scholarly journals Structure of a tRNA-specific deaminase with compromised deamination activity

2020 ◽  
Vol 477 (8) ◽  
pp. 1483-1497
Author(s):  
Huijuan Liu ◽  
Saibin Wu ◽  
Dewei Ran ◽  
Wei Xie
Keyword(s):  
Molecular Level ◽  
Evolutionary Strategy ◽  
Structural Basis ◽  
Mutant Form ◽  
Binding Assays ◽  
Natural Evolution ◽  
Dimer Interface ◽  
Mycoplasma Capricolum ◽  
Insight Into

Nucleotide 34 in tRNA is extensively modified to ensure translational fidelity and efficacy in cells. The deamination of adenosine at this site catalyzed by the enzyme TadA gives rise to inosine (I), which serves as a typical example of the wobble hypothesis due to its diverse basepairing capability. However, recent studies have shown that tRNAArgACG in Mycoplasma capricolum contains unmodified adenosine, in order to decode the CGG codon. The structural basis behind the poorly performing enzyme M. capricolum TadA (McTadA) is largely unclear. Here we present the structures of the WT and a mutant form of McTadA determined at high resolutions. Through structural comparison between McTadA and other active TadA enzymes as well as modeling efforts, we found that McTadA presents multiple structural conflicts with RNA substrates and thus offered support to previous studies from a structural perspective. These clashes would potentially lead to reduced substrate binding affinity of McTadA, consistent with our in vitro deamination activity and binding assays. To rescue the deamination activity of McTadA, we carried out two rounds of protein engineering through structure-guided design. The unsuccessful attempts of the activity restoration could be attributed to the altered dimer interface and stereo hindrance from the non-catalytic subunit of McTadA, which could be the inevitable outcome of the natural evolution. Our study provides structural insight into an alternative decoding and evolutionary strategy by a compromised TadA enzyme at a molecular level.

scholarly journals Structural basis for distinct inflammasome complex assembly by human NLRP1 and CARD8

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qin Gong ◽  
Kim Robinson ◽  
Chenrui Xu ◽  
Phuong Thao Huynh ◽  
Kelvin Han Chung Chong ◽  
...  
Keyword(s):  
Cell Death ◽  
Inner Core ◽  
Inflammatory Diseases ◽  
Structural Features ◽  
Structural Basis ◽  
Cultured Human Cells ◽  
Structural Insight ◽  
Sensor Proteins ◽  
Insight Into

AbstractNod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related sensor proteins NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, their mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to assemble distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core of oligomerized CARD surrounded by an outer ring of FIINDUPA. Biochemically, self-assembled NLRP1-CARD filaments are sufficient to drive ASC speck formation in cultured human cells—a process that is greatly enhanced by NLRP1-FIINDUPA which forms oligomers in vitro. The cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments, solved here at 3.7 Å, uncover unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide structural insight into the mechanisms of activation for human NLRP1 and CARD8 and reveal how highly specific signaling can be achieved by heterotypic CARD interactions within the inflammasome complexes.

scholarly journals Investigating crosstalk among PTMs provides novel insight into the structural basis underlying the differential effects of Nt17 PTMs on mutant Httex1 aggregation

2021 ◽  
Author(s):  
Anass Chiki ◽  
Zhidian Zhang ◽  
Kolla Rajasekhar ◽  
Luciano A. Abriata ◽  
Iman Rostami ◽  
...  
Keyword(s):  
Inhibitory Effect ◽  
Dynamics Simulation ◽  
Helical Conformation ◽  
Structural Basis ◽  
Similar Distribution ◽  
Post Translational Modifications ◽  
Differential Effects ◽  
The Impact ◽  
Insight Into

AbstractPost-translational modifications (PTMs) within the first 17 amino acids (Nt17) of the Huntingtin protein (Htt) have been shown to inhibit the aggregation and attenuate the toxicity of mutant Htt proteins in vitro and in various models of Huntington’s disease. Our group’s previous studies suggested that the Nt17 PTM code is a combinatorial code that involves a complex interplay between different PTMs. Here, we expand on these studies by investigating the effect of methionine 8 oxidation (oxM8) and crosstalk between this PTM and either lysine 6 acetylation (AcK6) or threonine 3 phosphorylation (pT3) on the aggregation of mutant Httex1. We show that M8 oxidation delays but does not inhibit the aggregation and has no effect on the final morphologies of mutant Httex1 aggregates. This delay in aggregation kinetics could be attributed to the transient accumulation of oligomeric aggregates, which disappear upon the formation of Httex1 oxM8 fibrils. Interestingly, the presence of both oxM8 and AcK6 resulted in dramatic inhibition of Httex1 fibrillization, whereas the presence of oxM8 did not influence the aggregation inhibitory effect of pT3. To gain insight into the structural basis underlying these proteins’ aggregation properties, we investigated the impact of each PTM and the combination of these PTMs on the conformational properties of the Nt17 peptide by circular dichroism spectroscopy and molecular dynamics simulation. These studies show that M8 oxidation decreases the helicity of the Nt17 in the presence or absence of PTMs and provides novel insight into the structural basis underlying the effects of different PTMs on mutant Httex1 aggregation. PTMs that lower the mutant Httex1 aggregation rate (oxM8, AcK6/oxM8, pT3, pT3/oxM8, and phosphorylation at Serine 13) result in stabilization and increased population of a short N-terminal helix (first eight residues) in Nt17 or decreased abundance of other helical forms, including long helix and short C-terminal helix. PTMs that did not alter the aggregation of mutant Httex1 exhibit a similar distribution of helical conformation as the unmodified peptides. These results show that the relative abundance of N- vs. C-terminal helical conformations and long helices, rather than the overall helicity of Nt17, better explains the effect of different Nt17 PTMs on mutant Httex1; thus, explaining the lack of correlation between the effect of PTMs on the overall helicity of Nt17 and mutant Httex1 aggregation in vitro. Taken together, our results provide novel structural insight into the differential effects of single PTMs and crosstalk between different PTMs in regulating mutant Httex1 aggregation.TOC Figure

scholarly journals Investigating Crosstalk Among PTMs Provides Novel Insight Into the Structural Basis Underlying the Differential Effects of Nt17 PTMs on Mutant Httex1 Aggregation

2021 ◽  
Vol 8 ◽  
Author(s):  
Anass Chiki ◽  
Zhidian Zhang ◽  
Kolla Rajasekhar ◽  
Luciano A. Abriata ◽  
Iman Rostami ◽  
...  
Keyword(s):  
Dynamics Simulation ◽  
Helical Conformation ◽  
Structural Basis ◽  
Similar Distribution ◽  
Simulation Studies ◽  
Post Translational Modifications ◽  
Differential Effects ◽  
Aggregation Rate ◽  
Insight Into

Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of the Huntingtin protein (Htt) have been shown to inhibit the aggregation and attenuate the toxicity of mutant Htt proteins in vitro and in various models of Huntington’s disease. Here, we expand on these studies by investigating the effect of methionine eight oxidation (oxM8) and its crosstalk with lysine 6 acetylation (AcK6) or threonine 3 phosphorylation (pT3) on the aggregation of mutant Httex1 (mHttex1). We show that M8 oxidation delays but does not inhibit the aggregation and has no effect on the final morphologies of mHttex1aggregates. The presence of both oxM8 and AcK6 resulted in dramatic inhibition of Httex1 fibrillization. Circular dichroism spectroscopy and molecular dynamics simulation studies show that PTMs that lower the mHttex1 aggregation rate (oxM8, AcK6/oxM8, pT3, pT3/oxM8, and pS13) result in increased population of a short N-terminal helix (first eight residues) in Nt17 or decreased abundance of other helical forms, including long helix and short C-terminal helix. PTMs that did not alter the aggregation rate (AcK6) of mHttex1 exhibit a similar distribution of helical conformation as the unmodified peptides. These results show that the relative abundance of N- vs. C-terminal helical conformations and long helices, rather than the overall helicity of Nt17, better explains the effect of different Nt17 PTMs on mHttex1; thus, explaining the lack of correlation between the effect of PTMs on the overall helicity of Nt17 and mHttex1 aggregation in vitro. Taken together, our results provide novel structural insight into the differential effects of single PTMs and crosstalk between different PTMs in regulating mHttex1 aggregation.

scholarly journals The SARS-CoV-2 Spike Variant D614G Favors an Open Conformational State

2020 ◽  
Author(s):  
Rachael A. Mansbach ◽  
Srirupa Chakraborty ◽  
Kien Nguyen ◽  
David C. Montefiori ◽  
Bette Korber ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Molecular Level ◽  
Receptor Binding Domain ◽  
Target Region ◽  
Infected People ◽  
Viral Loads ◽  
Rapid Transition ◽  
Open States ◽  
Insight Into

SummaryThe COVID-19 pandemic underwent a rapid transition with the emergence of a SARS-CoV-2 variant that carried the amino acid substitution D614G in the Spike protein that became globally prevalent. The G-form is both more infectious in vitro and associated with increased viral loads in infected people. To gain insight into the mechanism underlying these distinctive characteristics, we employed multiple replicas of microsecond all-atom simulations to probe the molecular-level impact of this substitution on Spike’s closed and open states. The open state enables Spike interactions with its human cellular receptor, ACE2. Here we show that changes in the inter-protomer energetics due to the D614G substitution favor a higher population of infection-capable (open) states. The inter-protomer interactions between S1 and S2 subunits in the open state of the D-form are asymmetric. This asymmetry is resolved in the G-form due to the release of tensile hydrogen bonds resulting in an increased population of open conformations. Thus, the increased infectivity of the G-form is likely due to a higher rate of profitable binding encounters with the host receptor. It is also predicted to be more neutralization sensitive due to enhanced exposure of the receptor binding domain, a key target region for neutralizing antibodies.

scholarly journals Thyroid Peroxidase Activity is Inhibited by Phenolic Compounds—Impact of Interaction

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2766
Author(s):  
Ewa Habza-Kowalska ◽  
Agnieszka A. Kaczor ◽  
Justyna Żuk ◽  
Dariusz Matosiuk ◽  
Urszula Gawlik-Dziki
Keyword(s):  
Chlorogenic Acid ◽  
Rosmarinic Acid ◽  
Molecular Level ◽  
Combination Index ◽  
3D Structure ◽  
Thyroid Peroxidase ◽  
Ic50 Values ◽  
Insight Into ◽  
Rutin And Quercetin

The aim of this study was to estimate the mode of thyroid peroxidase (TPO) inhibition by polyphenols: Chlorogenic acid, rosmarinic acid, quercetin, and rutin. All the tested polyphenols inhibited TPO; the IC50 values ranged from 0.004 mM to 1.44 mM (for rosmarinic acid and rutin, respectively). All these pure phytochemical substances exhibited different modes of TPO inhibition. Rutin and rosmarinic acid showed competitive, quercetin—uncompetitive and chlorogenic acid—noncompetitive inhibition effect on TPO. Homology modeling was used to gain insight into the 3D structure of TPO and molecular docking was applied to study the interactions of the inhibitors with their target at the molecular level. Moreover, the type and strength of mutual interactions between the inhibitors (expressed as the combination index, CI) were analyzed. Slight synergism, antagonism, and moderate antagonism were found in the case of the combined addition of the pure polyphenols. Rutin and quercetin as well as rutin and rosmarinic acid acted additively (CI = 0.096 and 1.06, respectively), while rutin and chlorogenic acid demonstrated slight synergism (CI = 0.88) and rosmarinic acid with quercetin and rosmarinic acid with chlorogenic acid showed moderate antagonism (CI = 1.45 and 1.25, respectively). The mixture of chlorogenic acid and quercetin demonstrated antagonism (CI = 1.79). All the polyphenols showed in vitro antiradical ability against 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), ABTS. The highest ability (expressed as IC50) was exhibited by rosmarinic acid (0.12 mM) and the lowest value was ascribed to quercetin (0.45 mM).

scholarly journals Molecular mechanisms and structural features of cardiomyopathy-causing troponin T mutants in the tropomyosin overlap region

2017 ◽  
Vol 114 (42) ◽  
pp. 11115-11120 ◽  
Author(s):  
Binnu Gangadharan ◽  
Margaret S. Sunitha ◽  
Souhrid Mukherjee ◽  
Ritu Roy Chowdhury ◽  
Farah Haque ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Troponin T ◽  
Point Mutations ◽  
Structural Features ◽  
Sarcomeric Proteins ◽  
Binding Assays ◽  
Genes Encoding ◽  
The Stability ◽  
Insight Into

Point mutations in genes encoding sarcomeric proteins are the leading cause of inherited primary cardiomyopathies. Among them are mutations in the TNNT2 gene that encodes cardiac troponin T (TnT). These mutations are clustered in the tropomyosin (Tm) binding region of TnT, TNT1 (residues 80–180). To understand the mechanistic changes caused by pathogenic mutations in the TNT1 region, six hypertrophic cardiomyopathy (HCM) and two dilated cardiomyopathy (DCM) mutants were studied by biochemical approaches. Binding assays in the absence and presence of actin revealed changes in the affinity of some, but not all, TnT mutants for Tm relative to WT TnT. HCM mutants were hypersensitive and DCM mutants were hyposensitive to Ca2+ in regulated actomyosin ATPase activities. To gain better insight into the disease mechanism, we modeled the structure of TNT1 and its interactions with Tm. The stability predictions made by the model correlated well with the affinity changes observed in vitro of TnT mutants for Tm. The changes in Ca2+ sensitivity showed a strong correlation with the changes in binding affinity. We suggest the primary reason by which these TNNT2 mutations between residues 92 and 144 cause cardiomyopathy is by changing the affinity of TnT for Tm within the TNT1 region.

scholarly journals NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors

2020 ◽  
Author(s):  
Bryon P. Mahler ◽  
Pawel J. Bujalowski ◽  
Huzhang Mao ◽  
Erik A. Dill ◽  
Paul J. Jardine ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Molecular Motors ◽  
Solution Structure ◽  
Nuclease Activity ◽  
Structural Basis ◽  
Binding Assays ◽  
Dna Viruses ◽  
Double Stranded Dna ◽  
Fluorescence Binding ◽  
Insight Into

SummaryDouble-stranded DNA viruses use ATP-powered molecular motors to package their genomes. To do so, these motors must efficiently transition between initiation, translocation, and termination modes. Here, we report structural and biophysical analyses of the C-terminal domain of the bacteriophage phi29 ATPase (CTD) that suggest a structural basis for these functional transitions. Sedimentation experiments show that the inter-domain linker in the full-length protein promotes dimerization and thus may play a role in assembly of the functional motor. The NMR solution structure of the CTD indicates it is a vestigial nuclease domain that likely evolved from conserved nuclease domains in phage terminases. Despite the loss of nuclease activity, fluorescence binding assays confirm the CTD retains its DNA binding capabilities and fitting the CTD into cryoEM density of the phi29 motor shows that the CTD directly binds DNA. However, the interacting residues differ from those identified by NMR titration in solution, suggesting that packaging motors undergo conformational changes to transition between initiation, translocation, and termination.

scholarly journals Structural basis for distinct inflammasome complex assembly by human NLRP1 and CARD8

2020 ◽  
Author(s):  
Gong Qin ◽  
Kim Robinson ◽  
Xu Chenrui ◽  
Zhang Jiawen ◽  
Boo Zhao Zhi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Inner Core ◽  
Inflammatory Diseases ◽  
Structural Features ◽  
Structural Basis ◽  
Card Domain ◽  
Domain Interactions ◽  
Cultured Human Cells ◽  
Insight Into

AbstractNod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related NLR proteins, NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, the molecular mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to trigger the assembly of distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core composed of oligomerized CARD domains and the outer layer consisting of FIINDUPA rings. Biochemically, oligomerized NLRP1-CARD is sufficient to drive ASC speck formation in cultured human cells via filament formation-a process that is greatly enhanced by NLRP1-FIINDUPA, which forms ring-like oligomers in vitro. In addition, we report the cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments at 3.7 Å, which uncovers unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide unique structural insight into the mechanisms of activation for human NLRP1 and CARD8, uncovering an unexpected level of specificity in inflammasome signaling mediated by heterotypic CARD domain interactions.

scholarly journals The structural basis of huntingtin (Htt) fibril polymorphism, revealed by cryo-EM of exon 1 Htt fibrils

2021 ◽  
Author(s):  
Sergey Nazarov ◽  
Anass Chiki ◽  
Driss Boudeffa ◽  
Hilal Lashuel
Keyword(s):  
Structural Heterogeneity ◽  
Structural Basis ◽  
Particle Analysis ◽  
Structural Determinants ◽  
Huntingtin Protein ◽  
Fibril Structure ◽  
Exon 1 ◽  
High Degree ◽  
Insight Into

The lack of detailed insight into the structure of aggregates formed by the huntingtin protein has hampered efforts to develop therapeutics and diagnostics targeting pathology formation in the brain of patients with Huntington's disease. To address this knowledge gap, we investigated the structural properties of in vitro generated fibrils from exon1 of the huntingtin protein by electron cryo-microscopy and single- particle analysis. We show that wildtype and mutant exon1 of the huntingtin protein form non-helical fibrils with a polygultamine amyloid core composed of β-hairpins with unique characteristics that have not been previously observed with other amyloid filaments. The stacks of β-hairpins form long planar β- sheets (protofilaments) with variable stacking angle and occasional out-of-register state of individual β-hairpins. These features and the propensity of protofilament to undergo lateral association results in a high degree of fibril polymorphism, including fibrils composed of varying numbers of protofilaments. Our results also represent the first direct observation of how the flanking domains are organized around the polyglutamine core of the fibril and provide insight into how they might affect huntingtin fibril structure, polymorphism, and stacking of β-hairpins within its core structure. Removal of the first 17 amino acids at the N-terminus resulted in surprising intra-fibril structural heterogeneity and reduced fibril's propensity to lateral associations. Overall, this work provides valuable insights that could guide future mechanistic studies to elucidate the sequence and structural determinants of huntingtin aggregation, as well as cryo- EM and structural studies of fibrils derived from huntingtin proteins and other disease-associated polyglutamine-containing proteins.

scholarly journals Structures of ABCB4 provide insight into phosphatidylcholine translocation

2021 ◽  
Vol 118 (33) ◽  
pp. e2106702118
Author(s):  
Kamil Nosol ◽  
Rose Bang-Sørensen ◽  
Rossitza N. Irobalieva ◽  
Satchal K. Erramilli ◽  
Bruno Stieger ◽  
...  
Keyword(s):  
Bile Salts ◽  
Structural Basis ◽  
Tryptophan Residue ◽  
Bile Canaliculi ◽  
Central Cavity ◽  
Translocation Assay ◽  
Specific Lipid ◽  
Insight Into

ABCB4 is expressed in hepatocytes and translocates phosphatidylcholine into bile canaliculi. The mechanism of specific lipid recruitment from the canalicular membrane, which is essential to mitigate the cytotoxicity of bile salts, is poorly understood. We present cryogenic electron microscopy structures of human ABCB4 in three distinct functional conformations. An apo-inward structure reveals how phospholipid can be recruited from the inner leaflet of the membrane without flipping its orientation. An occluded structure reveals a single phospholipid molecule in a central cavity. Its choline moiety is stabilized by cation-π interactions with an essential tryptophan residue, rationalizing the specificity of ABCB4 for phosphatidylcholine. In an inhibitor-bound structure, a posaconazole molecule blocks phospholipids from reaching the central cavity. Using a proteoliposome-based translocation assay with fluorescently labeled phosphatidylcholine analogs, we recapitulated the substrate specificity of ABCB4 in vitro and confirmed the role of the key tryptophan residue. Our results provide a structural basis for understanding an essential translocation step in the generation of bile and its sensitivity to azole drugs.

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