scholarly journals Mapping of a N-terminal α-helix domain required for human PINK1 stabilisation, Serine228 autophosphorylation and activation in cells

2021 ◽  
Author(s):  
Poonam Kakade ◽  
Hina Ojha ◽  
Olawale Raimi ◽  
Andrew Shaw ◽  
Andrew Waddell ◽  
...  
Keyword(s):  
Intramolecular Interaction ◽  
Human Fibroblasts ◽  
Bioinformatic Analysis ◽  
Kinase Domain ◽  
Major Mechanism ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Α Helix ◽  
Hdx Ms ◽  
In Cells

PINK1 encodes a mitochondrial localised protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function, however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain are unknown. We have employed mutagenesis studies of human PINK1 in cells to define the minimal region of PINK1, required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Bioinformatic analysis of the region spanning Ile111 to the kinase domain and inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal alpha-helical domain extension (NTE domain) within this region corroborated by hydrogen/deuterium exchange mass spectrometry (HDX-MS) of recombinant insect PINK1 protein. The AlphaFold structure also predicts the NTE domain forms an intramolecular interaction with the C-terminal extension (CTE). Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE:CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228); and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE domain mutants do not affect intrinsic catalytic kinase activity but do disrupt PINK1 stabilisation at the mitochondrial Translocase of outer membrane (TOM) complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE:CTE interface towards PINK1 stabilisation and activation and show that loss of NTE:CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.

scholarly journals Two helices control the dynamic crosstalk between the catalytic domains of LRRK2

2021 ◽  
Author(s):  
Jui-Hung Weng ◽  
Phillop C. Aoto ◽  
Robin Lorenz ◽  
Jian Wu ◽  
Sven H. Schmidt ◽  
...  
Keyword(s):  
Md Simulations ◽  
Kinase Domain ◽  
Pharmacological Intervention ◽  
Exchange Mass Spectrometry ◽  
Allosteric Sites ◽  
Hydrogen Deuterium ◽  
Wd40 Domain ◽  
Deuterium Exchange Mass Spectrometry ◽  
And Function ◽  
Hdx Ms

The two major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson’s Disease-related Leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Molecular Dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2 RCKW). We identified two helices that shield the kinase domain and regulate LRRK2 conformation and function. One docking helix in COR-B (Dk-Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its Activation Loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the Dk-Helix create a “cap” that regulates the N-Lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.

scholarly journals Conformational dynamics of FERM-mediated autoinhibition in Pyk2 tyrosine kinase

2019 ◽  
Author(s):  
Hanna S. Loving ◽  
Eric S. Underbakke
Keyword(s):  
Tyrosine Kinase ◽  
Conformational Dynamics ◽  
Kinase Domain ◽  
Ferm Domain ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Protein Scaffolding ◽  
Hdx Ms

AbstractPyk2 is a non-receptor tyrosine kinase that evolved from gene duplication of focal adhesion kinase (FAK) and subsequent functional specialization in the brain and hemopoietic cells. Pyk2 shares a domain organization with FAK, with an N-terminal regulatory FERM domain adjoining the kinase domain. FAK regulation involves integrin-mediated membrane clustering to relieve autoinhibitory interactions between FERM and kinase domains. Pyk2 regulation remains cryptic, involving Ca2+ influx and protein scaffolding. While the mechanism of the FAK FERM domain in autoinhibition is well-established, the regulatory role of the Pyk2 FERM is ambiguous. We probed the mechanisms of FERM-mediated autoinhibition of Pyk2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and kinase activity profiling. The results reveal FERM-kinase interfaces responsible for autoinhibition. Pyk2 autoinhibition impacts activation loop conformation. In addition, the autoinhibitory FERM-kinase interface exhibits allosteric linkage with the FERM basic patch conserved in both FAK and Pyk2.Table of Contents graphic

scholarly journals Activation of GCN2 by the ribosomal P-stalk

2019 ◽  
Vol 116 (11) ◽  
pp. 4946-4954 ◽  
Author(s):  
Alison J. Inglis ◽  
Glenn R. Masson ◽  
Sichen Shao ◽  
Olga Perisic ◽  
Stephen H. McLaughlin ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Principal Component ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Hdx Ms ◽  
Ribosomal P

Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange–mass spectrometry (HDX-MS) mapped GCN2–ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.

scholarly journals Cataract-Associated New Mutants S175G/H181Q of βΒ2-Crystallin and P24S/S31G of γD-Crystallin Are Involved in Protein Aggregation by Structural Changes

2020 ◽  
Vol 21 (18) ◽  
pp. 6504
Author(s):  
In-Kang Song ◽  
Seungjin Na ◽  
Eunok Paek ◽  
Kong-Joo Lee
Keyword(s):  
Structural Changes ◽  
Hydrogen Deuterium Exchange ◽  
Structural Protein ◽  
Exchange Mass Spectrometry ◽  
Human Lenses ◽  
Hydrogen Deuterium ◽  
Proteomic Dataset ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms ◽  
New Mutations

β/γ-Crystallins, the main structural protein in human lenses, have highly stable structure for keeping the lens transparent. Their mutations have been linked to cataracts. In this study, we identified 10 new mutations of β/γ-crystallins in lens proteomic dataset of cataract patients using bioinformatics tools. Of these, two double mutants, S175G/H181Q of βΒ2-crystallin and P24S/S31G of γD-crystallin, were found mutations occurred in the largest loop linking the distant β-sheets in the Greek key motif. We selected these double mutants for identifying the properties of these mutations, employing biochemical assay, the identification of protein modifications with nanoUPLC-ESI-TOF tandem MS and examining their structural dynamics with hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that both double mutations decrease protein stability and induce the aggregation of β/γ-crystallin, possibly causing cataracts. This finding suggests that both the double mutants can serve as biomarkers of cataracts.

scholarly journals Protein Backbone and Average Particle Dynamics in Reconstituted Discoidal and Spherical HDL Probed by Hydrogen Deuterium Exchange and Elastic Incoherent Neutron Scattering

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 121
Author(s):  
Valentin Gogonea ◽  
Judith Peters ◽  
Gary S. Gerstenecker ◽  
Celalettin Topbas ◽  
Liming Hou ◽  
...  
Keyword(s):  
Neutron Scattering ◽  
Deuterium Exchange ◽  
Average Particle ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Incoherent Neutron Scattering ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms ◽  
Incoherent Neutron

Lipoproteins are supramolecular assemblies of proteins and lipids with dynamic characteristics critically linked to their biological functions as plasma lipid transporters and lipid exchangers. Among them, spherical high-density lipoproteins are the most abundant forms of high-density lipoprotein (HDL) in human plasma, active participants in reverse cholesterol transport, and associated with reduced development of atherosclerosis. Here, we employed elastic incoherent neutron scattering (EINS) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to determine the average particle dynamics and protein backbone local mobility of physiologically competent discoidal and spherical HDL particles reconstituted with human apolipoprotein A-I (apoA-I). Our EINS measurements indicated that discoidal HDL was more dynamic than spherical HDL at ambient temperatures, in agreement with their lipid-protein composition. Combining small-angle neutron scattering (SANS) with contrast variation and MS cross-linking, we showed earlier that the most likely organization of the three apolipoprotein A-I (apoA-I) chains in spherical HDL is a combination of a hairpin monomer and a helical antiparallel dimer. Here, we corroborated those findings with kinetic studies, employing hydrogen-deuterium exchange mass spectrometry (HDX-MS). Many overlapping apoA-I digested peptides exhibited bimodal HDX kinetics behavior, suggesting that apoA-I regions with the same amino acid composition located on different apoA-I chains had different conformations and/or interaction environments.

scholarly journals Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

Life ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 286
Author(s):  
Oliver Ozohanics ◽  
Attila Ambrus
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Structural Biology ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Ligand Interaction ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) is a rapidly evolving technique for analyzing structural features and dynamic properties of proteins. It may stand alone or serve as a complementary method to cryo-electron-microscopy (EM) or other structural biology approaches. HDX-MS is capable of providing information on individual proteins as well as large protein complexes. Owing to recent methodological advancements and improving availability of instrumentation, HDX-MS is becoming a routine technique for some applications. When dealing with samples of low to medium complexity and sizes of less than 150 kDa, conformation and ligand interaction analyses by HDX-MS are already almost routine applications. This is also well supported by the rapid evolution of the computational (software) background that facilitates the analysis of the obtained experimental data. HDX-MS can cope at times with analytes that are difficult to tackle by any other approach. Large complexes like viral capsids as well as disordered proteins can also be analyzed by this method. HDX-MS has recently become an established tool in the drug discovery process and biopharmaceutical development, as it is now also capable of dissecting post-translational modifications and membrane proteins. This mini review provides the reader with an introduction to the technique and a brief overview of the most common applications. Furthermore, the most challenging likely applications, the analyses of glycosylated and membrane proteins, are also highlighted.

scholarly journals Modulation of PTH1R signaling by an ECD binding antibody results in inhibition of β-arrestin 2 coupling

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kaushik Sarkar ◽  
Lisa Joedicke ◽  
Marta Westwood ◽  
Rebecca Burnley ◽  
Michael Wright ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Receptor Activation ◽  
Parathyroid Hormone Receptor ◽  
Exchange Mass Spectrometry ◽  
Gpcr Activation ◽  
Hydrogen Deuterium ◽  
Binding Antibody ◽  
Hdx Ms

Abstract Parathyroid hormone receptor 1 (PTH1R) belongs to the secretin class of G protein coupled receptors (GPCRs) and natively binds parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP). Ligand binding to PTH1R involves binding to the large extracellular domain (ECD) and the orthosteric pocket, inducing conformational changes in the transmembrane domain and receptor activation. PTH1R regulates bone metabolism, signaling mainly through Gs and Gq/11 G-proteins. Here, we used phage display to generate PTH1R ECD-specific antibodies with the aim of modulating receptor functionality. We identified ECD-scFvhFc, which exhibited high affinity binding to both the isolated ECD and to the full-length receptor in styrene-maleic acid (SMA) lipid particles. Epitope mapping using hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the α1 helix of the ECD is ECD-scFvhFc’s epitope which may partially overlap with the known PTH (1–34) binding site. However, PTH (1–34)-mediated Gs activation is Undisturbed by ECD-scFvhFc binding. In contrast, ECD-scFvhFc potently inhibits β-arrestin-2 recruitment after PTH (1–34)-driven receptor activation and thus represents the first monoclonal antibody to selectively inhibit distinct PTH1R signaling pathways. Given the complexity of PTH1R signaling and the emerging importance of biased GPCR activation in drug development, ECD-scFvhFc could be a valuable tool to study PTH1R signaling bias.

Sign in / Sign up

Export Citation Format

Share Document