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

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mohammed Jamshad ◽  
Timothy J Knowles ◽  
Scott A White ◽  
Douglas G Ward ◽  
Fiyaz Mohammed ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Metal Binding ◽  
Ribosome Binding ◽  
Substrate Protein ◽  
Metal Binding Domain ◽  
Opposing Effects ◽  
Substrate Proteins ◽  
Binding Substrate

In bacteria, the translocation of proteins across the cytoplasmic membrane by the Sec machinery requires the ATPase SecA. SecA binds ribosomes and recognises nascent substrate proteins, but the molecular mechanism of nascent substrate recognition is unknown. We investigated the role of the C-terminal tail (CTT) of SecA in nascent polypeptide recognition. The CTT consists of a flexible linker (FLD) and a small metal-binding domain (MBD). Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the MBD only or the entire CTT had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function, suggesting that the CTT influences the conformation of SecA. Site-specific crosslinking indicated that F399 in SecA contacts ribosomal protein uL29, and binding to nascent chains disrupts this interaction. Structural studies provided insight into the CTT-mediated conformational changes in SecA. Our results suggest a mechanism for nascent substrate protein recognition.

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

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).

scholarly journals In vivo FRET analyses reveal a role of ATP hydrolysis–associated conformational changes in human P-glycoprotein

2020 ◽  
Vol 295 (15) ◽  
pp. 5002-5011 ◽  
Author(s):  
Ryota Futamata ◽  
Fumihiko Ogasawara ◽  
Takafumi Ichikawa ◽  
Atsushi Kodan ◽  
Yasuhisa Kimura ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Hek293 Cells ◽  
Atp Binding ◽  
Binding Domains ◽  
P Glycoprotein ◽  
Atp Binding And Hydrolysis

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

scholarly journals Iron is a ligand of SecA-like metal-binding domains in vivo

2020 ◽  
Vol 295 (21) ◽  
pp. 7516-7528
Author(s):  
Tamar Cranford-Smith ◽  
Mohammed Jamshad ◽  
Mark Jeeves ◽  
Rachael A. Chandler ◽  
Jack Yule ◽  
...  
Keyword(s):  
Sodium Azide ◽  
Metal Binding ◽  
Cytoplasmic Membrane ◽  
E Coli ◽  
Binding Domains ◽  
Equilibrium Binding ◽  
Plausible Model ◽  
Metal Binding Domain ◽  
Linker Domain

The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modeling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.

scholarly journals Overproduction of Polypeptides Corresponding to the Amino Terminus of the F-Box Proteins Cdc4p and Met30p Inhibits Ubiquitin Ligase Activities of Their SCF Complexes

2003 ◽  
Vol 2 (1) ◽  
pp. 123-133 ◽  
Author(s):  
Cheryl Dixon ◽  
Lee Ellen Brunson ◽  
Mary Margaret Roy ◽  
Dechelle Smothers ◽  
Michael G. Sehorn ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
Cellular Responses ◽  
Amino Terminus ◽  
Variable Component ◽  
Amino Terminal ◽  
F Box Protein ◽  
Substrate Proteins

ABSTRACT Ubiquitin ligases direct the transfer of ubiquitin onto substrate proteins and thus target the substrate for proteasome-dependent degradation. SCF complexes are a family of ubiquitin ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity. Distinct SCF complexes, defined by a particular F-box protein, target different substrate proteins for degradation. Although a few have been identified to be involved in important biological pathways, such as the cell division cycle and coordinating cellular responses to changes in environmental conditions, the role of the overwhelming majority of F-box proteins is not clear. Creating inhibitors that will block the in vivo activities of specific SCF ubiquitin ligases may provide identification of substrates of these uncharacterized F-box proteins. Using Saccharomyces cerevisiae as a model system, we demonstrate that overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p results in specific inhibition of their SCF complexes. Analyses of mutant amino-terminal alleles demonstrate that the interaction of these polypeptides with their full-length counterparts is an important step in the inhibitory process. These results suggest a common means to inhibit specific SCF complexes in vivo.

scholarly journals The LINC00961 transcript and its encoded micropeptide, small regulatory polypeptide of amino acid response, regulate endothelial cell function

2020 ◽  
Vol 116 (12) ◽  
pp. 1981-1994 ◽  
Author(s):  
Helen L Spencer ◽  
Rachel Sanders ◽  
Mounia Boulberdaa ◽  
Marco Meloni ◽  
Amy Cochrane ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Amino Acid ◽  
Full Length ◽  
Binding Partners ◽  
Full Length Transcript ◽  
Opposing Effects ◽  
Amino Acid Response

Abstract Aims Long non-coding RNAs (lncRNAs) play functional roles in physiology and disease, yet understanding of their contribution to endothelial cell (EC) function is incomplete. We identified lncRNAs regulated during EC differentiation and investigated the role of LINC00961 and its encoded micropeptide, small regulatory polypeptide of amino acid response (SPAAR), in EC function. Methods and results Deep sequencing of human embryonic stem cell differentiation to ECs was combined with Encyclopedia of DNA Elements (ENCODE) RNA-seq data from vascular cells, identifying 278 endothelial enriched genes, including 6 lncRNAs. Expression of LINC00961, first annotated as an lncRNA but reassigned as a protein-coding gene for the SPAAR micropeptide, was increased during the differentiation and was EC enriched. LINC00961 transcript depletion significantly reduced EC adhesion, tube formation, migration, proliferation, and barrier integrity in primary ECs. Overexpression of the SPAAR open reading frame increased tubule formation; however, overexpression of the full-length transcript did not, despite production of SPAAR. Furthermore, overexpression of an ATG mutant of the full-length transcript reduced network formation, suggesting a bona fide non-coding RNA function of the transcript with opposing effects to SPAAR. As the LINC00961 locus is conserved in mouse, we generated an LINC00961 locus knockout (KO) mouse that underwent hind limb ischaemia (HLI) to investigate the angiogenic role of this locus in vivo. In agreement with in vitro data, KO animals had a reduced capillary density in the ischaemic adductor muscle after 7 days. Finally, to characterize LINC00961 and SPAAR independent functions in ECs, we performed pull-downs of both molecules and identified protein-binding partners. LINC00961 RNA binds the G-actin sequestering protein thymosin beta-4x (Tβ4) and Tβ4 depletion phenocopied the overexpression of the ATG mutant. SPAAR binding partners included the actin-binding protein, SYNE1. Conclusion The LINC00961 locus regulates EC function in vitro and in vivo. The gene produces two molecules with opposing effects on angiogenesis: SPAAR and LINC00961.

NMR and EPR studies of membrane transporters

2009 ◽  
Vol 390 (8) ◽  
Author(s):  
Ute A. Hellmich ◽  
Clemens Glaubitz
Keyword(s):  
Membrane Proteins ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Conformational Dynamics ◽  
Crystallographic Analysis ◽  
Membrane Transporters ◽  
Paramagnetic Resonance ◽  
Substrate Protein ◽  
Electron Paramagnetic

AbstractIn order to fulfill their function, membrane transport proteins have to cycle through a number of conformational and/or energetic states. Thus, understanding the role of conformational dynamics seems to be the key for elucidation of the functional mechanism of these proteins. However, membrane proteins in general are often difficult to express heterologously and in sufficient amounts for structural studies. It is especially challenging to trap a stable energy minimum, e.g., for crystallographic analysis. Furthermore, crystallization is often only possible by subjecting the protein to conditions that do not resemble its native environment and crystals can only be snapshots of selected conformational states. Nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy are complementary methods that offer unique possibilities for studying membrane proteins in their natural membrane environment and for investigating functional conformational changes, lipid interactions, substrate-lipid and substrate-protein interactions, oligomerization states and overall dynamics of membrane transporters. Here, we review recent progress in the field including studies from primary and secondary active transporters.

scholarly journals Chaperonins

1998 ◽  
Vol 333 (2) ◽  
pp. 233-242 ◽  
Author(s):  
Neil A. RANSON ◽  
Helen E. WHITE ◽  
Helen R. SAIBIL
Keyword(s):  
Conformational Changes ◽  
Substrate Binding ◽  
Atp Binding ◽  
Dependent Manner ◽  
Oligomeric Proteins ◽  
Double Ring ◽  
Substrate Protein ◽  
Hydrophobic Binding ◽  
Substrate Binding Sites

The molecular chaperones are a diverse set of protein families required for the correct folding, transport and degradation of other proteins in vivo. There has been great progress in understanding the structure and mechanism of action of the chaperonin family, exemplified by Escherichia coli GroEL. The chaperonins are large, double-ring oligomeric proteins that act as containers for the folding of other protein subunits. Together with its co-protein GroES, GroEL binds non-native polypeptides and facilitates their refolding in an ATP-dependent manner. The action of the ATPase cycle causes the substrate-binding surface of GroEL to alternate in character between hydrophobic (binding/unfolding) and hydrophilic (release/folding). ATP binding initiates a series of dramatic conformational changes that bury the substrate-binding sites, lowering the affinity for non-native polypeptide. In the presence of ATP, GroES binds to GroEL, forming a large chamber that encapsulates substrate proteins for folding. For proteins whose folding is absolutely dependent on the full GroE system, ATP binding (but not hydrolysis) in the encapsulating ring is needed to initiate protein folding. Similarly, ATP binding, but not hydrolysis, in the opposite GroEL ring is needed to release GroES, thus opening the chamber. If the released substrate protein is still not correctly folded, it will go through another round of interaction with GroEL.

scholarly journals A Mutation in the PP2C Phosphatase Gene in a Staphylococcus aureus USA300 Clinical Isolate with Reduced Susceptibility to Vancomycin and Daptomycin

2012 ◽  
Vol 56 (10) ◽  
pp. 5212-5223 ◽  
Author(s):  
Karla D. Passalacqua ◽  
Sarah W. Satola ◽  
Emily K. Crispell ◽  
Timothy D. Read
Keyword(s):  
Staphylococcus Aureus ◽  
Metal Binding ◽  
De Novo ◽  
Surface Protein ◽  
Complex Nature ◽  
Insertion Mutation ◽  
Content Type ◽  
Metal Binding Domain ◽  
Multiple Routes

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) strains with reduced susceptibility to vancomycin (MIC of 4 to 8 μg/ml) are referred to as vancomycin-intermediateS. aureus(VISA). In this study, we characterized two isogenic USA300S. aureusisolates collected sequentially from a single patient with endocarditis where theS. aureusisolate changed from being susceptible to vancomycin (VSSA) (1 μg/ml) to VISA (8 μg/ml). In addition, the VISA isolate lost beta-lactamase activity and showed increased resistance to daptomycin and linezolid. The two strains did not differ in growth rate, but the VISA isolate had a thickened cell wall and was less autolytic. Transcriptome sequencing (RNA-seq) analysis comparing the two isolates grown to late exponential phase showed significant differences in transcription of cell surface protein genes (spa, SBI [second immunoglobulin-binding protein ofS. aureus], and fibrinogen-binding proteins), regulatory genes (agrBCA, RNAIII,sarT, andsaeRS), and others. Using whole-genome shotgun resequencing, we identified 6 insertion/deletion mutations between the VSSA and VISA isolates. A protein phosphatase 2C (PP2C) family phosphatase had a 6-bp (nonframeshift) insertion mutation in a highly conserved metal binding domain. Complementation of the clinical VISA isolate with a wild-type copy of the PP2C gene reduced the vancomycin and daptomycin MICs and increased autolytic activity, suggesting that this gene contributed to the reduced vancomycin susceptibility phenotype acquiredin vivo. Creation ofde novomutants from the VSSA strain resulted in different mutations, demonstrating that reduced susceptibility to vancomycin in USA300 strains can occur via multiple routes, highlighting the complex nature of the VISA phenotype.

scholarly journals The Effect of Dysfunctional Ubiquitin Enzymes in the Pathogenesis of Most Common Diseases

2020 ◽  
Vol 21 (17) ◽  
pp. 6335 ◽  
Author(s):  
Gizem Celebi ◽  
Hale Kesim ◽  
Ebru Ozer ◽  
Ozlem Kutlu
Keyword(s):  
Protein Quality ◽  
Ubiquitin Proteasome System ◽  
Deubiquitinating Enzymes ◽  
E3 Ligases ◽  
Substrate Protein ◽  
Common Diseases ◽  
Ubiquitin Proteasome ◽  
Ubiquitin Conjugating Enzymes ◽  
Substrate Proteins

Ubiquitination is a multi-step enzymatic process that involves the marking of a substrate protein by bonding a ubiquitin and protein for proteolytic degradation mainly via the ubiquitin–proteasome system (UPS). The process is regulated by three main types of enzymes, namely ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). Under physiological conditions, ubiquitination is highly reversible reaction, and deubiquitinases or deubiquitinating enzymes (DUBs) can reverse the effect of E3 ligases by the removal of ubiquitin from substrate proteins, thus maintaining the protein quality control and homeostasis in the cell. The dysfunction or dysregulation of these multi-step reactions is closely related to pathogenic conditions; therefore, understanding the role of ubiquitination in diseases is highly valuable for therapeutic approaches. In this review, we first provide an overview of the molecular mechanism of ubiquitination and UPS; then, we attempt to summarize the most common diseases affecting the dysfunction or dysregulation of these mechanisms.

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