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1.
Proc Natl Acad Sci U S A ; 121(11): e2318320121, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38457518

RESUMO

Coordinated carbon and nitrogen metabolism is crucial for bacteria living in the fluctuating environments. Intracellular carbon and nitrogen homeostasis is maintained by a sophisticated network, in which the widespread signaling protein PII acts as a major regulatory hub. In cyanobacteria, PII was proposed to regulate the nitrate uptake by an ABC (ATP-binding cassette)-type nitrate transporter NrtABCD, in which the nucleotide-binding domain of NrtC is fused with a C-terminal regulatory domain (CRD). Here, we solved three cryoelectron microscopy structures of NrtBCD, bound to nitrate, ATP, and PII, respectively. Structural and biochemical analyses enable us to identify the key residues that form a hydrophobic and a hydrophilic cavity along the substrate translocation channel. The core structure of PII, but not the canonical T-loop, binds to NrtC and stabilizes the CRD, making it visible in the complex structure, narrows the substrate translocation channel in NrtB, and ultimately locks NrtBCD at an inhibited inward-facing conformation. Based on these results and previous reports, we propose a putative transport cycle driven by NrtABCD, which is allosterically inhibited by PII in response to the cellular level of 2-oxoglutarate. Our findings provide a distinct regulatory mechanism of ABC transporter via asymmetrically binding to a signaling protein.


Assuntos
Cianobactérias , Transportadores de Nitrato , Nitratos/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Alostérica , Microscopia Crioeletrônica , Cianobactérias/metabolismo , Trifosfato de Adenosina/metabolismo , Nitrogênio/metabolismo , Carbono/metabolismo , Proteínas PII Reguladoras de Nitrogênio/genética , Proteínas PII Reguladoras de Nitrogênio/metabolismo
2.
Structure ; 32(1): 1-2, 2024 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-38181725

RESUMO

In this issue of Structure, Subramanian et al. present the cryo-EM structure of Shigella podophage HRP29, which possesses a T7-like tail complex surrounded by six P22/Sf6-like tailspikes and two unique decoration proteins. These colorful masks of HRP29 record the frequent events of horizontal gene transfer during evolution.


Assuntos
Bacteriófagos , Shigella , Shigella/virologia , Bacteriófagos/ultraestrutura
3.
Nat Commun ; 15(1): 2654, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38531972

RESUMO

The Myoviridae cyanophage A-1(L) specifically infects the model cyanobacteria Anabaena sp. PCC 7120. Following our recent report on the capsid structure of A-1(L), here we present the high-resolution cryo-EM structure of its intact tail machine including the neck, tail and attached fibers. Besides the dodecameric portal, the neck contains a canonical hexamer connected to a unique pentadecamer that anchors five extended bead-chain-like neck fibers. The 1045-Å-long contractile tail is composed of a helical bundle of tape measure proteins surrounded by a layer of tube proteins and a layer of sheath proteins, ended with a five-component baseplate. The six long and six short tail fibers are folded back pairwise, each with one end anchoring to the baseplate and the distal end pointing to the capsid. Structural analysis combined with biochemical assays further enable us to identify the dual hydrolytic activities of the baseplate hub, in addition to two host receptor binding domains in the tail fibers. Moreover, the structure of the intact A-1(L) also helps us to reannotate its genome. These findings will facilitate the application of A-1(L) as a chassis cyanophage in synthetic biology.


Assuntos
Anabaena , Myoviridae , Proteínas do Capsídeo/química , Capsídeo
4.
Nat Commun ; 15(1): 1061, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38316776

RESUMO

Bilirubin is mainly generated from the breakdown of heme when red blood cells reach the end of their lifespan. Accumulation of bilirubin in human body usually leads to various disorders, including jaundice and liver disease. Bilirubin is conjugated in hepatocytes and excreted to bile duct via the ATP-binding cassette transporter ABCC2, dysfunction of which would lead to Dubin-Johnson syndrome. Here we determine the structures of ABCC2 in the apo, substrate-bound and ATP/ADP-bound forms using the cryo-electron microscopy, exhibiting a full transporter with a regulatory (R) domain inserted between the two half modules. Combined with substrate-stimulated ATPase and transport activity assays, structural analysis enables us to figure out transport cycle of ABCC2 with the R domain adopting various conformations. At the rest state, the R domain binding to the translocation cavity functions as an affinity filter that allows the substrates of high affinity to be transported in priority. Upon substrate binding, the R domain is expelled from the cavity and docks to the lateral of transmembrane domain following ATP hydrolysis. Our findings provide structural insights into a transport mechanism of ABC transporters finely tuned by the R domain.


Assuntos
Bilirrubina , Proteína 2 Associada à Farmacorresistência Múltipla , Humanos , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Proteína 2 Associada à Farmacorresistência Múltipla/genética , Proteína 2 Associada à Farmacorresistência Múltipla/metabolismo
5.
Structure ; 32(4): 440-452.e4, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38340732

RESUMO

Nonribosomal peptide synthetases (NRPSs) are large multidomain enzymes for the synthesis of a variety of bioactive peptides in a modular and pipelined fashion. Here, we investigated how the condensation (C) domain and the adenylation (A) domain cooperate with each other for the efficient catalytic activity in microcystin NRPS modules. We solved two crystal structures of the microcystin NRPS modules, representing two different conformations in the NRPS catalytic cycle. Our data reveal that the dynamic interaction between the C and the A domains in these modules is mediated by the conserved "RXGR" motif, and this interaction is important for the adenylation activity. Furthermore, the "RXGR" motif-mediated dynamic interaction and its functional regulation are prevalent in different NRPSs modules possessing both the A and the C domains. This study provides new insights into the catalytic mechanism of NRPSs and their engineering strategy for synthetic peptides with different structures and properties.


Assuntos
Microcistinas , Peptídeo Sintases , Peptídeo Sintases/química , Conformação Molecular , Peptídeos
6.
Nat Plants ; 10(4): 661-672, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38589484

RESUMO

Carboxysomes are bacterial microcompartments that encapsulate the enzymes RuBisCO and carbonic anhydrase in a proteinaceous shell to enhance the efficiency of photosynthetic carbon fixation. The self-assembly principles of the intact carboxysome remain elusive. Here we purified α-carboxysomes from Prochlorococcus and examined their intact structures using single-particle cryo-electron microscopy to solve the basic principles of their shell construction and internal RuBisCO organization. The 4.2 Å icosahedral-like shell structure reveals 24 CsoS1 hexamers on each facet and one CsoS4A pentamer at each vertex. RuBisCOs are organized into three concentric layers within the shell, consisting of 72, 32 and up to 4 RuBisCOs at the outer, middle and inner layers, respectively. We uniquely show how full-length and shorter forms of the scaffolding protein CsoS2 bind to the inner surface of the shell via repetitive motifs in the middle and C-terminal regions. Combined with previous reports, we propose a concomitant 'outside-in' assembly principle of α-carboxysomes: the inner surface of the self-assembled shell is reinforced by the middle and C-terminal motifs of the scaffolding protein, while the free N-terminal motifs cluster to recruit RuBisCO in concentric, three-layered spherical arrangements. These new insights into the coordinated assembly of α-carboxysomes may guide the rational design and repurposing of carboxysome structures for improving plant photosynthetic efficiency.

7.
Microbiol Spectr ; 12(6): e0029824, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38695606

RESUMO

The cyanosiphophage Mic1 specifically infects the bloom-forming Microcystis aeruginosa FACHB 1339 from Lake Chaohu, China. Previous genomic analysis showed that its 92,627 bp double-stranded DNA genome consists of 98 putative open reading frames, 63% of which are of unknown function. Here, we investigated the transcriptome dynamics of Mic1 and its host using RNA sequencing. In the early, middle, and late phases of the 10 h lytic cycle, the Mic1 genes are sequentially expressed and could be further temporally grouped into two distinct clusters in each phase. Notably, six early genes, including gp49 that encodes a TnpB-like transposase, immediately reach the highest transcriptional level in half an hour, representing a pioneer cluster that rapidly regulates and redirects host metabolism toward the phage. An in-depth analysis of the host transcriptomic profile in response to Mic1 infection revealed significant upregulation of a polyketide synthase pathway and a type III-B CRISPR system, accompanied by moderate downregulation of the photosynthesis and key metabolism pathways. The constant increase of phage transcripts and relatively low replacement rate over the host transcripts indicated that Mic1 utilizes a unique strategy to gradually take over a small portion of host metabolism pathways after infection. In addition, genomic analysis of a less-infective Mic1 and a Mic1-resistant host strain further confirmed their dynamic interplay and coevolution via the frequent horizontal gene transfer. These findings provide insights into the mutual benefit and symbiosis of the highly polymorphic cyanobacteria M. aeruginosa and cyanophages. IMPORTANCE: The highly polymorphic Microcystis aeruginosa is one of the predominant bloom-forming cyanobacteria in eutrophic freshwater bodies and is infected by diverse and abundant cyanophages. The presence of a large number of defense systems in M. aeruginosa genome suggests a dynamic interplay and coevolution with the cyanophages. In this study, we investigated the temporal gene expression pattern of Mic1 after infection and the corresponding transcriptional responses of its host. Moreover, the identification of a less-infective Mic1 and a Mic1-resistant host strain provided the evolved genes in the phage-host coevolution during the multiple-generation cultivation in the laboratory. Our findings enrich the knowledge on the interplay and coevolution of M. aeruginosa and its cyanophages and lay the foundation for the future application of cyanophage as a potential eco-friendly and bio-safe agent in controlling the succession of harmful cyanobacterial blooms.


Assuntos
Bacteriófagos , Microcystis , Microcystis/virologia , Microcystis/genética , Microcystis/metabolismo , Bacteriófagos/genética , Bacteriófagos/fisiologia , China , Transcriptoma , Lagos/microbiologia , Lagos/virologia , Genoma Viral/genética , Evolução Molecular
8.
Nat Struct Mol Biol ; 31(2): 293-299, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38177666

RESUMO

Transcription factors respond to multilevel stimuli and co-occupy promoter regions of target genes to activate RNA polymerase (RNAP) in a cooperative manner. To decipher the molecular mechanism, here we report two cryo-electron microscopy structures of Anabaena transcription activation complexes (TACs): NtcA-TAC composed of RNAP holoenzyme, promoter and a global activator NtcA, and NtcA-NtcB-TAC comprising an extra context-specific regulator, NtcB. Structural analysis showed that NtcA binding makes the promoter DNA bend by ∼50°, which facilitates RNAP to contact NtcB at the distal upstream NtcB box. The sequential binding of NtcA and NtcB induces looping back of promoter DNA towards RNAP, enabling the assembly of a fully activated TAC bound with two activators. Together with biochemical assays, we propose a 'DNA looping' mechanism of cooperative transcription activation in bacteria.


Assuntos
Proteínas de Bactérias , Compostos Nitrosos , Tiazolidinas , Tiocianatos , Transativadores , Transativadores/genética , Ativação Transcricional , Microscopia Crioeletrônica , Sequência de Bases , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Gênica , Regulação Bacteriana da Expressão Gênica
9.
Structure ; 32(8): 1110-1120.e4, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-38823379

RESUMO

Carboxysomes are large self-assembled microcompartments that serve as the central machinery of a CO2-concentrating mechanism (CCM). Biogenesis of carboxysome requires the fine organization of thousands of individual proteins; however, the packaging pattern of internal RuBisCOs remains largely unknown. Here we purified the intact ß-carboxysomes from Synechococcus elongatus PCC 7942 and identified the protein components by mass spectrometry. Cryo-electron tomography combined with subtomogram averaging revealed the general organization pattern of internal RuBisCOs, in which the adjacent RuBisCOs are mainly arranged in three distinct manners: head-to-head, head-to-side, and side-by-side. The RuBisCOs in the outermost layer are regularly aligned along the shell, the majority of which directly interact with the shell. Moreover, statistical analysis enabled us to propose an ideal packaging model of RuBisCOs in the ß-carboxysome. These results provide new insights into the biogenesis of ß-carboxysomes and also advance our understanding of the efficient carbon fixation functionality of carboxysomes.


Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Ribulose-Bifosfato Carboxilase , Synechococcus , Synechococcus/metabolismo , Tomografia com Microscopia Eletrônica/métodos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Ribulose-Bifosfato Carboxilase/metabolismo , Ribulose-Bifosfato Carboxilase/química , Microscopia Crioeletrônica/métodos , Modelos Moleculares
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