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1.
Nature ; 606(7913): 414-419, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35650436

RESUMO

All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene1. This approach is fundamentally different from the biosynthesis of short-chain (C10-C25) terpenes that are formed from polyisoprenyl diphosphates2-4. In this study, two fungal chimeric class I TrTSs, Talaromyces verruculosus talaropentaene synthase (TvTS) and Macrophomina phaseolina macrophomene synthase (MpMS), were characterized. Both enzymes use dimethylallyl diphosphate and isopentenyl diphosphate or hexaprenyl diphosphate as substrates, representing the first examples, to our knowledge, of non-squalene-dependent triterpene biosynthesis. The cyclization mechanisms of TvTS and MpMS and the absolute configurations of their products were investigated in isotopic labelling experiments. Structural analyses of the terpene cyclase domain of TvTS and full-length MpMS provide detailed insights into their catalytic mechanisms. An AlphaFold2-based screening platform was developed to mine a third TrTS, Colletotrichum gloeosporioides colleterpenol synthase (CgCS). Our findings identify a new enzymatic mechanism for the biosynthesis of triterpenes and enhance understanding of terpene biosynthesis in nature.


Assuntos
Ascomicetos , Talaromyces , Triterpenos , Ascomicetos/enzimologia , Colletotrichum/enzimologia , Ciclização , Difosfatos/metabolismo , Esqualeno/química , Especificidade por Substrato , Talaromyces/enzimologia , Triterpenos/química , Triterpenos/metabolismo
2.
EMBO Rep ; 24(11): e56864, 2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37575008

RESUMO

Kinesin-driven intracellular transport is essential for various cell biological events and thus plays a crucial role in many pathological processes. However, little is known about the molecular basis of the specific and dynamic cargo-binding mechanism of kinesins. Here, an integrated structural analysis of the KIF3/KAP3 and KIF3/KAP3-APC complexes unveils the mechanism by which KIF3/KAP3 can dynamically grasp APC in a two-step manner, which suggests kinesin-cargo recognition dynamics composed of cargo loading, locking, and release. Our finding is the first demonstration of the two-step cargo recognition and stabilization mechanism of kinesins, which provides novel insights into the intracellular trafficking machinery.


Assuntos
Comunicação Celular , Cinesinas , Cinesinas/metabolismo , Transporte Biológico , Microtúbulos/metabolismo
3.
Biochem Biophys Res Commun ; 709: 149855, 2024 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-38579618

RESUMO

P-glycoprotein (P-gp) is an ATP-binding cassette transporter known for its roles in expelling xenobiotic compounds from cells and contributing to cellular drug resistance through multidrug efflux. This mechanism is particularly problematic in cancer cells, where it diminishes the therapeutic efficacy of anticancer drugs. P-gp inhibitors, such as elacridar, have been developed to circumvent the decrease in drug efficacy due to P-gp efflux. An earlier study reported the cryo-EM structure of human P-gp-Fab (MRK-16) complex bound by two elacridar molecules, at a resolution of 3.6 Å. In this study, we have obtained a higher resolution (2.5 Å) structure of the P-gp- Fab (UIC2) complex bound by three elacridar molecules. This finding, which exposes a larger space for compound-binding sites than previously acknowledged, has significant implications for the development of more selective inhibitors and enhances our understanding of the compound recognition mechanism of P-gp.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Acridinas , Tetra-Hidroisoquinolinas , Humanos , Acridinas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Microscopia Crioeletrônica
4.
J Biol Chem ; 298(5): 101827, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35293315

RESUMO

Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is one of the most diverse families of carbohydrate-active enzymes, containing various enzymes that act on α-glycosides. However, the function of some GH31 groups remains unknown, as their enzymatic activity is difficult to estimate due to the low amino acid sequence similarity between characterized and uncharacterized members. Here, we performed a phylogenetic analysis and discovered a protein cluster (GH31_u1) sharing low sequence similarity with the reported GH31 enzymes. Within this cluster, we showed that a GH31_u1 protein from Lactococcus lactis (LlGH31_u1) and its fungal homolog demonstrated hydrolytic activities against nigerose [α-D-Glcp-(1→3)-D-Glc]. The kcat/Km values of LlGH31_u1 against kojibiose and maltose were 13% and 2.1% of that against nigerose, indicating that LlGH31_u1 has a higher specificity to the α-1,3 linkage of nigerose than other characterized GH31 enzymes, including eukaryotic enzymes. Furthermore, the three-dimensional structures of LlGH31_u1 determined using X-ray crystallography and cryogenic electron microscopy revealed that LlGH31_u1 forms a hexamer and has a C-terminal domain comprising four α-helices, suggesting that it contributes to hexamerization. Finally, crystal structures in complex with nigerooligosaccharides and kojibiose along with mutational analysis revealed the active site residues involved in substrate recognition in this enzyme. This study reports the first structure of a bacterial GH31 α-1,3-glucosidase and provides new insight into the substrate specificity of GH31 enzymes and the physiological functions of bacterial and fungal GH31_u1 members.


Assuntos
Bactérias/enzimologia , Fungos/enzimologia , Glucosidases , Glicosídeo Hidrolases , Sequência de Aminoácidos , Bactérias/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Fungos/metabolismo , Glucosidases/metabolismo , Glicosídeo Hidrolases/metabolismo , Lactococcus lactis/enzimologia , Lactococcus lactis/metabolismo , Modelos Moleculares , Oligossacarídeos/metabolismo , Filogenia , Especificidade por Substrato
5.
J Am Chem Soc ; 145(1): 216-223, 2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36541447

RESUMO

Protein nanocages are of increasing interest for use as drug capsules, but the encapsulation and release of drug molecules at appropriate times require the reversible association and dissociation of the nanocages. One promising approach to addressing this challenge is the design of metal-dependent associating proteins. Such designed proteins typically have Cys or His residues at the protein surface for connecting the associating proteins through metal-ion coordination. However, Cys and His residues favor interactions with soft and borderline metal ions, such as Au+ and Zn2+, classified by the hard and soft acids and bases concept, restricting the types of metal ions available to drive association. Here, we show the alkaline earth (AE) metal-dependent association of the recently designed artificial protein nanocage TIP60, which is composed of 60-mer fusion proteins. The introduction of a Glu (hard base) mutation to the fusion protein (K67E mutant) prevented the formation of the 60-mer but formed the expected cage structure in the presence of Ca, Sr, or Ba ions (hard acids). Cryogenic electron microscopy (cryo-EM) analysis indicated a Ba ion at the interface of the subunits. Furthermore, we demonstrated the encapsulation and release of single-stranded DNA molecules using this system. Our results provide insights into the design of AE metal-dependent association and dissociation mechanisms for proteins.


Assuntos
Metais Alcalinoterrosos , Metais , Metais Alcalinoterrosos/química , Metais/química , Íons , DNA de Cadeia Simples
6.
J Am Chem Soc ; 145(29): 16160-16165, 2023 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-37435991

RESUMO

The steric zipper is a common hydrophobic packing structure of peptide side chains that forms between two adjacent ß-sheet layers in amyloid and related fibrils. Although previous studies have revealed that peptide fragments derived from native protein sequences exhibit steric zipper structures, their de novo designs have rarely been studied. Herein, steric zipper structures were artificially constructed in the crystalline state by metal-induced folding and assembly of tetrapeptide fragments Boc-3pa-X1-3pa-X2-OMe (3pa: ß-(3-pyridyl)-l-alanine; X1 and X2: hydrophobic amino acids). Crystallographic studies revealed two types of packing structures, interdigitation and hydrophobic contact, that result in a class 1 steric zipper geometry when the X1 and X2 residues contain alkyl side chains. Furthermore, a class 3 steric zipper geometry was also observed for the first time among any reported steric zippers when using tetrapeptide fragments with (X1, X2) = (Thr, Thr) and (Phe, Leu). The system could also be extended to a knob-hole-type zipper using a pentapeptide sequence.


Assuntos
Elétrons , Nanoestruturas , Raios X , Estrutura Secundária de Proteína , Modelos Moleculares , Peptídeos/química , Amiloide/química , Difração de Raios X
7.
Nucleic Acids Res ; 49(8): 4599-4612, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33849056

RESUMO

The eukaryotic replisome is comprised of three family-B DNA polymerases (Polα, δ and ϵ). Polα forms a stable complex with primase to synthesize short RNA-DNA primers, which are subsequently elongated by Polδ and Polϵ in concert with proliferating cell nuclear antigen (PCNA). In some species of archaea, family-D DNA polymerase (PolD) is the only DNA polymerase essential for cell viability, raising the question of how it alone conducts the bulk of DNA synthesis. We used a hyperthermophilic archaeon, Thermococcus kodakarensis, to demonstrate that PolD connects primase to the archaeal replisome before interacting with PCNA. Whereas PolD stably connects primase to GINS, a component of CMG helicase, cryo-EM analysis indicated a highly flexible PolD-primase complex. A conserved hydrophobic motif at the C-terminus of the DP2 subunit of PolD, a PIP (PCNA-Interacting Peptide) motif, was critical for the interaction with primase. The dissociation of primase was induced by DNA-dependent binding of PCNA to PolD. Point mutations in the alternative PIP-motif of DP2 abrogated the molecular switching that converts the archaeal replicase from de novo to processive synthesis mode.


Assuntos
Proteínas Arqueais/metabolismo , DNA Helicases/metabolismo , DNA Polimerase III/metabolismo , DNA Primase/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Thermococcus/metabolismo , Motivos de Aminoácidos , Proteínas Arqueais/química , Cromatografia em Gel , DNA Helicases/genética , DNA Polimerase III/química , DNA Primase/genética , DNA Primase/metabolismo , Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Mutagênese Sítio-Dirigida , Eletroforese em Gel de Poliacrilamida Nativa , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica , Proteínas Recombinantes , Ressonância de Plasmônio de Superfície , Thermococcus/genética
8.
Nucleic Acids Res ; 49(15): 8934-8946, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34352093

RESUMO

Giardia lamblia is a pathogenic unicellular eukaryotic parasite that causes giardiasis. Its genome encodes the canonical histones H2A, H2B, H3, and H4, which share low amino acid sequence identity with their human orthologues. We determined the structure of the G. lamblia nucleosome core particle (NCP) at 3.6 Å resolution by cryo-electron microscopy. G. lamblia histones form a characteristic NCP, in which the visible 125 base-pair region of the DNA is wrapped in a left-handed supercoil. The acidic patch on the G. lamblia octamer is deeper, due to an insertion extending the H2B α1 helix and L1 loop, and thus cannot bind the LANA acidic patch binding peptide. The DNA and histone regions near the DNA entry-exit sites could not be assigned, suggesting that these regions are asymmetrically flexible in the G. lamblia NCP. Characterization by thermal unfolding in solution revealed that both the H2A-H2B and DNA association with the G. lamblia H3-H4 were weaker than those for human H3-H4. These results demonstrate the uniformity of the histone octamer as the organizing platform for eukaryotic chromatin, but also illustrate the unrecognized capability for large scale sequence variations that enable the adaptability of histone octamer surfaces and confer internal stability.


Assuntos
Microscopia Crioeletrônica , Giardia lamblia/ultraestrutura , Histonas/genética , Nucleossomos/ultraestrutura , Sequência de Aminoácidos/genética , Cromatina/genética , Cromatina/ultraestrutura , Giardia lamblia/genética , Histonas/ultraestrutura , Humanos , Estrutura Molecular , Nucleossomos/genética
9.
J Biol Chem ; 297(3): 101028, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34339732

RESUMO

Ribonuclease P (RNase P) is an endoribonuclease that catalyzes the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). Ribonucleoprotein RNase P and protein-only RNase P (PRORP) in eukaryotes have been extensively studied, but the mechanism by which a prokaryotic nuclease recognizes and cleaves pre-tRNA is unclear. To gain insights into this mechanism, we studied homologs of Aquifex RNase P (HARPs), thought to be enzymes of approximately 23 kDa comprising only this nuclease domain. We determined the cryo-EM structure of Aq880, the first identified HARP enzyme. The structure unexpectedly revealed that Aq880 consists of both the nuclease and protruding helical (PrH) domains. Aq880 monomers assemble into a dimer via the PrH domain. Six dimers form a dodecamer with a left-handed one-turn superhelical structure. The structure also revealed that the active site of Aq880 is analogous to that of eukaryotic PRORPs. The pre-tRNA docking model demonstrated that 5' processing of pre-tRNAs is achieved by two adjacent dimers within the dodecamer. One dimer is responsible for catalysis, and the PrH domains of the other dimer are responsible for pre-tRNA elbow recognition. Our study suggests that HARPs measure an invariant distance from the pre-tRNA elbow to cleave the 5' leader sequence, which is analogous to the mechanism of eukaryotic PRORPs and the ribonucleoprotein RNase P. Collectively, these findings shed light on how different types of RNase P enzymes utilize the same pre-tRNA processing.


Assuntos
Precursores de RNA/metabolismo , RNA de Transferência/metabolismo , Ribonuclease P/química , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Microscopia Crioeletrônica , Dimerização , Simulação de Acoplamento Molecular , Ribonuclease P/metabolismo , Homologia de Sequência de Aminoácidos
10.
J Struct Biol ; 213(3): 107768, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34217801

RESUMO

Cu-containing nitrite reductases (NiRs) are 110 kDa enzymes that play central roles in denitrification. Although the NiRs have been well studied, with over 100 Protein Data Bank entries, such issues as crystal packing, photoreduction, and lack of high pH cases have impeded structural analysis of their catalytic mechanisms. Here we show the cryogenic electron microscopy (cryo-EM) structures of Achromobacter cycloclastes NiR (AcNiR) at pH 6.2 and 8.1. The optimization of 3D-reconstruction parameters achieved 2.99 and 2.85 Å resolution. Comprehensive comparisons with cryo-EM and 56 AcNiR crystal structures suggested crystallographic artifacts in residues 185-215 and His255' due to packing and photoreduction, respectively. We used a newly developed map comparison method to detect structural change around the type 2 Cu site. While the theoretical estimation of coordinate errors of cryo-EM structures remains difficult, combined analysis using X-ray and cryo-EM structures will allow deeper insight into the local structural changes of proteins.


Assuntos
Cobre , Nitrito Redutases , Achromobacter cycloclastes/metabolismo , Catálise , Cobre/química , Microscopia Crioeletrônica/métodos , Nitrito Redutases/química , Nitrito Redutases/metabolismo
11.
Angew Chem Int Ed Engl ; 60(26): 14554-14562, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-33783097

RESUMO

Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and peptidyl carrier protein (PCP) domains. It uses α-methyl-l-serine to synthesize a 4-methyloxazoline ring, probably with another Cy domain in the preceding module FmoA2. Here, we determined the head-to-tail homodimeric structures of FmoA3 by X-ray crystallography (apo-form, with adenylyl-imidodiphosphate and α-methyl-l-seryl-AMP) and cryogenic electron microscopy single particle analysis, and performed site-directed mutagenesis experiments. The data revealed that α-methyl-l-serine can be accommodated in the active site because of the extra space around Ala688. The Cy domains of FmoA2 and FmoA3 catalyze peptide bond formation and heterocyclization, respectively. FmoA3's Cy domain seems to lose its donor PCP binding activity. The collective data support a proposed catalytic cycle of FmoA3.


Assuntos
Oxazóis/metabolismo , Peptídeo Sintases/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Oxazóis/química , Peptídeo Sintases/química
12.
Protein Expr Purif ; 133: 50-56, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28259734

RESUMO

In vitro transcription systems have been utilized to elucidate detailed mechanisms of transcription. Purified RNA polymerase II (pol II) and general transcription factors (GTFs) are required for the in vitro reconstitution of eukaryotic transcription systems. Among GTFs, TFIID and TFIIA play critical roles in the early stage of transcription initiation; TFIID first binds to the DNA in transcription initiation and TFIIA regulates TFIID's DNA binding activity. Despite the important roles of TFIIA, the time-consuming steps required to purify it, such as denaturing and refolding, have hampered the preparation of in vitro transcription systems. Here, we report an improved method for soluble expression and rapid purification of yeast TFIIA. The subunits of TFIIA, TOA1 and TOA2, were bacterially expressed as fusion proteins in soluble form, then processed by the PreScission protease and co-purified. TFIIA's heterodimer formation was confirmed by size exclusion chromatography-multiangle light scattering (SEC-MALS). The hydrodynamic radius (Rh) and radius of gyration (Rg) were measured by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS), respectively. The Rg/Rh value implied that the intrinsically disordered region of TOA1 might not have an extended structure in solution. Our improved method provides highly purified TFIIA of sufficient quality for biochemical, biophysical, and structural analyses of eukaryotic transcription systems.


Assuntos
Escherichia coli/metabolismo , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIA , Escherichia coli/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Solubilidade , Fator de Transcrição TFIIA/biossíntese , Fator de Transcrição TFIIA/química , Fator de Transcrição TFIIA/genética
13.
Chem Commun (Camb) ; 60(34): 4605-4608, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38586927

RESUMO

A split-protein system is a simple approach to introduce new termini which are useful as modification sites in protein engineering, but has been adapted mainly for monomeric proteins. Here we demonstrate the design of split subunits of the 60-mer artificial fusion-protein nanocage TIP60. The subunit fragments successfully reformed the cage structure in the same manner as prior to splitting. One of the newly introduced terminals at the interior surface can be modified using a tag peptide and green fluorescent protein. Therefore, the termini could serve as a versatile modification site for incorporating a wide variety of functional peptides and proteins.

14.
Structure ; 2024 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-39321803

RESUMO

During drug discovery, it is crucial to exclude compounds with toxic effects. The human ether-à-go-go-related gene (hERG) channel is essential for maintaining cardiac repolarization and is a critical target in drug safety evaluation due to its role in drug-induced arrhythmias. Inhibition of the hERG channel can lead to severe cardiac issues, including Torsades de Pointes tachycardia. Understanding hERG inhibition mechanisms is essential to avoid these toxicities. Several structural studies have elucidated the interactions between inhibitors and hERG. However, orientation and resolution issues have so far limited detailed insights. Here, we used digitonin to analyze the apo state of hERG, which resolved orientation issues and improved the resolution. We determined the structure of hERG bound to astemizole, showing a clear map in the pore pathway. Using this strategy, we also analyzed the binding modes of E-4031 and pimozide. These insights into inhibitor interactions with hERG may aid safer drug design and enhance cardiac safety.

15.
Org Lett ; 26(20): 4302-4307, 2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38728049

RESUMO

A plant used in an Indonesian traditional herbal medicine as a diabetes treatment and known locally as "Jampu Salo" was collected on Sulawesi Island, Indonesia. It was identified as Syzygium oblanceolatum (C. B. Rob.) Merr. (Myrtaceae) and found for the first time in Sulawesi; it was previously reported only in the eastern Philippines and Borneo. A phytochemical study of S. oblanceolatum led to the isolation of three unprecedented meroterpenoids, syzygioblanes A-C (1-3, respectively). These compounds might be biosynthesized through [4+2] cycloaddition of various germacrane-based cyclic sesquiterpenoids with the flavone desmethoxymatteucinol to form a spiro skeleton. The unique and complex structures were elucidated by microcrystal electron diffraction analysis in addition to general analytical techniques such as high-resolution mass spectrometry, various nuclear magnetic resonance methods, and infrared spectroscopy. Synchrotron X-ray diffraction and calculations of electronic circular dichroism spectra helped to determine the absolute configurations. The newly isolated compounds exhibited collateral sensitivity to more strongly inhibit the growth of a multidrug resistant tumor cell line compared to a chemosensitive tumor cell line.


Assuntos
Sesquiterpenos , Syzygium , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Sesquiterpenos/isolamento & purificação , Syzygium/química , Estrutura Molecular , Indonésia , Humanos , Flavanonas/química , Flavanonas/farmacologia , Flavanonas/isolamento & purificação , Medicina Tradicional , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais , Linhagem Celular Tumoral
16.
Proc Natl Acad Sci U S A ; 107(18): 8153-8, 2010 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-20393127

RESUMO

Nucleosomes around the promoter region are disassembled for transcription in response to various signals, such as acetylation and methylation of histones. Although the interactions between histone-acetylation-recognizing bromodomains and factors involved in nucleosome disassembly have been reported, no structural basis connecting histone modifications and nucleosome disassembly has been obtained. Here, we determined at 3.3 A resolution the crystal structure of histone chaperone cell cycle gene 1 (CCG1) interacting factor A/antisilencing function 1 (CIA/ASF1) in complex with the double bromodomain in the CCG1/TAF1/TAF(II)250 subunit of transcription factor IID. Structural, biochemical, and biological studies suggested that interaction between double bromodomain and CIA/ASF1 is required for their colocalization, histone eviction, and pol II entry at active promoter regions. Furthermore, the present crystal structure has characteristics that can connect histone acetylation and CIA/ASF1-mediated histone eviction. These findings suggest that the molecular complex between CIA/ASF1 and the double bromodomain plays a key role in site-specific histone eviction at active promoter regions. The model we propose here is the initial structure-based model of the biological signaling from histone modifications to structural change of the nucleosome (hi-MOST model).


Assuntos
Proteínas de Ciclo Celular/química , Histonas/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Histonas/metabolismo , Humanos , Modelos Moleculares , Chaperonas Moleculares , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
17.
ACS Omega ; 8(12): 11583-11587, 2023 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-37008077

RESUMO

Squalene bearing 18-crown-6 was synthesized and formed unilamellar vesicles with a membrane thickness of about 6 nm and a diameter of about 0.32 µm. In the wake of the recognition of alkali metal cations, squalene unilamellar vesicles become larger as multilamellar vesicles or smaller while maintaining unilamellar vesicles depending on cations.

18.
Nat Commun ; 14(1): 730, 2023 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-36792917

RESUMO

Prasiola crispa, an aerial green alga, forms layered colonies under the severe terrestrial conditions of Antarctica. Since only far-red light is available at a deep layer of the colony, P. crispa has evolved a molecular system for photosystem II (PSII) excitation using far-red light with uphill energy transfer. However, the molecular basis underlying this system remains elusive. Here, we purified a light-harvesting chlorophyll (Chl)-binding protein complex from P. crispa (Pc-frLHC) that excites PSII with far-red light and revealed its ring-shaped structure with undecameric 11-fold symmetry at 3.13 Šresolution. The primary structure suggests that Pc-frLHC evolved from LHCI rather than LHCII. The circular arrangement of the Pc-frLHC subunits is unique among eukaryote LHCs and forms unprecedented Chl pentamers at every subunit‒subunit interface near the excitation energy exit sites. The Chl pentamers probably contribute to far-red light absorption. Pc-frLHC's unique Chl arrangement likely promotes PSII excitation with entropy-driven uphill excitation energy transfer.


Assuntos
Fotossíntese , Complexo de Proteína do Fotossistema I , Regiões Antárticas , Complexo de Proteína do Fotossistema I/metabolismo , Tilacoides/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Transferência de Energia , Complexos de Proteínas Captadores de Luz/metabolismo , Clorofila/metabolismo
19.
FEBS J ; 290(2): 412-427, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36007953

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S protein) is highly N-glycosylated, and a "glycan shield" is formed to limit the access of other molecules; however, a small open area coincides with the interface to the host's receptor and also neutralising antibodies. Most of the variants of concern have mutations in this area, which could reduce the efficacy of existing antibodies. In contrast, N-glycosylation sites are relatively invariant, and some are essential for infection. Here, we observed that the S proteins of the ancestral (Wuhan) and Omicron strains bind with Pholiota squarrosa lectin (PhoSL), a 40-amino-acid chemically synthesised peptide specific to core-fucosylated N-glycans. The affinities were at a low nanomolar level, which were ~ 1000-fold stronger than those between PhoSL and the core-fucosylated N-glycans at the micromolar level. We demonstrated that PhoSL inhibited infection by both strains at similar submicromolar levels, suggesting its broad-spectrum effect on SARS-CoV-2 variants. Cryogenic electron microscopy revealed that PhoSL caused an aggregation of the S protein, which was likely due to the multivalence of both the trimeric PhoSL and S protein. This characteristic is likely relevant to the inhibitory mechanism. Structural modelling of the PhoSL-S protein complex indicated that PhoSL was in contact with the amino acids of the S protein, which explains the enhanced affinity with S protein and also indicates the significant potential for developing specific binders by the engineering of PhoSL.


Assuntos
Antivirais , Lectinas , SARS-CoV-2 , Humanos , COVID-19 , Fucose/química , Lectinas/farmacologia , Polissacarídeos/química , SARS-CoV-2/efeitos dos fármacos , Antivirais/farmacologia , Pholiota/química
20.
Nat Commun ; 13(1): 5097, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-36042318

RESUMO

Cyanophycin is a natural biopolymer consisting of equimolar amounts of aspartate and arginine as the backbone and branched sidechain, respectively. It is produced by a single enzyme, cyanophycin synthetase (CphA1), and accumulates as a nitrogen reservoir during N2 fixation by most cyanobacteria. A recent structural study showed that three constituent domains of CphA1 function as two distinct catalytic sites and an oligomerization interface in cyanophycin synthesis. However, it remains unclear how the ATP-dependent addition of aspartate to cyanophycin is initiated at the catalytic site of the glutathione synthetase-like domain. Here, we report the cryogenic electron microscopy structures of CphA1, including a complex with aspartate, cyanophycin primer peptide, and ATP analog. These structures reveal the aspartate binding mode and phosphate-binding loop movement to the active site required for the reaction. Furthermore, structural and mutational data show a potential role of protein dynamics in the catalytic efficiency of the arginine condensation reaction.


Assuntos
Ácido Aspártico , Cianobactérias , Trifosfato de Adenosina/metabolismo , Arginina/metabolismo , Ácido Aspártico/metabolismo , Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Peptídeo Sintases/metabolismo , Proteínas de Plantas/metabolismo , Polimerização
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