Structural insights on the effects of mutation of a charged binding pocket residue on phosphopeptide binding to 14‐3‐3ζ Protein

AbstractSerotonin receptors (5-HT3AR) play a crucial role in regulating gut movement, and are the principal target of setrons, a class of high-affinity competitive antagonists, used in the management of nausea and vomiting associated with radiation and chemotherapies. Structural insights into setron-binding poses and their inhibitory mechanisms are just beginning to emerge. Here, we present high-resolution cryo-EM structures of full-length 5-HT3AR in complex with palonosetron, ondansetron, and alosetron. Each structure reveals a distinct interaction fingerprint between the setron and binding-pocket residues that may underlie their diverse affinities. In addition, setrons elicit varying degrees of conformational change throughout the channel that, quite surprisingly, lie along the channel activation pathway, suggesting a novel mechanism of competitive inhibition. Molecular dynamic simulations were used to assess binding-poses and the drug-target interaction dynamics. Together, this study provides a molecular basis for setron binding affinities and their inhibitory effects.

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Structural insights into WcbI, a novel polysaccharide-biosynthesis enzyme

IUCrJ ◽

10.1107/s205225251302695x ◽

2013 ◽

Vol 1 (1) ◽

pp. 28-38 ◽

Cited By ~ 4

Author(s):

Mirella Vivoli ◽

Emily Ayres ◽

Edward Beaumont ◽

Michail N. Isupov ◽

Nicholas J. Harmer

Keyword(s):

Sequence Similarity ◽

Functional Module ◽

Binding Pocket ◽

Capsular Polysaccharides ◽

Biochemical Tests ◽

Polysaccharide Biosynthesis ◽

Tier 1 ◽

Significant Attenuation ◽

Protective Structures ◽

Structural Insights

Capsular polysaccharides (CPSs) are protective structures on the surfaces of many Gram-negative bacteria. The principal CPS of the human pathogen and Tier 1 select agentBurkholderia pseudomalleiconsists of a linear repeat of –3)-2-O-acetyl-6-deoxy-β-D-manno-heptopyranose-(1–. This CPS is critical to the virulence of this emerging pathogen and represents a key target for the development of novel therapeutics.wcbIis one of several genes in the CPS biosynthetic cluster whose deletion leads to significant attenuation of the pathogen; unlike most others, it has no homologues of known function and no detectable sequence similarity to any protein with an extant structure. Here, the crystal structure of WcbI bound to its proposed product, coenzyme A, is reported at 1.38 Å resolution, solved using the halide-soak method with multiple anomalous dispersion. This structure reveals that WcbI incorporates a previously described 100-amino-acid subdomain into a novel, principally helical fold (310 amino acids). This fold adopts a cradle-like structure, with a deep binding pocket for CoA in the loop-rich cradle. Structural analysis and biophysical assays suggest that WcbI functions as an acetyltransferase enzyme, whilst biochemical tests suggest that another functional module might be required to assist its activity in forming the matureB. pseudomalleicapsule.

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Structural Insights into the ATP Binding Pocket of the Anaplastic Lymphoma Kinase by Site-Directed Mutagenesis, Inhibitor Binding Analysis, and Homology Modeling

Journal of Medicinal Chemistry ◽

10.1021/jm060380k ◽

2006 ◽

Vol 49 (19) ◽

pp. 5759-5768 ◽

Cited By ~ 23

Author(s):

Rosalind H. Gunby ◽

Shaheen Ahmed ◽

Roberta Sottocornola ◽

Marc Gasser ◽

Sara Redaelli ◽

...

Keyword(s):

Homology Modeling ◽

Anaplastic Lymphoma Kinase ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Atp Binding ◽

Inhibitor Binding ◽

Anaplastic Lymphoma ◽

Binding Analysis ◽

Structural Insights

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Biochemical and structural insights into the activation of PBP1b by the essential domain of FtsN

10.1101/2020.06.05.136150 ◽

2020 ◽

Author(s):

Adrien Boes ◽

Frederic Kerff ◽

Raphael Herman ◽

Thierry Touze ◽

Eefjan Breukink ◽

...

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Cell Wall ◽

Cell Division ◽

Binding Pocket ◽

Nonpermissive Temperature ◽

Multiprotein Complex ◽

Division Site ◽

Structural Insights

AbstractPeptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division PG synthesis localizes at mid-cell under the control of a multiprotein complex, the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. The region L75 to Q93 of FtsN (EFtsN) was shown to be essential and sufficient for its functioning in vivo but the specific target and the molecular mechanism remained unknown. Here, we show that EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its GTase activity. We also report the crystal structure of PBP1b in complex with EFtsN which provides structural insights into the mode of binding of EFtsN at the junction between the GTase and UB2H domains of PBP1b. Interestingly, the mutations R141A/R397A of PBP1b, within the EFtsN binding pocket, reduce the activation of PBP1b by FtsN. This mutant was unable to rescue ΔponB-ponAts strain at nonpermissive temperature and induced a mild cell chaining phenotype and cell lysis. Altogether, the results show that PBP1b is a target of EFtsN and suggest that binding of FtsN to PBP1b contributes to trigger septal PG synthesis and cell constriction.

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Structural insights into ligand-binding pocket formation in Nurr1 by molecular dynamics simulations

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.2018.1559099 ◽

2019 ◽

Vol 37 (17) ◽

pp. 4651-4657 ◽

Cited By ~ 5

Author(s):

Björn Windshügel

Keyword(s):

Molecular Dynamics ◽

Ligand Binding ◽

Molecular Dynamics Simulations ◽

Binding Pocket ◽

Ligand Binding Pocket ◽

Dynamics Simulations ◽

Pocket Formation ◽

Structural Insights

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Crystal structures ofN-carbamoyl-D-amino-acid amidohydrolase-substrate complexes: structural insights into the binding pocket

Acta Crystallographica Section A Foundations of Crystallography ◽

10.1107/s0108767302089845 ◽

2002 ◽

Vol 58 (s1) ◽

pp. c120-c120

Author(s):

W. Wang ◽

C. Chen ◽

J. Liu

Keyword(s):

Amino Acid ◽

Crystal Structures ◽

Binding Pocket ◽

Structural Insights ◽

Acid Amidohydrolase

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Structural insights into the functional versatility of an FHA domain protein in mycobacterial signaling

Science Signaling ◽

10.1126/scisignal.aav9504 ◽

2019 ◽

Vol 12 (580) ◽

pp. eaav9504 ◽

Cited By ~ 6

Author(s):

Tristan Wagner ◽

Gwénaëlle André-Leroux ◽

Valérie Hindie ◽

Nathalie Barilone ◽

María-Natalia Lisa ◽

...

Keyword(s):

Metabolic Flux ◽

Central Element ◽

Binding Pocket ◽

Signaling Cascades ◽

Binding Modes ◽

Allosteric Site ◽

Fha Domain ◽

Protein Partners ◽

Intramolecular Association ◽

Structural Insights

Forkhead-associated (FHA) domains are modules that bind to phosphothreonine (pThr) residues in signaling cascades. The FHA-containing mycobacterial protein GarA is a central element of a phosphorylation-dependent signaling pathway that redirects metabolic flux in response to amino acid starvation or cell growth requirements. GarA acts as a phosphorylation-dependent ON/OFF molecular switch. In its nonphosphorylated ON state, the GarA FHA domain engages in phosphorylation-independent interactions with various metabolic enzymes that orchestrate nitrogen flow, such as 2-oxoglutarate decarboxylase (KGD). However, phosphorylation at the GarA N-terminal region by the protein kinase PknB or PknG triggers autoinhibition through the intramolecular association of the N-terminal domain with the FHA domain, thus blocking all downstream interactions. To investigate these different FHA binding modes, we solved the crystal structures of the mycobacterial upstream (phosphorylation-dependent) complex PknB-GarA and the downstream (phosphorylation-independent) complex GarA-KGD. Our results show that the phosphorylated activation loop of PknB serves as a docking site to recruit GarA through canonical FHA-pThr interactions. However, the same GarA FHA–binding pocket targets an allosteric site on nonphosphorylated KGD, where a key element of recognition is a phosphomimetic aspartate. Further enzymatic and mutagenesis studies revealed that GarA acted as a dynamic allosteric inhibitor of KGD by preventing crucial motions in KGD that are necessary for catalysis. Our results provide evidence for physiological phosphomimetics, supporting numerous mutagenesis studies using such approaches, and illustrate how evolution can shape a single FHA-binding pocket to specifically interact with multiple phosphorylated and nonphosphorylated protein partners.

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Structural insights into the recognition of mono- and di-acetylated histones by the ATAD2B bromodomain

10.1101/263624 ◽

2018 ◽

Author(s):

Jonathan T. Lloyd ◽

Kyle McLaughlin ◽

Mulu Y. Lubula ◽

Jamie C. Gay ◽

Andrea Dest ◽

...

Keyword(s):

Binding Pocket ◽

List Type ◽

Lysine Acetylation ◽

Ligand Specificity ◽

Inhibitor Binding ◽

Functional Relationships ◽

Functional Features ◽

Key Residues ◽

Structural Insights ◽

Alternate Splice Variant

ABSTRACTBromodomains are chromatin reader modules that recognize acetylated lysine. Different bromodomains exhibit a preference for specific patterns of lysine acetylation marks on core and variant histone proteins, however, the functional relationships that exist between histone acetyllysine ligands and bromodomain recognition remain poorly understood. In this study, we examined the ligand specificity of the ATAD2B bromodomain and compared it to its closely related paralog in ATAD2. We show that the ATAD2B bromodomain selects for mono- and di-acetylated histones, and structural analysis identified key residues in the acetyllysine binding pocket that dictate ligand binding specificity. The X-ray crystal structure of the ATAD2B bromodomain in complex with an ATAD2 bromodomain inhibitor was solved at 2.4 Å resolution. This structure demonstrated that critical contacts required for bromodomain inhibitor coordination are conserved between the ATAD2/B bromodomains, and many of these residues play a dual role in acetyllysine recognition. We further characterized a variant of the ATAD2B bromodomain that through alternative splicing loses critical amino acids required for histone ligand and inhibitor coordination. Altogether our results outline the structural and functional features of the ATAD2B bromodomain and identify a novel mechanism important for regulating the interaction of the ATAD2B protein with chromatin.HIGHLIGHTSThe ATAD2B bromodomain recognizes mono- and di-acetylated histone ligands.Chemical shift perturbations outline the ATAD2B bromodomain acetyllysine binding pocket.An ATAD2B bromodomain-inhibitor complex reveals important binding contacts.An alternate splice variant in the ATAD2B bromodomain abolishes histone and inhibitor binding.

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High-resolution structures of multiple 5-HT3AR-setron complexes reveal a novel mechanism of competitive inhibition

eLife ◽

10.7554/elife.57870 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Sandip Basak ◽

Arvind Kumar ◽

Steven Ramsey ◽

Eric Gibbs ◽

Abhijeet Kapoor ◽

...

Keyword(s):

High Resolution ◽

Serotonin Receptors ◽

Drug Target ◽

Competitive Inhibition ◽

Binding Pocket ◽

Dynamic Simulations ◽

Inhibitory Mechanisms ◽

Lipid Environment ◽

The Stability ◽

Structural Insights

Serotonin receptors (5-HT3AR) play a crucial role in regulating gut movement, and are the principal target of setrons, a class of high-affinity competitive antagonists, used in the management of nausea and vomiting associated with radiation and chemotherapies. Structural insights into setron-binding poses and their inhibitory mechanisms are just beginning to emerge. Here, we present high-resolution cryo-EM structures of full-length 5-HT3AR in complex with palonosetron, ondansetron, and alosetron. Molecular dynamic simulations of these structures embedded in a fully-hydrated lipid environment assessed the stability of ligand-binding poses and drug-target interactions over time. Together with simulation results of apo- and serotonin-bound 5-HT3AR, the study reveals a distinct interaction fingerprint between the various setrons and binding-pocket residues that may underlie their diverse affinities. In addition, varying degrees of conformational change in the setron-5-HT3AR structures, throughout the channel and particularly along the channel activation pathway, suggests a novel mechanism of competitive inhibition.

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