scholarly journals Structures of trehalose-6-phosphate phosphatase from pathogenic fungi reveal the mechanisms of substrate recognition and catalysis

2016 ◽  
Vol 113 (26) ◽  
pp. 7148-7153 ◽  
Author(s):  
Yi Miao ◽  
Jennifer L. Tenor ◽  
Dena L. Toffaletti ◽  
Erica J. Washington ◽  
Jiuyu Liu ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Fungal Pathogens ◽  
Elevated Temperatures ◽  
Catalytic Mechanism ◽  
Substrate Recognition ◽  
Complex Structure ◽  
Pathogenic Fungi ◽  
Hydroxyl Group ◽  
Structural Basis ◽  
Phosphatase Domain

Trehalose is a disaccharide essential for the survival and virulence of pathogenic fungi. The biosynthesis of trehalose requires trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. Here, we report the structures of the N-terminal domain of Tps2 (Tps2NTD) fromCandida albicans, a transition-state complex of the Tps2 C-terminal trehalose-6-phosphate phosphatase domain (Tps2PD) bound to BeF3and trehalose, and catalytically dead Tps2PD(D24N) fromCryptococcus neoformansbound to trehalose-6-phosphate (T6P). The Tps2NTD closely resembles the structure of Tps1 but lacks any catalytic activity. The Tps2PD–BeF3–trehalose and Tps2PD(D24N)–T6P complex structures reveal a “closed” conformation that is effected by extensive interactions between each trehalose hydroxyl group and residues of the cap and core domains of the protein, thereby providing exquisite substrate specificity. Disruption of any of the direct substrate–protein residue interactions leads to significant or complete loss of phosphatase activity. Notably, the Tps2PD–BeF3–trehalose complex structure captures an aspartyl-BeF3covalent adduct, which closely mimics the proposed aspartyl-phosphate intermediate of the phosphatase catalytic cycle. Structures of substrate-free Tps2PD reveal an “open” conformation whereby the cap and core domains separate and visualize the striking conformational changes effected by substrate binding and product release and the role of two hinge regions centered at approximately residues 102–103 and 184–188. Significantly,tps2Δ,tps2NTDΔ, andtps2D705Nstrains are unable to grow at elevated temperatures. Combined, these studies provide a deeper understanding of the substrate recognition and catalytic mechanism of Tps2 and provide a structural basis for the future design of novel antifungal compounds against a target found in three major fungal pathogens.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

Structural basis for the dimerization-dependent CRISPR-Cas12f nuclease

2020 ◽  
Author(s):  
Renjian Xiao ◽  
Zhuang Li ◽  
Shukun Wang ◽  
Ruijie Han ◽  
Leifu Chang
Keyword(s):  
Genome Editing ◽  
Conformational Changes ◽  
Dna Cleavage ◽  
Substrate Recognition ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Target Dna ◽  
Type V ◽  
Underlying Substrate

ABSTRACTCas12f, also known as Cas14, is an exceptionally small type V-F CRISPR-Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 Å and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR-Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.

scholarly journals Structural basis for the transport mechanism of the human glutamine transporter SLC1A5 (ASCT2)

2019 ◽  
Author(s):  
Xiaodi Yu ◽  
Olga Plotnikova ◽  
Paul D. Bonin ◽  
Timothy A. Subashi ◽  
Thomas J. McLellan ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Conformational Changes ◽  
Transport Mechanism ◽  
Body Movement ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Glutamine Transporter ◽  
Mtorc1 Signaling ◽  
Transport Cycle ◽  
Transport Domain

AbstractAlanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) is the primary transporter of glutamine in cancer cells and regulates the mTORC1 signaling pathway. The SLC1A5 function involves finely tuned orchestration of two domain movements that include the substrate-binding transport domain and the scaffold domain. Here, we present cryo-EM structures of human SLC1A5 and its complex with the substrate, L-glutamine in an outward-facing conformation. These structures reveal insights into the conformation of the critical ECL2a loop which connects the two domains, thus allowing rigid body movement of the transport domain throughout the transport cycle. Furthermore, the structures provide new insights into substrate recognition, which involves conformational changes in the HP2 loop. A putative cholesterol binding site was observed near the domain interface in the outward-facing state. Comparison with the previously determined inward-facing structure of SCL1A5 provides a basis for a more integrated understanding of substrate recognition and transport mechanism in the SLC1 family. Our structures are likely to aid the development of potent and selective SLC1A5 inhibitors for the treatment of cancer and autoimmune disorders.

scholarly journals Complex structure of type VI peptidoglycan muramidase effector and a cognate immunity protein

2013 ◽  
Vol 69 (10) ◽  
pp. 1889-1900 ◽  
Author(s):  
Tianyu Wang ◽  
Jinjing Ding ◽  
Ying Zhang ◽  
Da-Cheng Wang ◽  
Wei Liu
Keyword(s):  
Calcium Binding ◽  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Complex Structure ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Type Vi Secretion System ◽  
Lytic Enzymes ◽  
Lytic Transglycosylase

The type VI secretion system (T6SS) is a bacterial protein-export machine that is capable of delivering virulence effectors between Gram-negative bacteria. The T6SS ofPseudomonas aeruginosatransports two lytic enzymes, Tse1 and Tse3, to degrade cell-wall peptidoglycan in the periplasm of rival bacteria that are competing for nichesviaamidase and muramidase activities, respectively. Two cognate immunity proteins, Tsi1 and Tsi3, are produced by the bacterium to inactivate the two antibacterial effectors, thereby protecting its siblings from self-intoxication. Recently, Tse1–Tsi1 has been structurally characterized. Here, the structure of the Tse3–Tsi3 complex is reported at 1.9 Å resolution. The results reveal that Tse3 contains a C-terminal catalytic domain that adopts a soluble lytic transglycosylase (SLT) fold in which three calcium-binding sites were surprisingly observed close to the catalytic Glu residue. The electrostatic properties of the substrate-binding groove are also distinctive from those of known structures with a similar fold. All of these features imply that a unique catalytic mechanism is utilized by Tse3 in cleaving glycosidic bonds. Tsi3 comprises a single domain showing a β-sandwich architecture that is reminiscent of the immunoglobulin fold. Three loops of Tsi3 insert deeply into the groove of Tse3 and completely occlude its active site, which forms the structural basis of Tse3 inactivation. This work is the first crystallographic report describing the three-dimensional structure of the Tse3–Tsi3 effector–immunity pair.

scholarly journals Structural basis of catalysis and substrate recognition by the NAD(H)-dependent α-d-glucuronidase from the glycoside hydrolase family 4

2021 ◽  
Vol 478 (4) ◽  
pp. 943-959
Author(s):  
Samar Ballabha Mohapatra ◽  
Narayanan Manoj
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Glycoside Hydrolase ◽  
Metal Ion ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Glycoside Hydrolase Family ◽  
Diverse Range ◽  
Molecular Features ◽  
Hydrolase Family

Members of the glycoside hydrolase family 4 (GH4) employ an unusual glycosidic bond cleavage mechanism utilizing NAD(H) and a divalent metal ion, under reducing conditions. These enzymes act upon a diverse range of glycosides, and unlike most other GH families, homologs here are known to accommodate both α- and β-anomeric specificities within the same active site. Here, we report the catalytic properties and the crystal structures of TmAgu4B, an α-d-glucuronidase from the hyperthermophile Thermotoga maritima. The structures in three different states include the apo form, the NADH bound holo form, and the ternary complex with NADH and the reaction product d-glucuronic acid, at 2.15, 1.97 and 1.85 Å resolutions, respectively. These structures reveal the step-wise route of conformational changes required in the active site to achieve the catalytically competent state, and illustrate the direct role of residues that determine the reaction mechanism. Furthermore, a structural transition of a helical region in the active site to a turn geometry resulting in the rearrangement of a unique arginine residue governs the exclusive glucopyranosiduronic acid recognition in TmAgu4B. Mutational studies show that modifications of the glycone binding site geometry lead to catalytic failure and indicate overlapping roles of specific residues in catalysis and substrate recognition. The data highlight hitherto unreported molecular features and associated active site dynamics that determine the structure–function relationships within the unique GH4 family.

scholarly journals Half Way to Hypusine—Structural Basis for Substrate Recognition by Human Deoxyhypusine Synthase

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 522 ◽  
Author(s):  
Elżbieta Wątor ◽  
Piotr Wilk ◽  
Przemysław Grudnik
Keyword(s):  
Conformational Changes ◽  
Bound States ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Dependent Manner ◽  
Post Translational Modification ◽  
Deoxyhypusine Synthase ◽  
Recognition Mechanism ◽  
Eukaryotic Translation ◽  
Potential Applications

Deoxyhypusine synthase (DHS) is a transferase enabling the formation of deoxyhypusine, which is the first, rate-limiting step of a unique post-translational modification: hypusination. DHS catalyses the transfer of a 4-aminobutyl moiety of polyamine spermidine to a specific lysine of eukaryotic translation factor 5A (eIF5A) precursor in a nicotinamide adenine dinucleotide (NAD)-dependent manner. This modification occurs exclusively on one protein, eIF5A, and it is essential for cell proliferation. Malfunctions of the hypusination pathway, including those caused by mutations within the DHS encoding gene, are associated with conditions such as cancer or neurodegeneration. Here, we present a series of high-resolution crystal structures of human DHS. Structures were determined as the apoprotein, as well as ligand-bound states at high-resolutions ranging from 1.41 to 1.69 Å. By solving DHS in complex with its natural substrate spermidine (SPD), we identified the mode of substrate recognition. We also observed that other polyamines, namely spermine (SPM) and putrescine, bind DHS in a similar manner as SPD. Moreover, we performed activity assays showing that SPM could to some extent serve as an alternative DHS substrate. In contrast to previous studies, we demonstrate that no conformational changes occur in the DHS structure upon spermidine-binding. By combining mutagenesis and a light-scattering approach, we show that a conserved “ball-and-chain” motif is indispensable to assembling a functional DHS tetramer. Our study substantially advances our knowledge of the substrate recognition mechanism by DHS and may aid the design of pharmacological compounds for potential applications in cancer therapy.

scholarly journals Structural Basis of Eco1-Mediated Cohesin Acetylation

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
William C. H. Chao ◽  
Benjamin O. Wade ◽  
Céline Bouchoux ◽  
Andrew W. Jones ◽  
Andrew G. Purkiss ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Substrate Specificity ◽  
Binding Site ◽  
Conformational Changes ◽  
Sister Chromatid ◽  
Molecular Basis ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Chromatid Cohesion

Abstract Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension.

scholarly journals Structural basis for protein glutamylation by the Legionella pseudokinase SidJ

2021 ◽  
Author(s):  
Michael Adams ◽  
Rahul Sharma ◽  
Thomas Colby ◽  
Felix Weis ◽  
Ivan Matic ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Substrate Recognition ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Effector Proteins ◽  
Structural Basis ◽  
Rab Gtpases ◽  
Binding Pockets

Legionella pneumophila (LP) avoids phagocytosis by secreting nearly 300 effector proteins into the host cytosol. SidE family of effectors (SdeA, SdeB, SdeC and SidE) employ phosphoribosyl serine ubiquitination to target an array of host Rab GTPases and innate immune factors. To suppress the deleterious toxicity of SidE enzymes in a timely manner, LP employs a metaeffector named SidJ. Upon activation by host Calmodulin (CaM), SidJ executes an ATP-dependent glutamylation to modify the catalytic residue Glu860 in the mono-ADP-ribosyl transferase (mART) domain of SdeA. SidJ is a unique glutamylase that adopts a kinase-like fold but contains two nucleotide-binding pockets. There is a lack of consensus about the substrate recognition and catalytic mechanism of SidJ. Here, we determined the cryo-EM structure of SidJ in complex with its substrate SdeA in two different states of catalysis. Our structures reveal that both phosphodiesterase (PDE) and mART domains of SdeA make extensive contacts with SidJ. In the pre-glutamylation state structure of the SidJ-SdeA complex, adenylylated E860 of SdeA is inserted into the non-canonical (migrated) nucleotide-binding pocket of SidJ. Structure-based mutational analysis indicates that SidJ employs its migrated pocket for the glutamylation of SdeA. Finally, using mass spectrometry, we identified several transient autoAMPylation sites close to both the catalytic pockets of SidJ. Our data provide unique insights into the substrate recognition and the mechanism of protein glutamylation by the pseudokinase SidJ.

scholarly journals Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR–Cas12f nuclease

2021 ◽  
Vol 49 (7) ◽  
pp. 4120-4128
Author(s):  
Renjian Xiao ◽  
Zhuang Li ◽  
Shukun Wang ◽  
Ruijie Han ◽  
Leifu Chang
Keyword(s):  
Genome Editing ◽  
Conformational Changes ◽  
Dna Cleavage ◽  
Substrate Recognition ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Target Dna ◽  
Type V ◽  
Underlying Substrate

Abstract Cas12f, also known as Cas14, is an exceptionally small type V-F CRISPR–Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR–Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.

scholarly journals Structural basis of FANCD2 deubiquitination by USP1-UAF1

2020 ◽  
Author(s):  
Martin L. Rennie ◽  
Connor Arkinson ◽  
Viduth K. Chaugule ◽  
Rachel Toth ◽  
Helen Walden
Keyword(s):  
Dna Repair ◽  
Fanconi Anemia ◽  
Conformational Changes ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Interstrand Crosslinks ◽  
Biochemical Assays ◽  
Dna Interstrand Crosslinks ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

AbstractUbiquitin-Specific Protease 1 (USP1), together with the cofactor UAF1, acts during DNA repair processes to specifically to remove mono-ubiquitin signals. The mono-ubiquitinated FANCI-FANCD2 heterodimer is one such substrate and is involved in the repair of DNA interstrand crosslinks via the Fanconi Anemia pathway. Here we determine structures of human USP1-UAF1 with and without ubiquitin, and bound to mono-ubiquitinated FANCI-FANCD2 substrate. Crystal structures of USP1-UAF1 reveal plasticity in USP1 and key differences to USP12-UAF1 and USP46-UAF1. A cryoEM reconstruction of USP1-UAF1 in complex mono-ubiquitinated FANCI-FANCD2, highlights a highly orchestrated deubiquitination process with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for their requirement despite neither being directly involved in catalysis. Overall, our data provide molecular details of USP1-UAF1 regulation and substrate recognition.

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