RESUMO
The BisI family of restriction endonucleases is unique in requiring multiple methylated or hydroxymethylated cytosine residues within a short recognition sequence (GCNGC), and in cleaving directly within this sequence, rather than at a distance. Here, we report that the number of modified cytosines that are required for cleavage can be tuned by the salt concentration. We present crystal structures of two members of the BisI family, NhoI and Eco15I_Ntd (N-terminal domain of Eco15I), in the absence of DNA and in specific complexes with tetra-methylated GCNGC target DNA. The structures show that NhoI and Eco15I_Ntd sense modified cytosine bases in the context of double-stranded DNA (dsDNA) without base flipping. In the co-crystal structures of NhoI and Eco15I_Ntd with DNA, the internal methyl groups (G5mCNGC) interact with the side chains of an (H/R)(V/I/T/M) di-amino acid motif near the C-terminus of the distal enzyme subunit and arginine residue from the proximal subunit. The external methyl groups (GCNG5mC) interact with the proximal enzyme subunit, mostly through main chain contacts. Surface plasmon resonance analysis for Eco15I_Ntd shows that the internal and external methyl binding pockets contribute about equally to sensing of cytosine methyl groups.
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The Protein Data Bank (PDB) includes a carefully curated treasury of experimentally derived structural data on biological macromolecules and their various complexes. Such information is fundamental for a multitude of projects that involve large-scale data mining and/or detailed evaluation of individual structures of importance to chemistry, biology and, most of all, to medicine, where it provides the foundation for structure-based drug discovery. However, despite extensive validation mechanisms, it is almost inevitable that among the â¼215â 000 entries there will occasionally be suboptimal or incorrect structure models. It is thus vital to apply careful verification procedures to those segments of the PDB that are of direct medicinal interest. Here, such an analysis was carried out for crystallographic models of L-asparaginases, enzymes that include approved drugs for the treatment of certain types of leukemia. The focus was on the adherence of the atomic coordinates to the rules of stereochemistry and their agreement with the experimental electron-density maps. Whereas the current clinical application of L-asparaginases is limited to two bacterial proteins and their chemical modifications, the field of investigations of such enzymes has expanded tremendously in recent years with the discovery of three entirely different structural classes and with numerous reports, not always quite reliable, of the anticancer properties of L-asparaginases of different origins.
Assuntos
Asparaginase , Bases de Dados de Proteínas , Asparaginase/química , Humanos , Modelos Moleculares , Cristalografia por Raios X/métodos , Conformação ProteicaRESUMO
ReAV, the inducible Class-3 L-asparaginase from the nitrogen-fixing symbiotic bacterium Rhizobium etli, is an interesting candidate for optimizing its enzymatic potential for antileukemic applications. Since it has no structural similarity to known enzymes with this activity, it may offer completely new ways of approach. Also, as an unrelated protein, it would evade the immunological response elicited by other asparaginases. The crystal structure of ReAV revealed a uniquely assembled protein homodimer with a highly specific C135/K138/C189 zinc binding site in each subunit. It was also shown before that the Zn2+ cation at low and optimal concentration boosts the ReAV activity and improves substrate specificity, which indicates its role in substrate recognition. However, the detailed catalytic mechanism of ReAV is still unknown. In this work, we have applied site-directed mutagenesis coupled with enzymatic assays and X-ray structural analysis to elucidate the role of the residues in the zinc coordination sphere in catalysis. Almost all of the seven ReAV muteins created in this campaign lost the ability to hydrolyze L-asparagine, confirming our predictions about the significance of the selected residues in substrate hydrolysis. We were able to crystallize five of the ReAV mutants and solve their crystal structures, revealing some intriguing changes in the active site area as a result of the mutations. With alanine substitutions of Cys135 or Cys189, the zinc coordination site fell apart and the mutants were unable to bind the Zn2+ cation. Moreover, the absence of Lys138 induced atomic shifts and conformational changes of the neighboring residues from two active-site Ser-Lys tandems. Ser48 from one of the tandems, which is hypothesized to be the catalytic nucleophile, usually changes its hydration pattern in response to the mutations. Taken together, the results provide many useful clues about the catalytic mechanism of the enzyme, allowing one to cautiously postulate a possible enzymatic scenario.
RESUMO
The genome of Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes two L-asparaginases, ReAIV and ReAV, that have no similarity to the well characterized enzymes of class 1 (bacterial type) and class 2 (plant type). It has been hypothesized that ReAIV and ReAV might belong to the same structural class 3 despite their low level of sequence identity. When the crystal structure of the inducible and thermolabile protein ReAV was solved, this hypothesis gained a stronger footing because the key residues of ReAV are also present in the sequence of the constitutive and thermostable ReAIV protein. High-resolution crystal structures of ReAIV now confirm that it is a class 3 L-asparaginase that is structurally similar to ReAV but with important differences. The most striking differences concern the peculiar hydration patterns of the two proteins, the presence of three internal cavities in ReAIV and the behavior of the zinc-binding site. ReAIV has a high pH optimum (9-11) and a substrate affinity of â¼1.3â mM at pH 9.0. These parameters are not suitable for the direct application of ReAIV as an antileukemic drug, although its thermal stability and lack of glutaminase activity would be of considerable advantage. The five crystal structures of ReAIV presented in this work allow a possible enzymatic scenario to be postulated in which the zinc ion coordinated in the active site is a dispensable element. The catalytic nucleophile seems to be Ser47, which is part of two Ser-Lys tandems in the active site. The structures of ReAIV presented here may provide a basis for future enzyme-engineering experiments to improve the kinetic parameters for medicinal applications.
Assuntos
Asparaginase , Rhizobium etli , Asparaginase/química , Rhizobium etli/química , Rhizobium etli/genética , Catálise , Sítios de Ligação , Plantas/metabolismo , ZincoRESUMO
The self-complementary L-d(CGCGCG)2 purine/pyrimidine hexanucleotide was crystallized in complex with the polyamine cadaverine and potassium cations. Since the oligonucleotide contained the enantiomeric 2'-deoxy-L-ribose, the Z-DNA duplex is right-handed, as confirmed by the ultrahigh-resolution crystal structure determined at 0.69â Å resolution. Although the X-ray diffraction data were collected at a very short wavelength (0.7085â Å), where the anomalous signal of the P and K atoms is very weak, the signal was sufficiently outstanding to clearly indicate the wrong hand when the structure was mistakenly solved assuming the presence of 2'-deoxy-D-ribose. The electron density clearly shows the entire cadaverinium dication, which has an occupancy of 0.53 and interacts with one Z-DNA duplex. The K+ cation, with an occupancy of 0.32, has an irregular coordination sphere that is formed by three OP atoms of two symmetry-related Z-DNA duplexes and one O5' hydroxyl O atom, and is completed by three water sites, one of which is twofold disordered. The K+ site is complemented by a partial water molecule, the hydrogen bonds of which have the same lengths as the K-O bonds. The sugar-phosphate backbone assumes two conformations, but the base pairs do not show any sign of disorder.
Assuntos
DNA Forma Z , Conformação de Ácido Nucleico , Modelos Moleculares , Difração de Raios X , ÁguaRESUMO
This work reports the results of random mutagenesis of the Escherichia coli class 2 L-asparaginase EcAIII belonging to the Ntn-hydrolase family. New variants of EcAIII were studied using structural, biophysical and bioinformatic methods. Activity tests revealed that the L-asparaginase activity is abolished in all analyzed mutants with the absence of Arg207, but some of them retained the ability to undergo the autoproteolytic maturation process. The results of spectroscopic studies and the determined crystal structures showed that the EcAIII fold is flexible enough to accept different types of mutations; however, these mutations may have a diverse impact on the thermal stability of the protein. The conclusions from the experiments are grouped into six lessons focused on (i) the adaptation of the EcAIII fold to new substitutions, (ii) the role of Arg207 in EcAIII activity, (iii) a network of residues necessary for autoprocessing, (iv) the complexity of the autoprocessing reaction, (v) the conformational changes observed in enzymatically inactive variants and (vi) the cooperativity of the EcAIII dimer subunits. Additionally, the structural requirements (pre-maturation checkpoints) that are necessary for the initiation of the autocleavage of Ntn-hydrolases have been classified. The findings reported in this work provide useful hints that should be considered before planning enzyme-engineering experiments aimed at the design of proteins for therapeutic applications. This is especially important for L-asparaginases that can be utilized in leukemia therapy, as alternative therapeutics are urgently needed to circumvent the severe side effects associated with the currently used enzymes.
Assuntos
Asparaginase , Escherichia coli , Asparaginase/química , Modelos Moleculares , Mutagênese , MutaçãoRESUMO
Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes an essential L-asparaginase (ReAV) with no sequence homology to known enzymes with this activity. High-resolution crystal structures of ReAV show indeed a structurally distinct, dimeric enzyme, with some resemblance to glutaminases and ß-lactamases. However, ReAV has no glutaminase or lactamase activity, and at pH 9 its allosteric asparaginase activity is relatively high, with Km for L-Asn at 4.2 mM and kcat of 438 s-1. The active site of ReAV, deduced from structural comparisons and confirmed by mutagenesis experiments, contains a highly specific Zn2+ binding site without a catalytic role. The extensive active site includes residues with unusual chemical properties. There are two Ser-Lys tandems, all connected through a network of H-bonds to the Zn center, and three tightly bound water molecules near Ser48, which clearly indicate the catalytic nucleophile.
Assuntos
Asparaginase/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Rhizobium etli/enzimologia , Asparaginase/química , Asparaginase/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Biocatálise , Cátions/química , Cátions/metabolismo , Cristalografia por Raios X , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Metais/química , Metais/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Rhizobium etli/genética , TemperaturaRESUMO
The X-ray crystal structure of the d(CGCGCG)2/putrescine(2+)/K+ complex has been determined at 0.60â Å resolution. Stereochemical restraints were used only for the putrescinium dication, and 23 bonds and 18 angles of the Z-DNA nucleotides with dual conformation. The N atoms of the putrescine(2+) dication form three direct hydrogen bonds with the N7_G atoms of three different Z-DNA molecules, plus three water-mediated hydrogen bonds with cytosine, guanine and phosphate acceptors. A unique potassium cation was also unambiguously identified in the structure, albeit at a â¼0.5 occupation site shared with a water molecule, providing the first example of such a complex with Z-DNA. The K+ cation has coordination number of eight and an irregular coordination sphere, formed by four water molecules and four O atoms from two phosphate groups of the Z-DNA, including ligands present at fractional occupancy. The structural disorder of the Z-DNA duplex is manifested by the presence of alternate conformations along the DNA backbone. Comparison of the position and interactions of putrescine(2+) in the present structure with other ultra-high-resolution structures of Z-DNA in complexes with Mn2+ and Zn2+ ions shows that the dicationic putrescinium moiety can effectively substitute these metal ions for stabilization of Z-type DNA duplexes. Furthermore, this comparison also suggests that the spermine(4+) tetracation has a higher affinity for Z-DNA than K+.
Assuntos
DNA Forma Z/química , Potássio/química , Putrescina/química , Cátions/química , Cristalografia por Raios X , Modelos MolecularesRESUMO
As part of the global mobilization to combat the present pandemic, almost 100â 000 COVID-19-related papers have been published and nearly a thousand models of macromolecules encoded by SARS-CoV-2 have been deposited in the Protein Data Bank within less than a year. The avalanche of new structural data has given rise to multiple resources dedicated to assessing the correctness and quality of structural data and models. Here, an approach to evaluate the massive amounts of such data using the resource https://covid19.bioreproducibility.org is described, which offers a template that could be used in large-scale initiatives undertaken in response to future biomedical crises. Broader use of the described methodology could considerably curtail information noise and significantly improve the reproducibility of biomedical research.
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The appearance at the end of 2019 of the new SARS-CoV-2 coronavirus led to an unprecedented response by the structural biology community, resulting in the rapid determination of many hundreds of structures of proteins encoded by the virus. As part of an effort to analyze and, if necessary, remediate these structures as deposited in the Protein Data Bank (PDB), this work presents a detailed analysis of 81 crystal structures of the main protease 3CLpro, an important target for the design of drugs against COVID-19. The structures of the unliganded enzyme and its complexes with a number of inhibitors were determined by multiple research groups using different experimental approaches and conditions; the resulting structures span 13 different polymorphs representing seven space groups. The structures of the enzyme itself, all determined by molecular replacement, are highly similar, with the exception of one polymorph with a different inter-domain orientation. However, a number of complexes with bound inhibitors were found to pose significant problems. Some of these could be traced to faulty definitions of geometrical restraints for ligands and to the general problem of a lack of such information in the PDB depositions. Several problems with ligand definition in the PDB itself were also noted. In several cases extensive corrections to the models were necessary to adhere to the evidence of the electron-density maps. Taken together, this analysis of a large number of structures of a single, medically important protein, all determined within less than a year using modern experimental tools, should be useful in future studies of other systems of high interest to the biomedical community.
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Mason-Pfizer monkey virus protease (PR) was crystallized in complex with two pepstatin-based inhibitors in P1 space group. In both crystal structures, the extended flap loops that lock the inhibitor/substrate over the active site, are visible in the electron density either completely or with only small gaps, providing the first observation of the conformation of the flap loops in dimeric complex form of this retropepsin. The H-bond network in the active site (with D26N mutation) differs from that reported for the P21 crystal structures and is similar to a rarely occurring system in HIV-1 PR.
Assuntos
Vírus dos Macacos de Mason-Pfizer/enzimologia , Pepstatinas/química , Peptídeo Hidrolases/química , Inibidores de Proteases/química , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Substituição de Aminoácidos , Vírus dos Macacos de Mason-Pfizer/genética , Mutação de Sentido Incorreto , Peptídeo Hidrolases/genética , Estrutura Secundária de Proteína , Proteínas Virais/genéticaRESUMO
The COVID-19 pandemic has triggered numerous scientific activities aimed at understanding the SARS-CoV-2 virus and ultimately developing treatments. Structural biologists have already determined hundreds of experimental X-ray, cryo-EM, and NMR structures of proteins and nucleic acids related to this coronavirus, and this number is still growing. To help biomedical researchers, who may not necessarily be experts in structural biology, navigate through the flood of structural models, we have created an online resource, covid19.bioreproducibility.org, that aggregates expert-verified information about SARS-CoV-2-related macromolecular models. In this article, we describe this web resource along with the suite of tools and methodologies used for assessing the structures presented therein.
Assuntos
COVID-19/genética , Internet , SARS-CoV-2/ultraestrutura , Proteínas Virais/ultraestrutura , COVID-19/virologia , Bases de Dados de Compostos Químicos , Humanos , Modelos Estruturais , Pandemias , Pesquisa , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
A supramolecular complex, formed by encapsulation of C60 fullerene in a molecular container built from two resorcin[4]arene rims zipped together by peptidic arms hydrogen bonded into a cylindrical ß-sheet, was studied by X-ray crystallography, solid-state and solution NMR, EPR spectroscopy and differential scanning calorimetry (DSC). The crystal structure, determined at 100â K, reveals that the complex occupies 422 site symmetry, which is compatible with the molecular symmetry of the container but not of the fullerene molecule, which has only 222 symmetry. The additional crystallographic symmetry leads to a complicated but discrete disorder, which could be resolved and modelled using advanced features of the existing refinement software. Solid-state NMR measurements at 184-333â K indicate that the thermal motion of C60 in this temperature range is fast but has different activation energies at different temperatures, which was attributed to a phase transition, which was confirmed by DSC. Intriguingly, the activation energy for reorientations of C60 in the solid state is very similar for the free and encaged molecules. Also, the rotational diffusion coefficients seem to be very similar or even slightly higher for the encaged fullerene compared to the free molecule. We also found that chemical shift anisotropy (CSA) is not the main relaxation mechanism for the 13C spins of C60 in the studied complex.
RESUMO
Superstructure modulation, with violation of the strict short-range periodic order of consecutive crystal unit cells, is well known in small-molecule crystallography but is rarely reported for macromolecular crystals. To date, one modulated macromolecular crystal structure has been successfully determined and refined for a pathogenesis-related class 10 protein from Hypericum perforatum (Hyp-1) crystallized as a complex with 8-anilinonaphthalene-1-sulfonate (ANS) [Sliwiak et al. (2015), Acta Cryst. D71, 829-843]. The commensurate modulation in that case was interpreted in a supercell with sevenfold expansion along c. When crystallized in the additional presence of melatonin, the Hyp-1-ANS complex formed crystals with a different pattern of structure modulation, in which the supercell shows a ninefold expansion of c, manifested in the diffraction pattern by a wave of reflection-intensity modulation with crests at l = 9n and l = 9n ± 4. Despite complicated tetartohedral twinning, the structure has been successfully determined and refined to 2.3â Å resolution using a description in a ninefold-expanded supercell, with 36 independent Hyp-1 chains and 156 ANS ligands populating the three internal (95 ligands) and five interstitial (61 ligands) binding sites. The commensurate superstructures and ligand-binding sites of the two crystal structures are compared, with a discussion of the effect of melatonin on the co-crystallization process.
Assuntos
Naftalenossulfonato de Anilina/química , Hypericum/química , Melatonina/química , Proteínas de Plantas/química , Sítios de Ligação , Cristalografia , Ligantes , Estrutura Molecular , Ligação ProteicaRESUMO
A bifunctional molecule containing both a bidentate binding site for metal ions and an aminopyrimidine H-bond donor-acceptor site has been synthesized, and its properties, in its free and coordinated forms, have been established in solution and in the solid state by analytical and spectroscopic methods as well as by X-ray structure determinations. Structural characterization has shown that it forms a one-dimensional H-bonded polymeric assembly in the solid state, while spectroscopic measurements indicate that it also aggregates in solution. The reaction of a simple Fe(II) salt with this assembly results in the emergence of two geometrical isomers of the complex: [FeL3](BF4)2·9H2O-C1 (meridional, mer) and [FeL3]2(SiF6)(BF4)2·12H2O-C2 (facial, fac). While, complex C1 in the solid state generates a one-dimensional H-bonded polymer involving just two ligands on each Fe center, with the chirality of the complex units alternating along the polymer chain, the structure of complex C2 shows NH···N interactions seen in both the ligand and mer complex (C1) structures to be completely absent. Physicochemical properties of the free and complexed ligand differ substantially.
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A bright spot in the SARS-CoV-2 (CoV-2) coronavirus pandemic has been the immediate mobilization of the biomedical community, working to develop treatments and vaccines for COVID-19. Rational drug design against emerging threats depends on well-established methodology, mainly utilizing X-ray crystallography, to provide accurate structure models of the macromolecular drug targets and of their complexes with candidates for drug development. In the current crisis, the structural biological community has responded by presenting structure models of CoV-2 proteins and depositing them in the Protein Data Bank (PDB), usually without time embargo and before publication. Since the structures from the first-line research are produced in an accelerated mode, there is an elevated chance of mistakes and errors, with the ultimate risk of hindering, rather than speeding up, drug development. In the present work, we have used model-validation metrics and examined the electron density maps for the deposited models of CoV-2 proteins and a sample of related proteins available in the PDB as of April 1, 2020. We present these results with the aim of helping the biomedical community establish a better-validated pool of data. The proteins are divided into groups according to their structure and function. In most cases, no major corrections were necessary. However, in several cases significant revisions in the functionally sensitive area of protein-inhibitor complexes or for bound ions justified correction, re-refinement, and eventually reversioning in the PDB. The re-refined coordinate files and a tool for facilitating model comparisons are available at https://covid-19.bioreproducibility.org. DATABASE: Validated models of CoV-2 proteins are available in a dedicated, publicly accessible web service https://covid-19.bioreproducibility.org.
Assuntos
Enzima de Conversão de Angiotensina 2/química , Antivirais/química , Proteases 3C de Coronavírus/química , Receptores Virais/química , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , Sítios de Ligação , COVID-19/virologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/genética , Proteases 3C de Coronavírus/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Bases de Dados de Proteínas/normas , Desenho de Fármacos , Humanos , Ligantes , Modelos Moleculares , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/antagonistas & inibidores , Receptores Virais/genética , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , TermodinâmicaRESUMO
Tryptophan synthase catalyzes the last two steps of tryptophan biosynthesis in plants, fungi and bacteria. It consists of two protein chains, designated α and ß, encoded by trpA and trpB genes, that function as an αßßα complex. Structural and functional features of tryptophan synthase have been extensively studied, explaining the roles of individual residues in the two active sites in catalysis and allosteric regulation. TrpA serves as a model for protein-folding studies. In 1969, Jackson and Yanofsky observed that the typically monomeric TrpA forms a small population of dimers. Dimerization was postulated to take place through an exchange of structural elements of the monomeric chains, a phenomenon later termed 3D domain swapping. The structural details of the TrpA dimer have remained unknown. Here, the crystal structure of the Streptococcus pneumoniae TrpA homodimer is reported, demonstrating 3D domain swapping in a TIM-barrel fold for the first time. The N-terminal domain comprising the H0-S1-H1-S2 elements is exchanged, while the hinge region corresponds to loop L2 linking strand S2 to helix H2'. The structural elements S2 and L2 carry the catalytic residues Glu52 and Asp63. As the S2 element is part of the swapped domain, the architecture of the catalytic apparatus in the dimer is recreated from two protein chains. The homodimer interface overlaps with the α-ß interface of the tryptophan synthase αßßα heterotetramer, suggesting that the 3D domain-swapped dimer cannot form a complex with the ß subunit. In the crystal, the dimers assemble into a decamer comprising two pentameric rings.
Assuntos
Multimerização Proteica , Streptococcus pneumoniae/enzimologia , Triptofano Sintase/química , Regulação Alostérica , Catálise , Domínio Catalítico , Estrutura Molecular , Domínios Proteicos , Dobramento de ProteínaRESUMO
Stereochemical restraints are commonly used to aid the refinement of macromolecular structures obtained by experimental methods at lower resolution. The standard restraint library for nucleic acids has not been updated for over two decades and needs revision. In this paper, geometrical restraints for nucleic acids sugars are derived using information from high-resolution crystal structures in the Cambridge Structural Database. In contrast to the existing restraints, this work shows that different parts of the sugar moiety form groups of covalent geometry dependent on various chemical and conformational factors, such as the type of ribose or the attached nucleobase, and ring puckering or rotamers of the glycosidic (χ) or side-chain (γ) torsion angles. Moreover, the geometry of the glycosidic link and the endocyclic ribose bond angles are functionally dependent on χ and sugar pucker amplitude (τm), respectively. The proposed restraints have been positively validated against data from the Nucleic Acid Database, compared with an ultrahigh-resolution Z-DNA structure in the Protein Data Bank, and tested by re-refining hundreds of crystal structures in the Protein Data Bank. The conformation-dependent sugar restraints presented in this work are publicly available in REFMAC, PHENIX and SHELXL format through a dedicated RestraintLib web server with an API function.
Assuntos
Ácidos Nucleicos/química , Polinucleotídeos/química , Proteínas/química , Açúcares/química , Cristalografia por Raios X , Bases de Dados de Ácidos Nucleicos , Bases de Dados de Proteínas , Modelos Moleculares , Estrutura Molecular , Ácidos Nucleicos/genética , Conformação Proteica , Proteínas/classificação , SoftwareRESUMO
Retroviral proteases (RPs) are of high interest owing to their crucial role in the maturation process of retroviral particles. RPs are obligatory homodimers, with a pepsin-like active site built around two aspartates (in DTG triads) that activate a water molecule, as the nucleophile, under two flap loops. Mason-Pfizer monkey virus (M-PMV) is unique among retroviruses as its protease is also stable in the monomeric form, as confirmed by an existing crystal structure of a 13â kDa variant of the protein (M-PMV PR) and its previous biochemical characterization. In the present work, two mutants of M-PMV PR, D26N and C7A/D26N/C106A, were crystallized in complex with a peptidomimetic inhibitor and one mutant (D26N) was crystallized without the inhibitor. The crystal structures were solved at resolutions of 1.6, 1.9 and 2.0â Å, respectively. At variance with the previous study, all of the new structures have the canonical dimeric form of retroviral proteases. The protomers within a dimer differ mainly in the flap-loop region, with the most extreme case observed in the apo structure, in which one flap loop is well defined while the other flap loop is not defined by electron density. The presence of the inhibitor molecules in the complex structures was assessed using polder maps, but some details of their conformations remain ambiguous. In all of the presented structures the active site contains a water molecule buried deeply between the Asn26-Thr27-Gly28 triads of the protomers. Such a water molecule is completely unique not only in retropepsins but also in aspartic proteases in general. The C7A and C106A mutations do not influence the conformation of the protein. The Cys106 residue is properly placed at the homodimer interface area for a disulfide cross-link, but the reducing conditions of the crystallization experiment prevented S-S bond formation. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Acta_Cryst_D:S2059798319011355.
Assuntos
Endopeptidases/química , Vírus dos Macacos de Mason-Pfizer/enzimologia , Multimerização Proteica , Estrutura Quaternária de Proteína , Endopeptidases/genética , Mutação , Peptidomiméticos/química , Inibidores de Proteases/químicaRESUMO
Directional self-assembly of uncharged molecules in water is a major challenge in supramolecular chemistry. Herein, it is demonstrated that peptide-based cavitands wrap around a hydrophobic core (fullerene C60 ) by a combination of the hydrophobic effect and hydrogen-bonding interactions to form highly ordered three-component complexes in water that resemble the molten-globule stage of protein folding. The complexes were characterized by DOSY NMR spectroscopy, small-angle X-ray scattering, and circular dichroism, and their structures were confirmed by X-ray crystallography. Enhancement of the CD signals by nearly one order of magnitude and increased hydrolytic stability of hydrazone bonds of the complexes relative to the nonassembled species were observed. In contrast, DMSO and DMSO/water mixtures were found to be highly disintegrative for these complexes. Interestingly, some cavitands can only be synthesized in the presence of the hydrophobic template followed by disassembly of the complexes.