Cargo modulates the conformation of the nuclear pore in living cells

Mapping Intimacies ◽

10.1101/2020.07.27.223081 ◽

2020 ◽

Author(s):

Joan Pulupa ◽

Harriet Prior ◽

Daniel S. Johnson ◽

Sanford M. Simon

Keyword(s):

Conformational Changes ◽

Nuclear Pore Complex ◽

Conformational Dynamics ◽

Living Cells ◽

Nuclear Pore ◽

Static Structure ◽

X Ray ◽

X Ray Crystallography

AbstractWhile the static structure of the nuclear pore complex (NPC) continues to be refined with cryo-EM and x-ray crystallography, the in vivo conformational dynamics of the NPC remain under-explored. We developed sensors that report on the orientation of NPC components by rigidly conjugating mEGFP to different NPC proteins. Our studies show conformational changes to select domains of Nups within the inner ring (Nup54, Nup58, Nup62) when transport through the NPC is perturbed and no conformational changes to Nups elsewhere in the NPC. Our results suggest that select components of the NPC are flexible and undergo conformational changes upon engaging with cargo.

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Conformation of the nuclear pore in living cells is modulated by transport state

eLife ◽

10.7554/elife.60654 ◽

2020 ◽

Vol 9 ◽

Author(s):

Joan Pulupa ◽

Harriet Prior ◽

Daniel S Johnson ◽

Sanford M Simon

Keyword(s):

Conformational Changes ◽

Nuclear Pore Complex ◽

Living Cells ◽

Nuclear Pore ◽

Static Structure ◽

X Ray ◽

X Ray Crystallography

While the static structure of the nuclear pore complex (NPC) continues to be refined with cryo-EM and x-ray crystallography, in vivo conformational changes of the NPC remain under-explored. We developed sensors that report on the orientation of NPC components by rigidly conjugating mEGFP to different NPC proteins. Our studies show conformational changes to select domains of nucleoporins (Nups) within the inner ring (Nup54, Nup58, Nup62) when transport through the NPC is perturbed and no conformational changes to Nups elsewhere in the NPC. Our results suggest that select components of the NPC are flexible and undergo conformational changes upon engaging with cargo.

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The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity

10.1101/389460 ◽

2018 ◽

Author(s):

Mohammed Jamshad ◽

Timothy J. Knowles ◽

Scott A. White ◽

Douglas G. Ward ◽

Fiyaz Mohammed ◽

...

Keyword(s):

Conformational Changes ◽

Metal Binding ◽

Cytoplasmic Membrane ◽

Protein Recognition ◽

X Ray ◽

Ribosome Binding ◽

X Ray Crystallography ◽

Substrate Protein ◽

Metal Binding Domain

AbstractIn bacteria, the translocation of a subset of proteins across the cytoplasmic membrane by the Sec machinery requires SecA. Although SecA can recognise nascent polypeptides, the mechanism of cotranslational substrate protein recognition is not known. Here, we investigated the role of the C-terminal tail (CTT) of SecA, which consists of a flexible linker (FLD) and a small metal-binding domain (MBD), in its interaction with nascent polypeptides. Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the entire CTT or the MBD alone had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function. Autophotocrosslinking, mass spectrometry, x-ray crystallography and small-angle x-ray scattering experiments provided insight into the CTT-mediated conformational changes in SecA. Finally, photocrosslinking experiments indicated that binding of SecA to substrate protein affected its interaction with the ribosome. Taken together, our results suggest a mechanism for substrate protein recognition.Impact StatementSecA is an evolutionarily conserved ATPase that is required for the translocation of a subset of proteins across the cytoplasmic membrane in bacteria. We investigated how SecA recognises its substrate proteins at the ribosome as they are still being synthesised (i.e. cotranslationally).

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Integrative Structure Determination of the Components of the Nuclear Pore Complex by X-Ray Crystallography, Small Angle X-Ray Scattering, Electron Microscopy, NMR, and Comparative Modeling

Biophysical Journal ◽

10.1016/j.bpj.2011.11.3371 ◽

2012 ◽

Vol 102 (3) ◽

pp. 619a

Author(s):

Seung Joong Kim ◽

Parthasarathy Sampathkumar ◽

Javier Fernandez-Martinez ◽

Jeremy Phillips ◽

Dina Schneidman ◽

...

Keyword(s):

Electron Microscopy ◽

Nuclear Pore Complex ◽

Structure Determination ◽

Small Angle ◽

Comparative Modeling ◽

Nuclear Pore ◽

X Ray ◽

X Ray Crystallography ◽

X Ray Scattering

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Shining light on cysteine modification: connecting protein conformational dynamics to catalysis and regulation

Journal of Synchrotron Radiation ◽

10.1107/s160057751900568x ◽

2019 ◽

Vol 26 (4) ◽

pp. 958-966 ◽

Cited By ~ 3

Author(s):

Henry van den Bedem ◽

Mark A Wilson

Keyword(s):

Conformational Changes ◽

Conformational Dynamics ◽

Covalent Bond ◽

Covalent Modification ◽

Cysteine Residues ◽

Cysteine Oxidation ◽

X Ray ◽

Photo Oxidation ◽

X Ray Crystallography ◽

Important Amino Acid

Cysteine is a rare but functionally important amino acid that is often subject to covalent modification. Cysteine oxidation plays an important role in many human disease processes, and basal levels of cysteine oxidation are required for proper cellular function. Because reactive cysteine residues are typically ionized to the thiolate anion (Cys-S−), their formation of a covalent bond alters the electrostatic and steric environment of the active site. X-ray-induced photo-oxidation to sulfenic acids (Cys-SOH) can recapitulate some aspects of the changes that occur under physiological conditions. Here we propose how site-specific cysteine photo-oxidation can be used to interrogate ensuing changes in protein structure and dynamics at atomic resolution. Although this powerful approach can connect cysteine covalent modification to global protein conformational changes and function, careful biochemical validation must accompany all such studies to exclude misleading artifacts. New types of X-ray crystallography experiments and powerful computational methods are creating new opportunities to connect conformational dynamics to catalysis for the large class of systems that use covalently modified cysteine residues for catalysis or regulation.

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Rescue of Conformational Dynamics in Enzyme Catalysis by Directed Evolution

10.1101/185009 ◽

2017 ◽

Author(s):

Renee Otten ◽

Lin Liu ◽

Lillian R. Kenner ◽

Michael W. Clarkson ◽

David Mavor ◽

...

Keyword(s):

Directed Evolution ◽

Conformational Changes ◽

Rational Design ◽

Room Temperature ◽

Energy Landscape ◽

Conformational Dynamics ◽

Biological Macromolecules ◽

Evolutionary Trajectory ◽

X Ray ◽

X Ray Crystallography

AbstractRational design and directed evolution have proved to be successful approaches to increase catalytic efficiencies of both natural and artificial enzymes1-3. However, a comprehensive understanding of how evolution shapes the energy landscape of catalysis remains a fundamental challenge. Protein dynamics is widely recognized as important, but due to the inherent flexibility of biological macromolecules it is often difficult to distinguish which conformational changes are directly related to function. Here, we used directed evolution on an impaired mutant of the human proline isomerase cyclophilin A (CypA) and identify two second-shell mutations that partially restore its catalytic activity. We show both kinetically, using NMR spectroscopy, and structurally, by room-temperature X-ray crystallography, how local perturbations propagate through a large allosteric network to facilitate conformational dynamics. The increased catalysis selected for in the evolutionary screen could be rationalized entirely by accelerated interconversion between the two catalytically essential conformational sub-states, which are both captured in the high-resolution X-ray ensembles at room temperature. Our data provide a glimpse of the evolutionary trajectory of an enzyme’s energy landscape and shows how subtle changes can fine-tune its function.

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Tele-Substitution Reactions in the Synthesis of a Promising Class of Antimalarials

10.26434/chemrxiv.12212927 ◽

2020 ◽

Author(s):

Marat Korsik ◽

Edwin Tse ◽

David Smith ◽

William Lewis ◽

Peter J. Rutledge ◽

...

Keyword(s):

Heterocyclic Ring ◽

Ring System ◽

Substitution Reactions ◽

Laboratory Notebook ◽

Polar Solvents ◽

X Ray ◽

X Ray Crystallography ◽

The Core ◽

Nmr Data

We have discovered and studied a telesubstitution reaction in a biologically important heterocyclic ring system. Conditions that favour the tele-substitution pathway were identified: the use of increased equivalents of the nucleophile or decreased equivalents of base, or the use of softer nucleophiles, less polar solvents and larger halogens on the electrophile. Using results from X-ray crystallography and isotope labelling experiments a mechanism for this unusual transformation is proposed. We focused on this triazolopyrazine as it is the core structure of the in vivo active anti-plasmodium compounds of Series 4 of the Open Source Malaria consortium. Archive of the electronic laboratory notebook with the description of all conducted experiments and raw NMR data could be accessed via following link <a href="https://ses.library.usyd.edu.au/handle/2123/21890">https://ses.library.usyd.edu.au/handle/2123/21890</a> . For navigation between entries of laboratory notebook please use file "Strings for compounds in the article.pdf" that works as a reference between article codes and notebook codes, also this file contain SMILES for these compounds.

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The Odd Faces of Oligomers: The Case of TRAF2-C, A Trimeric C-Terminal Domain of TNF Receptor-Associated Factor

International Journal of Molecular Sciences ◽

10.3390/ijms22115871 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5871

Author(s):

Almerinda Di Venere ◽

Eleonora Nicolai ◽

Velia Minicozzi ◽

Anna Maria Caccuri ◽

Luisa Di Paola ◽

...

Keyword(s):

Conformational Changes ◽

Strong Dependence ◽

Conformational Dynamics ◽

Receptor Interaction ◽

Tnf Receptor ◽

Oligomeric State ◽

Dynamic Simulations ◽

Associated Factor ◽

Terminal Domain

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.

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