Structure of the Acinetobacter baumannii PmrA receiver domain and insights into clinical mutants affecting DNA-binding and promoting colistin resistance

2021 ◽  
Author(s):  
Samantha Palethorpe ◽  
Morgan E Milton ◽  
Everett C Pesci ◽  
John Cavanagh
Keyword(s):  
Dna Binding ◽  
Acinetobacter Baumannii ◽  
Structural Information ◽  
Response Regulator ◽  
Regulatory System ◽  
Nosocomial Pathogen ◽  
Colistin Resistance ◽  
X Ray ◽  
X Ray Crystallography ◽  
Receiver Domain

Abstract Acinetobacter baumannii is an insidious emerging nosocomial pathogen that has developed resistance to all available antimicrobials, including the last resort antibiotic, colistin. Colistin resistance often occurs due to mutations in the PmrAB two component regulatory system. To better understand the regulatory mechanisms contributing to colistin resistance, we have biochemically characterized the A. baumannii PmrA response regulator. Initial DNA-binding analysis shows that A. baumannii PmrA bound to the Klebsiella pneumoniae PmrA box motif. This prompted analysis of the putative A. baumannii PmrAB regulon which indicated that the A. baumannii PmrA consensus box is 5′- HTTAAD N5 HTTAAD. Additionally, we provide the first structural information for the A. baumannii PmrA N-terminal domain through X-ray crystallography, and we present a full-length model using molecular modeling. From these studies, we were able to infer the effects of two critical PmrA mutations, PmrA::I13M and PmrA::P102R, both of which confer increased colistin resistance. Based on these data, we suggest structural and dynamic reasons for how these mutations can affect PmrA function and hence encourage resistive traits. Understanding these mechanisms will aid in the development of new targeted antimicrobial therapies.

scholarly journals Deciphering the activation and recognition mechanisms of Staphylococcus aureus response regulator ArlR

2019 ◽  
Vol 47 (21) ◽  
pp. 11418-11429 ◽  
Author(s):  
Zhenlin Ouyang ◽  
Fang Zheng ◽  
Jared Y Chew ◽  
Yingmei Pei ◽  
Jinhong Zhou ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Crystal Structures ◽  
Response Regulator ◽  
Regulatory System ◽  
Repeat Sequence ◽  
Effector Domain ◽  
Receiver Domain ◽  
Regulation Mechanisms ◽  
Significant Conformational Change

Abstract Staphylococcus aureus ArlRS is a key two-component regulatory system necessary for adhesion, biofilm formation, and virulence. The response regulator ArlR consists of a C-terminal DNA-binding effector domain and an N-terminal receiver domain that is phosphorylated by ArlS, the cognate transmembrane sensor histidine kinase. We demonstrate that the receiver domain of ArlR adopts the canonical α5β5 response regulator assembly, which dimerizes upon activation, using beryllium trifluoride as an aspartate phosphorylation mimic. Activated ArlR recognizes a 20-bp imperfect inverted repeat sequence in the ica operon, which is involved in intercellular adhesion polysaccharide production. Crystal structures of the inactive and activated forms reveal that activation induces a significant conformational change in the β4-α4 and β5-α5-connecting loops, in which the α4 and α5 helices constitute the homodimerization interface. Crystal structures of the DNA-binding ArlR effector domain indicate that it is able to dimerize via a non-canonical β1–β2 hairpin domain swapping, raising the possibility of a new mechanism for signal transduction from the receiver domain to effector domain. Taken together, the current study provides structural insights into the activation of ArlR and its recognition, adding to the diversity of response regulation mechanisms that may inspire novel antimicrobial strategies specifically targeting Staphylococcus.

Acinetobacter baumannii regulates its stress responses via the BfmRS two-component regulatory system

2021 ◽  
Author(s):  
Samantha Palethorpe ◽  
John M. Farrow ◽  
Greg Wells ◽  
Morgan E. Milton ◽  
Luis A. Actis ◽  
...  
Keyword(s):  
Stress Response ◽  
Acinetobacter Baumannii ◽  
Stress Responses ◽  
Capsular Polysaccharide ◽  
Response Regulator ◽  
Regulatory System ◽  
Hospital Environment ◽  
Nosocomial Pathogen ◽  
Polysaccharide Biosynthesis ◽  
Two Component

Acinetobacter baumannii is a common nosocomial pathogen that utilizes numerous mechanisms to aid its survival in both the environment and in the host. Coordination of such mechanisms requires an intricate regulatory network. We report here that A. baumannii can directly regulate several stress-related pathways via the two-component regulatory system, BfmRS. Similar to previous studies, results from transcriptomic analysis showed that mutation of the BfmR response regulator causes dysregulation of genes required for the oxidative stress response, the osmotic stress response, the misfolded protein/heat shock response, Csu pili/fimbriae production, and capsular polysaccharide biosynthesis. We also found that the BfmRS system is involved in controlling siderophore biosynthesis and transport, and type IV pili production. We provide evidence that BfmR binds to various stress-related promoter regions and show that BfmR alone can directly activate transcription of some stress-related genes. Additionally, we show that the BfmS sensor kinase acts as a BfmR phosphatase to negatively regulate BfmR activity. This work highlights the importance of the BfmRS system in promoting survival of A. baumannii . Importance Acinetobacter baumannii is a nosocomial pathogen that has extremely high rates of multidrug resistance. This organism’s ability to endure stressful conditions is a key part of its ability to spread in the hospital environment and cause infections. Unlike other members of the γ-proteobacteria, A. baumannii does not encode a homolog of the RpoS sigma factor to coordinate its stress response. Here, we demonstrate that the BfmRS two-component system directly controls the expression of multiple stress resistance genes. Our findings suggest that BfmRS is central to a unique scheme of general stress response regulation by A. baumannii .

Examining the structure of the mature amyloid fibril

2002 ◽  
Vol 30 (4) ◽  
pp. 521-525 ◽  
Author(s):  
O. S. Makin ◽  
L. C. Serpell
Keyword(s):  
Self Assembly ◽  
Rational Design ◽  
Amyloid Fibrils ◽  
Structural Information ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Fibre Diffraction ◽  
Complementary Techniques ◽  
Mature Fibril

The pathogenesis of the group of diseases known collectively as the amyloidoses is characterized by the deposition of insoluble amyloid fibrils. These are straight, unbranching structures about 70–120 å (1 å = 0.1 nm) in diameter and of indeterminate length formed by the self-assembly of a diverse group of normally soluble proteins. Knowledge of the structure of these fibrils is necessary for the understanding of their abnormal assembly and deposition, possibly leading to the rational design of therapeutic agents for their prevention or disaggregation. Structural elucidation is impeded by fibril insolubility and inability to crystallize, thus preventing the use of X-ray crystallography and solution NMR. CD, Fourier-transform infrared spectroscopy and light scattering have been used in the study of the mechanism of fibril formation. This review concentrates on the structural information about the final, mature fibril and in particular the complementary techniques of cryo-electron microscopy, solid-state NMR and X-ray fibre diffraction.

scholarly journals Colistin heteroresistance and the involvement of the PmrAB regulatory system in Acinetobacter baumannii

2018 ◽  
Author(s):  
Yannick Charretier ◽  
Seydina M. Diene ◽  
Damien Baud ◽  
Sonia Chatellier ◽  
Emmanuelle Santiago-Allexant ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Population Analysis ◽  
Regulatory System ◽  
Clinical Problem ◽  
Multidrug Resistant ◽  
Regulatory Pathway ◽  
Colistin Resistance ◽  
Bacterial Killing ◽  
One Step

AbstractMultidrug-resistant Acinetobacter baumannii infection has recently emerged as a worldwide clinical problem and colistin is increasingly being used as last resort therapy. Despite its favorable bacterial killing, resistance and heteroresistance to colistin have been described. Mutations in the PmrAB regulatory pathway have been already associated with colistin resistance whereas the mechanisms for heteroresistance remain largely unknown. The purpose of the present study is to investigate the role of PmrAB in laboratory-selected mutants representative of global epidemic strains. During brief colistin exposure, colistin resistant and colistin heteroresistant mutants were selected in a one-step strategy. Population Analysis Profiling (PAP) was performed to confirm the suspected phenotype. Upon withdrawal of selective pressure, compensatory mutations were evaluated in another one-step strategy. A trans-complementation assay was designed to delineate the involvement of the PmrAB regulatory system using qPCR and PAP. Mutations in the PmrAB regulatory pathway were associated with colistin resistance and colistin heteroresistance as well. The transcomplementation assay provides a proof for the role played by changes in the PmrAB regulatory pathway. The level of colistin resistance is correlated to the level of expression of pmrC. The resistance phenotype was partially restored since the complemented strain became heteroresistant. This report shows the role of different mutations in the PmrAB regulatory pathway and warns on the development of colistin heteroresistance that could be present but not easily detected with routine testing.

Molecular characterization of lpxACD and pmrA/B two-component regulatory system in the colistin resistance Acinetobacter baumannii clinical isolates

Gene Reports ◽  
2020 ◽  
Vol 21 ◽  
pp. 100952
Author(s):  
Mohammad Reza Kandehkar Ghahraman ◽  
Hossein Hosseini-Nave ◽  
Omid Azizi ◽  
Mohammad Reza Shakibaie ◽  
Hamid Reza Mollaie ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Molecular Characterization ◽  
Regulatory System ◽  
Clinical Isolates ◽  
Colistin Resistance ◽  
Two Component

scholarly journals 20 YEARS OF LEPTIN: Insights into signaling assemblies of the leptin receptor

2014 ◽  
Vol 223 (1) ◽  
pp. T9-T23 ◽  
Author(s):  
Frank Peelman ◽  
Lennart Zabeau ◽  
Kedar Moharana ◽  
Savvas N Savvides ◽  
Jan Tavernier
Keyword(s):  
Electron Microscopy ◽  
Energy Homeostasis ◽  
Leptin Receptor ◽  
Structural Information ◽  
Cell Types ◽  
Activation Mechanism ◽  
Peripheral Cell ◽  
X Ray ◽  
X Ray Crystallography ◽  
X Ray Scattering

Leptin plays a central role in the control of body weight and energy homeostasis, but is a pleiotropic cytokine with activities on many peripheral cell types. In this review, we discuss the interaction of leptin with its receptor, and focus on the structural and mechanistic aspects of the extracellular aspects of leptin receptor (LR) activation. We provide an extensive overview of all structural information that has been obtained for leptin and its receptor via X-ray crystallography, electron microscopy, small-angle X-ray scattering, homology modeling, and mutagenesis studies. The available knowledge is integrated into putative models toward a recapitulation of the LR activation mechanism.

scholarly journals Crystallization and preliminary X-ray analysis of the receiver domain of a putative response regulator, BPSL0128, fromBurkholderia pseudomallei

2012 ◽  
Vol 68 (8) ◽  
pp. 917-922
Author(s):  
Abd Ghani Abd Aziz ◽  
Svetlana E. Sedelnikova ◽  
Sergey N. Ruzheinikov ◽  
Simon Thorpe ◽  
Rahmah Mohamed ◽  
...  
Keyword(s):  
Response Regulator ◽  
X Ray ◽  
Receiver Domain

The Signaling Pathway in Histidine Kinase and the Response Regulator Complex Revealed by X-ray Crystallography and Solution Scattering

2006 ◽  
Vol 362 (1) ◽  
pp. 123-139 ◽  
Author(s):  
Seiji Yamada ◽  
Shuji Akiyama ◽  
Hiroshi Sugimoto ◽  
Hideyuki Kumita ◽  
Kazuki Ito ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Histidine Kinase ◽  
Response Regulator ◽  
X Ray ◽  
X Ray Crystallography

scholarly journals Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

IUCrJ ◽  
2020 ◽  
Vol 7 (2) ◽  
pp. 306-323 ◽  
Author(s):  
Alexander M. Wolff ◽  
Iris D. Young ◽  
Raymond G. Sierra ◽  
Aaron S. Brewster ◽  
Michael W. Martynowycz ◽  
...  
Keyword(s):  
Electron Diffraction ◽  
Structural Information ◽  
Biological Macromolecules ◽  
Structural Studies ◽  
Macromolecular Crystallography ◽  
Delivery Methods ◽  
X Ray ◽  
X Ray Crystallography ◽  
Physical Conditions ◽  
Serial Crystallography

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.

scholarly journals Allosteric Regulation of the EphA2 Receptor Intracellular Region by Serine/Threonine Kinases

2021 ◽  
Author(s):  
Bernhard C. Lechtenberg ◽  
Marina P. Gehring ◽  
Taylor P. Light ◽  
Mike W. Matsumoto ◽  
Kalina Hristova ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Cell Communication ◽  
Kinase Domain ◽  
Eph Receptor ◽  
X Ray ◽  
X Ray Crystallography ◽  
Exchange Mass Spectrometry ◽  
Intracellular Region

ABSTRACTEph receptor tyrosine kinases play a key role in cell-cell communication. However, lack of structural information on the entire multi-domain intracellular region of any Eph receptor has hindered detailed understanding of their signaling mechanisms. Here, we use an integrative structural biology approach combining X-ray crystallography, small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry, to gain the first insights into the structure and dynamics of the entire EphA2 intracellular region. EphA2 promotes cancer malignancy through a poorly understood non-canonical form of signaling that depends on serine/threonine phosphorylation of the linker connecting the EphA2 kinase and SAM domains. We uncovered two distinct molecular mechanisms that may function in concert to mediate the effects of linker phosphorylation through an orchestrated allosteric regulatory network. The first involves a shift in the equilibrium between a “closed” configuration of the EphA2 intracellular region and an “open” more extended configuration induced by the accumulation of phosphorylation sites in the linker. This implies that cooperation of multiple serine/threonine kinase signaling networks is necessary to promote robust EphA2 non-canonical signaling. The second involves allosteric rearrangements in the kinase domain and juxtamembrane segment induced by phosphorylation of some linker residues, suggesting a link between EphA2 non-canonical signaling and canonical signaling through tyrosine phosphorylation. Given the key role of EphA2 in cancer malignancy, this new knowledge can inform therapeutic strategies.

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