Molecular insight into substrate recognition by human cytosolic sialidase NEU2

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.

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Insight into the molecular basis of substrate recognition by the wall teichoic acid glycosyltransferase TagA

Journal of Biological Chemistry ◽

10.1016/j.jbc.2021.101464 ◽

2021 ◽

pp. 101464

Author(s):

Orlando E. Martinez ◽

Brendan J. Mahoney ◽

Andrew K. Goring ◽

Sung-Wook Yi ◽

Denise P. Tran ◽

...

Keyword(s):

Molecular Basis ◽

Substrate Recognition ◽

Teichoic Acid ◽

Wall Teichoic Acid ◽

Insight Into

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012698 ◽

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.

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Structural evidence for a proline-specific glycopeptide recognition domain in an O-glycopeptidase

Glycobiology ◽

10.1093/glycob/cwaa095 ◽

2020 ◽

Author(s):

Ilit Noach ◽

Alisdair B Boraston

Keyword(s):

Active Sites ◽

Substrate Recognition ◽

Peptide Bond ◽

Multidomain Protein ◽

Threonine Residue ◽

Tyrosine Residues ◽

Host Pathogen Interaction ◽

A Site ◽

Insight Into

Abstract The glycosylation of proteins is typically considered as a stabilizing modification, including resistance to proteolysis. A class of peptidases, referred to as glycopeptidases or O-glycopeptidases, circumvent the protective effect of glycans against proteolysis by accommodating the glycans in their active sites as specific features of substrate recognition. IMPa from Pseudomonas aeruginosa is such an O-glycopeptidase that cleaves the peptide bond immediately preceding a site of O-glycosylation, and through this glycoprotein-degrading function contributes to the host-pathogen interaction. IMPa, however, is a relatively large multidomain protein and how its additional domains may contribute to its function remains unknown. Here, through the determination of a crystal structure of IMPa in complex with an O-glycopeptide, we reveal that the N-terminal domain of IMPa, which is classified in Pfam as IMPa_N_2, is a proline recognition domain that also shows the properties of recognizing an O-linked glycan on the serine/threonine residue following the proline. The proline is bound in the center of a bowl formed by four functionally conserved aromatic amino acid side chains while the glycan wraps around one of the tyrosine residues in the bowl to make classic aromatic ring-carbohydrate CH-π interactions. This structural evidence provides unprecedented insight into how the ancillary domains in glycoprotein-specific peptidases can noncatalytically recognize specific glycosylated motifs that are common in mucin and mucin-like molecules.

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Crystal Structures of the Histidine Acid Phosphatase from Francisella tularensis Provide Insight into Substrate Recognition

Journal of Molecular Biology ◽

10.1016/j.jmb.2009.10.009 ◽

2009 ◽

Vol 394 (5) ◽

pp. 893-904 ◽

Cited By ~ 10

Author(s):

Harkewal Singh ◽

Richard L. Felts ◽

Jonathan P. Schuermann ◽

Thomas J. Reilly ◽

John J. Tanner

Keyword(s):

Acid Phosphatase ◽

Crystal Structures ◽

Francisella Tularensis ◽

Substrate Recognition ◽

Histidine Acid Phosphatase ◽

Insight Into

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Structural studies on dehydroshikimate dehydratase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314095242 ◽

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.

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Box C/D guide RNAs recognize a maximum of 10 nt of substrates

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1604872113 ◽

2016 ◽

Vol 113 (39) ◽

pp. 10878-10883 ◽

Cited By ~ 14

Author(s):

Zuxiao Yang ◽

Jinzhong Lin ◽

Keqiong Ye

Keyword(s):

Substrate Recognition ◽

Active Conformation ◽

Base Pairs ◽

Recognition Mechanism ◽

Site Specific ◽

Guide Rnas ◽

Modification Activity ◽

Guide Sequence ◽

Protein Channel ◽

Insight Into

Box C/D RNAs guide site-specific 2′-O-methylation of RNAs in archaea and eukaryotes. The spacer regions between boxes C to D′ and boxes C′ to D contain the guide sequence that can form a stretch of base pairs with substrate RNAs. The lengths of spacer regions and guide-substrate duplexes are variable among C/D RNAs. In a previously determined structure of C/D ribonucleoprotein (RNP), a 12-nt-long spacer forms 10 bp with the substrate. How spacers and guide–substrate duplexes of other lengths are accommodated remains unknown. Here we analyze how the lengths of spacers and guide-substrate duplexes affect the modification activity and determine three structures of C/D RNPs assembled with different spacers and substrates. We show that the guide can only form a duplex of a maximum of 10 bp with the substrate during modification. Slightly shorter duplexes are tolerated, but longer duplexes must be unwound to fit into a capped protein channel for modification. Spacers with <12 nucleotides are defective, mainly because they cannot load the substrate in the active conformation. For spacers with >12 nucleotides, the excessive unpaired sequences near the box C/C′ side are looped out. Our results provide insight into the substrate recognition mechanism of C/D RNA and refute the RNA-swapped model for dimeric C/D RNP.

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Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

eLife ◽

10.7554/elife.62518 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

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.

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