping its C-terminal beta-strand, thus revealing the first example of three-dimensional (3D) domain swapping taking place in different parts of the same protein. This feature permits RNase A to form tightly bonded higher oligomers. The hinge loop of the major dimer, connecting the swapped beta-strand to the protein core, resembles a short segment of the polar zipper proposed by Perutz and suggests a model for aggregate formation by 3D domain swapping with a polar zipper.     

Structures of the two 3D domain-swapped RNase A trimers

When concentrated in mildly acidic solutions, bovine pancreatic ribonuclease (RNase A) forms long-lived oligomers including two types of dimer, two types of trimer, and higher oligomers. In previous crystallographic work, we found that the major dimeric component forms by a swapping of the C-terminal beta-strands between the monomers, and that the minor dimeric component forms by swapping the N-terminal alpha-helices of the monomers. On the basis of these structures, we proposed that a linear RNase A trimer can form from a central molecule that simultaneously swaps its N-terminal helix with a second RNase A molecule and its C-terminal strand with a third molecule. Studies by dissociation are consistent with this model for the major trimeric component: the major trimer dissociates into both the major and the minor dimers, as well as monomers. In contrast, the minor trimer component dissociates into the monomer and the major dimer. This suggests that the minor trimer is cyclic, formed from three monomers that swap their C-terminal beta-strands into identical molecules. These conclusions are supported by cross-linking of lysyl residues, showing that the major trimer swaps its N-terminal helix, and the minor trimer does not. We verified by X-ray crystallography the proposed cyclic structure for the minor trimer, with swapping of the C-terminal beta-strands. This study thus expands the variety of domain-swapped oligomers by revealing the first example of a protein that can form both a linear and a cyclic domain-swapped oligomer. These structures permit interpretation of the enzymatic activities of the RNase A oligomers on double-stranded RNA.     

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.     

Coupling of Domain Swapping to Kinetic Stability in a Thioredoxin Mutant

The thioredoxin (Trx) fold is a small monomeric domain that is ubiquitous in redox-active enzymes. Trxs are characterized by a typical WCGPC active-site sequence motif. A single active-site mutation of the tryptophan to an alanine in Staphylococcus aureus Trx converts the oxidized protein into a biologically inactive domain-swapped dimer. While the monomeric protein unfolds reversibly in a two-state manner, the oxidized dimeric form is kinetically stable and converts to the monomeric form upon refolding. After reduction, the half-life of the dimer decreases many orders of magnitude to ∼ 4.3 h, indicating that the active-site disulfide between Cys29 and Cys32 is an important determinant for the kinetics of unfolding. We propose kinetic stability as a possible evolutionary strategy in the evolution of multimeric proteins from their monomeric ancestors by domain swapping, which, for this biologically inactive Trx mutant, turned out to be an evolutionary dead end.     

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.