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1.
    
Cytochrome c′′ from the obligate methylotroph Methylophilus methylotrophus is a 15 kDa monohaem protein which has a c‐type haem covalently linked to the protein chain. Two histidine residues are the axial ligands of the Fe atom in the oxidized form. This cytochrome is one of the few known haem proteins which undergoes a change of spin state of the Fe atom upon reduction, with the detachment of an axial histidine ligand. Initial crystallization conditions involved the utilization of cadmium chloride as an additive and resulted in highly mosaic crystals with poor diffraction properties. Optimization of the crystallization conditions was achieved by slowing the nucleation process utilizing agarose gels and viscous additives such as PEG, ethylene glycol and glycerol. Addition of glycerol to the crystallization buffer produced crystals suitable for X‐ray diffraction, with a reduced solvent content and mosaicity, which diffracted to a maximum resolution of 1.19 Å using synchrotron radiation. The crystals obtained under these conditions were employed for structure solution using the multiwavelength anomalous dispersion method at the Fe K edge.  相似文献   

2.
    
It is crucial to assign the correct redox and ligand states to crystal structures of proteins with an active redox centre to gain valid functional information and prevent the misinterpretation of structures. Single‐crystal spectroscopies, particularly when applied in situ at macromolecular crystallography beamlines, allow spectroscopic investigations of redox and ligand states and the identification of reaction intermediates in protein crystals during the collection of structural data. Single‐crystal resonance Raman spectroscopy was carried out in combination with macromolecular crystallography on Swiss Light Source beamline X10SA using cytochrome c′ from Alcaligenes xylosoxidans. This allowed the fingerprinting and validation of different redox and ligand states, identification of vibrational modes and identification of intermediates together with monitoring of radiation‐induced changes. This combined approach provides a powerful tool to obtain complementary data and correctly assign the true oxidation and ligand state(s) in redox‐protein crystals.  相似文献   

3.
    
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.  相似文献   

4.
    
Fam96a mRNA, which encodes a mammalian DUF59 protein, is enriched in macrophages. Recombinant human Fam96a forms stable monomers and dimers in solution. Crystal structures of these two forms revealed that each adopts a distinct type of domain‐swapped dimer, one of which is stabilized by zinc binding. Two hinge loops control Fam96a domain swapping; both are flexible and highly conserved, suggesting that domain swapping may be a common feature of eukaryotic but not bacterial DUF59 proteins. The derived monomer fold of Fam96a diverges from that of bacterial DUF59 counterparts in that the C‐terminal region of Fam96a is much longer and is positioned on the opposite side of the N‐terminal core fold. The putative metal‐binding site of bacterial DUF59 proteins is not conserved in Fam96a, but Fam96a interacts tightly in vitro with Ciao1, the cytosolic iron‐assembly protein. Moreover, Fam96a and Ciao1 can be co‐immunoprecipitated, suggesting that the interaction also occurs in vivo. Although predicted to have a signal peptide, it is shown that Fam96a is cytoplasmic. The data reveal that eukaryotic DUF59 proteins share intriguing characteristics with amyloidogenic proteins.  相似文献   

5.
    
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6.
    
We have investigated the mechanism and the evolutionary pathway of protein dimerization through analysis of experimental structures of dimers. We propose that the evolution of dimers may have multiple pathways, including (1) formation of a functional dimer directly without going through an ancestor monomer, (2) formation of a stable monomer as an intermediate followed by mutations of its surface residues, and (3), a domain swapping mechanism, replacing one segment in a monomer by an equivalent segment from an identical chain in the dimer. Some of the dimers which are governed by a domain swapping mechanism may have evolved at an earlier stage of evolution via the second mechanism. Here, we follow the theory that the kinetic pathway reflects the evolutionary pathway. We analyze the structure-kinetics-evolution relationship for a collection of symmetric homodimers classified into three groups: (1) 14 dimers, which were referred to as domain swapping dimers in the literature; (2) nine 2-state dimers, which have no measurable intermediates in equilibrium denaturation; and (3), eight 3-state dimers, which have stable intermediates in equilibrium denaturation. The analysis consists of the following stages: (i) The dimer is divided into two structural units, which have twofold symmetry. Each unit contains a contiguous segment from one polypeptide chain of the dimer, and its complementary contiguous segment from the other chain. (ii) The division is repeated progressively, with different combinations of the two segments in each unit. (iii) The coefficient of compactness is calculated for the units in all divisions. The coefficients obtained for different cuttings of a dimer form a compactness profile. The profile probes the structural organization of the two chains in a dimer and the stability of the monomeric state. We describe the features of the compactness profiles in each of the three dimer groups. The profiles identify the swapping segments in domain swapping dimers, and can usually predict whether a dimer has domain swapping. The kinetics of dimerization indicates that some dimers which have been assigned in the literature as domain swapping cases, dimerize through the 2-state kinetics, rather than through swapping segments of performed monomers. The compactness profiles indicate a wide spectrum in the kinetics of dimerization: dimers having no intermediate stable monomers; dimers having an intermediate with a stable monomer structure; and dimers having an intermediate with a stable structure in part of the monomer. These correspond to the multiple evolutionary pathways for dimer formation. The evolutionary mechanisms proposed here for dimers are applicable to other oligomers as well.  相似文献   

7.
We have previously attempted to simulate domain creation in early protein evolution by recombining polypeptide segments from non-homologous proteins, and we have described the structure of one such de novo protein, 1b11, a segment-swapped tetramer with novel architecture. Here, we have analyzed the thermodynamic stability and folding kinetics of the 1b11 tetramer and its monomeric and dimeric intermediates, and of 1b11 mutants with changes at the domain interface. Denatured 1b11 polypeptides fold into transient, folded monomers with marginal stability (DeltaG<1kcalmol(-1)) which convert rapidly ( approximately 6x10(4)M(-1)s(-1)) into dimers (DeltaG=9.8kcal/mol) and then more slowly ( approximately 3M(-1)s(-1)) into tetramers (DeltaG=28kcalmol(-1)). Segment swapping takes place during dimerization, as suggested by mass spectroscopic analysis of covalently linked peptides derived from proteolysis of a disulfide-linked dimer. Our results confirm that segment swapping and associated oligomerization are both powerful ways of stabilizing proteins, and we suggest that this may have been a feature of early protein evolution.  相似文献   

8.
    
Domain swapping is a widespread oligomerization process that is observed in a large variety of protein families. In the large superfamily of substrate‐binding proteins, non‐monomeric members have rarely been reported. The arginine‐binding protein from Thermotoga maritima (TmArgBP), a protein endowed with a number of unusual properties, presents a domain‐swapped structure in its dimeric native state in which the two polypeptide chains mutually exchange their C‐terminal helices. It has previously been shown that mutations in the region connecting the last two helices of the TmArgBP structure lead to the formation of a variety of oligomeric states (monomers, dimers, trimers and larger aggregates). With the aim of defining the structural determinants of domain swapping in TmArgBP, the monomeric form of the P235GK mutant has been structurally characterized. Analysis of this arginine‐bound structure indicates that it consists of a closed monomer with its C‐terminal helix folded against the rest of the protein, as typically observed for substrate‐binding proteins. Notably, the two terminal helices are joined by a single nonhelical residue (Gly235). Collectively, the present findings indicate that extending the hinge region and conferring it with more conformational freedom makes the formation of a closed TmArgBP monomer possible. On the other hand, the short connection between the helices may explain the tendency of the protein to also adopt alternative oligomeric states (dimers, trimers and larger aggregates). The data reported here highlight the importance of evolutionary control to avoid the uncontrolled formation of heterogeneous and potentially harmful oligomeric species through domain swapping.  相似文献   

9.
    
Purified preparations of the recombinant b′x domain fragment of human protein‐disulphide isomerase (PDI), which are homogeneous by mass spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis, comprise more than one species when analyzed by ion‐exchange chromatography and nondenaturing polyacrylamide gel electrophoresis. These species were resolved and shown to be monomer and dimer by analytical ultracentrifugation and analytical size‐exclusion chromatography. Spectroscopic properties indicate that the monomeric species corresponds to the “capped” conformation observed in the x‐ray structure of the I272A mutant of b′x (Nguyen, Wallis, Howard, Haapalainen, Salo, Saaranen, Sidhu, Wierenga, Freedman, Ruddock, and Williamson, J Mol Biol 2008;383:1144‐1155) in which the x region binds to a hydrophobic patch on the surface of the b′ domain; conversely, the dimeric species has an “open” or “uncapped” conformation in which the x region does not bind to this surface. The larger bb′x fragment of human PDI shows very similar behavior to b′x and can be resolved into a capped monomeric species and an uncapped dimer. Preparations of recombinant b′ domain of human PDI and of the bb′ domain pair are found exclusively as dimers. Full‐length PDI is known to comprise a mixture of monomeric and dimeric species, whereas the isolated a , b , and a′ domains of PDI are found exclusively as monomers. These results show that the b′ domain of human PDI tends to form homodimers—both in isolation and in other contexts—and that this tendency is moderated by the adjacent x region, which can bind to a surface patch on the b′ domain.  相似文献   

10.
    
Human zonula occludens 2 (ZO‐2) protein is a multi‐domain protein that consists of an SH3 domain, a GK domain and three copies of a PDZ domain with slight divergence. The three PDZ domains act as protein‐recognition modules that may mediate protein assembly and subunit localization. The crystal structure of the second PDZ domain of ZO‐2 (ZO‐2 PDZ2) was determined by molecular replacement at 1.75 Å resolution, revealing a dimer in the asymmetric unit. The dimer is stabilized by extensive symmetrical domain‐swapping of the β1 and β2 strands. Structural comparison shows that the ZO‐2 PDZ2 homodimer may have a similar ligand‐binding pattern to the ZO‐1 PDZ2–connexin 43 complex.  相似文献   

11.
    
In addition to binding intracellular fatty acids, fatty‐acid‐binding proteins (FABPs) have recently been reported to also transport the endocannabinoids anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG), arachidonic acid derivatives that function as neurotransmitters and mediate a diverse set of physiological and psychological processes. To understand how the endocannabinoids bind to FABPs, the crystal structures of FABP5 in complex with AEA, 2‐AG and the inhibitor BMS‐309403 were determined. These ligands are shown to interact primarily with the substrate‐binding pocket via hydrophobic interactions as well as a common hydrogen bond to the Tyr131 residue. This work advances our understanding of FABP5–endocannabinoid interactions and may be useful for future efforts in the development of small‐molecule inhibitors to raise endocannabinoid levels.  相似文献   

12.
    
Random‐acceleration molecular‐dynamics (RAMD) simulations with models of homodimeric 6‐ligated distal‐NO and 5‐ligated proximal‐NO cytochrome c′ complexes, in TIP3 H2O, showed two distinct, non‐intercommunicating worlds. In the framework of a long cavity formed by four protein helices with heme at one extremity, NO was observed to follow different pathways with the two complexes to reach the solvent. With the 6‐ligated complex, NO was observed to progress by exploiting protein internal channels created by thermal fluctuations, and be temporarily trapped into binding pockets before reaching the preferred gate at the heme end of the cavity. In contrast, with the 5‐ligated complex, NO was observed to surface the solvent‐exposed helix 7, up to a gate at the other extremity of the protein, only occasionally finding an earlier, direct way out toward the solvent. That only bulk NO gets involved in forming the 5‐ligated proximal‐NO complex is in agreement with previous experimental observations, while the occurrence of binding pockets suggests that also reservoir NO might play a role with the distal‐NO complex.  相似文献   

13.
    
Many protein architectures exhibit evidence of internal rotational symmetry postulated to be the result of gene duplication/fusion events involving a primordial polypeptide motif. A common feature of such structures is a domain‐swapped arrangement at the interface of the N‐ and C‐termini motifs and postulated to provide cooperative interactions that promote folding and stability. De novo designed symmetric protein architectures have demonstrated an ability to accommodate circular permutation of the N‐ and C‐termini in the overall architecture; however, the folding requirement of the primordial motif is poorly understood, and tolerance to circular permutation is essentially unknown. The β‐trefoil protein fold is a threefold‐symmetric architecture where the repeating ~42‐mer “trefoil‐fold” motif assembles via a domain‐swapped arrangement. The trefoil‐fold structure in isolation exposes considerable hydrophobic area that is otherwise buried in the intact β‐trefoil trimeric assembly. The trefoil‐fold sequence is not predicted to adopt the trefoil‐fold architecture in ab initio folding studies; rather, the predicted fold is closely related to a compact “blade” motif from the β‐propeller architecture. Expression of a trefoil‐fold sequence and circular permutants shows that only the wild‐type N‐terminal motif definition yields an intact β‐trefoil trimeric assembly, while permutants yield monomers. The results elucidate the folding requirements of the primordial trefoil‐fold motif, and also suggest that this motif may sample a compact conformation that limits hydrophobic residue exposure, contains key trefoil‐fold structural features, but is more structurally homologous to a β‐propeller blade motif.  相似文献   

14.
    
Crystallins are long-lived proteins packed inside eye lens fiber cells that are essential in maintaining the transparency and refractive power of the eye lens. Members of the two-domain betagamma-crystallin family assemble into an array of oligomer sizes, forming intricate higher-order networks in the lens cell. Here we describe the 1.4 angstroms resolution crystal structure of a truncated version of human betaB1 that resembles an in vivo age-related truncation. The structure shows that unlike its close homolog, betaB2-crystallin, the homodimer is not domain swapped, but its domains are paired intramolecularly, as in more distantly related monomeric gamma-crystallins. However, the four-domain dimer resembles one half of the crystallographic bovine betaB2 tetramer and is similar to the engineered circular permuted rat betaB2. The crystal structure shows that the truncated betaB1 dimer is extremely well suited to form higher-order lattice interactions using its hydrophobic surface patches, linker regions, and sequence extensions.  相似文献   

15.
    
The folding of a multi‐domain trimeric α‐helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two‐state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter‐subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate‐limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrBΔloop, from the SDS unfolded state. The CD spectrum of the refolded AcrBΔloop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re‐association of the trimer might be the limiting factor to obtain folded wild‐type AcrB.  相似文献   

16.
    
Gene duplication and fusion events in protein evolution are postulated to be responsible for the common protein folds exhibiting internal rotational symmetry. Such evolutionary processes can also potentially yield regions of repetitive primary structure. Repetitive primary structure offers the potential for alternative definitions of critical regions, such as the folding nucleus (FN). In principle, more than one instance of the FN potentially enables an alternative folding pathway in the face of a subsequent deleterious mutation. We describe the targeted mutation of the carboxyl‐terminal region of the (internally located) FN of the de novo designed purely‐symmetric β‐trefoil protein Symfoil‐4P. This mutation involves wholesale replacement of a repeating trefoil‐fold motif with a “blade” motif from a β‐propeller protein, and postulated to trap that region of the Symfoil‐4P FN in a nonproductive folding intermediate. The resulting protein (termed “Bladefoil”) is shown to be cooperatively folding, but as a trimeric oligomer. The results illustrate how symmetric protein architectures have potentially diverse folding alternatives available to them, including oligomerization, when preferred pathways are perturbed.  相似文献   

17.
    
It is generally accepted that naturally existing functional domains can serve as building blocks for complex protein structures, and that novel functions can arise from assembly of different combinations of these functional domains. To inform our understanding of protein evolution and explore the modular nature of protein structure, two model enzymes were chosen for study, purT‐encoded glycinamide ribonucleotide formyltransferase (PurT) and purK‐encoded N5‐carboxylaminoimidazole ribonucleotide synthetase (PurK). Both enzymes are found in the de novo purine biosynthetic pathway of Escherichia coli. In spite of their low sequence identity, PurT and PurK share significant similarity in terms of tertiary structure, active site organization, and reaction mechanism. Their characteristic three domain structures categorize both PurT and PurK as members of the ATP‐grasp protein superfamily. In this study, we investigate the exchangeability of individual protein domains between these two enzymes and the in vivo and in vitro functional properties of the resulting hybrids. Six domain‐swapped hybrids were unable to catalyze full wild‐type reactions, but each hybrid protein could catalyze partial reactions. Notably, an additional loop replacement in one of the domain‐swapped hybrid proteins was able to restore near wild‐type PurK activity. Therefore, in this model system, domain‐swapped proteins retained the ability to catalyze partial reactions, but further modifications were required to efficiently couple the reaction intermediates and achieve catalysis of the full reaction. Implications for understanding the role of domain swapping in protein evolution are discussed.  相似文献   

18.
    
Designed armadillo repeat proteins (dArmRP) are promising modular proteins for the engineering of binding molecules that recognize extended polypeptide chains. We determined the structure of a dArmRP containing five internal repeats and 3rd generation capping repeats in three different states by X‐ray crystallography: without N‐terminal His6‐tag and in the presence of calcium (YM5A/Ca2+), without N‐terminal His6‐tag and in the absence of calcium (YM5A), and with N‐terminal His6‐tag and in the presence of calcium (His‐YM5A/Ca2+). All structures show different quaternary structures and superhelical parameters. His‐YM5A/Ca2+ forms a crystallographic dimer, which is bridged by the His6‐tag, YM5A/Ca2+ forms a domain‐swapped tetramer, and only in the absence of calcium and the His6‐tag, YM5A forms a monomer. The changes of superhelical parameters are a consequence of calcium binding, because calcium ions interact with negatively charged residues, which can also participate in the modulation of helix dipole moments between adjacent repeats. These observations are important for further optimizations of dArmRPs and provide a general illustration of how construct design and crystallization conditions can influence the exact structure of the investigated protein.  相似文献   

19.
In the process of oligomeric structure formation through a mechanism of three-dimensional domain swapping, one domain of a monomeric protein is replaced by the same domain from an identical monomer. The swapped domain can represent an entire tertiary globular domain or an element of secondary protein structure, such as an -helix or a -strand. Different examples of three-dimensional domain swapping are reviewed; the functional importance of this phenomenon and its role in the development of new properties by some proteins in the process of evolution are considered. The contribution of three-dimensional domain swapping to the formation of linear protein polymers and amyloids is discussed.  相似文献   

20.
    
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