Modeling of DNA binding to the condensin hinge domain using molecular dynamics simulations guided by atomic force microscopy

The condensin protein complex compacts chromatin during mitosis using its DNA-loop extrusion activity. Previous studies proposed scrunching and loop-capture models as molecular mechanisms for the loop extrusion process, both of which assume the binding of double-strand (ds) DNA to the hinge domain formed at the interface of the condensin subunits Smc2 and Smc4. However, how the hinge domain contacts dsDNA has remained unknown. Here, we conducted atomic force microscopy imaging of the budding yeast condensin holo-complex and used this data as basis for coarse-grained molecular dynamics simulations to model the hinge structure in a transient open conformation. We then simulated the dsDNA binding to open and closed hinge conformations, predicting that dsDNA binds to the outside surface when closed and to the outside and inside surfaces when open. Our simulations also suggested that the hinge can close around dsDNA bound to the inside surface. Based on these simulation results, we speculate that the conformational change of the hinge domain might be essential for the dsDNA binding regulation and play roles in condensin-mediated DNA-loop extrusion

Importance of tyrosine residues of Bacillus stearothermophilus serine hydroxymethyltransferase in cofactor binding and L-allo-Thr cleavage

Serine hydroxymethyltransferase (SHMT) from Bacillus stearothermophilus (bsSHMT) is a pyridoxal 5′-phosphate-dependent enzyme that catalyses the conversion of L-serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. In addition, the enzyme catalyses the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids and transamination. In this article, we have examined the mechanism of the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids by SHMT. The three-dimensional structure and biochemical properties of Y51F and Y61A bsSHMTs and their complexes with substrates, especially L-allo-Thr, show that the cleavage of 3-hydroxy amino acids could proceed via Cα proton abstraction rather than hydroxyl proton removal. Both mutations result in a complete loss of tetrahydrofolate-dependent and tetrahydrofolate-independent activities. The mutation of Y51 to F strongly affects the binding of pyridoxal 5′-phosphate, possibly as a consequence of a change in the orientation of the phenyl ring in Y51F bsSHMT. The mutant enzyme could be completely reconstituted with pyridoxal 5′-phosphate. However, there was an alteration in the λmax value of the internal aldimine (396 nm), a decrease in the rate of reduction with NaCNBH3 and a loss of the intermediate in the interaction with methoxyamine (MA). The mutation of Y61 to A results in the loss of interaction with Cα and Cβ of the substrates. X-Ray structure and visible CD studies show that the mutant is capable of forming an external aldimine. However, the formation of the quinonoid intermediate is hindered. It is suggested that Y61 is involved in the abstraction of the Cα proton from 3-hydroxy amino acids. A new mechanism for the cleavage of 3-hydroxy amino acids via Cα proton abstraction by SHMT is proposed

Crystal structures of open and closed forms of D-serine deaminase from Salmonella typhimurium - implications on substrate specificity and catalysis

Metabolism of D-amino acids is of considerable interest due to their key importance in cell structure and function. Salmonella typhimurium D-serine deaminase (StDSD) is a pyridoxal 5' phosphate (PLP) dependent enzyme that catalyses degradation of D-Ser to pyruvate and ammonia. The first crystal structure of D-serine deaminase described here reveals a typical Foldtype II or tryptophan synthase beta subunit fold of PLP-dependent enzymes. Although holoenzyme was used for crystallization of both wild-type StDSD (WtDSD) and selenomethionine labelled StDSD (SeMetDSD), significant electron density was not observed for the cofactor, indicating that the enzyme has a low affinity for the cofactor under crystallization conditions. Interestingly, unexpected conformational differences were observed between the two structures. The WtDSD was in an open conformation while SeMetDSD, crystallized in the presence of isoserine, was in a closed conformation suggesting that the enzyme is likely to undergo conformational changes upon binding of substrate as observed in other Foldtype II PLP-dependent enzymes. Electron density corresponding to a plausible sodium ion was found near the active site of the closed but not in the open state of the enzyme. Examination of the active site and substrate modelling suggests that Thr166 may be involved in abstraction of proton from the C alpha atom of the substrate. Apart from the physiological reaction, StDSD catalyses a, b elimination of D-Thr, D-Allothr and L-Ser to the corresponding alpha-keto acids and ammonia. The structure of StDSD provides a molecular framework necessary for understanding differences in the rate of reaction with these substrates

Crystal structures of open and closed forms of d-serine deaminase from Salmonella typhimurium–implications on substrate specificity and catalysis

Metabolism of d-amino acids is of considerable interest due to their key importance in cell structure and function. Salmonella typhimuriumd-serine deaminase (StDSD) is a pyridoxal 5′ phosphate (PLP) dependent enzyme that catalyses degradation of d-Ser to pyruvate and ammonia. The first crystal structure of d-serine deaminase described here reveals a typical Foldtype II or tryptophan synthase β subunit fold of PLP-dependent enzymes. Although holoenzyme was used for crystallization of both wild-type StDSD (WtDSD) and selenomethionine labelled StDSD (SeMetDSD), significant electron density was not observed for the cofactor, indicating that the enzyme has a low affinity for the cofactor under crystallization conditions. Interestingly, unexpected conformational differences were observed between the two structures. The WtDSD was in an open conformation while SeMetDSD, crystallized in the presence of isoserine, was in a closed conformation suggesting that the enzyme is likely to undergo conformational changes upon binding of substrate as observed in other Foldtype II PLP-dependent enzymes. Electron density corresponding to a plausible sodium ion was found near the active site of the closed but not in the open state of the enzyme. Examination of the active site and substrate modelling suggests that Thr166 may be involved in abstraction of proton from the Cα atom of the substrate. Apart from the physiological reaction, StDSD catalyses α, β elimination of d-Thr, d-Allothr and l-Ser to the corresponding α-keto acids and ammonia. The structure of StDSD provides a molecular framework necessary for understanding differences in the rate of reaction with these substrates

Mechanistic Insights into the Neutralization of Cytotoxic Abrin by the Monoclonal Antibody D6F10

<div><p>Abrin, an A/B toxin obtained from the <i>Abrus precatorius</i> plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74–123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. <i>In silico</i> analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin <i>in vitro</i>. At 1∶10 molar concentration of abrin:antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin’s toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin.</p></div

Electrospray mass spectrometric characterization of hemoglobin Q (Hb Q-India) and a double mutant hemoglobin S/D in clinical samples

The clinical analysis of hemoglobin by ion exchange chromatography can result in ambiguities in identification of the nature of the globin chain present in patient samples. LC/ESI-MS provides rapid and precise determination of globin chain masses

The mAb D6F10 reduces the binding of abrin on HeLa cells at high concentrations.

<p>(A) 500 ng/ml of Alexa-488 labelled abrin was incubated with varying molar concentrations of the mAbs D6F10 or F5B10 or unlabelled abrin for 1 h at 4°C. 0.2 million HeLa cells were treated with the pre-incubated samples for 1 h on ice, washed with ice cold PBS and analysed by FACScan. Significant reduction in cell attachment of abrin was observed with unlabelled abrin (positive control) and mAb D6F10 (at 50 and 100 fold molar concentration) but not with F5B10 (isotype control). The figure is a representative of three separate experiments performed. (B) 0.04 million HeLa cells adhered on a cover slip were treated with Alexa-488 labelled abrin in the presence of 100 fold molar excess of unlabelled abrin, mAbs D6F10 or F5B10 for 1 h. Cells were then fixed with paraformaldehyde, stained with Hoechst dye, mounted on slides and observed under a Zeiss confocal scanning microscope.</p

The core epitope corresponding to the mAb D6F10 includes the residues Thr112, Gly114 and Arg118 of ABA.

<p>The uninduced (Un) and induced (In) samples of the chimeric proteins (∼45 kDa) of ABA and APA A chain were subjected to immunoblot analysis with mAb D6F10 or anti-GST antibody. (A) The recombinant protein ABA_1–123APA_124–175 bound the mAb D6F10 whereas no binding was observed with the proteins APA_1–123ABA_124–175 and ABA_1–73APA_74–175. (B) Sequence alignment of the amino acids 76–123 of ABA with the corresponding residues of APA A chain. Each box represents one recombinant clone obtained where the ABA residues highlighted in red are mutated to the corresponding amino acids of APA A chain, also highlighted in red. (C) Mutants of the truncated chimeric protein ABA_1–123APA_124–175 namely, T₈₂Q₈₃H₈₅ to SEF, L₈₇D₈₉ to FN, S₉₂D₉₆ to AT and D₁₀₃H₁₀₅ to QY bound the mAb D6F10 whereas the mutants namely, Y₁₁₀T₁₁₂G₁₁₄R₁₁₈ to DNDK and T₁₁₂G₁₁₄R₁₁₈ to NDK showed very little binding to the antibody.</p

The epitope corresponding to the mAb D6F10 is present close to the active site cleft of ABA.

<p>Surface diagram of ABA showing the active site cleft. The residues Tyr74, Tyr113, Glu164, Arg167 and Trp198 (in green) are the active site residues of ABA whereas the amino acids Thr112, Gly114 and Arg118 (in blue) are involved in the formation of the epitope. The figure clearly illustrates that Thr112 and Gly114 residues are present very close to the active site cleft of ABA.</p

The mAb D6F10 blocks abrin’s enzymatic activity/cytotoxicity.

<p>(A) Rescue of inhibition of protein synthesis by recombinant ABA (rABA) in cell-free system. Rabbit reticulocyte lysate containing amino acids and luciferase mRNA was treated with either 200 pM recombinant ABA (rABA) alone or in the presence of varying molar concentrations of the mAbs D6F10, F5B10 or 1C3E4 for 1 h at 37°C followed by addition of the luciferin substrate. The rescue of inhibition of protein synthesis in the test samples by the mAb D6F10 was evaluated as the luminescence obtained in comparison with the rABA control. The figure represents data obtained from three different experiments. (B) Rescue of inhibition of protein synthesis by abrin in HeLa cells. 0.2 million HeLa cells were cultured with either 200 pM abrin alone or in the presence of varying molar concentrations of the mAbs D6F10 or F5B10 for 7 h, starved in leucine-free RPMI for 2 h and pulsed with [³H] leucine for 1 h. Rescue of inhibition of protein synthesis by mAb D6F10 in abrin treated cells was evaluated as the radioactivity incorporated into test samples compared with the untreated control. The assay was performed in duplicates and carried out three times. Statistical analysis was performed using One way ANOVA followed by Tukey’s multiple comparison tests (*p<0.05).</p