Domain swapping: a mathematical model for quantitative assessment of structural effects.

The domain-swapping mechanism involves the exchange of structural elements within a secondary or supersecondary structure between two (or more) proteins. The present paper proposes to interpret the domain-swapping mechanism using a model that assesses the structure of proteins (and complexes) based on building the structure of a common hydrophobic core in a micelle-like arrangement (a central hydrophobic core with a polar shell in contact with polar water), which has a considerable impact on the stabilisation of the domain structure built by domain swapping. Domains with a hydrophobicity system that is incompatible with the micelle-like structure have also been identified. This incompatibility is the form of structural codes related to biological function.

Structural studies of KCTD1 and its disease-causing mutant P20S provide insights into the protein function and misfunction.

Members of the KCTD protein family play key roles in fundamental physio-pathological processes including cancer, neurodevelopmental/neuropsychiatric, and genetic diseases. Here, we report the crystal structure of the KCTD1 P20S mutant, which causes the scalp-ear-nipple syndrome, and molecular dynamics (MD) data on the wild-type protein. Surprisingly, the structure unravels that the N-terminal region, which precedes the BTB domain (preBTB) and bears the disease-associated mutation, adopts a folded polyproline II (PPII) state. The KCTD1 pentamer is characterized by an intricate architecture in which the different subunits mutually exchange domains to generate a closed domain swapping motif. Indeed, the BTB of each chain makes peculiar contacts with the preBTB and the C-terminal domain (CTD) of an adjacent chain. The BTB-preBTB interaction consists of a PPII-PPII recognition motif whereas the BTB-CTD contacts are mediated by an unusual (+/-) helix discontinuous association. The inspection of the protein structure, along with the data emerged from the MD simulations, provides an explanation of the pathogenicity of the P20S mutation and unravels the role of the BTB-preBTB interaction in the insurgence of the disease. Finally, the presence of potassium bound to the central cavity of the CTD pentameric assembly provides insights into the role of KCTD1 in metal homeostasis.

The crystal structure of Shethna protein II (FeSII) from Azotobacter vinelandii suggests a domain swap.

The Azotobacter vinelandii FeSII protein forms an oxygen-resistant complex with the nitrogenase MoFe and Fe proteins. FeSII is an adrenodoxin-type ferredoxin that forms a dimer in solution. Previously, the crystal structure was solved [Schlesier et al. (2016), J. Am. Chem. Soc. 138, 239-247] with five copies in the asymmetric unit. One copy is a normal adrenodoxin domain that forms a dimer with its crystallographic symmetry mate. The other four copies are in an `open' conformation with a loop flipped out exposing the 2Fe-2S cluster. The open and closed conformations were interpreted as oxidized and reduced, respectively, and the large conformational change in the open configuration allowed binding to nitrogenase. Here, the structure of FeSII was independently solved in the same crystal form. The positioning of the atoms in the unit cell is similar to the earlier report. However, the interpretation of the structure is different. The `open' conformation is interpreted as the product of a crystallization-induced domain swap. The 2Fe-2S cluster is not exposed to solvent, but in the crystal its interacting helix is replaced by the same helix residues from a crystal symmetry mate. The domain swap is complicated, as it is unusual in being in the middle of the protein rather than at a terminus, and it creates arrangements of molecules that can be interpreted in multiple ways. It is also cautioned that crystal structures should be interpreted in terms of the contents of the entire crystal rather than of one asymmetric unit.

Inter domain linker region affects properties of CBM6 in GH5_34 arabinoxylanases and alters oligosaccharide product profile.

Understanding the relation between enzyme domain structure and catalytic activity is crucial for optimal engineering of novel enzymes for lignocellulose bioconversion. Xylanases with varying specificities are commonly used to valorise the hemicellulose arabinoxylan (AX), yet characterization of specific arabinoxylanases remain limited. Two homologous GH5_34 arabinoxylanases, HhXyn5A and CtXyn5A, in which the two domains are connected by a 40-residue linker, exhibit distinct activity on AX, yielding different reaction product patterns, despite high sequence identity, conserved active sites and similar domain composition. In this study, the carbohydrate binding module 6 (CBM6), or the inter domain linker together with CBM6, were swapped to investigate their influence on hydrolytic activity and oligosaccharide product pattern on cereal AXs. The variants, with only CBM6 swapped, displayed reduced activity on commercial wheat and rye AX, as well as on extracted oat fibre, compared to the original enzymes. Additionally, exchange of both linker and CBM6 resulted in a reduced ratio of enzyme produced in soluble form in Escherichia coli cultivations, causing loss of activity of both HhXyn5A and CtXyn5A variants. Analysis of oligosaccharide product patterns applying HPAEC-PAD revealed a decreased number of reaction products for CtXyn5A with swapped CBM6, which resembled the product pattern of HhXyn5A. These findings emphasize the importance of the CBM6 interactions with the linker and the catalytic domain for enzyme activity and specificity, and underlines the role of the linker in enzyme structure organisation and product formation, where alterations in linker interactions with the catalytic and/or CBM6 domains, influence enzyme-substrate association and specificity.

Dissecting the structural and functional consequences of the evolutionary proline-glycine deletion in the wing 1 region of the forkhead domain of human FoxP1.

The human FoxP transcription factors dimerize via three-dimensional domain swapping, a unique feature among the human Fox family, as result of evolutionary sequence adaptations in the forkhead domain. This is the case for the conserved glycine and proline residues in the wing 1 region, which are absent in FoxP proteins but present in most of the Fox family. In this work, we engineered both glycine (G) and proline-glycine (PG) insertion mutants to evaluate the deletion events in FoxP proteins in their dimerization, stability, flexibility, and DNA-binding ability. We show that the PG insertion only increases protein stability, whereas the single glycine insertion decreases the association rate and protein stability and promotes affinity to the DNA ligand.

Antigen-binding fragments with improved crystal lattice packing and enhanced conformational flexibility at the elbow region as crystallization chaperones.

It has been shown previously that a set of three modifications-termed S1, Crystal Kappa, and elbow-act synergistically to improve the crystallizability of an antigen-binding fragment (Fab) framework. Here, we prepared a phage-displayed library and performed crystallization screenings to identify additional substitutions-located near the heavy-chain elbow region-which cooperate with the S1, Crystal Kappa, and elbow modifications to increase expression and improve crystallizability of the Fab framework even further. One substitution (K141Q) supports the signature Crystal Kappa-mediated Fab:Fab crystal lattice packing interaction. Another substitution (E172G) improves the compatibility of the elbow modification with the Fab framework by alleviating some of the strain incurred by the shortened and bulkier elbow linker region. A third substitution (F170W) generates a split-Fab conformation, resulting in a powerful crystal lattice packing interaction comprising the biological interaction interface between the variable heavy and light chain domains. In sum, we have used K141Q, E172G, and F170W substitutions-which complement the S1, Crystal Kappa, and elbow modifications-to generate a set of highly crystallizable Fab frameworks that can be used as chaperones to enable facile elucidation of Fab:antigen complex structures by x-ray crystallography.

Domain Swapping in Abiotic Foldamers.

Foldamer sequences that adopt tertiary helix-turn-helix folds mediated by helix-helix hydrogen bonding in organic solvents have been previously reported. In an attempt to create genuine abiotic quaternary structures, i.e. assemblies of tertiary structures, new sequences were prepared that possess additional hydrogen bond donors at positions that may promote an association between the tertiary folds. However, a solid state structure and extensive solution state investigations by Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) show that, instead of forming a quaternary structure, the tertiary folds assemble into stable domain-swapped dimer motifs. Domain swapping entails a complete reorganization of the arrays of hydrogen bonds and changes in relative helix orientation and handedness that can all be rationalized.

A domain swapping strategy to create modular transcriptional regulators for novel topology in genetic network.

Transcriptional regulators generate connections between biological signals and genetic outputs. They are used robustly for sensing input signals in building genetic circuits. However, each regulator can only generate a fixed connection, which generates constraints in linking multiple signals for more complex processes. Recent studies discovered that a domain swapping strategy can be applied to various regulator families to create modular regulators for new signal-output connections, significantly broadening possibilities in circuit design. Here we review the development of this emerging strategy, the use of resulting modular regulators for creating novel genetic response behaviors, and current limitations and solutions for further advancing the design of modular regulators.

Structure-based design, biophysical characterization, and biochemical application of the heterodimeric affinity purification tag based on the Schistosoma japonicum glutathione-S-transferase (SjGST) homodimer.

Schistosoma japonicum glutathione-S-transferase (SjGST), the so-called GST-tag, is one of the most widely used protein tags for the purification of recombinant proteins by affinity chromatography. Attachment of SjGST enables the purification of a protein of interest (POI) using commercially available glutathione-immobilizing resins. Here we produced an SjGST mutant pair that forms heterodimers by adjusting the salt bridge pairs in the homodimer interface of SjGST. An MD study confirmed that the SjGST mutant pair did not disrupt the heterodimer formation. The modified SjGST protein pair coexpressed in Escherichia coli was purified by glutathione-immobilized resin. The stability of the heterodimeric form of the SjGST mutant pair was further confirmed by size exclusion chromatography. Surface plasmon resonance measurements unveiled the selective formation of heterodimers within the pair, accompanied by a significant suppression of homodimerization. The heterodimeric SjGST exhibited enzymatic activity in assays employing a commercially available fluorescent substrate. By fusing one member of the heterodimeric SjGST pair with a fluorescent protein and the other with the POI, we were able to conveniently and sensitively detect protein-protein interactions using fluorescence spectroscopy in the pull-down assays. Thus, utilization of the heterodimeric SjGST would be a useful tag for protein science.

Crystal structure and interconversion of monomers and domain-swapped dimers of the walnut tree phytocystatin.

Biotechnological applications of phytocystatins have garnered significant interest due to their potential applications in crop protection and improve crop resistance to abiotic stress factors. Cof1 and Wal1 are phytocystatins derived from Coffea arabica and Juglans regia, respectively. These plants hold significant economic value due to coffee's global demand and the walnut tree's production of valuable timber and widely consumed walnuts with culinary and nutritional benefits. The study involved the heterologous expression in E. coli Lemo 21(DE3), purification by immobilized metal ion affinity and size exclusion chromatography, and biophysical characterization of both phytocystatins, focusing on isolating and interconverting their monomers and dimers. The crystal structure of the domain-swapped dimer of Wal1 was determined revealing two domain-swapped dimers in the asymmetric unit, an arrangement reminiscent of the human cystatin C structure. Alphafold models of monomers and Alphafold-Multimer models of domain-swapped dimers of Cof1 and Wal1 were analyzed in the context of the crystal structure. The methodology and data presented here contribute to a deeper understanding of the oligomerization mechanisms of phytocystatins and their potential biotechnological applications in agriculture.

ß-Strand-mediated Domain-swapping in the Absence of Hydrophobic Core Repacking.

Domain swapping is a process wherein a portion of a protein is exchanged with its counterpart in another copy of the molecule, resulting in the formation of homo-oligomers with concomitant repacking of a hydrophobic core. Here, we report domain swapping triggered upon modifying a ß-hairpin sequence within a single-layer ß-sheet (SLB) of a model protein, OspA that did not involve the formation of a reorganized hydrophobic core. The replacement of two ß-hairpin sequences with a Gly-Gly and shorteing of a ß-hairpin resulted in a protein that formed two distinct crystal structures under similar conditions: one was monomeric, similar to the parental molecule, whereas the other was a domain-swapped dimer, mediated by an intermolecular ß-sheet in the SLB portion. Based on the dimer interface structure, we replaced the Gly-Gly sequence with three-residue sequences that enable the formation of a consecutive intermolecular ß-sheet, including the Cys-Thr-Cys sequence that formed a stable disulfide-linked dimer. These results provide new insights into protein folding, evolution, and the designability of protein structure.

The Structure and Nucleotide-Binding Characteristics of Regulated Cystathionine ß-Synthase Domain-Containing Pyrophosphatase without One Catalytic Domain.

Regulatory adenine nucleotide-binding cystathionine ß-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is scarce. The difficulty in obtaining structural data for such proteins is ascribed to their unusual flexibility and propensity to form higher-order oligomeric structures. In this study, we deleted the most movable domain from the catalytic part of a CBS domain-containing bacterial inorganic pyrophosphatase (CBS-PPase) and characterized the deletion variant both structurally and functionally. The truncated CBS-PPase was inactive but retained the homotetrameric structure of the full-size enzyme and its ability to bind a fluorescent AMP analog (inhibitor) and diadenosine tetraphosphate (activator) with the same or greater affinity. The deletion stabilized the protein structure against thermal unfolding, suggesting that the deleted domain destabilizes the structure in the full-size protein. A "linear" 3D structure with an unusual type of domain swapping predicted for the truncated CBS-PPase by Alphafold2 was confirmed by single-particle electron microscopy. The results suggest a dual role for the CBS domains in CBS-PPase regulation: they allow for enzyme tetramerization, which impedes the motion of one catalytic domain, and bind adenine nucleotides to mitigate or aggravate this effect.

REC protein family expansion by the emergence of a new signaling pathway.

IMPORTANCE: We explore when and why large classes of proteins expand into new sequence space. We used an unsupervised machine learning approach to observe the sequence landscape of REC domains of bacterial response regulator proteins. We find that within-gene recombination can switch effector domains and, consequently, change the regulatory context of the duplicated protein.

Unraveling the Functional Significance of Unstructured Regions in G Protein-Coupled Receptors.

Unstructured regions in functional proteins have gained attention in recent years due to advancements in informatics tools and biophysical methods. G protein-coupled receptors (GPCRs), a large family of cell surface receptors, contain unstructured regions in the form of the i3 loop and C-terminus. This review provides an overview of the functional significance of these regions in GPCRs. GPCRs transmit signals from the extracellular environment to the cell interior, regulating various physiological processes. The i3 loop, located between the fifth and sixth transmembrane helices, and the C-terminus, connected to the seventh transmembrane helix, are determinant of interactions with G proteins and with other intracellular partners such as arrestins. Recent studies demonstrate that the i3 loop and C-terminus play critical roles in allosterically regulating GPCR activation. They can act as autoregulators, adopting conformations that, by restricting G protein access, modulate receptor coupling specificity. The length and unstructured nature of the i3 loop and C-terminus provide unique advantages in GPCR interactions with intracellular protein partners. They act as "fishing lines", expanding the radius of interaction and enabling GPCRs to tether scaffolding proteins, thus facilitating receptor stability during cell membrane movements. Additionally, the i3 loop may be involved in domain swapping between GPCRs, generating novel receptor dimers with distinct binding and coupling characteristics. Overall, the i3 loop and C-terminus are now widely recognized as crucial elements in GPCR function and regulation. Understanding their functional roles enhances our comprehension of GPCR structure and signaling complexity and holds promise for advancements in receptor pharmacology and drug development.

First 3-D structural evidence of a native-like intertwined dimer in the acylphosphatase family.

Acylphosphatase (AcP, EC 3.6.1.7) is a small model protein conformed by a ferredoxin-like fold, profoundly studied to get insights into protein folding and aggregation processes. Numerous studies focused on the aggregation and/or amyloidogenic properties of AcPs suggest the importance of edge-ß-strands in the process. In this work, we present the first crystallographic structure of Escherichia coli AcP (EcoAcP), showing notable differences with the only available NMR structure for this enzyme. EcoAcP is crystalised as an intertwined dimer formed by replacing a single C-terminal ß-strand between two protomers, suggesting a flexible character of the C-terminal edge of EcoAcP. Despite numerous works where AcP from different sources have been used as a model system for protein aggregation, our domain-swapped EcoAcP structure is the first 3-D structural evidence of native-like aggregated species for any AcP reported to date, providing clues on molecular determinants unleashing aggregation.

Human RNase 1 can extensively oligomerize through 3D domain swapping thanks to the crucial contribution of its C-terminus.

Human ribonuclease (RNase) 1 and bovine RNase A are the proto-types of the secretory "pancreatic-type" (pt)-RNase super-family. RNase A can oligomerize through the 3D domain swapping (DS) mechanism upon acetic acid (HAc) lyophilisation, producing enzymatically active oligomeric conformers by swapping both N- and C-termini. Also some RNase 1 mutants were found to self-associate through 3D-DS, however forming only N-swapped dimers. Notably, enzymatically active dimers and larger oligomers of wt-RNase 1 were collected here, in higher amount than RNase A, from HAc lyophilisation. In particular, RNase 1 self-associates through the 3D-DS of its N-terminus and, at a higher extent, of the C-terminus. Since RNase 1 is four-residues longer than RNase A, we further analyzed its oligomerization tendency in a mutant lacking the last four residues. The C-terminus role has been investigated also in amphibian onconase (ONC®), a pt-RNase that can form only a N-swapped dimer, since its C-terminus, that is three-residues longer than RNase A, is locked by a disulfide bond. While ONC mutants designed to unlock or cut this constraint were almost unable to dimerize, the RNase 1 mutant self-associated at a higher extent than the wt, suggesting a specific role of the C-terminus in the oligomerization of different RNases. Overall, RNase 1 reaches here the highest ability, among pt-RNases, to extensively self-associate through 3D-DS, paving the way for new investigations on the structural and biological properties of its oligomers.

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.

Monomeric crystal structure of the vaccine carrier protein CRM197 and implications for vaccine development.

CRM197 is a genetically detoxified mutant of diphtheria toxin (DT) that is widely used as a carrier protein in conjugate vaccines. Protective immune responses to several bacterial diseases are obtained by coupling CRM197 to glycans from these pathogens. Wild-type DT has been described in two oligomeric forms: a monomer and a domain-swapped dimer. Their proportions depend on the chemical conditions and especially the pH, with a large kinetic barrier to interconversion. A similar situation occurs in CRM197, where the monomer is preferred for vaccine synthesis. Despite 30 years of research and the increasing application of CRM197 in conjugate vaccines, until now all of its available crystal structures have been dimeric. Here, CRM197 was expressed as a soluble, intracellular protein in an Escherichia coli strain engineered to have an oxidative cytoplasm. The purified product, called EcoCRM, remained monomeric throughout crystallization. The structure of monomeric EcoCRM is reported at 2.0 Šresolution with the domain-swapping hinge loop (residues 379-387) in an extended, exposed conformation, similar to monomeric wild-type DT. The structure enables comparisons across expression systems and across oligomeric states, with implications for monomer-dimer interconversion and for the optimization of conjugation.

[Three ways for protein aggregation and the control strategies].

Protein aggregation is a critical issue in the production of biopharmaceuticals. During protein production, transport and storage, various factors can lead to protein aggregation. With the in-depth study, different ways of protein aggregation and various influencing factors were identified. This includes physical and chemical factors, translation modifications and protein structure. Since protein aggregation exerts major impact on the activity and homogeneity of proteins, it is of great importance to study the ways of protein aggregation and how to control it to obtain high-quality proteins. The review focuses on three ways of protein aggregation, namely 3D domain swapping, salt bridge formation, and oxidative stress, as well as methods to control protein aggregation during protein production, transport and storage. This may facilitate reducing the loss caused by the formation of protein aggregation and improving the purity and homogeneity of protein in research and commercial production.

Structures of permuted halves of a modern ribose-binding protein.

Periplasmic binding proteins (PBPs) are a class of proteins that participate in the cellular transport of various ligands. They have been used as model systems to study mechanisms in protein evolution, such as duplication, recombination and domain swapping. It has been suggested that PBPs evolved from precursors half their size. Here, the crystal structures of two permuted halves of a modern ribose-binding protein (RBP) from Thermotoga maritima are reported. The overexpressed proteins are well folded and show a monomer-dimer equilibrium in solution. Their crystal structures show partially noncanonical PBP-like fold type I conformations with structural deviations from modern RBPs. One of the half variants forms a dimer via segment swapping, suggesting a high degree of malleability. The structural findings on these permuted halves support the evolutionary hypothesis that PBPs arose via a duplication event of a flavodoxin-like protein and further support a domain-swapping step that might have occurred during the evolution of the PBP-like fold, a process that is necessary to generate the characteristic motion of PBPs essential to perform their functions.