Crystal Structure of the Cysteine Desulfurase DndA from Streptomyces lividans Which Is Involved in DNA Phosphorothioation

DNA phosphorothioation is widespread among prokaryotes, and might function to restrict gene transfer among different kinds of bacteria. There has been little investigation into the structural mechanism of the DNA phosphorothioation process. DndA is a cysteine desulfurase which is involved in the first step of DNA phosphorothioation. In this study, we determined the crystal structure of Streptomyces lividans DndA in complex with its covalently bound cofactor PLP, to a resolution of 2.4 Å. Our structure reveals the molecular mechanism that DndA employs to recognize its cofactor PLP, and suggests the potential binding site for the substrate L-cysteine on DndA. In contrast to previously determined structures of cysteine desulfurases, the catalytic cysteine of DndA was found to reside on a β strand. This catalytic cysteine is very far away from the presumable location of the substrate, suggesting that a conformational change of DndA is required during the catalysis process to bring the catalytic cysteine close to the substrate cysteine. Moreover, our in vitro enzymatic assay results suggested that this conformational change is unlikely to be a simple result of random thermal motion, since moving the catalytic cysteine two residues forward or backward in the primary sequence completely disabled the cysteine desulfurase activity of DndA

Satellite-based Estimates of Dust Deposition Into Tropical Atlantic Ocean

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Allosteric Autoinhibition Pathway in Transcription Factor ERG: Dynamics Network and Mutant Experimental Evaluations

Allosteric autoinhibition exists in many transcription factors. The ERG proteins exhibit autoinhibition on DNA binding by the C-terminal and N-terminal inhibitory domains (CID and NID). However, the autoinhibition mechanism and allosteric pathway of ERG are unknown. In this study we intend to elucidate the residue-level allosteric mechanism and pathway via a combined approach of computational and experimental analyses. Specifically computational residue-level fluctuation correlation data was analyzed to reveal detailed dynamics signatures in the allosteric autoinhibition process. A hypothesis of "NID/CID binding induced allostery" is proposed to link similar structures and different protein functions, which is subsequently validated by perturbation and mutation analyses in both computation and experiment. Two possible allosteric autoinhibition pathways of L286-L382-A379-G377-I360-Y355-R353 and L286-L382-A379-G377-I360-Y355- A351-K347-R350 were identified computationally and were confirmed by the computational and experimental mutations. Specifically we identified two mutation sites on the allosteric inhibition pathways, L286P/Q383P (NID/CID binding site) and I360G (pathway junction), which completely restore the wild type DNA binding affinity. These results suggest that the putative protein structure-function relationship may be augmented with a general relationship of protein "structure/fluctuation-correlation/function" for more thorough analyses of protein functions

Correlation of sLOX-1 Levels and MR Characteristics of Culprit Plaques in Intracranial Arteries with Stroke Recurrence

(1) Background: Symptomatic intracranial artery atherosclerosis (sICAS) is an important cause of acute ischaemic stroke (AIS) and is associated with a high risk of stroke recurrence. High-resolution magnetic resonance vessel wall imaging (HR-MR-VWI) is an effective method for evaluating atherosclerotic plaque characteristics. Soluble lectin-like oxidised low-density lipoprotein receptor-1 (sLOX-1) is closely associated with plaque formation and rupture. We aim to explore the correlation between sLOX-1 levels and culprit plaque characteristics, based on HR-MR-VWI, with stroke recurrence in patients with sICAS. (2) Methods: A total of 199 patients with sICAS underwent HR-MR-VWI between June 2020 and June 2021 in our hospital. The culprit vessel and plaque characteristics were assessed according to HR-MR-VWI, and sLOX-1 levels were measured by ELISA (enzyme linked immunosorbent assay). Outpatient follow-up was performed 3, 6, 9, and 12 months after discharge. (3) Results: sLOX-1 levels were significantly higher in the recurrence group than in the non-recurrence group (p p = 0.003, p = 0.014 and p = 0.010, respectively). The incidence of hyperintensity on T1WI, positive remodelling and significant enhancement (p p = 0.003 and p = 0.027, respectively) was higher in the recurrence group than in the non-recurrence group. Kaplan–Meier curves showed that patients with sLOX-1 levels > 912.19 pg/mL and hyperintensity on T1WI in the culprit plaque had a higher risk of stroke recurrence (both p 912.19 pg/mL (HR = 2.583, 95%CI 1.142, 5.846, p = 0.023) and hyperintensity on T1WI in the culprit plaque (HR = 2.632, 95% CI 1.197, 5.790, p = 0.016) were independent risk factors for stroke recurrence. sLOX-1 levels were significantly associated with the culprit plaque thickness (r = 0.162, p = 0.022), degree of stenosis (r = 0.217, p = 0.002), plaque burden (r = 0.183, p = 0.010), hyperintensity on T1WI (F = 14.501, p p p < 0.001) (4) Conclusions: sLOX-1 levels were associated with vulnerability of the culprit plaque and can be used as a supplement to HR-MR-VWI to predict stroke recurrence

Allosteric Autoinhibition Pathway in Transcription Factor ERG: Dynamics Network and Mutant Experimental Evaluations

Allosteric autoinhibition exists in many transcription factors. The ERG proteins exhibit autoinhibition on DNA binding by the C-terminal and N-terminal inhibitory domains (CID and NID). However, the autoinhibition mechanism and allosteric pathway of ERG are unknown. In this study we intend to elucidate the residue-level allosteric mechanism and pathway via a combined approach of computational and experimental analyses. Specifically computational residue-level fluctuation correlation data was analyzed to reveal detailed dynamics signatures in the allosteric autoinhibition process. A hypothesis of “NID/CID binding induced allostery” is proposed to link similar structures and different protein functions, which is subsequently validated by perturbation and mutation analyses in both computation and experiment. Two possible allosteric autoinhibition pathways of L286-L382-A379-G377-I360-Y355-R353 and L286-L382-A379-G377-I360-Y355- A351-K347-R350 were identified computationally and were confirmed by the computational and experimental mutations. Specifically we identified two mutation sites on the allosteric inhibition pathways, L286P/Q383P (NID/CID binding site) and I360G (pathway junction), which completely restore the wild type DNA binding affinity. These results suggest that the putative protein structure–function relationship may be augmented with a general relationship of protein “structure/fluctuation–correlation/function” for more thorough analyses of protein functions

Crystal Structure of StnA for the Biosynthesis of Antitumor Drug Streptonigrin Reveals a Unique Substrate Binding Mode.

Streptonigrin methylesterase A (StnA) is one of the tailoring enzymes that modify the aminoquinone skeleton in the biosynthesis pathway of Streptomyces species. Although StnA has no significant sequence homology with the reported α/β-fold hydrolases, it shows typical hydrolytic activity in vivo and in vitro. In order to reveal its functional characteristics, the crystal structures of the selenomethionine substituted StnA (SeMet-StnA) and the complex (S185A mutant) with its substrate were resolved to the resolution of 2.71 Å and 2.90 Å, respectively. The overall structure of StnA can be described as an α-helix cap domain on top of a common α/β hydrolase domain. The substrate methyl ester of 10'-demethoxystreptonigrin binds in a hydrophobic pocket that mainly consists of cap domain residues and is close to the catalytic triad Ser185-His349-Asp308. The transition state is stabilized by an oxyanion hole formed by the backbone amides of Ala102 and Leu186. The substrate binding appears to be dominated by interactions with several specific hydrophobic contacts and hydrogen bonds in the cap domain. The molecular dynamics simulation and site-directed mutagenesis confirmed the important roles of the key interacting residues in the cap domain. Structural alignment and phylogenetic tree analysis indicate that StnA represents a new subfamily of lipolytic enzymes with the specific binding pocket located at the cap domain instead of the interface between the two domains

Crystal Structure of StnA for the Biosynthesis of Antitumor Drug Streptonigrin Reveals a Unique Substrate Binding Mode.

Streptonigrin methylesterase A (StnA) is one of the tailoring enzymes that modify the aminoquinone skeleton in the biosynthesis pathway of Streptomyces species. Although StnA has no significant sequence homology with the reported α/β-fold hydrolases, it shows typical hydrolytic activity in vivo and in vitro. In order to reveal its functional characteristics, the crystal structures of the selenomethionine substituted StnA (SeMet-StnA) and the complex (S185A mutant) with its substrate were resolved to the resolution of 2.71 Å and 2.90 Å, respectively. The overall structure of StnA can be described as an α-helix cap domain on top of a common α/β hydrolase domain. The substrate methyl ester of 10'-demethoxystreptonigrin binds in a hydrophobic pocket that mainly consists of cap domain residues and is close to the catalytic triad Ser185-His349-Asp308. The transition state is stabilized by an oxyanion hole formed by the backbone amides of Ala102 and Leu186. The substrate binding appears to be dominated by interactions with several specific hydrophobic contacts and hydrogen bonds in the cap domain. The molecular dynamics simulation and site-directed mutagenesis confirmed the important roles of the key interacting residues in the cap domain. Structural alignment and phylogenetic tree analysis indicate that StnA represents a new subfamily of lipolytic enzymes with the specific binding pocket located at the cap domain instead of the interface between the two domains

Control of chrysanthemum flowering through integration with an aging pathway

The mechanisms by which plant age regulates flowering remain incompletely understood. Here the authors show that age dependent regulation of SPL transcription factors by miR156 influence flowering via control of NF-YB8 expression in Chrysanthemum