scholarly journals Common sequence motifs of nascent chains engage the ribosome surface and trigger factor

2021 ◽  
Vol 118 (52) ◽  
pp. e2103015118
Author(s):  
Annika Deckert ◽  
Anaïs M. E. Cassaignau ◽  
Xiaolin Wang ◽  
Tomasz Włodarski ◽  
Sammy H. S. Chan ◽  
...  
Keyword(s):  
Surface Interactions ◽  
Trigger Factor ◽  
Structural Basis ◽  
Sequence Motifs ◽  
Nascent Chain ◽  
Dynamics Simulations ◽  
Polypeptide Chains ◽  
Common Sequence

In the cell, the conformations of nascent polypeptide chains during translation are modulated by both the ribosome and its associated molecular chaperone, trigger factor. The specific interactions that underlie these modulations, however, are still not known in detail. Here, we combine protein engineering, in-cell and in vitro NMR spectroscopy, and molecular dynamics simulations to explore how proteins interact with the ribosome during their biosynthesis before folding occurs. Our observations of α-synuclein nascent chains in living Escherichia coli cells reveal that ribosome surface interactions dictate the dynamics of emerging disordered polypeptides in the crowded cytosol. We show that specific basic and aromatic motifs drive such interactions and directly compete with trigger factor binding while biasing the direction of the nascent chain during its exit out of the tunnel. These results reveal a structural basis for the functional role of the ribosome as a scaffold with holdase characteristics and explain how handover of the nascent chain to specific auxiliary proteins occurs among a host of other factors in the cytosol.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Peptidyl Transferase ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
In Vitro Reconstitution

AbstractRibosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

scholarly journals Structural and mechanistic basis for translation inhibition by macrolide and ketolide antibiotics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bertrand Beckert ◽  
Elodie C. Leroy ◽  
Shanmugapriya Sothiselvam ◽  
Lars V. Bock ◽  
Maxim S. Svetlov ◽  
...  
Keyword(s):  
Antibiotic Resistance Genes ◽  
Structural Basis ◽  
Sequence Motifs ◽  
Specific Sequence ◽  
Bacterial Ribosome ◽  
Translational Arrest ◽  
Exit Tunnel ◽  
Mechanistic Basis ◽  
Dynamics Simulations ◽  
Context Specific

AbstractMacrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics.

scholarly journals Analysis of the structural requirements of sugar binding to the liver, brain and insulin-responsive glucose transporters expressed in oocytes

1993 ◽  
Vol 294 (3) ◽  
pp. 753-760 ◽  
Author(s):  
C A Colville ◽  
M J Seatter ◽  
G W Gould
Keyword(s):  
Hydrogen Bond ◽  
Binding Sites ◽  
Glucose Transporters ◽  
Binding Pocket ◽  
Structural Basis ◽  
Sugar Binding ◽  
Glucose Analogues ◽  
Sugar Recognition

We have expressed the liver (GLUT 2), brain (GLUT 3) and insulin-responsive (GLUT 4) glucose transporters in oocytes from Xenopus laevis by microinjection of in vitro-transcribed mRNA. Using a range of halogeno- and deoxy-glucose analogues, and other hexoses, we have studied the structural basis of sugar binding to these different isoforms. We show that a hydrogen bond to the C-3 position is involved in sugar binding for all three isoforms, but that the direction of this hydrogen bond is different in GLUT 2 from either GLUT 1, 3 or 4. Hydrogen-bonding at the C-4 position is also involved in sugar recognition by all three isoforms, but we propose that in GLUT 3 this hydrogen bond plays a less significant role than in GLUT 2 and 4. In all transporters we propose that the C-4 position is directed out of the sugar-binding pocket. The role of the C-6 position is also discussed. In addition, we have analysed the ability of fructopyranose and fructofuranose analogues to inhibit the transport mediated by GLUT2. We show that fructofuranose analogues, but not fructopyranose analogues, are efficient inhibitors of transport mediated by GLUT 2, and therefore suggest that GLUT 2 accommodates D-glucose as a pyranose ring, but D-fructose as a furanose ring. Models for the binding sites of GLUT 2, 3 and 4 are presented.

scholarly journals A Conserved Tryptophan in the Ebola Virus Matrix Protein C-Terminal Domain Is Required for Efficient Virus-Like Particle Formation

Pathogens ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 402 ◽  
Author(s):  
Kristen A. Johnson ◽  
Rudramani Pokhrel ◽  
Melissa R. Budicini ◽  
Bernard S. Gerstman ◽  
Prem P. Chapagain ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Membrane Localization ◽  
Virus Like Particles ◽  
Plasma Membrane Localization ◽  
The Matrix ◽  
Dynamics Simulations

The Ebola virus (EBOV) harbors seven genes, one of which is the matrix protein eVP40, a peripheral protein that is sufficient to induce the formation of virus-like particles from the host cell plasma membrane. eVP40 can form different structures to fulfil different functions during the viral life cycle, although the structural dynamics of eVP40 that warrant dimer, hexamer, and octamer formation are still poorly understood. eVP40 has two conserved Trp residues at positions 95 and 191. The role of Trp95 has been characterized in depth as it serves as an important residue in eVP40 oligomer formation. To gain insight into the functional role of Trp191 in eVP40, we prepared mutations of Trp191 (W191A or W191F) to determine the effects of mutation on eVP40 plasma membrane localization and budding as well as eVP40 oligomerization. These in vitro and cellular experiments were complemented by molecular dynamics simulations of the wild-type (WT) eVP40 structure versus that of W191A. Taken together, Trp is shown to be a critical amino acid at position 191 as mutation to Ala reduces the ability of VP40 to localize to the plasma membrane inner leaflet and form new virus-like particles. Further, mutation of Trp191 to Ala or Phe shifted the in vitro equilibrium to the octamer form by destabilizing Trp191 interactions with nearby residues. This study has shed new light on the importance of interdomain interactions in stability of the eVP40 structure and the critical nature of timing of eVP40 oligomerization for plasma membrane localization and viral budding.

scholarly journals Structural basis of the interplay between α-synuclein and Tau in regulating pathological amyloid aggregation

2020 ◽  
Vol 295 (21) ◽  
pp. 7470-7480 ◽  
Author(s):  
Jinxia Lu ◽  
Shengnan Zhang ◽  
Xiaojuan Ma ◽  
Chunyu Jia ◽  
Zhenying Liu ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Neurodegenerative Diseases ◽  
Direct Interaction ◽  
Structural Basis ◽  
Amyloid Aggregation ◽  
C Terminus ◽  
Amyloid Fibrillation

Amyloid aggregation of pathological proteins is closely associated with a variety of neurodegenerative diseases, and α-synuclein (α-syn) deposition and Tau tangles are considered hallmarks of Parkinson's disease and Alzheimer's disease, respectively. Intriguingly, α-syn and Tau have been found to co-deposit in the brains of individuals with dementia and parkinsonism, suggesting a potential role of cross-talk between these two proteins in neurodegenerative pathologies. Here we show that monomeric α-syn and the two variants of Tau, Tau23 and K19, synergistically promote amyloid fibrillation, leading to their co-aggregation in vitro. NMR spectroscopy experiments revealed that α-syn uses its highly negatively charged C terminus to directly interact with Tau23 and K19. Deletion of the C terminus effectively abolished its binding to Tau23 and K19 as well as its synergistic effect on promoting their fibrillation. Moreover, an S129D substitution of α-syn, mimicking C-terminal phosphorylation of Ser129 in α-syn, which is commonly observed in the brains of Parkinson's disease patients with elevated α-syn phosphorylation levels, significantly enhanced the activity of α-syn in facilitating Tau23 and K19 aggregation. These results reveal the molecular basis underlying the direct interaction between α-syn and Tau. We proposed that this interplay might contribute to pathological aggregation of α-syn and Tau in neurodegenerative diseases.

scholarly journals The chaperone Hsp70 is a BH3 receptor activated by the pro-apoptotic Bim to stabilize anti-apoptotic clients

2020 ◽  
Vol 295 (37) ◽  
pp. 12900-12909
Author(s):  
Zongwei Guo ◽  
Ting Song ◽  
Ziqian Wang ◽  
Donghai Lin ◽  
Keke Cao ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Cell Fate ◽  
Protein Misfolding ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Allosteric Site ◽  
Transverse Relaxation ◽  
Bh3 Domain

The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, 1H-15N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins.

scholarly journals Molecular determinant of substrate binding and specificity of cytochrome P450 2J2

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Liang Xu ◽  
Liao Y. Chen
Keyword(s):  
Arachidonic Acid ◽  
Cytochrome P450 ◽  
Active Site ◽  
Structural Model ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Structural Basis ◽  
Molecular Determinant ◽  
Dynamics Simulations

AbstractCytochrome P450 2J2 (CYP2J2) is responsible for the epoxidation of endogenous arachidonic acid, and is involved in the metabolism of exogenous drugs. To date, no crystal structure of CYP2J2 is available, and the proposed structural basis for the substrate recognition and specificity in CYP2J2 varies with the structural models developed using different computational protocols. In this study, we developed a new structural model of CYP2J2, and explored its sensitivity to substrate binding by molecular dynamics simulations of the interactions with chemically similar fluorescent probes. Our results showed that the induced-fit binding of these probes led to the preferred active poses ready for the catalysis by CYP2J2. Divergent conformational dynamics of CYP2J2 due to the binding of each probe were observed. However, a stable hydrophobic clamp composed of residues I127, F310, A311, V380, and I487 was identified to restrict any substrate access to the active site of CYP2J2. Molecular docking of a series of compounds including amiodarone, astemizole, danazol, ebastine, ketoconazole, terfenadine, terfenadone, and arachidonic acid to CYP2J2 confirmed the role of those residues in determining substrate binding and specificity of CYP2J2. In addition to the flexibility of CYP2J2, the present work also identified other factors such as electrostatic potential in the vicinity of the active site, and substrate strain energy and property that have implications for the interpretation of CYP2J2 metabolism.

Involvement of the consensus sequence motif at coil 2b in the assembly and stability of vimentin filaments

1992 ◽  
Vol 102 (1) ◽  
pp. 31-41 ◽  
Author(s):  
P.D. Kouklis ◽  
P. Traub ◽  
S.D. Georgatos
Keyword(s):  
Consensus Sequence ◽  
Sequence Motif ◽  
Sequence Motifs ◽  
3T3 Cells ◽  
Filament Assembly ◽  
In Vitro Assembly ◽  
Vimentin Filaments

Nearly all intermediate filament (IF) proteins share two sequence motifs located at the N- and the C-terminal ends of their helical rod domain (‘coil 1a’ and ‘coil 2b’, respectively). To examine the structural role of the coil 2b motif, we have performed in vitro assembly studies and in vivo microinjection experiments employing two site-specific reagents: (a) a 20-residue synthetic peptide (C-2) representing the conserved motif itself and (b) a monoclonal antibody (anti-IFA) that recognises an epitope within the conserved coil 2b sequence. We demonstrate here that vimentin protofilaments, when induced to assemble in the presence of C-2 or anti-IFA, show a lower propensity to polymerise and yield various abberant structures. The few filaments that are formed under these conditions appear much shorter than normal IFs and are unravelled or aggregated. Furthermore, when preformed vimentin filaments are exposed to C-2 or anti-IFA, most of the normal IFs are converted into shorter filamentous forms that possess an abberant morphology. None of these effects is seen when vimentin subunits are coincubated with control peptides. Microinjection of anti-IFA into the cytoplasm of interphasic 3T3 cells provokes collapse of vimentin IFs into a juxtanuclear mass and formation of numerous amorphous aggregates distributed throughout the cytoplasm. These two effects are not seen when the anti-IFA is microinjected into the cell nucleus. Our results provide experimental evidence supporting previous suggestions for a role for the conserved coil 2b sequence in filament assembly. We propose that this region is interacting with other sites along the vimentin molecule and that these interactions are essential for proper protofilament-protofilament alignment and filament stability.

scholarly journals Structural basis for the role of serine-rich repeat proteins from Lactobacillus reuteri in gut microbe–host interactions

2018 ◽  
Vol 115 (12) ◽  
pp. E2706-E2715 ◽  
Author(s):  
Saannya Sequeira ◽  
Devon Kavanaugh ◽  
Donald A. MacKenzie ◽  
Tanja Šuligoj ◽  
Samuel Walpole ◽  
...  
Keyword(s):  
Lactobacillus Reuteri ◽  
Structural Information ◽  
Bacterial Species ◽  
Surface Proteins ◽  
Structural Basis ◽  
Gene Encoding ◽  
Rhamnogalacturonan I ◽  
Host Microbe Interactions ◽  
Dynamics Simulations

Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates, displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens, but no structural information is available in commensal bacteria. Here we report the 2.00-Å and 1.92-Å crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique β-solenoid fold in this important adhesin family. SRRP53608-BR bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of SRRP53608-BR with PGA. Long molecular dynamics simulations showed that SRRP53608-BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host–microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens.

Gentamicin inhibits rat renal cortical homotypic endosomal fusion: role of megalin

1997 ◽  
Vol 272 (1) ◽  
pp. F117-F123 ◽  
Author(s):  
T. G. Hammond ◽  
R. R. Majewski ◽  
J. H. Kaysen ◽  
F. O. Goda ◽  
G. L. Navar ◽  
...  
Keyword(s):  
Damage Mechanism ◽  
Repeat Sequence ◽  
Sequence Motifs ◽  
Polyaspartic Acid ◽  
In Vitro Reconstitution ◽  
Proximal Tubular ◽  
Renal Proximal Tubular Cells ◽  
Renal Proximal Tubular

Megalin, a giant glycoprotein receptor heavily concentrated in the early endosomal pathway of renal proximal tubular cells, binds gentamicin with high affinity and delivers the drug to lysosomes. Utilizing an in vitro reconstitution assay we tested whether gentamicin-induced vacuolation is associated with inhibition of early endosomal fusion, as well as whether megalin plays a role in mediating these effects. Pretreatment of rats with gentamicin inhibited rat renal proximal tubular homotypic endosomal fusion. Administered simultaneously, gentamicin and polymers of polyaspartic acid, which protect against the hemodynamic effects of gentamicin nephrotoxicity, had no net effect on fusion. Polyaspartic acid alone had no effect on fusion. Antisera to the tail of the megalin/gentamicin receptor inhibited fusion, whereas non-specific controls had no effect. Peptides matching homologous NPXY repeat sequence motifs in the cytosolic tail stimulated endosomal fusion, whereas reverse sequence control peptides had no effect. These data suggest that gentamicin inhibition of endosomal fusion in the renal proximal tubule is a damage mechanism mediated by specific peptide sequences in the cytosolic tail of the giant gentamicin-binding receptor megalin and that receptors can effect the fusion properties of membranes in which they reside.

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