scholarly journals Sensor Histidine Kinase NarQ Activates via Helical Rotation, Diagonal Scissoring, and Eventually Piston-Like Shifts

2020 ◽  
Vol 21 (9) ◽  
pp. 3110
Author(s):  
Ivan Gushchin ◽  
Philipp Orekhov ◽  
Igor Melnikov ◽  
Vitaly Polovinkin ◽  
Anastasia Yuzhakova ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Degrees Of Freedom ◽  
Response Regulator ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Crystallographic Structure ◽  
Signaling Systems ◽  
Ligand Free ◽  
Dynamics Simulations ◽  
Cognate Response Regulator

Membrane-embedded sensor histidine kinases (HKs) and chemoreceptors are used ubiquitously by bacteria and archaea to percept the environment, and are often crucial for their survival and pathogenicity. The proteins can transmit the signal from the sensor domain to the catalytic kinase domain reliably over the span of several hundreds of angstroms, and regulate the activity of the cognate response regulator proteins, with which they form two-component signaling systems (TCSs). Several mechanisms of transmembrane signal transduction in TCS receptors have been proposed, dubbed (swinging) piston, helical rotation, and diagonal scissoring. Yet, despite decades of studies, there is no consensus on whether these mechanisms are common for all TCS receptors. Here, we extend our previous work on Escherichia coli nitrate/nitrite sensor kinase NarQ. We determined a crystallographic structure of the sensor-TM-HAMP fragment of the R50S mutant, which, unexpectedly, was found in a ligand-bound-like conformation, despite an inability to bind nitrate. Subsequently, we reanalyzed the structures of the ligand-free and ligand-bound NarQ and NarX sensor domains, and conducted extensive molecular dynamics simulations of ligand-free and ligand-bound wild type and mutated NarQ. Based on the data, we show that binding of nitrate to NarQ causes, first and foremost, helical rotation and diagonal scissoring of the α-helices at the core of the sensor domain. These conformational changes are accompanied by a subtle piston-like motion, which is amplified by a switch in the secondary structure of the linker between the sensor and TM domains. We conclude that helical rotation, diagonal scissoring, and piston are simply different degrees of freedom in coiled-coil proteins and are not mutually exclusive in NarQ, and likely in other nitrate sensors and TCS proteins as well.

scholarly journals Comparative analysis of two paradigm bacteriophytochromes reveals opposite functionalities in two-component signaling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Elina Multamäki ◽  
Rahul Nanekar ◽  
Dmitry Morozov ◽  
Topias Lievonen ◽  
David Golonka ◽  
...  
Keyword(s):  
Histidine Kinase ◽  
Deinococcus Radiodurans ◽  
Response Regulator ◽  
Red Light ◽  
Kinase Domain ◽  
Structural Level ◽  
Signaling Systems ◽  
Two Component ◽  
Cognate Response Regulator ◽  
Absorbing States

AbstractBacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (Agp1). Whereas Agp1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While Agp1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes suggest the use of light-controllable histidine kinases and phosphatases for optogenetics.

scholarly journals Illuminating a Phytochrome Paradigm – a Light-Activated Phosphatase in Two-Component Signaling Uncovered

2020 ◽  
Author(s):  
Elina Multamäki ◽  
Rahul Nanekar ◽  
Dmitry Morozov ◽  
Topias Lievonen ◽  
David Golonka ◽  
...  
Keyword(s):  
Histidine Kinase ◽  
Deinococcus Radiodurans ◽  
Response Regulator ◽  
Red Light ◽  
Kinase Domain ◽  
Structural Level ◽  
Signaling Systems ◽  
Two Component ◽  
Cognate Response Regulator ◽  
Absorbing States

ABSTRACTBacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (AgP1). Whereas AgP1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While AgP1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes inform the use of light-controllable histidine kinases and phosphatases for optogenetics.

scholarly journals New insights on human IRE1 tetramer structures based on molecular modeling

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Antonio Carlesso ◽  
Johanna Hörberg ◽  
Anna Reymer ◽  
Leif A. Eriksson
Keyword(s):  
Conformational Changes ◽  
Three Dimensional ◽  
Protein Docking ◽  
Activation Mechanism ◽  
Crystallographic Structure ◽  
Face To Face ◽  
Unfolded Protein ◽  
Dynamics Simulations ◽  
The Unfolded Protein Response ◽  
Protein Response

Abstract Inositol-Requiring Enzyme 1α (IRE1α; hereafter IRE1) is a transmembrane kinase/ribonuclease protein related with the unfolded protein response (UPR) signaling. Experimental evidence suggests that IRE1 forms several three dimensional (3D) structural variants: dimers, tetramers and higher order oligomers, where each structural variant can contain different IRE1 conformers in different arrangements. For example, studies have shown that two sets of IRE1 dimers exist; a face-to-face dimer and a back-to-back dimer, with the latter considered the important unit for UPR signaling propagation. However, the structural configuration and mechanistic details of the biologically important IRE1 tetramers are limited. Here, we combine protein–protein docking with molecular dynamics simulations to derive human IRE1 tetramer models and identify a molecular mechanism of IRE1 activation. To validate the derived models of the human IRE1 tetramer, we compare the dynamic behavior of the models with the yeast IRE1 tetramer crystallographic structure. We show that IRE1 tetramer conformational changes could be linked to the initiation of the unconventional splicing of mRNA encoding X-box binding protein-1 (XBP1), which allows for the expression of the transcription factor XBP1s (XBP1 spliced). The derived IRE1 tetrameric models bring new mechanistic insights about the IRE1 molecular activation mechanism by describing the IRE1 tetramers as active protagonists accommodating the XBP1 substrate.

scholarly journals Membrane-induced Lever Arm Expansion Allows Myosin VI to Walk with Large and Variable Step Sizes

2012 ◽  
Vol 287 (42) ◽  
pp. 35021-35035 ◽  
Author(s):  
Cong Yu ◽  
Jizhong Lou ◽  
Jingjing Wu ◽  
Lifeng Pan ◽  
Wei Feng ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Lipid Membranes ◽  
Lipid Membrane ◽  
Coiled Coil ◽  
Myosin Vi ◽  
Iq Motif ◽  
Helix Bundle ◽  
Lever Arm ◽  
Variable Step ◽  
Dynamics Simulations

Myosin VI, the only known minus-ended actin filament-dependent motor, plays diverse cellular roles both as a processive motor and as a mechanical anchor. Although myosin VI has a short lever arm containing only one “IQ-motif” and a unique insertion for CaM binding, the motor walks with large and variable step sizes of ∼30–36 nm. Here, we show that the previously predicted coiled-coil domain immediately following the IQ-motifs (referred to as the lever arm extension (LAE)) adopts a stable monomeric, three-helix bundle fold in solution. Importantly, the LAE can undergo reversible, lipid membrane-dependent conformational changes. Upon exposure to lipid membranes, the LAE adopts a partially extended rod shape, and the removal of lipids from the LAE converts it back into the compact helix bundle structure. Molecular dynamics simulations indicate that lipid membrane binding may initiate unfolding and thereby trigger the LAE expansion. This reversible, lipid membrane-dependent expansion of the LAE provides a mechanistic base for myosin VI to walk with large and variable step sizes.

scholarly journals Dual control of RegX3 transcriptional activity by SenX3 and PknB

2019 ◽  
Vol 294 (28) ◽  
pp. 11023-11034 ◽  
Author(s):  
Eun-Jin Park ◽  
Yu-Mi Kwon ◽  
Jin-Won Lee ◽  
Ho-Young Kang ◽  
Jeong-Il Oh
Keyword(s):  
Conformational Changes ◽  
Transcriptional Activity ◽  
Response Regulator ◽  
Regulatory System ◽  
Kinase Domain ◽  
Two Component System ◽  
Mobility Shift ◽  
Cellular Replication ◽  
Electrophoretic Mobility Shift Assays

The mycobacterial SenX3–RegX3 two-component system consists of the SenX3 sensor histidine kinase and its cognate RegX3 response regulator. This system is a phosphorelay-based regulatory system involved in sensing environmental Pi levels and induction of genes required for Pi acquisition under Pi-limiting conditions. Here we demonstrate that overexpression of the kinase domain of Mycobacterium tuberculosis PknB (PknB-KDMtb) inhibits the transcriptional activity of RegX3 of both M. tuberculosis and Mycobacterium smegmatis (RegX3Mtb and RegX3Ms, respectively). Mass spectrometry results, along with those of in vitro phosphorylation and complementation analyses, revealed that PknB kinase activity inhibits the transcriptional activity of RegX3Mtb through phosphorylation events at Thr-100, Thr-191, and Thr-217. Electrophoretic mobility shift assays disclosed that phosphorylation of Thr-191 and Thr-217 abolishes the DNA-binding ability of RegX3Mtb and that Thr-100 phosphorylation likely prevents RegX3Mtb from being activated through conformational changes induced by SenX3-mediated phosphorylation. We propose that the convergence of the PknB and SenX3-RegX3 signaling pathways might enable mycobacteria to integrate environmental Pi signals with the cellular replication state to adjust gene expression in response to Pi availability.

scholarly journals Structural and mechanistic insights into mechanoactivation of focal adhesion kinase

2019 ◽  
Vol 116 (14) ◽  
pp. 6766-6774 ◽  
Author(s):  
Magnus Sebastian Bauer ◽  
Fabian Baumann ◽  
Csaba Daday ◽  
Pilar Redondo ◽  
Ellis Durner ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Single Molecule ◽  
Conformational Changes ◽  
Cell Attachment ◽  
Focal Adhesions ◽  
Kinase Domain ◽  
Initial Rupture ◽  
Dynamics Simulations ◽  
Force Range

Focal adhesion kinase (FAK) is a key signaling molecule regulating cell adhesion, migration, and survival. FAK localizes into focal adhesion complexes formed at the cytoplasmic side of cell attachment to the ECM and is activated after force generation via actomyosin fibers attached to this complex. The mechanism of translating mechanical force into a biochemical signal is not understood, and it is not clear whether FAK is activated directly by force or downstream to the force signal. We use experimental and computational single-molecule force spectroscopy to probe the mechanical properties of FAK and examine whether force can trigger activation by inducing conformational changes in FAK. By comparison with an open and active mutant of FAK, we are able to assign mechanoactivation to an initial rupture event in the low-force range. This activation event occurs before FAK unfolding at forces within the native range in focal adhesions. We are also able to assign all subsequent peaks in the force landscape to partial unfolding of FAK modules. We show that binding of ATP stabilizes the kinase domain, thereby altering the unfolding hierarchy. Using all-atom molecular dynamics simulations, we identify intermediates along the unfolding pathway, which provide buffering to allow extension of FAK in focal adhesions without compromising functionality. Our findings strongly support that forces in focal adhesions applied to FAK via known interactions can induce conformational changes, which in turn, trigger focal adhesion signaling.

COMPUTATIONAL STUDY OF THE DYNAMICS OF PEPTIDE DEFORMYLASE COMPLEX FROM LEPTOSPIRA INTERROGANS: EXPLORING THE CONFORMATIONAL CHANGES OF THE SUBSTRATE POCKET

2008 ◽  
Vol 07 (05) ◽  
pp. 911-922
Author(s):  
QIANG WANG ◽  
JIANWU WANG ◽  
ZHENGTING CAI ◽  
WEIREN XU
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Computational Study ◽  
Dynamics Simulation ◽  
Peptide Deformylase ◽  
Leptospira Interrogans ◽  
Hydrophobic Cluster ◽  
Open Conformation ◽  
Ligand Free ◽  
Dynamics Simulations

The peptide deformylase from Leptospira interrogans (LiPDF) shows many unusual characteristics. The substrate pocket of formate-bound complex adopts an open conformation. However, in the actinonin-bound LiPDF complex, a slightly open substrate pocket is observed. The opening is not large enough for the inhibitor, because the CD-loop restricts the access to the active site. To explore the conformational changes of the substrate pocket, we perform a 16,000 ps molecular dynamics simulation separately on the ligand-free LiPDF and actinonin-bound LiPDF. During the molecular dynamics simulations, extensive conformational changes have taken place. The comparison of the two MD results shows that the CD-loop, hydrophilic inhibitor, and hydrophobic cluster are necessary for the reopening of the substrate pocket. In addition, Tyr71 plays an important role in mediating the movements of CD-loop, and the transition of the substrate pocket from open to semi-open only occurs in the presence of an inhibitor, which are consistent with the experiment very well.

scholarly journals Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

2021 ◽  
Vol 118 (23) ◽  
pp. e2100844118
Author(s):  
Sven H. Schmidt ◽  
Jui-Hung Weng ◽  
Phillip C. Aoto ◽  
Daniela Boassa ◽  
Sebastian Mathea ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Subcellular Location ◽  
Kinase Domain ◽  
Activation Process ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Kinase Domain Mutations ◽  
Dynamics Simulations

To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson’s disease (PD), we used a multitiered approach. Most mutations mimic Rab-mediated activation by “unleashing” kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2RCKW retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2RCKW and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

Molecular Basis of Glucose Transport Mechanism in Plants

2019 ◽  
Author(s):  
Balaji Selvam ◽  
Ya-Chi Yu ◽  
Liqing Chen ◽  
Diwakar Shukla
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Transport Mechanism ◽  
Conformational Dynamics ◽  
Site Directed Mutagenesis ◽  
Free Energy Barrier ◽  
Transport Cycle ◽  
Dynamics Simulations

<p>The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.</p>

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