scholarly journals PE5-PPE4-EspG3 trimer structure from mycobacterial ESX-3 secretion system gives insight into cognate substrate recognition by ESX systems

2020 ◽  
Author(s):  
Zachary A. Williamson ◽  
Catherine T. Chaton ◽  
William A. Ciocca ◽  
Natalia Korotkova ◽  
Konstantin V. Korotkov
Keyword(s):  
Cell Envelope ◽  
Substrate Recognition ◽  
Secretion System ◽  
Multiple Factors ◽  
Type Vii Secretion ◽  
Shape Complementarity ◽  
Numerous Type ◽  
Unique Shape ◽  
Insight Into

ABSTRACTMycobacterium tuberculosis (Mtb) has evolved numerous type VII secretion (ESX) systems to secrete multiple factors important for both growth and virulence across their cell envelope. Three such systems; ESX-1, ESX-3, and ESX-5; have been shown to each secrete a unique set of substrates. A large class of these substrates secreted by these three systems are the PE and PPE families of proteins. Proper secretion of the PE-PPE proteins requires the presence of EspG, with each system encoding its own unique copy. There is no cross-talk between any of the ESX systems and how each EspG is recognizing its subset of PE-PPE proteins is currently unknown. The only current structural characterization of PE-PPE-EspG trimers is from the ESX-5 system. Here we present the crystal structure of the PE5mt-PPE4mt-EspG3mm trimer, from the ESX-3 system. Our trimer reveals that EspG3mm interacts exclusively with PPE4mt in a similar manner to EspG5, shielding the hydrophobic tip of PPE4mt from solvent. The C-terminal helical domain of EspG3mm is dynamic, alternating between an ‘open’ and ‘closed’ form, and this movement is likely functionally relevant in the unloading of PE-PPE heterodimers at the secretion machinery. In contrast to the previously solved ESX-5 trimers, the PE-PPE heterodimer of our ESX-3 trimer is interacting with it’s chaperone at a drastically different angle, and presents different faces of the PPE protein to the chaperone. We conclude that the PPE-EspG interface from each ESX system has a unique shape complementarity that allows each EspG to discriminate amongst non-cognate PE-PPE pairs.

scholarly journals PE5–PPE4–EspG3 heterotrimer structure from mycobacterial ESX-3 secretion system gives insight into cognate substrate recognition by ESX systems

2020 ◽  
Vol 295 (36) ◽  
pp. 12706-12715
Author(s):  
Zachary A. Williamson ◽  
Catherine T. Chaton ◽  
William A. Ciocca ◽  
Natalia Korotkova ◽  
Konstantin V. Korotkov
Keyword(s):  
Cell Envelope ◽  
Substrate Recognition ◽  
Secretion System ◽  
Multiple Factors ◽  
Type Vii Secretion ◽  
Shape Complementarity ◽  
Numerous Type ◽  
Unique Shape ◽  
Insight Into

Mycobacterium tuberculosis has evolved numerous type VII secretion (ESX) systems to secrete multiple factors important for both growth and virulence across their cell envelope. ESX-1, ESX-3, and ESX-5 systems have been shown to each secrete a distinct set of substrates, including PE and PPE families of proteins, named for conserved Pro-Glu and Pro-Pro-Glu motifs in their N termini. Proper secretion of the PE–PPE proteins requires the presence of EspG, with each system encoding its own unique copy. There is no cross-talk between any of the ESX systems, and how each EspG recognizes its subset of PE–PPE proteins is currently unknown. The only current structural characterization of PE–PPE–EspG heterotrimers is from the ESX-5 system. Here we present the crystal structure of the PE5mt–PPE4mt–EspG3mm heterotrimer from the ESX-3 system. Our heterotrimer reveals that EspG3mm interacts exclusively with PPE4mt in a similar manner to EspG5, shielding the hydrophobic tip of PPE4mt from solvent. The C-terminal helical domain of EspG3mm is dynamic, alternating between “open” and “closed” forms, and this movement is likely functionally relevant in the unloading of PE–PPE heterodimers at the secretion machinery. In contrast to the previously solved ESX-5 heterotrimers, the PE–PPE heterodimer of our ESX-3 heterotrimer is interacting with its chaperone at a drastically different angle and presents different faces of the PPE protein to the chaperone. We conclude that the PPE–EspG interface from each ESX system has a unique shape complementarity that allows each EspG to discriminate among noncognate PE–PPE pairs.

scholarly journals Structural studies on dehydroshikimate dehydratase

2014 ◽  
Vol 70 (a1) ◽  
pp. C475-C475
Author(s):  
James Peek ◽  
Dinesh Christendat
Keyword(s):  
Sole Carbon Source ◽  
Catalytic Mechanism ◽  
Substrate Recognition ◽  
Structural Studies ◽  
Sugar Phosphate ◽  
X Ray ◽  
Structural Insight ◽  
Functional Relevance ◽  
Insight Into

The soil bacterium, Pseudomonas putida, is capable of using the alicyclic compound quinate as a sole carbon source. During this process, quinate is converted to 3-dehydroshikimate, which subsequently undergoes a dehydration to form protocatechuate. The latter transformation is performed by the enzyme dehydroshikimate dehydratase (DSD). We have recombinantly produced DSD from P. putida and are currently performing x-ray crystallographic studies on the enzyme to gain structural insight into its catalytic mechanism and mode of substrate recognition. Initial crystals of DSD diffracted to 2.7 Ä resolution, but exhibited strong twinning. A redesigned construct has recently yielded crystals that diffract to similar resolution, but with a significantly reduced tendency toward twinning. Interestingly, sequence analysis of P. putida DSD reveals that the protein is in fact a fusion of two distinct domains: an N-terminal sugar phosphate isomerase-like domain associated with DSD activity, and a C-terminal hydroxyphenylpyruvate dioxygenase (HPPD)-like domain with unknown functional significance. Structural characterization of the protein may provide novel insight into the functional relevance of the unusual HPPD-like domain.

scholarly journals Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Nicolas Coudray ◽  
Georgia L Isom ◽  
Mark R MacRae ◽  
Mariyah N Saiduddin ◽  
Gira Bhabha ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Sequence Similarity ◽  
Substrate Recognition ◽  
Membrane Integrity ◽  
Transport Pathway ◽  
Mechanistic Insight ◽  
Membrane Barrier ◽  
Phospholipid Transport ◽  
Insight Into

In double-membraned bacteria, phospholipid transport across the cell envelope is critical to maintain the outer membrane barrier, which plays a key role in virulence and antibiotic resistance. An MCE transport system called Mla has been implicated in phospholipid trafficking and outer membrane integrity, and includes an ABC transporter, MlaFEDB. The transmembrane subunit, MlaE, has minimal sequence similarity to other transporters, and the structure of the entire inner-membrane MlaFEDB complex remains unknown. Here, we report the cryo-EM structure of MlaFEDB at 3.05 Å resolution, revealing distant relationships to the LPS and MacAB transporters, as well as the eukaryotic ABCA/ABCG families. A continuous transport pathway extends from the MlaE substrate-binding site, through the channel of MlaD, and into the periplasm. Unexpectedly, two phospholipids are bound to MlaFEDB, suggesting that multiple lipid substrates may be transported each cycle. Our structure provides mechanistic insight into substrate recognition and transport by MlaFEDB.

scholarly journals Species-specific secretion of ESX-5 type VII substrates is determined by the linker 2 of EccC5

2019 ◽  
Author(s):  
C. M. Bunduc ◽  
R. Ummels ◽  
W. Bitter ◽  
E.N.G. Houben
Keyword(s):  
Cell Envelope ◽  
Substrate Recognition ◽  
Effector Proteins ◽  
Specific Substrate ◽  
Secretion Systems ◽  
Binding Domains ◽  
Type Vii Secretion ◽  
Wide Range ◽  
Species Specific ◽  
System 1

AbstractType VII secretion systems (T7SSs) are used by mycobacteria to translocate a wide range of effector proteins across their diderm cell envelope. These systems, also known as ESX systems, have crucial roles for the viability and/or virulence of mycobacterial pathogens, including Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. We previously observed species-specificity in the secretion of the PE_PGRS proteins by the ESX-5 system [1], in that the M. tuberculosis ESX-5 system was unable to fully complement an M. marinum esx-5 mutant. In this study, we established that the responsible factor for this is the central membrane ATPase EccC5, which has three nucleotide binding domains (NBDs). By creating chimeric M. marinum/M. tuberculosis EccC5 constructs, we observed that PE_PGRS secretion is mediated only in the presence of an EccC5 containing the cognate linker 2, irrespective of the origin of the EccC5 backbone. This region is responsible for linking the first two NBDs and for keeping the first NBD in an inhibited state. Notably, this region is disordered in a EccC crystal structure and is particularly extended in EccC proteins of the different ESX-5 systems. These results indicate that this region is involved in species-specific substrate recognition and might therefore be an additional substrate recognition site of EccC5.

Structure and characterization of amidase from Rhodococcus sp. N-771: Insight into the molecular mechanism of substrate recognition

2010 ◽  
Vol 1804 (1) ◽  
pp. 184-192 ◽  
Author(s):  
Akashi Ohtaki ◽  
Kensuke Murata ◽  
Yuichi Sato ◽  
Keiichi Noguchi ◽  
Hideyuki Miyatake ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Substrate Recognition ◽  
Insight Into ◽  
Rhodococcus Sp

scholarly journals Structure of the mycobacterial ESX-5 Type VII Secretion System hexameric pore complex

2020 ◽  
Author(s):  
Kathrine S. H. Beckham ◽  
Christina Ritter ◽  
Grzegorz Chojnowski ◽  
Edukondalu Mullapudi ◽  
Mandy Rettel ◽  
...  
Keyword(s):  
Cell Envelope ◽  
Secretion System ◽  
Transmembrane Helices ◽  
Membrane Protein Complex ◽  
Virulence Proteins ◽  
The Core ◽  
Type Vii Secretion ◽  
Cryo Electron Microscopy ◽  
Core Components ◽  
Type Vii Secretion System

AbstractTo establish an infection, pathogenic mycobacteria use the Type VII secretion or ESX system to secrete virulence proteins across their cell envelope. The five ESX systems (ESX-1 to ESX-5) have evolved diverse functions in the cell, with the ESX-5 found almost exclusively in pathogens. Here we present a high-resolution cryo-electron microscopy structure of the hexameric ESX-5 Type VII secretion system. This 2.1 MDa membrane protein complex is built by a total of 30 subunits from six protomeric units, which are composed of the core components EccB5, EccC5, two copies of EccD5, and EccE5. The hexameric assembly of the overall ESX-5 complex is defined by specific inter-protomer interactions mediated by EccB5 and EccC5. The central transmembrane pore is formed by six pairs of EccC5 transmembrane helices that adopt a closed conformation in the absence of substrate in our structure. On the periplasmic face of the ESX-5 complex, we observe an extended arrangement of the six EccB5 subunits around a central cleft. Our structural findings provide molecular details of ESX-5 assembly and observations of the central secretion pore, which reveal insights into possible gating mechanisms used to regulate the transport of substrates.

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