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FASEB J ; 36 Suppl 12022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35552858


Secretion is an essential cellular function in which proteins and lipids are transported from the Golgi to the plasma membrane via secretory vesicles. The formation of secretory vesicles is regulated by activation of the Rab GTPase Rab11. The activation of Rab GTPases at membrane compartments is dependent on the localization of their specific Guanosine Exchange Factors (GEFs), which catalyze the exchange of GDP with GTP and stabilize the active GTPase on the membrane. The multisubunit TRAPPII complex acts as Rab11 GEF and recruits it to the trans-Golgi network (TGN) membrane. More than 60 different Rabs are present in mammals and budding yeast has 11 different Rabs. Specific localization of each of these Rabs to different membrane compartments is important for determination of organelle identity and membrane organization of these compartments. While multiple GTPases and GEFs localize at the TGN, the mechanistic details of how TRAPPII specifically activates Rab11, and not other GTPases, is unknown. Moreover, another related GEF, the TRAPPIII complex, shares the same catalytic subunits with TRAPPII and yet activates another GTPase, Rab1. A steric gating mechanism has been proposed in which TRAPPII selects against Rab1 based on the length of the C-terminal hypervariable domain. We determined the structure of yeast TRAPPII bound to its substrate Rab11 using cryo-EM at an overall resolution of 3.7Å. Analysis of the atomic structure has revealed specific interactions between Rab11 and the TRAPPII complex, and how TRAPPII interacts with the membrane surface. We tested the physiological relevance of these observed interactions using in vivo functional studies and in vitro reconstitution of nucleotide exchange. We show that the TRAPPII subunit Trs130 provides a 'leg' which lifts the TRAPP catalytic site above the membrane, thereby preventing access to the catalytic site by Rab1. TRAPPIII complex selects against Rab11 based on repulsive interactions with the catalytic subunits. We also show an alternative conformation of the TRAPPII complex which may facilitate access of Rab11 to the TRAPP catalytic site. Taken together, these experiments reveal the mechanism of specific activation of Rab11 on the membrane by TRAPPII, a key step for initiation of secretory vesicle formation. As Rab11 and the TRAPPII complex are conserved throughout eukaryotes, this mechanism may be widely conserved across all eukaryotic secretory systems.

Sci Adv ; 8(19): eabn7446, 2022 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-35559680


Rab1 and Rab11 are essential regulators of the eukaryotic secretory and endocytic recycling pathways. The transport protein particle (TRAPP) complexes activate these guanosine triphosphatases via nucleotide exchange using a shared set of core subunits. The basal specificity of the TRAPP core is toward Rab1, yet the TRAPPII complex is specific for Rab11. A steric gating mechanism has been proposed to explain TRAPPII counterselection against Rab1. Here, we present cryo-electron microscopy structures of the 22-subunit TRAPPII complex from budding yeast, including a TRAPPII-Rab11 nucleotide exchange intermediate. The Trs130 subunit provides a "leg" that positions the active site distal to the membrane surface, and this leg is required for steric gating. The related TRAPPIII complex is unable to activate Rab11 because of a repulsive interaction, which TRAPPII surmounts using the Trs120 subunit as a "lid" to enclose the active site. TRAPPII also adopts an open conformation enabling Rab11 to access and exit from the active site chamber.

Science ; 374(6573): eabm4805, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34762488


Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning­based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.

Aprendizado Profundo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mapeamento de Interação de Proteínas , Proteoma/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Aciltransferases/química , Aciltransferases/metabolismo , Segregação de Cromossomos , Biologia Computacional , Simulação por Computador , Reparo do DNA , Evolução Molecular , Recombinação Homóloga , Ligases/química , Ligases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Biossíntese de Proteínas , Conformação Proteica , Mapas de Interação de Proteínas , Proteoma/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Ubiquitina/química , Ubiquitina/metabolismo
Science ; 374(6568): 723-729, 2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34735234


Type I modular polyketide synthases are homodimeric multidomain assembly line enzymes that synthesize a variety of polyketide natural products by performing polyketide chain extension and ß-keto group modification reactions. We determined the 2.4-angstrom-resolution x-ray crystal structure and the 3.1-angstrom-resolution cryo­electron microscopy structure of the Lsd14 polyketide synthase, stalled at the transacylation and condensation steps, respectively. These structures revealed how the constituent domains are positioned relative to each other, how they rearrange depending on the step in the reaction cycle, and the specific interactions formed between the domains. Like the evolutionarily related mammalian fatty acid synthase, Lsd14 contains two reaction chambers, but only one chamber in Lsd14 has the full complement of catalytic domains, indicating that only one chamber produces the polyketide product at any given time.

Policetídeo Sintases/química , Streptomyces/enzimologia , Proteína de Transporte de Acila/química , Acilação , Aciltransferases/química , Domínio Catalítico , Microscopia Crioeletrônica , Cristalografia por Raios X , Hidroliases/química , Hidroliases/metabolismo , Hidroliases/ultraestrutura , Lasalocida/biossíntese , Modelos Moleculares , Policetídeo Sintases/metabolismo , Policetídeo Sintases/ultraestrutura , Conformação Proteica , Domínios Proteicos , Multimerização Proteica
Nature ; 598(7881): 515-520, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34588691


Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array1. Spacer insertion is carried out by the Cas1-Cas2 integrase complex2-4. A substantial fraction of CRISPR-Cas systems use a Fe-S cluster containing Cas4 nuclease to ensure that spacers are acquired from DNA flanked by a protospacer adjacent motif (PAM)5,6 and inserted into the CRISPR array unidirectionally, so that the transcribed CRISPR RNA can guide target searching in a PAM-dependent manner. Here we provide a high-resolution mechanistic explanation for the Cas4-assisted PAM selection, spacer biogenesis and directional integration by type I-G CRISPR in Geobacter sulfurreducens, in which Cas4 is naturally fused with Cas1, forming Cas4/Cas1. During biogenesis, only DNA duplexes possessing a PAM-embedded 3'-overhang trigger Cas4/Cas1-Cas2 assembly. During this process, the PAM overhang is specifically recognized and sequestered, but is not cleaved by Cas4. This 'molecular constipation' prevents the PAM-side prespacer from participating in integration. Lacking such sequestration, the non-PAM overhang is trimmed by host nucleases and integrated to the leader-side CRISPR repeat. Half-integration subsequently triggers PAM cleavage and Cas4 dissociation, allowing spacer-side integration. Overall, the intricate molecular interaction between Cas4 and Cas1-Cas2 selects PAM-containing prespacers for integration and couples the timing of PAM processing with the stepwise integration to establish directionality.

Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Endonucleases/metabolismo , Geobacter/enzimologia , Bases de Dados Genéticas , Modelos Moleculares , Conformação Molecular , Motivos de Nucleotídeos
ACS Chem Biol ; 13(11): 3072-3077, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30354045


During polyketide and fatty acid biosynthesis, the growing acyl chain is attached to the acyl carrier protein via a thioester linkage. The acyl carrier protein interacts with other enzymes that perform chain elongation and chain modification on the bound acyl chain. Most type I polyketide synthases and fatty acid synthases contain only one acyl carrier protein. However, polyunsaturated fatty acid synthases from deep-sea bacteria contain anywhere from two to nine acyl carrier proteins. Recent studies have shown that this tandem acyl carrier protein feature is responsible for the unusually high fatty acid production rate of deep-sea bacteria. To investigate if a similar strategy can be used to increase the production rate of type I polyketide synthases, a 3×ACP domain was rationally designed and genetically installed in module 6 of 6-deoxyerythronolide B synthase, which is a prototypical type I modular polyketide synthase that naturally harbors just one acyl carrier protein. This modification resulted in an ∼2.5-fold increase in the total amount of polyketide produced in vitro, demonstrating that installing a tandem acyl carrier domain in a type I polyketide synthase is an effective strategy for enhancing the rate of polyketide natural product biosynthesis.

Proteína de Transporte de Acila/química , Policetídeo Sintases/química , Policetídeos/síntese química , Domínios Proteicos , Proteína de Transporte de Acila/genética , Sequência de Aminoácidos , Escherichia coli/genética , Cinética , Policetídeo Sintases/genética , Domínios Proteicos/genética , Engenharia de Proteínas/métodos , Saccharopolyspora/enzimologia