Protein surface charge effect on 3D domain swapping in cells for c-type cytochromes

Many c-type cytochromes (cyts) can form domain-swapped oligomers. The positively charged Hydrogenobacter thermophilus (HT) cytochrome (cyt) c₅₅₂ forms domain-swapped oligomers during expression in the Escherichia coli (E. coli) expression system, but the factors influencing the oligomerization remain unrevealed. Here, we found that the dimer of the negatively charged Shewanella violacea (SV) cyt c₅ exhibits a domain-swapped structure, in which the N-terminal helix is exchanged between protomers, similar to the structures of the HT cyt c₅₅₂ and Pseudomonas aeruginosa (PA) cyt c₅₅₁ domain-swapped dimers. Positively charged horse cyt c and HT cyt c₅₅₂ domain swapped during expression in E. coli, whereas negatively charged PA cyt c₅₅₁ and SV cyt c₅ did not. Oligomers were formed during expression in E. coli for HT cyt c₅₅₂ attached to either a co- or post-translational signal peptide for transportation through the cytoplasm membrane, but not for PA cyt c₅₅₁ attached to either signal peptide. HT cyt c₅₅₂ formed oligomers in E. coli in the presence and absence of rare codons. More oligomers were obtained from the in vitro folding of horse cyt c and HT cyt c₅₅₂ by the addition of negatively charged liposomes during folding, whereas the amount of oligomers for the in vitro folding of PA cyt c₅₅₁ and SV cyt c₅ did not change significantly by the addition. These results indicate that the protein surface charge affects the oligomerization of c-type cyts in cells; positively charged c-type cyts assemble on a negatively charged membrane, inducing formation of domain-swapped oligomers during folding.     

Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping

Cytochrome c₅₅₅ from hyperthermophilic bacteria Aquifex aeolicus (AA cyt c₅₅₅) is a hyperstable protein belonging to the cyt c protein family, which possesses a unique long 3₁₀‐α‐3₁₀ helix containing the heme‐ligating Met61. Herein, we show that AA cyt c₅₅₅ forms dimers by swapping the region containing the extra 3₁₀‐α‐3₁₀ helix and C‐terminal α‐helix. The asymmetric unit of the crystal of dimeric AA cyt c₅₅₅ contained two dimer structures, where the structure of the hinge region (Val53–Lys57) was different among all four protomers. Dimeric AA cyt c₅₅₅ dissociated to monomers at 92 ± 1°C according to DSC measurements, showing that the dimer was thermostable. According to CD measurements, the secondary structures of dimeric AA cyt c₅₅₅ were maintained at pH 2.2–11.0. CN^‐ and CO bound to dimeric AA cyt c₅₅₅ in the ferric and ferrous states, respectively, owing to the flexibility of the hinge region close to Met61 in the dimer, whereas these ligands did not bind to the monomer under the same conditions. In addition, CN^‐ and CO bound to the oxidized and reduced dimer at neutral pH and a wide range of pH (pH 2.2–11.0), respectively, in a wide range of temperature (25–85°C), owing to the thermostability and pH tolerance of the dimer. These results show that the ligand binding character of hyperstable AA cyt c₅₅₅ changes upon dimerization by domain swapping.     

Cytochrome components of denitrifyingPseudomonas stutzeri

Cytochromesc-551,c-552,c-554,cd, ac type with low α/β peaks, and an acidicc-type cytochrome were detected in extracts ofPseudomonas stutzeri. The first four were purified and physically characterized. Light absorption spectra indicate a probably histidine-methionine liganding of heme iron inc-551 andc-554, but an absence of methionine in the ligand ofc-552 heme iron. In displaying two separately reduciblec-hemes, thec-552 appears homologous with that fromP. perfectomarinus.     

SERR Spectroelectrochemical Study of Cytochrome cd 1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c 552 on Biocompatible Metal Electrodes

Cytochrome cd1 nitrite reductases (cd ₁NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd ₁NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd ₁NiR from Marinobacter hydrocarbonoclasticus (Mhcd ₁) depends on the presence of its physiological redox partner, cytochrome c ₅₅₂ (cyt c ₅₅₂), we show here a detailed surface enhanced resonance Raman characterization of Mhcd ₁ and cyt c ₅₅₂ attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd ₁ and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd ₁ // cyt c ₅₅₂ and Ag // SAM // cyt c ₅₅₂ // Mhcd ₁ constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c ₅₅₂ retains its native properties, while the redox potential of apparently intact Mhcd ₁ undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd ₁, reinforcing the idea that subtle and very specific interactions between Mhcd ₁ and cyt c ₅₅₂ govern efficient intermolecular electron transfer and catalytic activity of Mhcd ₁.     

Crystal Structure of the Electron Carrier Domain of the Reaction Center Cytochrome cz Subunit from Green Photosynthetic Bacterium Chlorobium tepidum

In green sulfur photosynthetic bacteria, the cytochrome c_z (cyt c_z) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt c_z subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt c_z subunit (C-cyt c_z) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-Å resolution. The overall fold of C-cyt c_z consists of four α-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt c_z supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt c_z.     

Effects of axial methionine coordination on the in-plane asymmetry of the heme electronic structure of cytochrome<Emphasis Type="Italic"> c</Emphasis>

The paramagnetic susceptibility () tensors of the oxidized forms of thermophile Hydrogenobacter thermophilus cytochrome c₅₅₂ (Ht cyt c₅₅₂) and a quintuple mutant (F7A/V13 M/F34Y/E43Y/V78I; qm) of mesophile Pseudomonas aeruginosa cytochrome c₅₅₁ (Pa cyt c₅₅₁) have been determined on the basis of the redox-dependent ¹H NMR shift changes of the main-chain NH and CH proton resonances of non-coordinated amino acid residues and the NMR structures of the reduced forms of the corresponding proteins (J. Hasegawa, T. Yoshida, T. Yamazaki, Y. Sambongi, Y. Yu, Y. Igarashi, T. Kodama, K. Yamazaki, Y. Kyogoku, Y. Kobayashi (1998) Biochemistry 37:9641–9649; J. Hasegawa, S. Uchiyama, Y. Tanimoto, M. Mizutani, Y. Kobayashi, Y. Sambongi,Y. Igarashi (2000) J Biol Chem 275:37824–37828). From the tensors determined, we obtained the contact shifts for heme methyl proton resonances, which provided the heme electronic structures of the oxidized forms of Ht cyt c₅₅₂ and qm. We also characterized the heme electronic structure of the cyanide adducts of the proteins, where the axial Met was replaced by an exogenous cyanide ion, through the analysis of ¹H NMR spectra. The results indicated that the heme electronic structures of both the proteins in their oxidized forms with axial His and Met coordination are largely different to each other, while those in their cyanide adducts are similar to each other. These results demonstrated that the orientation of the axial Met sulfur lone pair, with respect to heme, predominantly contributes to the spin delocalization into the porphyrin- system of heme in the oxidized proteins with axial His and Met coordination.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations COSY correlation spectroscopy - DQF-COSY double quantum filtered COSY - TOCSY total correlation spectroscopy - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect correlated spectroscopy - Cyt c cytochrome c - Pa cyt c₅₅₁ Pseudomonas aeruginosa cytochrome c₅₅₁ - Ht cyt c₅₅₂ Hydrogenobacter thermophilus cytochrome c₅₅₂ - _obs observed shift - _para paramagnetic shift - _dia diamagnetic shift - _con contact shift - _pc pseudo-contact shift     

Purification of two pressure-regulated c-type cytochromes from a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F

From a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F, a membrane-bound cytochrome c-551 and a cytoplasmic cytochrome c-552 were purified. The cytochrome c-551 contained 44.2 nmol of heme c mg protein⁻¹ and cytochrome c-552 contained 31.3 nmol of heme c mg protein⁻¹. The CO difference spectrum of cytochrome c-551 showed a peak at 413.7 nm and troughs at 423.2, 522 and 552 nm which indicated that this cytochrome combined with CO. Cytochrome c-551 was found to consist of two subunits with molecular masses of 29.1 kDa and 14.7 kDa, respectively, and each subunit contained one heme c molecule. Cytochrome c-552 also consisted of two subunits with molecular masses of 16.9 kDa and 14.7 kDa, respectively, and only one of these subunits contained heme c. Cytochrome c-551 was constitutively synthesized when the cells were grown at pressures of either 0.1 MPa or 60 MPa, whereas cytochrome c-552 was synthesized only at 0.1 MPa. These results together with the results of analysis of membrane-associated catalytic activities suggest that the respiratory system of DB-172F is regulated by pressure and may be intimately related to the baroadaptability mechanism of this deep-sea bacterium.     

The dual role of a loop with low loop contact distance in folding and domain swapping

Alpha helices, beta strands, and loops are the basic building blocks of protein structure. The folding kinetics of alpha helices and beta strands have been investigated extensively. However, little is known about the formation of loops. Experimental studies show that for some proteins, the formation of a single loop is the rate-determining step for folding, whereas for others, a loop (or turn) can misfold to serve as the hinge loop region for domain-swapped species. Computer simulations of an all-atom model of fragment B of Staphylococcal protein A found that the formation of a single loop initiates the dominant folding pathway. On the other hand, the stability analysis of intermediates suggests that the same loop is a likely candidate to serve as a hinge loop for domain swapping. To interpret the simulation result, we developed a simple structural parameter: the loop contact distance (LCD), or the sequence distance of contacting residues between a loop and the rest of the protein. The parameter is applied to a number of other proteins, including SH3 domains and prion protein. The results suggest that a locally interacting loop (low LCD) can either promote folding or serve as the hinge region for domain swapping. Thus, there is an intimate connection between folding and domain swapping, a possible cause of misfolding and aggregation.     

Analysis of domain-swapped oligomers reveals local sequence preferences and structural imprints at the linker regions and swapped interfaces

Background

3D domain swapping is an oligomerization process in which structural elements get exchanged between subunits. This mechanism grasped interest of many researchers due to its association with neurodegenerative diseases like Alzheimer''s disease, spongiform encephalopathy etc. Despite the biomedical relevance, very little is known about understanding this mechanism. The quest for ruling principles behind this curious phenomenon that could enable early prediction provided an impetus for our bioinformatics studies.

Methodology

A novel method, HIDE, has been developed to find non-domain-swapped homologues and to identify hinge from domain-swapped oligomers. Non-domain-swapped homologues were identified from the protein structural databank for majority of the domain-swapped entries and hinge boundaries could be recognised automatically by means of successive superposition techniques. Different sequence and structural features in domain-swapped proteins and related proteins have also been analysed.

Conclusions

The HIDE algorithm was able to identify hinge region in 83% cases. Sequence and structural analyses of hinge and interfaces reveal amino acid preferences and specific conformations of residues at hinge regions, while comparing the domain-swapped and non-domain-swapped states. Interactions differ significantly between regular dimeric interfaces and interface formed at the site of domain-swapped examples. Such preferences of residues, conformations and interactions could be of predictive value.     

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides: I. EQUILIBRIUM AND MODELING STUDIES*

We have used imidazole (Im) and N-methylimidazole (MeIm) as probes of the heme-binding cavity of membrane-bound cytochrome (cyt) c₁ in detergent-solubilized bc₁ complex from Rhodobacter sphaeroides. Imidazole binding to cyt c₁ substantially lowers the midpoint potential of the heme and fully inhibits bc₁ complex activity. Temperature dependences showed that binding of Im (K_d ≈ 330 μm, 25 °C, pH 8) is enthalpically driven (ΔH⁰ = −56 kJ/mol, ΔS⁰ = −121 J/mol/K), whereas binding of MeIm is 30 times weaker (K_d ≈ 9.3 mm) and is entropically driven (ΔH⁰ = 47 kJ/mol, ΔS⁰° = 197 J/mol/K). The large enthalpic and entropic contributions suggest significant structural and solvation changes in cyt c₁ triggered by ligand binding. Comparison of these results with those obtained previously for soluble cyts c and c₂ suggested that Im binding to cyt c₁ is assisted by formation of hydrogen bonds within the heme cleft. This was strongly supported by molecular dynamics simulations of Im adducts of cyts c, c₂, and c₁, which showed hydrogen bonds formed between the N_δH of Im and the cyt c₁ protein, or with a water molecule sequestered with the ligand in the heme cleft.     

The structure and function of the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>2</Subscript>: reaction center electron transfer complex from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

In the photosynthetic bacterium, Rhodobacter sphaeroides, the mobile electron carrier, cytochrome c₂ (cyt c₂) transfers an electron from reduced heme to the photooxidized bacteriochlorophyll dimer in the membrane bound reaction center (RC) as part of the light induced cyclic electron transfer chain. A complex between these two proteins that is active in electron transfer has been crystallized and its structure determined by X-ray diffraction. The structure of the cyt:RC complex shows the cyt c₂ (cyt c₂) positioned at the center of the periplasmic surface of the RC. The exposed heme edge from cyt c₂ is in close tunneling contact with the electron acceptor through an intervening bridging residue, Tyr L162 located on the RC surface directly above the bacteriochlorophyll dimer. The binding interface between the two proteins can be divided into two regions: a short-range interaction domain and a long-range interaction domain. The short-range domain includes residues immediately surrounding the tunneling contact region around the heme and Tyr L162 that display close intermolecular contacts optimized for electron transfer. These include a small number of hydrophobic interactions, hydrogen bonds and a pi-cation interaction. The long-range interaction domain consists of solvated complementary charged residues; positively charged residues from the cyt and negatively charged residues from the RC that provide long range electrostatic interactions that can steer the two proteins into position for rapid association.     

Evolutionary alkaline transition in human cytochrome <Emphasis Type="Italic">c</Emphasis>

Conformational transitions in cytochrome c (cyt c) are being realized to be responsible for its multi-functions. Among a number of conformational transitions in cyt c, the alkaline transition has attracted much attention. The cDNA of human cyt c is cloned by RT-PCR and a high-effective expression system for human cyt c has been developed in this study. The equilibrium and kinetics of the alkaline transition of human cyt c have been systematically investigated for the first time, and compared with those of yeast and horse cyt c from an evolutionary perspective. The pK_a value for the alkaline transition of human cyt c is apparently higher than that of yeast and horse. Kinetic studies suggest that it is increasingly difficult for the alkaline transition of cyt c from yeast, horse and human. Molecular modeling of human cyt c shows that the omega loop where the lysine residue is located apparently further away from heme in human cyt c than in yeast iso-1 and horse heart cyt c. These results regarding alkaline conformational transition provide valuable information for understanding the molecular basis for the biological multi-functions of cyt c.     

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.     

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 Intrinsically Disordered Domain of the Antitoxin Phd Chaperones the Toxin Doc against Irreversible Inactivation and Misfolding

The toxin Doc from the phd/doc toxin-antitoxin module targets the cellular translation machinery and is inhibited by its antitoxin partner Phd. Here we show that Phd also functions as a chaperone, keeping Doc in an active, correctly folded conformation. In the absence of Phd, Doc exists in a relatively expanded state that is prone to dimerization through domain swapping with its active site loop acting as hinge region. The domain-swapped dimer is not capable of arresting protein synthesis in vitro, whereas the Doc monomer is. Upon binding to Phd, Doc becomes more compact and is secured in its monomeric state with a neutralized active site.     

Kinetics of cyanide binding as a probe of local stability/flexibility of cytochrome c

Effect of anions of the Hofmeister series (thiocyanate, perchlorate, iodide, bromide, nitrate, chloride, sulfate, and phosphate) on local and global stability and flexibility of horse heart ferricytochrome c (cyt c) has been studied. Global stability of cyt c was determined by iso/thermal denaturations monitored by change in ellipticity in the far-UV region and its local stability was determined from absorbance changes in the Soret region. Particularly, relative stability/flexibility of the Met80–heme iron bond has been assessed by analysis of binding of cyanide into the heme iron. Both global and local stabilities of cyt c exhibited monotonous increase induced by a change of anion from chaotropic to kosmotropic species. However, this monotonous dependence was not observed for the rate constants of cyanide association with cyt c. As expected more chaotropic ions induced lower stability of protein and faster binding of cyanide but this correlation was reversed for kosmotropic anions. We propose that the unusual bell-shaped dependence of the rate constant of cyanide association is a result of modulation of Met80–heme iron bond strength and/or flexibility of heme region by Hofmeister anions independently on global stability of cyt c. Further, our results demonstrate sensitivity of cyanide binding to local change in stability/flexibility in the heme region of cyt c.     

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.     

Five amino acid residues responsible for the high stability of Hydrogenobacter thermophilus cytochrome c552: reciprocal mutation analysis

Five amino acid residues responsible for extreme stability have been identified in cytochrome c(552) (HT c(552)) from a thermophilic bacterium, Hydrogenobacter thermophilus. The five residues, which are spatially distributed in three regions of HT c(552), were replaced with the corresponding residues in the homologous but less stable cytochrome c(551) (PA c(551)) from Pseudomonas aeruginosa. The quintuple HT c(552) variant (A7F/M13V/Y34F/Y43E/I78V) showed the same stability against guanidine hydrochloride denaturation as that of PA c(551), suggesting that the five residues in HT c(552) necessarily and sufficiently contribute to the overall stability. In the three HT c(552) variants carrying mutations in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by -13.3 kJ mol(-1), followed by A7F/M13V (-3.3 kJ mol(-1)) and then I78V (-1.5 kJ mol(-1)). The order of destabilization in HT c(552) was the same as that of stabilization in PA c(551) with reverse mutations such as F34Y/E43Y, F7A/V13M, and V78I (13.4, 10.3, and 0.3 kJ mol(-1), respectively). The results of guanidine hydrochloride denaturation were consistent with those of thermal denaturation for the same variants. The present study established a method for reciprocal mutation analysis. The effects of side-chain contacts were experimentally evaluated by swapping the residues between the two homologous proteins that differ in stability. A comparative study of the two proteins was a useful tool for assessing the amino acid contribution to the overall stability.     

Free radical fragmentation of cardiolipin by cytochrome c

The effect of cytochrome c (cyt c) on degradation of cardiolipin in its polar part was investigated in cardiolipin/phosphatidylcholine (CL/PC) liposomes incubated with cyt c/H₂O₂/and (or) ascorbate by high-performance thin layer chromatography and MALDI-TOF mass spectrometry. It has been shown that phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were formed in the system under conditions where hydrogen peroxide favours a release of heme iron from cyt c. The formation of PA and PHA occurs via an OH-induced fragmentation taking place in the polar moiety of cardiolipin. Formation of fragmentation products correlated with the loss of CL in CL/PC liposomes incubated with cyt c/H₂O₂/ascorbate or with Cu²⁺/H₂O₂/ascorbate.     

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.