Molecular mechanism of the calcium-induced conformational change in the spectrin EF-hands.

AbstractWhile genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

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A microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1)

Molecular Biology of the Cell ◽

10.1091/mbc.e17-11-0689 ◽

2018 ◽

Vol 29 (9) ◽

pp. 1060-1074 ◽

Cited By ~ 25

Author(s):

Tomohiro Kubo ◽

Yuqing Hou ◽

Deborah A. Cochran ◽

George B. Witman ◽

Toshiyuki Oda

Keyword(s):

Conformational Change ◽

Molecular Mechanism ◽

Wild Type ◽

Sliding Direction ◽

Large Conformational Change ◽

Dynein Arms ◽

Biochemical Analyses

Motility of cilia/flagella is generated by a coordinated activity of thousands of dyneins. Inner dynein arms (IDAs) are particularly important for the formation of ciliary/flagellar waveforms, but the molecular mechanism of IDA regulation is poorly understood. Here we show using cryoelectron tomography and biochemical analyses of Chlamydomonas flagella that a conserved protein FAP44 forms a complex that tethers IDA f (I1 dynein) head domains to the A-tubule of the axonemal outer doublet microtubule. In wild-type flagella, IDA f showed little nucleotide-dependent movement except for a tilt in the f β head perpendicular to the microtubule-sliding direction. In the absence of the tether complex, however, addition of ATP and vanadate caused a large conformational change in the IDA f head domains, suggesting that the movement of IDA f is mechanically restricted by the tether complex. Motility defects in flagella missing the tether demonstrates the importance of the IDA f-tether interaction in the regulation of ciliary/flagellar beating.

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Structural Analysis of an L-Cysteine Desulfurase from an Ssp DNA Phosphorothioation System

mBio ◽

10.1128/mbio.00488-20 ◽

2020 ◽

Vol 11 (2) ◽

Author(s):

Liqiong Liu ◽

Susu Jiang ◽

Mai Xing ◽

Chao Chen ◽

Chongde Lai ◽

...

Keyword(s):

Crystal Structure ◽

Conformational Change ◽

Molecular Mechanism ◽

Sulfur Atom ◽

Nucleophilic Attack ◽

Nonbridging Oxygen ◽

Long Distance ◽

Cysteine Desulfurase ◽

Modification System ◽

Pt Modification

ABSTRACT DNA phosphorothioate (PT) modification, in which the nonbridging oxygen in the sugar-phosphate backbone is substituted by sulfur, is catalyzed by DndABCDE or SspABCD in a double-stranded or single-stranded manner, respectively. In Dnd and Ssp systems, mobilization of sulfur in PT formation starts with the activation of the sulfur atom of cysteine catalyzed by the DndA and SspA cysteine desulfurases, respectively. Despite playing the same biochemical role, SspA cannot be functionally replaced by DndA, indicating its unique physiological properties. In this study, we solved the crystal structure of Vibrio cyclitrophicus SspA in complex with its natural substrate, cysteine, and cofactor, pyridoxal phosphate (PLP), at a resolution of 1.80 Å. Our solved structure revealed the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor, suggesting a common binding mode shared by cysteine desulfurases. In addition, although the distance between the catalytic Cys314 and the substrate cysteine is 8.9 Å, which is too far for direct interaction, our structural modeling and biochemical analysis revealed a conformational change in the active site region toward the cysteine substrate to move them close to each other to facilitate the nucleophilic attack. Finally, the pulldown analysis showed that SspA could form a complex with SspD, an ATP pyrophosphatase, suggesting that SspD might potentially accept the activated sulfur atom directly from SspA, providing further insights into the biochemical pathway of Ssp-mediated PT modification. IMPORTANCE Apart from its roles in Fe-S cluster assembly, tRNA thiolation, and sulfur-containing cofactor biosynthesis, cysteine desulfurase serves as a sulfur donor in the DNA PT modification, in which a sulfur atom substitutes a nonbridging oxygen in the DNA phosphodiester backbone. The initial sulfur mobilization from l-cysteine is catalyzed by the SspA cysteine desulfurase in the SspABCD-mediated DNA PT modification system. By determining the crystal structure of SspA, the study presents the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor. To overcome the long distance (8.9 Å) between the catalytic Cys314 and the cysteine substrate, a conformational change occurs to bring Cys314 to the vicinity of the substrate, allowing for nucleophilic attack.

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Molecular mechanism of multispecific recognition of Calmodulin through conformational changes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615949114 ◽

2017 ◽

Vol 114 (20) ◽

pp. E3927-E3934 ◽

Cited By ~ 18

Author(s):

Fei Liu ◽

Xiakun Chu ◽

H. Peter Lu ◽

Jin Wang

Keyword(s):

Protein Binding ◽

Conformational Change ◽

Molecular Mechanism ◽

Conformational Changes ◽

Electrostatic Interaction ◽

Hydrophobic Interaction ◽

High Specificity ◽

Binding Peptide ◽

Conformational Selection ◽

High Affinity

Calmodulin (CaM) is found to have the capability to bind multiple targets. Investigations on the association mechanism of CaM to its targets are crucial for understanding protein–protein binding and recognition. Here, we developed a structure-based model to explore the binding process between CaM and skMLCK binding peptide. We found the cooperation between nonnative electrostatic interaction and nonnative hydrophobic interaction plays an important role in nonspecific recognition between CaM and its target. We also found that the conserved hydrophobic anchors of skMLCK and binding patches of CaM are crucial for the transition from high affinity to high specificity. Furthermore, this association process involves simultaneously both local conformational change of CaM and global conformational changes of the skMLCK binding peptide. We found a landscape with a mixture of the atypical “induced fit,” the atypical “conformational selection,” and “simultaneously binding–folding,” depending on the synchronization of folding and binding. Finally, we extend our discussions on multispecific binding between CaM and its targets. These association characteristics proposed for CaM and skMLCK can provide insights into multispecific binding of CaM.

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Molecular mechanism of quorum sensing inhibition in Streptococcus by the phage protein paratox

10.1101/2021.06.03.446943 ◽

2021 ◽

Author(s):

Nicole R. Rutbeek ◽

Hanieh Rezasoltani ◽

Trushar R. Patel ◽

Mazdak Khajehpour ◽

Gerd Prehna

Keyword(s):

Quorum Sensing ◽

Conformational Change ◽

Molecular Mechanism ◽

Streptococcus Pyogenes ◽

Scattering Data ◽

Gram Positive ◽

Quorum Sensing Inhibition ◽

X Ray ◽

Phage Protein ◽

Phage Proteins

Streptococcus pyogenes, or Group A Streptococcus, is a Gram-positive bacterium that can be both a human commensal and pathogen. Central to this dichotomy are temperate bacteriophages that incorporate into the bacterial genome as a prophage. These genetic elements encode both the phage proteins as well as toxins harmful to the human host. One such conserved phage protein paratox (Prx) is always found encoded adjacent to the toxin genes and this linkage is preserved during transduction. Within Streptococcus pyogenes, Prx functions to inhibit the quorum-sensing ComRS receptor-signal pair that is the master regulator of natural competence, or the ability to uptake endogenous DNA. Specifically, Prx directly binds and inhibits the receptor ComR by unknown mechanism. To understand how Prx inhibits ComR at the molecular level we pursued an X-ray crystal structure of Prx bound to ComR. The structural data supported by solution X-ray scattering data demonstrate that Prx induces a conformational change in ComR to directly access the DNA binding domain. Furthermore, electromobility shift assays and competition binding assays reveal that Prx effectively uncouples the inter-domain conformational change that is required for activation of ComR by the signaling molecule XIP. Although to our knowledge the molecular mechanism of quorum-sensing inhibition by Prx is unique, it is analogous to the mechanism employed by the phage protein Aqs1 in Pseudomonas aeruginosa. Together, this demonstrates an example of convergent evolution between Gram-positive and Gram-negative phages to inhibit quorum-sensing, and highlights the versatility of small phage proteins.

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A Molecular Mechanism for Lys49-Phospholipase A2Activity Based on Ligand-induced Conformational Change

Journal of Biological Chemistry ◽

10.1074/jbc.m410588200 ◽

2004 ◽

Vol 280 (8) ◽

pp. 7326-7335 ◽

Cited By ~ 49

Author(s):

Andre L. B. Ambrosio ◽

M. Cristina Nonato ◽

Heloísa S. Selistre de Araújo ◽

Raghuvir Arni ◽

Richard J. Ward ◽

...

Keyword(s):

Conformational Change ◽

Molecular Mechanism

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Insights into Genome Recoding from the Mechanism of a Classic +1-Frameshifting tRNA

10.1101/2020.12.31.424971 ◽

2021 ◽

Author(s):

Howard Gamper ◽

Haixing Li ◽

Isao Masuda ◽

D. Miklos Robkis ◽

Thomas Christian ◽

...

Keyword(s):

Amino Acids ◽

Conformational Change ◽

Molecular Mechanism ◽

Late Stage ◽

Anticodon Loop ◽

Pairing Mechanism ◽

Extra Nucleotide ◽

In Cells

ABSTRACTWhile genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

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The interaction of α2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism

Biochemical Journal ◽

10.1042/bj1330709 ◽

1973 ◽

Vol 133 (4) ◽

pp. 709-724 ◽

Cited By ~ 764

Author(s):

Alan J. Barrett ◽

Phyllis M. Starkey

Keyword(s):

Conformational Change ◽

Molecular Mechanism ◽

Active Site ◽

Peptide Bond ◽

Serine Proteinases ◽

Equimolar Ratio ◽

Binding Characteristics ◽

Sensitive Region ◽

Physiological Importance ◽

Α2 Macroglobulin

1. α2-Macroglobulin is known to bind and inhibit a number of serine proteinases. We show that it binds thiol and carboxyl proteinases, and there is now reason to believe that α2-macroglobulin can bind essentially all proteinases. 2. Radiochemically labelled trypsin, chymotrypsin, cathepsin B1 and papain are bound by α2-macroglobulin in an approximately equimolar ratio. Equimolar binding was confirmed for trypsin by activesite titration. 3. Pretreatment of α2-macroglobulin with a saturating amount of one proteinase prevented the subsequent binding of another. We conclude that each molecule of α2-macroglobulin is able to react with one molecule of proteinase only. 4. α2-Macroglobulin did not react with exopeptidases, non-proteolytic hydrolases or inactive forms of endopeptidases. 5. The literature on binding and inhibition of proteinases by α2-macroglobulin is reviewed, and from consideration of this and our own work several general characteristics of the interaction can be discerned. 6. A model is proposed for the molecular mechanism of the interaction of α2-macroglobulin with proteinases. It is suggested that the enzyme cleaves a peptide bond in a sensitive region of the macroglobulin, and that this results in a conformational change in the α2-macroglobulin molecule that traps the enzyme irreversibly. Access of substrates to the active site of the enzyme becomes sterically hindered, causing inhibition that is most pronounced with large substrate molecules. 7. The possible physiological importance of the unique binding characteristics of α2-macroglobulin is discussed.

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Arsenic Binding to RBCC Domain Modulates Oncogenic PML-RARa and Wild-Type PML as a Key Molecular Mechanism of Arsenic Trioxide Treatment for APL.

Blood ◽

10.1182/blood.v114.22.592.592 ◽

2009 ◽

Vol 114 (22) ◽

pp. 592-592

Author(s):

Xiaowei Zhang ◽

Xiaojing Yan ◽

Feifei Yang ◽

Ziren Zhou ◽

Ziyu Wu ◽

...

Keyword(s):

Conformational Change ◽

Molecular Mechanism ◽

Metal Binding ◽

Conflicts Of Interest ◽

Periodic Fever ◽

Coiled Coil ◽

Wild Type ◽

Selective Degradation ◽

Enzymatic Machinery

Abstract Abstract 592 Arsenicals represent one group of the oldest drugs used in both traditional Chinese medicine (TCM) and Western medicine since 2,000 years ago to treat a variety of ailments from periodic fever to cancer. Recently, this ancient remedy has been revived due to its remarkable therapeutic efficacy for acute promyelocytic leukemia (APL) through selective degradation of the leukemogenic PML-RARaƒn as well as the wild-type PML protein. However, the precise molecular mechanism leading to arsenic-initiated modulationƒn of the target proteins remained unclear. Here we show that arsenic directly binds to PML and PML-RARaƒn through their RBCC (RING-B box-coiled coil) domain which contains conserved cysteine/histidine residues with metal-binding ability. Among RBCC domain, the RING and B2 motif are responsible for arsenic binding in cells, with recombinant RING motif showing the highest affinity to arsenic binding in vitro. We also observed that arsenic tends to coordinate with three sulfur atoms from the three conserved cysteines in the RING zinc finger. Arsenic binding alters the native structure of RING coordinated with zinc and induces its oligomerization through arsenic-mediated conformational change by intramolecular coordination as well as cross-linking between two RING motifs. Following conformational change and oligomerization of PML RBCC with arsenic binding, PML and PML-RARa undergoes SUMOylation through enhanced interaction with Ubc9, the E2 ligase for SUMOylation. Our findings provide the evidence that the PML RBCC domain is the direct target of arsenic and that the structural change of PML RBCC induced by arsenic binding facilitates the enhanced interaction with the cellular enzymatic machinery for protein SUMOylation/ubiquitination, which ultimately leads to the degradation of PML-RARa and cell differentiation and/or apoptosis. This mechanism sheds new insights into the mechanism of action of As2O3 for APL treatment, a model of targeted cancer therapy. Disclosures: No relevant conflicts of interest to declare.

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