Three-dimensional Domain Swapping Research Articles

Experimental and Computational Studies on Domain-Swapped Structure Stabilization of an Antibody Light Chain by Disulfide Bond Introduction.

Development of different platforms would be useful for designing functional antibodies to improve the efficiency of antibody-based drugs. Three-dimensional domain swapping (3D-DS) may occur in the variable region of antibody light chain #4C214A, and a pair of domain-swapped dimers may interact with each other to form a tetramer. In this study, to stabilize the 3D-DS dimer structure in #4C214A, Val2 in strand A (swapping region) and Thr97 in strand G were replaced with Cys residues, generating #4 V2C/T97C/C214A with a Cys2-Cys97 disulfide bond that cross-links strands A and G of different protomers. The #4 V2C/T97C/C214A tetramer did not dissociate into monomers at low protein concentration (6 μM); however, some of the tetramers were converted to monomers by disulfide bond reduction. Two-dimensional free energy profile analysis for the tetramerization of two 3D-DS dimers was performed by molecular dynamics simulation. These results show that disulfide bond introduction is useful for controlling the dimerization/dissociation of the variable region through 3D-DS.

Unraveling the folding and dimerization properties of the human FoxP subfamily of transcription factors.

Human FoxP proteins share a highly conserved DNA-binding domain that dimerizes via three-dimensional domain swapping, although showing varying oligomerization propensities among its members. Here, we present an experimental and computational characterization of all human FoxP proteins to unravel how their amino acid substitutions impact their folding and dimerization mechanism. We solved the crystal structure of the forkhead domain of FoxP4 to then perform a comparison across all members, finding that their sequence changes impact not only the structural heterogeneity of their forkhead domains but also the protein-protein association energy barrier. Lastly, we demonstrate that the accumulation of a monomeric intermediate is an oligomerization-dependent feature rather than a common aspect of monomers and dimers in this protein subfamily.

Domain swapping of the C-terminal helix promotes the dimerization of a novel ribonuclease protein from Mycobacterium tuberculosis.

Polyketide metabolism-associated proteins in Mycobacterium tuberculosis play an essential role in the survival of the bacterium, which makes them potential drug targets for the treatment of tuberculosis (TB). The novel ribonuclease protein Rv1546 is predicted to be a member of the steroidogenic acute regulatory protein-related lipid-transfer (START) domain superfamily, which comprises bacterial polyketide aromatase/cyclases (ARO/CYCs). Here, we determined the crystal structure of Rv1546 in a V-shaped dimer. The Rv1546 monomer consists of four α-helices and seven antiparallel β-strands. Interestingly, in the dimeric state, Rv1546 forms a helix-grip fold, which is present in START domain proteins, via three-dimensional domain swapping. Structural analysis revealed that the conformational change of the C-terminal α-helix of Rv1546 might contribute to the unique dimer structure. Site-directed mutagenesis followed by in vitro ribonuclease activity assays was performed to identify catalytic sites of the protein. This experiment suggested that surface residues R63, K84, K88, and R113 are important in the ribonuclease function of Rv1546. In summary, this study presents the structural and functional characterization of Rv1546 and supplies new perspectives for exploiting Rv1546 as a novel drug target for TB treatment.

Slow Evolution toward “Super-Aggregation” of the Oligomers Formed through the Swapping of RNase A N-Termini: A Wish for Amyloidosis?

Natively monomeric RNase A can oligomerize upon lyophilization from 40% acetic acid solutions or when it is heated at high concentrations in various solvents. In this way, it produces many dimeric or oligomeric conformers through the three-dimensional domain swapping (3D-DS) mechanism involving both RNase A N- or/and C-termini. Here, we found many of these oligomers evolving toward not negligible amounts of large derivatives after being stored for up to 15 months at 4 °C in phosphate buffer. We call these species super-aggregates (SAs). Notably, SAs do not originate from native RNase A monomer or from oligomers characterized by the exclusive presence of the C-terminus swapping of the enzyme subunits as well. Instead, the swapping of at least two subunits’ N-termini is mandatory to produce them. Through immunoblotting, SAs are confirmed to derive from RNase A even if they retain only low ribonucleolytic activity. Then, their interaction registered with Thioflavin-T (ThT), in addition to TEM analyses, indicate SAs are large and circular but not “amyloid-like” derivatives. This confirms that RNase A acts as an “auto-chaperone”, although it displays many amyloid-prone short segments, including the 16–22 loop included in its N-terminus. Therefore, we hypothesize the opening of RNase A N-terminus, and hence its oligomerization through 3D-DS, may represent a preliminary step favoring massive RNase A aggregation. Interestingly, this process is slow and requires low temperatures to limit the concomitant oligomers’ dissociation to the native monomer. These data and the hypothesis proposed are discussed in the light of protein aggregation in general, and of possible future applications to contrast amyloidosis.

Human FoxP Transcription Factors as Tractable Models of the Evolution and Functional Outcomes of Three-Dimensional Domain Swapping.

The association of two or more proteins to adopt a quaternary complex is one of the most widespread mechanisms by which protein function is modulated. In this scenario, three-dimensional domain swapping (3D-DS) constitutes one plausible pathway for the evolution of protein oligomerization that exploits readily available intramolecular contacts to be established in an intermolecular fashion. However, analysis of the oligomerization kinetics and thermodynamics of most extant 3D-DS proteins shows its dependence on protein unfolding, obscuring the elucidation of the emergence of 3D-DS during evolution, its occurrence under physiological conditions, and its biological relevance. Here, we describe the human FoxP subfamily of transcription factors as a feasible model to study the evolution of 3D-DS, due to their significantly faster dissociation and dimerization kinetics and lower dissociation constants in comparison to most 3D-DS models. Through the biophysical and functional characterization of FoxP proteins, relevant structural aspects highlighting the evolutionary adaptations of these proteins to enable efficient 3D-DS have been ascertained. Most biophysical studies on FoxP suggest that the dynamics of the polypeptide chain are crucial to decrease the energy barrier of 3D-DS, enabling its fast oligomerization under physiological conditions. Moreover, comparison of biophysical parameters between human FoxP proteins in the context of their minute sequence differences suggests differential evolutionary strategies to favor homoassociation and presages the possibility of heteroassociations, with direct impacts in their gene regulation function.

Dimerization of Human Angiogenin and of Variants Involved in Neurodegenerative Diseases.

Human Angiogenin (hANG, or ANG, 14.1 kDa) promotes vessel formation and is also called RNase 5 because it is included in the pancreatic-type ribonuclease (pt-RNase) super-family. Although low, its ribonucleolytic activity is crucial for angiogenesis in tumor tissues but also in the physiological development of the Central Nervous System (CNS) neuronal progenitors. Nevertheless, some ANG variants are involved in both neurodegenerative Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Notably, some pt-RNases acquire new biological functions upon oligomerization. Considering neurodegenerative diseases correlation with massive protein aggregation, we analyzed the aggregation propensity of ANG and of three of its pathogenic variants, namely H13A, S28N, and R121C. We found no massive aggregation, but wt-ANG, as well as S28N and R121C variants, can form an enzymatically active dimer, which is called ANG-D. By contrast, the enzymatically inactive H13A-ANG does not dimerize. Corroborated by a specific cross-linking analysis and by the behavior of H13A-ANG that in turn lacks one of the two His active site residues necessary for pt-RNases to self-associate through the three-dimensional domain swapping (3D-DS), we demonstrate that ANG actually dimerizes through 3D-DS. Then, we deduce by size exclusion chromatography (SEC) and modeling that ANG-D forms through the swapping of ANG N-termini. In light of these novelties, we can expect future investigations to unveil other ANG determinants possibly related with the onset and/or development of neurodegenerative pathologies.

CirPred, the first structure modeling and linker design system for circularly permuted proteins

BackgroundThis work aims to help develop new protein engineering techniques based on a structural rearrangement phenomenon called circular permutation (CP), equivalent to connecting the native termini of a protein followed by creating new termini at another site. Although CP has been applied in many fields, its implementation is still costly because of inevitable trials and errors.ResultsHere we present CirPred, a structure modeling and termini linker design method for circularly permuted proteins. Compared with state-of-the-art protein structure modeling methods, CirPred is the only one fully capable of both circularly-permuted modeling and traditional co-linear modeling. CirPred performs well when the permutant shares low sequence identity with the native protein and even when the permutant adopts a different conformation from the native protein because of three-dimensional (3D) domain swapping. Linker redesign experiments demonstrated that the linker design algorithm of CirPred achieved subangstrom accuracy.ConclusionsThe CirPred system is capable of (1) predicting the structure of circular permutants, (2) designing termini linkers, (3) performing traditional co-linear protein structure modeling, and (4) identifying the CP-induced occurrence of 3D domain swapping. This method is supposed helpful for broadening the application of CP, and its web server is available at http://10.life.nctu.edu.tw/CirPred/ and http://lo.life.nctu.edu.tw/CirPred/.

RNase A Domain-Swapped Dimers Produced Through Different Methods: Structure-Catalytic Properties and Antitumor Activity.

Upon oligomerization, RNase A can acquire important properties, such as cytotoxicity against leukemic cells. When lyophilized from 40% acetic acid solutions, the enzyme self-associates through the so-called three-dimensional domain swapping (3D-DS) mechanism involving both N- and/or C-terminals. The same species are formed if the enzyme is subjected to thermal incubation in various solvents, especially in 40% ethanol. We evaluated here if significant structural modifications might occur in RNase A N- or C-swapped dimers and/or in the residual monomer(s), as a function of the oligomerization protocol applied. We detected that the monomer activity vs. ss-RNA was partly affected by both protocols, although the protein does not suffer spectroscopic alterations. Instead, the two N-swapped dimers showed differences in the fluorescence emission spectra but almost identical enzymatic activities, while the C-swapped dimers displayed slightly different activities vs. both ss- or ds-RNA substrates together with not negligible fluorescence emission alterations within each other. Besides these results, we also discuss the reasons justifying the different relative enzymatic activities displayed by the N-dimers and C-dimers. Last, similarly with data previously registered in a mouse model, we found that both dimeric species significantly decrease human melanoma A375 cell viability, while only N-dimers reduce human melanoma MeWo cell growth.

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 (Mpro) 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 Mpro (MproC) locks Mpro 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 Mpro have stalled. Isolated MproC can exist as a monomer or a domain-swapped dimer. Here, we investigate the mechanism of domain swapping of MproC using coarse-grained structure-based models and molecular dynamics simulations. Our simulations recapitulate several experimental features of MproC folding. Further, we find that a contact between a tryptophan in the MproC 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 Mpro in the early-stage viral replication of both viruses.

Oligomerization of cytochrome c, myoglobin, and related heme proteins by 3D domain swapping

Oligomerization of heme proteins is useful for construction of new materials with cooperative and systematic functions; thus, diverse methods have been applied for construction of artificial heme protein oligomers. Three-dimensional (3D) domain swapping is a protein oligomerization phenomenon that exchanges the same domain or secondary structural element between molecules. 3D domain swapping was first reported in 1994; since then many proteins have been reported to domain swap. Our research group has been showing that various heme proteins domain swap. We also found that domain swapping of heme proteins occurs at the early stage of protein folding, and utilized it to construct various heme protein assemblies, including nanorings, cages, hetero dimers with different active sites, and a ligand-binding reversible monomer–polymer system. In this review, the basics and applications of domain swapping of heme proteins are summarized.

Exploring the Roles of Proline in Three-Dimensional Domain Swapping from Structure Analysis and Molecular Dynamics Simulations.

Three-dimensional (3D) domain swapping is a mechanism to form protein oligomers. It has been proposed that several factors, including proline residues in the hinge region, may affect the occurrence of 3D domain swapping. Although introducing prolines into the hinge region has been found to promote domain swapping for some proteins, the opposite effect has also been observed in several studies. So far, how proline affects 3D domain swapping remains elusive. In this work, based on a large set of 3D domain-swapped structures, we performed a systematic analysis to explore the correlation between the presence of proline in the hinge region and the occurrence of 3D domain swapping. We further analyzed the conformations of proline and pre-proline residues to investigate the roles of proline in 3D domain swapping. We found that more than 40% of the domain-swapped structures contained proline residues in the hinge region. Unexpectedly, conformational transitions of proline residues were rarely observed upon domain swapping. Our analyses showed that hinge regions containing proline residues preferred more extended conformations, which may be beneficial for the occurrence of domain swapping by facilitating opening of the exchanged segments.

Onconase dimerization through 3D domain swapping: structural investigations and increase in the apoptotic effect in cancer cells.

Onconase® (ONC), a protein extracted from the oocytes of the Rana pipiens frog, is a monomeric member of the secretory 'pancreatic-type' RNase superfamily. Interestingly, ONC is the only monomeric ribonuclease endowed with a high cytotoxic activity. In contrast with other monomeric RNases, ONC displays a high cytotoxic activity. In this work, we found that ONC spontaneously forms dimeric traces and that the dimer amount increases about four times after lyophilization from acetic acid solutions. Differently from RNase A (bovine pancreatic ribonuclease) and the bovine seminal ribonuclease, which produce N- and C-terminal domain-swapped conformers, ONC forms only one dimer, here named ONC-D. Cross-linking with divinylsulfone reveals that this dimer forms through the three-dimensional domain swapping of its N-termini, being the C-terminus blocked by a disulfide bond. Also, a homology model is proposed for ONC-D, starting from the well-known structure of RNase A N-swapped dimer and taking into account the results obtained from spectroscopic and stability analyses. Finally, we show that ONC is more cytotoxic and exerts a higher apoptotic effect in its dimeric rather than in its monomeric form, either when administered alone or when accompanied by the chemotherapeutic drug gemcitabine. These results suggest new promising implications in cancer treatment.

Gelsolin pathogenic Gly167Arg mutation promotes domain-swap dimerization of the protein.

AGel amyloidosis is a genetic degenerative disease characterized by the deposition of insoluble gelsolin protein aggregates in different tissues. Until recently, this disease was associated with two mutations of a single residue (Asp187 to Asn/Tyr) in the second domain of the protein. The general opinion is that pathogenic variants are not per se amyloidogenic but rather that the mutations trigger an aberrant proteolytic cascade, which results in the production of aggregation prone fragments. Here, we report the crystal structure of the second domain of gelsolin carrying the recently identified Gly167Arg mutation. This mutant dimerizes through a three-dimensional domain swapping mechanism, forming a tight but flexible assembly, which retains the structural topology of the monomer. To date, such dramatic conformational changes of this type have not been observed. Structural and biophysical characterizations reveal that the Gly167Arg mutation alone is responsible for the monomer to dimer transition and that, even in the context of the full-length protein, the pathogenic variant is prone to form dimers. These data suggest that, in addition to the well-known proteolytic-dependent mechanism, an alternative oligomerization pathway may participate in gelsolin misfolding and aggregation. We propose to integrate this alternative pathway into the current model of the disease that may also be relevant for other types of AGel amyloidosis, and other related diseases with similar underlying pathological mechanisms.

Rational Design of Domain-Swapping-Based c-Type Cytochrome Heterodimers by Using Chimeric Proteins.

The design of protein oligomers with multiple active sites has been gaining interest, owing to their potential use for biomaterials, which has encouraged researchers to develop a new design method. Three-dimensional domain swapping is the unique phenomenon in which protein molecules exchange the same structural region between each other. Herein, to construct oligomeric heme proteins with different active sites by utilizing domain swapping, two c-type cytochrome-based chimeric proteins have been constructed and the domains swapped. According to X-ray crystallographic analysis, the two chimeric proteins formed a domain-swapped dimer with two His/Met coordinated hemes. By mutating the heme coordination structure of one of the two chimeric proteins, a domainswapped heterodimer with His/Met and His/H2 O coordinated hemes was formed. Binding of an oxygen molecule to the His/H2 O site of the heterodimer was confirmed by resonance Raman spectroscopy, in which the Fe-O2 stretching band was observed at 580 cm-1 for the reduced/oxygenated heterodimer (at 554 cm-1 under an 18 O2 atmosphere). These results show that domain swapping is a useful method to design multiheme proteins.

Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1

The forkhead family of transcription factors (Fox) controls gene transcription during key processes such as regulation of metabolism, embryogenesis, and immunity. Structurally, Fox proteins feature a conserved DNA-binding domain known as forkhead. Interestingly, solved forkhead structures of members from the P subfamily (FoxP) show that they can oligomerize by three-dimensional domain swapping, whereby structural elements are exchanged between adjacent subunits, leading to an intertwined dimer. Recent evidence has largely stressed the biological relevance of domain swapping in FoxP, as several disease-causing mutations have been related to impairment of this process. Here, we explore the equilibrium folding and binding mechanism of the forkhead domain of wild-type FoxP1, and of two mutants that hinder DNA-binding (R53H) and domain swapping (A39P), using size-exclusion chromatography, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. Our results show that domain swapping of FoxP1 occurs at micromolar protein concentrations within hours of incubation and is energetically favored, in contrast to classical domain-swapping proteins. Also, DNA-binding mutations do not significantly affect domain swapping. Remarkably, equilibrium unfolding of dimeric FoxP1 follows a three-state N2 ↔ 2I ↔ 2U folding mechanism in which dimer dissociation into a monomeric intermediate precedes protein unfolding, in contrast to the typical two-state model described for most domain-swapping proteins, whereas the A39P mutant follows a two-state N ↔ U folding mechanism consistent with the second transition observed for dimeric FoxP1. Also, the free-energy change of the N ↔ U in A39P FoxP1 is ∼2 kcal⋅mol−1 larger than the I ↔ U transition of both wild-type and R53H FoxP1. Finally, hydrogen-deuterium exchange mass spectrometry reveals that the intermediate strongly resembles the native state. Our results suggest that domain swapping in FoxP1 is at least partially linked to monomer folding stability and follows an unusual three-state folding mechanism, which might proceed via transient structural changes rather than requiring complete protein unfolding as do most domain-swapping proteins.

Structural transition in Bcl-xL and its potential association with mitochondrial calcium ion transport.

Bcl-2 family proteins are key regulators for cellular homeostasis in response to apoptotic stimuli. Bcl-xL, an antiapoptotic Bcl-2 family member, undergoes conformational transitions, which leads to two conformational states: the cytoplasmic and membrane-bound. Here we present the crystal and small-angle X-ray scattering (SAXS) structures of Bcl-xL treated with the mild detergent n-Octyl β-D-Maltoside (OM). The detergent-treated Bcl-xL forms a dimer through three-dimensional domain swapping (3DDS) by swapping helices α6-α8 between two monomers. Unlike Bax, a proapoptotic member of the Bcl-2 family, Bcl-xL is not converted to 3DDS homodimer upon binding BH3 peptides and ABT-737, a BH3 mimetic drug. We also designed Bcl-xL mutants which cannot dimerize and show that these mutants reduced mitochondrial calcium uptake in MEF cells. This illustrates the structural plasticity in Bcl-xL providing hints toward the probable molecular mechanism for Bcl-xL to play a regulatory role in mitochondrial calcium ion transport.

New Insights into the Molecular Mechanism of E-Cadherin-Mediated Cell Adhesion by Free Energy Calculations.

Three-dimensional domain swapping is an important mode of protein association leading to the formation of stable dimers. Monomers associating via this mechanism mutually exchange a domain to form a homodimer. Classical cadherins, an increasingly important target for anticancer therapy, use domain swapping to mediate cell adhesion. However, despite its importance, the molecular mechanism of domain swapping is still debated. Here, we study the conformational changes that lead to activation and dimerization via domain swapping of E-cadherin. Using state-of-the-art enhanced sampling atomistic simulations, we reconstruct its conformational free energy landscape, obtaining the free energy profile connecting the inactive and active form. Our simulations predict that the E-cadherin monomer populates the open and closed forms almost equally, which is in agreement with the proposed "selected fit" mechanism in which monomers in an active conformational state bind to form a homodimer, analogous to the conformational selection mechanism often observed in ligand-target binding. Moreover, we find that the open state population is increased in the presence of calcium ions at the extracellular boundary, suggesting their possible role as allosteric activators of the conformational change.

Landscape of Intertwined Associations in Multi-Domain Homo-Oligomeric Proteins

This study charts the landscape of multi-domain protein structures that form intertwined homodimers by exchanging structural domains between subunits. A representative dataset of such homodimers was derived from the Protein Data Bank, and their structural and topological properties were compared to those of a representative set of non-intertwined homodimers. Most of the intertwined dimers form closed assemblies with head-to-tail arrangements, where the subunit interface involves contacts between dissimilar domains. In contrast, the non-intertwined dimers form preferentially head-to-head arrangements, where the subunit interface involves contacts between identical domains. Most of these contacts engage only one structural domain from each subunit, leaving the remaining domains free to form other associations. Remarkably, we find that multi-domain proteins closely related to the intertwined homodimers are significantly more likely than relatives of the non-intertwined versions to adopt alternative intramolecular domain arrangements. In ~40% of the intertwined dimers, the plasticity in domain arrangements among relatives affords maintenance of the head-to-head or head-to-tail topology and conservation of the corresponding subunit interface. This property seems to be exploited in several systems to regulate DNA binding. In ~58%, however, intramolecular domain re-arrangements are associated with changes in oligomeric states and poorly conserved interfaces among relatives. This time, the corresponding structural plasticity appears to be exploited by evolution to modulate function by switching between active and inactive states of the protein. Surprisingly, in total, only three systems were found to undergo the classical monomer to intertwined dimer conversion associated with three-dimensional domain swapping.

Micelle-catalyzed domain swapping in the GlpG rhomboid protease cytoplasmic domain.

Three-dimensional domain swapping is a mode of self-interaction that can give rise to altered functional states and has been identified as the trigger event in some protein deposition diseases, yet rates of interconversion between oligomeric states are usually slow, with the requirement for transient disruption of an extensive network of interactions giving rise to a large kinetic barrier. Here we demonstrate that the cytoplasmic domain of the Escherichia coli GlpG rhomboid protease undergoes slow dimerization via domain swapping and that micromolar concentrations of micelles can be used to enhance monomer-dimer exchange rates by more than 1000-fold. Detergents bearing a phosphocholine headgroup are shown to be true catalysts, with hexadecylphosphocholine reducing the 26 kcal/mol free energy barrier by >11 kcal/mol while preserving the 5 kcal/mol difference between monomer and dimer states. Catalysis involves the formation of a micelle-bound intermediate with a partially unfolded structure that is primed for domain swapping. Taken together, these results are the first to demonstrate true catalysis for domain swapping, by using micelles that work in a chaperonin-like fashion to unfold a kinetically trapped state and allow access to the domain-swapped form.

Three-dimensional domain swapping and supramolecular protein assembly: insights from the X-ray structure of a dimeric swapped variant of human pancreatic RNase

The deletion of five residues in the loop connecting the N-terminal helix to the core of monomeric human pancreatic ribonuclease leads to the formation of an enzymatically active domain-swapped dimer (desHP). The crystal structure of desHP reveals the generation of an intriguing fibril-like aggregate of desHP molecules that extends along the c crystallographic axis. Dimers are formed by three-dimensional domain swapping. Tetramers are formed by the aggregation of swapped dimers with slightly different quaternary structures. The tetramers interact in such a way as to form an infinite rod-like structure that propagates throughout the crystal. The observed supramolecular assembly captured in the crystal predicts that desHP fibrils could form in solution; this has been confirmed by atomic force microscopy. These results provide new evidence that three-dimensional domain swapping can be a mechanism for the formation of elaborate large assemblies in which the protein, apart from the swapping, retains its original fold.