scholarly journals Crystal structures of human MGST2 reveal synchronized conformational changes regulating catalysis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Madhuranayaki Thulasingam ◽  
Laura Orellana ◽  
Emmanuel Nji ◽  
Shabbir Ahmad ◽  
Agnes Rinaldo-Matthis ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Drug Targets ◽  
Active Sites ◽  
Oxidative Dna Damage ◽  
Glutathione S Transferase ◽  
Concerted Mechanism ◽  
Related Drug ◽  
Stress Oxidative ◽  
And Control

AbstractMicrosomal glutathione S-transferase 2 (MGST2) produces leukotriene C4, key for intracrine signaling of endoplasmic reticulum (ER) stress, oxidative DNA damage and cell death. MGST2 trimer restricts catalysis to only one out of three active sites at a time, but the molecular basis is unknown. Here, we present crystal structures of human MGST2 combined with biochemical and computational evidence for a concerted mechanism, involving local unfolding coupled to global conformational changes that regulate catalysis. Furthermore, synchronized changes in the biconical central pore modulate the hydrophobicity and control solvent influx to optimize reaction conditions at the active site. These unique mechanistic insights pertain to other, structurally related, drug targets.

scholarly journals Basis for drug selectivity of plasmepsin IX and X inhibition for Plasmodium falciparum and vivax

2021 ◽  
Author(s):  
Anthony N. Hodder ◽  
Stephen Scally ◽  
Tony Triglia ◽  
Anna Ngo ◽  
Richard W. Birkinshaw ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Crystal Structures ◽  
Drug Targets ◽  
Active Sites ◽  
Drug Binding ◽  
Dual Inhibitor ◽  
3 Dimensional ◽  
Protein Substrates ◽  
Binding Cleft ◽  
Parasite Egress

Abstract Plasmepsin IX (PMIX) and X (PMX) are aspartyl proteases of Plasmodium spp. that play essential roles in parasite egress, invasion and development. Consequently, they are important drug targets for Plasmodium falciparum and P. vivax. WM4 and WM382 are potent inhibitors of PMIX and PMX that block invasion of liver and blood stages and transmission to mosquitoes. WM4 specifically inhibits PMX whilst WM382 is a dual inhibitor of PMIX and PMX. To understand the function of PMIX and PMX proteases we identified new protein substrates in P. falciparum and together with detailed kinetic analyses and structural analyses identified key molecular interactions in the active site responsible for the specificity of WM4 and WM382 inhibition. The crystal structures of PMX apo enzyme and the protease/drug complexes of PMX/WM382 and PMX/WM4 for P. falciparum and P. vivax have been solved. We show PMIX and PMX have similar substrate selectivity, however, there are distinct differences for both peptide and full-length protein substrates through differences in localised 3-dimensional structures for the enzyme substrate-binding cleft and substrate interface. The differences in affinities of WM4 and WM382 binding for PMIX and PMX map to variations in surface interactions with each protease in the S' region of the active sites. Crystal structures of PMX reveal interactions and mechanistic detail on the selectivity of drug binding which will be important for further development of clinical candidates against these important molecular targets.

scholarly journals Dynamics of human protein kinase Aurora A linked to drug selectivity

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Warintra Pitsawong ◽  
Vanessa Buosi ◽  
Renee Otten ◽  
Roman V Agafonov ◽  
Adelajda Zorba ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Conformational Changes ◽  
Drug Targets ◽  
Active Sites ◽  
Kinase Inhibitors ◽  
Bound State ◽  
Drug Binding ◽  
Aurora A ◽  
Drug Selectivity ◽  
Approved Drugs

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinase Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.

Crystal Structures of a Mutant (βK87T) Tryptophan Synthase α2β2Complex with Ligands Bound to the Active Sites of the α- and β-Subunits Reveal Ligand-Induced Conformational Changes‡

Biochemistry ◽  
1997 ◽  
Vol 36 (25) ◽  
pp. 7664-7680 ◽  
Author(s):  
Sangkee Rhee ◽  
Kevin D. Parris ◽  
C. Craig Hyde ◽  
S. Ashraf Ahmed ◽  
Edith Wilson Miles ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Active Sites ◽  
Tryptophan Synthase ◽  
Α And Β Subunits

scholarly journals Dynamics of human protein kinases linked to drug selectivity

2018 ◽  
Author(s):  
Warintra Pitsawong ◽  
Vanessa Buosi ◽  
Renee Otten ◽  
Roman V. Agafonov ◽  
Adelajda Zorba ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Protein Kinases ◽  
Conformational Changes ◽  
Drug Targets ◽  
Active Sites ◽  
Kinase Inhibitors ◽  
Drug Binding ◽  
Inhibition Mechanism ◽  
Aurora A ◽  
Drug Selectivity

AbstractProtein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.eLife digestThe Ser/Thr kinase Aurora A is an important target for the development of new anticancer therapies. A longstanding question is how to specifically and effectively inhibit only this kinase in a background of over 550 protein kinases with very similar structures. To this end, understanding the inhibition mechanism of Aurora A by different drugs is essential. Here, we characterize the kinetic mechanism of three distinct kinase drugs, Gleevec (Imatinib), Danusertib (PHA739358) and AT9283 (Pyrazol-4-yl Urea) for Aurora A. We show that inhibitor affinities do not rely exclusively on the recognition of a specific conformation of the Asp-Phe-Gly loop of the kinase. Our quantitative kinetics data put forward an opposing mechanism in which a slow conformational change after drug binding (i.e., induced-fit step) dictates drug affinity.

scholarly journals Basis for drug selectivity of plasmepsin IX and X inhibition for Plasmodium falciparum and vivax

2021 ◽  
Author(s):  
Alan Cowman ◽  
Anthony Hodder ◽  
Janni Christensen ◽  
Stephen Scally ◽  
Tony Triglia ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Crystal Structures ◽  
Drug Targets ◽  
Active Sites ◽  
Drug Binding ◽  
Dual Inhibitor ◽  
3 Dimensional ◽  
Protein Substrates ◽  
Binding Cleft ◽  
Parasite Egress

Abstract Plasmepsin IX (PMIX) and X (PMX) are aspartyl proteases of Plasmodium spp. that play essential roles in parasite egress, invasion and development. Consequently, they are important drug targets for Plasmodium falciparum and P. vivax. WM4 and WM382 are potent inhibitors of PMIX and PMX that block invasion of liver and blood stages and transmission to mosquitoes. WM4 specifically inhibits PMX whilst WM382 is a dual inhibitor of PMIX and PMX. To understand the function of PMIX and PMX proteases we identified new protein substrates in P. falciparum and together with detailed kinetic analyses and structural analyses identified key molecular interactions in the active site responsible for the specificity of WM4 and WM382 inhibition. The crystal structures of PMX apo enzyme and the protease/drug complexes of PMX/WM382 and PMX/WM4 for P. falciparum and P. vivax have been solved. We show PMIX and PMX have similar substrate selectivity, however, there are distinct differences for both peptide and full-length protein substrates through differences in localised 3-dimensional structures for the enzyme substrate-binding cleft and substrate interface. The differences in affinities of WM4 and WM382 binding for PMIX and PMX map to variations in surface interactions with each protease in the S' region of the active sites. Crystal structures of PMX reveal interactions and mechanistic detail on the selectivity of drug binding which will be important for further development of clinical candidates against these important molecular targets.

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.

scholarly journals Metals in Calluna vulgaris, Empetrum nigrum, Festuca vivipara and Thymus praecox ssp. arcticus in the geothermal areas of Iceland

2021 ◽  
Author(s):  
Adam Rajsz ◽  
Bronisław Wojtuń ◽  
Aleksandra Samecka-Cymerman ◽  
Paweł Wąsowicz ◽  
Lucyna Mróz ◽  
...  
Keyword(s):  
Active Sites ◽  
Calluna Vulgaris ◽  
Bioaccumulation Factor ◽  
The Other ◽  
Extreme Situation ◽  
Empetrum Nigrum ◽  
Geothermal Activity ◽  
High Concentrations ◽  
And Control ◽  
Festuca Vivipara

AbstractThis investigation was conducted to identify the content of metals in Calluna vulgaris (family Ericaceae), Empetrum nigrum (family Ericaceae), Festuca vivipara (family Poaceae) and Thymus praecox subsp. arcticus (family Lamiaceae), as well as in the soils where they were growing in eight geothermal heathlands in Iceland. Investigation into the vegetation of geothermal areas is crucial and may contribute to their proper protection in the future and bring more understanding under what conditions the plants respond to an ecologically more extreme situation. Plants from geothermally active sites were enriched with metals as compared to the same species from non-geothermal control sites (at an average from about 150 m from geothermal activity). The enriched metals consisted of Cd, Co, Cu, Fe and Ni in C. vulgaris; Cd, Mn and Ti in E. nigrum; Hg and Pb in F. vivipara; and Cd, Fe and Hg in T. praecox. Notably, C. vulgaris, E. nigrum, F. vivipara and T. praecox had remarkably high concentrations of Ti at levels typical of toxicity thresholds. Cd and Pb (except for C. vulgaris and F. vivipara) were not accumulated in the shoots of geothermal plants. C. vulgaris from geothermal and control sites was characterised by the highest bioaccumulation factor (BF) of Ti and Mn; E. nigrum and F. vivipara by the highest BF of Ti and Cr; and T. praecox by the highest BF of Ti and Zn compared to the other elements. In comparison with the other examined species, F. vivipara from geothermal sites had the highest concentration of Ti in above-ground parts at any concentration of plant-available Ti in soil.

scholarly journals Structural studies of phosphorylation-dependent interactions between the V2R receptor and arrestin-2

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qing-Tao He ◽  
Peng Xiao ◽  
Shen-Ming Huang ◽  
Ying-Li Jia ◽  
Zhong-Liang Zhu ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Nmr Spectrum ◽  
H Nmr ◽  
Interaction Sites ◽  
Receptor Interactions ◽  
Remote Sites ◽  
Genetic Code Expansion ◽  
G Protein Coupled ◽  
Interaction Change

AbstractArrestins recognize different receptor phosphorylation patterns and convert this information to selective arrestin functions to expand the functional diversity of the G protein-coupled receptor (GPCR) superfamilies. However, the principles governing arrestin-phospho-receptor interactions, as well as the contribution of each single phospho-interaction to selective arrestin structural and functional states, are undefined. Here, we determined the crystal structures of arrestin2 in complex with four different phosphopeptides derived from the vasopressin receptor-2 (V2R) C-tail. A comparison of these four crystal structures with previously solved Arrestin2 structures demonstrated that a single phospho-interaction change results in measurable conformational changes at remote sites in the complex. This conformational bias introduced by specific phosphorylation patterns was further inspected by FRET and 1H NMR spectrum analysis facilitated via genetic code expansion. Moreover, an interdependent phospho-binding mechanism of phospho-receptor-arrestin interactions between different phospho-interaction sites was unexpectedly revealed. Taken together, our results provide evidence showing that phospho-interaction changes at different arrestin sites can elicit changes in affinity and structural states at remote sites, which correlate with selective arrestin functions.

scholarly journals In silico characterization and structural modeling of bacterial metalloprotease of family M4

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Rajnee Hasan ◽  
Md. Nazmul Haq Rony ◽  
Rasel Ahmed
Keyword(s):  
Drug Targets ◽  
Active Sites ◽  
Pathogenic Bacteria ◽  
Tertiary Structure ◽  
Transmembrane Helix ◽  
Confidence Score ◽  
Potential Drug ◽  
Protein Protein Interaction ◽  
Industrial Level ◽  
Potential Drug Targets

Abstract Background The M4 family of metalloproteases is comprised of a large number of zinc-containing metalloproteases. A large number of these enzymes are important virulence factors of pathogenic bacteria and therefore potential drug targets. Whereas some enzymes have potential for biotechnological applications, the M4 family of metalloproteases is known almost exclusively from bacteria. The aim of the study was to identify the structure and properties of M4 metalloprotease proteins. Results A total of 31 protein sequences of M4 metalloprotease retrieved from UniProt representing different species of bacteria have been characterized for various physiochemical properties. They were thermostable, hydrophillic protein of a molecular mass ranging from 38 to 66 KDa. Correlation on the basis of both enzymes and respective genes has also been studied by phylogenetic tree. B. cereus M4 metalloprotease (PDB ID: 1NPC) was selected as a representative species for secondary and tertiary structures among the M4 metalloprotease proteins. The secondary structure displaying 11 helices (H1-H11) is involved in 15 helix-helix interactions, while 4 β-sheet motifs composed of 15 β-strands in PDBsum. Possible disulfide bridges were absent in most of the cases. The tertiary structure of B. cereus M4 metalloprotease was validated by QMEAN4 and SAVES server (Ramachandran plot, verify 3D, and ERRAT) which proved the stability, reliability, and consistency of the tertiary structure of the protein. Functional analysis was done in terms of membrane protein topology, disease-causing region prediction, proteolytic cleavage sites prediction, and network generation. Transmembrane helix prediction showed absence of transmembrane helix in protein. Protein-protein interaction networks demonstrated that bacillolysin of B. cereus interacted with ten other proteins in a high confidence score. Five disorder regions were identified. Active sites analysis showed the zinc-binding residues—His-143, His-147, and Glu-167, with Glu-144 acting as the catalytic residues. Conclusion Moreover, this theoretical overview will help researchers to get a details idea about the protein structure and it may also help to design enzymes with desirable characteristics for exploiting them at industrial level or potential drug targets.

scholarly journals Crystal structures of aconitase X enzymes from bacteria and archaea provide insights into the molecular evolution of the aconitase superfamily

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Seiya Watanabe ◽  
Yohsuke Murase ◽  
Yasunori Watanabe ◽  
Yasuhiro Sakurai ◽  
Kunihiko Tajima
Keyword(s):  
Molecular Evolution ◽  
Agrobacterium Tumefaciens ◽  
Crystal Structures ◽  
Epr Spectroscopy ◽  
Binding Sites ◽  
Active Sites ◽  
Type I ◽  
Type Ii ◽  
Thermococcus Kodakarensis ◽  
Evolutionary Scenario

AbstractAconitase superfamily members catalyze the homologous isomerization of specific substrates by sequential dehydration and hydration and contain a [4Fe-4S] cluster. However, monomeric and heterodimeric types of function unknown aconitase X (AcnX) have recently been characterized as a cis-3-hydroxy-L-proline dehydratase (AcnXType-I) and mevalonate 5-phosphate dehydratase (AcnXType-II), respectively. We herein elucidated the crystal structures of AcnXType-I from Agrobacterium tumefaciens (AtAcnX) and AcnXType-II from Thermococcus kodakarensis (TkAcnX) without a ligand and in complex with substrates. AtAcnX and TkAcnX contained the [2Fe-2S] and [3Fe-4S] clusters, respectively, conforming to UV and EPR spectroscopy analyses. The binding sites of the [Fe-S] cluster and substrate were clearlydifferent from those that were completely conserved in other aconitase enzymes; however, theoverall structural frameworks and locations of active sites were partially similar to each other.These results provide novel insights into the evolutionary scenario of the aconitase superfamilybased on the recruitment hypothesis.

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