scholarly journals Mechanism of FtsZ assembly dynamics revealed by filament structures in different nucleotide states

2021 ◽  
Author(s):  
Federico M. Ruiz ◽  
Sonia Huecas ◽  
Alicia Santos-Aledo ◽  
Elena A. Prim ◽  
José M. Andreu ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Mechanistic Model ◽  
Bacterial Cell Division ◽  
Cellular Functions ◽  
Nucleotide Hydrolysis ◽  
Subunit Interface ◽  
Water Shell ◽  
Biochemical Analyses ◽  
Ftsz Assembly

Treadmilling protein filaments perform essential cellular functions by growing from one end while shrinking from the other, driven by nucleotide hydrolysis. Bacterial cell division relies on the primitive tubulin homolog FtsZ, a target for antibiotic discovery that assembles into single treadmilling filaments that hydrolyse GTP at an active site formed upon subunit association. We determined high-resolution filament structures of FtsZ from the pathogen Staphylococcus aureus in complex with different nucleotide analogues and cations, including mimetics of the ground and transition states of catalysis. Together with mutational and biochemical analyses, our structures reveal interactions made by the GTP γ-phosphate and Mg2+ at the subunit interface, a K+ ion stabilizing loop T7 for co-catalysis, new roles of key residues at the active site and a nearby crosstalk area, and rearrangements of a dynamic water shell bridging adjacent subunits upon GTP hydrolysis. We propose a mechanistic model that integrates nucleotide hydrolysis signalling with assembly-associated conformational changes and filament treadmilling. Equivalent assembly mechanisms may apply to more complex tubulin and actin cytomotive filaments that share analogous features with FtsZ.

scholarly journals Direct Observation of Topoisomerase IA Gate Dynamics

2018 ◽  
Author(s):  
Maria Mills ◽  
Yuk-Ching Tse-Dinh ◽  
Keir C. Neuman
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Mechanistic Model ◽  
Cellular Functions ◽  
Single Stranded Dna ◽  
Gate Opening ◽  
Type Ia ◽  
Dna Strand ◽  
Dynamics Simulations ◽  
Strand Passage

AbstractType IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although it is assumed type IA topoisomerases accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the E. coli type IA topoisomerases I and III. We found that following cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allow us to develop a mechanistic model of type IA topoisomerase-ssDNA interactions.

scholarly journals Electrostatic interactions affecting the active site of class Sigma glutathione S-transferase

2000 ◽  
Vol 347 (1) ◽  
pp. 193-197 ◽  
Author(s):  
Julie M. STEVENS ◽  
Richard N. ARMSTRONG ◽  
Heini W. DIRR
Keyword(s):  
Ionic Strength ◽  
Conformational Changes ◽  
Active Site ◽  
Electrostatic Interactions ◽  
Size Exclusion ◽  
Tryptophan Residue ◽  
Glutathione S Transferase ◽  
Protein Fluorescence ◽  
Subunit Interface ◽  
Α Helix

We have shown previously that the solvent-induced equilibrium unfolding mechanism of class Sigma glutathione S-transferase (GST) is strongly affected by ionic strength [Stevens, Hornby, Armstrong and Dirr (1998) Biochemistry 37, 15534-15541]. The protein is dimeric and has a hydrophilic subunit interface. Here we show that ionic strength alone has significant effects on the conformation of the protein, in particular at the active site. With the use of NaCl at up to 2 M under equilibrium conditions, the protein lost 60% of its catalytic activity and the single tryptophan residue per subunit became partly exposed. The effect was independent of protein concentration, eliminating the dissociation of the dimer as a possibility for the conformational changes. This was confirmed by size-exclusion HPLC. There was no significant change in the secondary structure of the protein according to far-UV CD data. Manual-mixing and stopped-flow kinetics experiments showed a slow single-exponential salt-induced change in protein fluorescence. For equilibrium and kinetics experiments, the addition of an active-site ligand (S-hexylglutathione) completely protected the protein from the ionic-strength-induced conformational changes. This suggests that the change occurs at or near the active site. Possible structural reasons for these novel effects are proposed, such as the flexibility of the α-helix 2 region as well as the hydrophilic subunit interface, highlighting the importance of electrostatic interactions in maintaining the structure of the active site of this GST.

scholarly journals Conformational changes in Apolipoprotein N-acyltransferase (Lnt)

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Benjamin Wiseman ◽  
Martin Högbom
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Cell Envelope ◽  
Covalent Modification ◽  
Covalent Attachment ◽  
Gram Negative Bacteria ◽  
Cellular Functions ◽  
Crystal Forms ◽  
Active Site Cysteine ◽  
Open Conformation

AbstractLipoproteins are important components of the cell envelope and are responsible for many essential cellular functions. They are produced by the post-translational covalent attachment of lipids that occurs via a sequential 3-step process controlled by three integral membrane enzymes. The last step of this process, unique to Gram-negative bacteria, is the N-acylation of the terminal cysteine by Apolipoprotein N-acyltransferase (Lnt) to form the final mature lipoprotein. Here we report 2 crystal forms of Lnt from Escherichia coli. In one form we observe a highly dynamic arm that is able to restrict access to the active site as well as a covalent modification to the active site cysteine consistent with the thioester acyl-intermediate. In the second form, the enzyme crystallized in an open conformation exposing the active site to the environment. In total we observe 3 unique Lnt molecules that when taken together suggest the movement of essential loops and residues are triggered by substrate binding that could control the interaction between Lnt and the incoming substrate apolipoprotein. The results provide a dynamic context for residues shown to be central for Lnt function and provide further insights into its mechanism.

scholarly journals Active site lysine backbone undergoes conformational changes in the bacteriorhodopsin photocycle.

1994 ◽  
Vol 269 (10) ◽  
pp. 7387-7389
Author(s):  
H. Takei ◽  
Y. Gat ◽  
Z. Rothman ◽  
A. Lewis ◽  
M. Sheves
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Bacteriorhodopsin Photocycle

New insights into the structural basis of integrin activation

Blood ◽  
2003 ◽  
Vol 102 (4) ◽  
pp. 1155-1159 ◽  
Author(s):  
Jian-Ping Xiong ◽  
Thilo Stehle ◽  
Simon L. Goodman ◽  
M. Amin Arnaout
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Structural Basis ◽  
Integrin Activation ◽  
Cellular Functions ◽  
Electron Microscopic ◽  
The Past ◽  
Intracellular Signals ◽  
Bidirectional Signaling ◽  
New Hypothesis

Abstract Integrins are cell adhesion receptors that communicate biochemical and mechanical signals in a bidirectional manner across the plasma membrane and thus influence most cellular functions. Intracellular signals switch integrins into a ligand-competent state as a result of elicited conformational changes in the integrin ectodomain. Binding of extracellular ligands induces, in turn, structural changes that convey distinct signals to the cell interior. The structural basis of this bidirectional signaling has been the focus of intensive study for the past 3 decades. In this perspective, we develop a new hypothesis for integrin activation based on recent crystallographic, electron microscopic, and biochemical studies.

MinD directly interacting with FtsZ at the H10 helix suggests a model for robust activation of MinC to destabilize FtsZ polymers

2017 ◽  
Vol 474 (18) ◽  
pp. 3189-3205 ◽  
Author(s):  
Ashoka Chary Taviti ◽  
Tushar Kant Beuria
Keyword(s):  
Cell Division ◽  
Single Point ◽  
Point Mutations ◽  
Direct Interaction ◽  
Ring Formation ◽  
Z Ring ◽  
Single Point Mutations ◽  
Biochemical Analyses ◽  
Ftsz Assembly ◽  
The Mind

Cell division in bacteria is a highly controlled and regulated process. FtsZ, a bacterial cytoskeletal protein, forms a ring-like structure known as the Z-ring and recruits more than a dozen other cell division proteins. The Min system oscillates between the poles and inhibits the Z-ring formation at the poles by perturbing FtsZ assembly. This leads to an increase in the FtsZ concentration at the mid-cell and helps in Z-ring positioning. MinC, the effector protein, interferes with Z-ring formation through two different mechanisms mediated by its two domains with the help of MinD. However, the mechanism by which MinD triggers MinC activity is not yet known. We showed that MinD directly interacts with FtsZ with an affinity stronger than the reported MinC–FtsZ interaction. We determined the MinD-binding site of FtsZ using computational, mutational and biochemical analyses. Our study showed that MinD binds to the H10 helix of FtsZ. Single-point mutations at the charged residues in the H10 helix resulted in a decrease in the FtsZ affinity towards MinD. Based on our findings, we propose a novel model for MinCD–FtsZ interaction, where MinD through its direct interaction with FtsZ would trigger MinC activity to inhibit FtsZ functions.

scholarly journals Specificity of Human Sulfiredoxin for Reductant and Peroxiredoxin Oligomeric State

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 946
Author(s):  
Tom E. Forshaw ◽  
Julie A. Reisz ◽  
Kimberly J. Nelson ◽  
Rajesh Gumpena ◽  
J. Reed Lawson ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Antioxidant Enzymes ◽  
Hydrogen Sulfide ◽  
Active Site ◽  
Relative Efficacy ◽  
Oligomeric Structure ◽  
Cellular Functions ◽  
Oligomeric State ◽  
Structural Requirements ◽  
First Time

Human peroxiredoxins (Prx) are a family of antioxidant enzymes involved in a myriad of cellular functions and diseases. During the reaction with peroxides (e.g., H2O2), the typical 2-Cys Prxs change oligomeric structure between higher order (do)decamers and disulfide-linked dimers, with the hyperoxidized inactive state (-SO2H) favoring the multimeric structure of the reduced enzyme. Here, we present a study on the structural requirements for the repair of hyperoxidized 2-Cys Prxs by human sulfiredoxin (Srx) and the relative efficacy of physiological reductants hydrogen sulfide (H2S) and glutathione (GSH) in this reaction. The crystal structure of the toroidal Prx1-Srx complex shows an extended active site interface. The loss of this interface within engineered Prx2 and Prx3 dimers yielded variants more resistant to hyperoxidation and repair by Srx. Finally, we reveal for the first time Prx isoform-dependent use of and potential cooperation between GSH and H2S in supporting Srx activity.

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