scholarly journals A splice site-sensing conformational switch in U2AF2 is modulated by U2AF1 and its recurrent myelodysplasia-associated mutation

2020 ◽  
Vol 48 (10) ◽  
pp. 5695-5709
Author(s):  
Chandani Warnasooriya ◽  
Callen F Feeney ◽  
Kholiswa M Laird ◽  
Dmitri N Ermolenko ◽  
Clara L Kielkopf
Keyword(s):  
Single Molecule ◽  
Splice Site ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Splicing Factors ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Recognition Motifs ◽  
Rna Complexes

Abstract An essential heterodimer of the U2AF1 and U2AF2 pre-mRNA splicing factors nucleates spliceosome assembly at polypyrimidine (Py) signals preceding the major class of 3′ splice sites. U2AF1 frequently acquires an S34F-encoding mutation among patients with myelodysplastic syndromes (MDS). The influence of the U2AF1 subunit and its S34F mutation on the U2AF2 conformations remains unknown. Here, we employ single molecule Förster resonance energy transfer (FRET) to determine the influence of wild-type or S34F-substituted U2AF1 on the conformational dynamics of U2AF2 and its splice site RNA complexes. In the absence of RNA, the U2AF1 subunit stabilizes a high FRET value, which by structure-guided mutagenesis corresponds to a closed conformation of the tandem U2AF2 RNA recognition motifs (RRMs). When the U2AF heterodimer is bound to a strong, uridine-rich splice site, U2AF2 switches to a lower FRET value characteristic of an open, side-by-side arrangement of the RRMs. Remarkably, the U2AF heterodimer binds weak, uridine-poor Py tracts as a mixture of closed and open U2AF2 conformations, which are modulated by the S34F mutation. Shifts between open and closed U2AF2 may underlie U2AF1-dependent splicing of degenerate Py tracts and contribute to a subset of S34F-dysregulated splicing events in MDS patients.

scholarly journals A Splice Site-Sensing Conformational Switch in U2AF2 is Modulated by U2AF1 and its Recurrent Myelodysplasia-Associated Mutation

2019 ◽  
Author(s):  
Chandani Warnasooriya ◽  
Eliezra Glasser ◽  
Callen F. Feeney ◽  
Kholiswa M. Laird ◽  
Jermaine L. Jenkins ◽  
...  
Keyword(s):  
Single Molecule ◽  
Splice Site ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Splicing Factors ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Recognition Motifs ◽  
Rna Complexes

ABSTRACTAn essential heterodimer of the U2AF1 and U2AF2 pre-mRNA splicing factors nucleates spliceosome assembly at polypyrimidine (Py) signals preceding the major class of 3ʹ splice sites. Among myelodysplastic syndromes (MDS), U2AF1 frequently acquires an S34F-encoding mutation. The influence of the U2AF1 subunit and its S34F mutation on the U2AF2 conformations remains unknown. Here, we employ single molecule Förster resonance energy transfer (FRET) to determine the influence of wild-type or S34F-substituted U2AF1 on the conformational dynamics of U2AF2 and its splice site RNA complexes. In the absence of RNA, the U2AF1 subunit stabilizes a high FRET value, which by structure-guided mutagenesis corresponds to a closed conformation of the tandem U2AF2 RNA recognition motifs (RRMs). When the U2AF heterodimer is bound to a strong, uridine-rich splice site, U2AF2 switches to a lower FRET value characteristic of an open, side-by-side arrangement of the RRMs. Remarkably, the U2AF heterodimer binds weak, uridine-poor Py tracts as a mixture of closed and open U2AF2 conformations, which are modulated by the S34F mutation. Shifts between open and closed U2AF2 may underlie U2AF1-dependent splicing of degenerate Py tracts and contribute to a subset of S34F-dysregulated splicing events in MDS patients.

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

2018 ◽  
Author(s):  
Alexander Carl DeHaven
Keyword(s):  
Energy Transfer ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
U2 Snrna ◽  
Folding Dynamics ◽  
The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

scholarly journals Allosteric modulation of the SARS-CoV-2 spike conformation

2021 ◽  
Author(s):  
Marco A Diaz-Salinas ◽  
Qi Li ◽  
Monir Ejemel ◽  
Yang Wang ◽  
James B Munro
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Data ◽  
Correlation Spectroscopy ◽  
Host Cells ◽  
Parallel Solution ◽  
Real Time Analysis ◽  
Multiple Conformations

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through binding to angiotensin-converting enzyme 2 (ACE2), which is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural data and real-time analysis of conformational dynamics have shown that S can adopt multiple conformations, which mediate the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We found that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicated antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

scholarly journals Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

2018 ◽  
Author(s):  
Robert B. Quast ◽  
Fataneh Fatemi ◽  
Michel Kranendonk ◽  
Emmanuel Margeat ◽  
Gilles Truan
Keyword(s):  
Single Molecule ◽  
Conformational Equilibrium ◽  
Fluorescent Dyes ◽  
Photophysical Properties ◽  
Accurate Determination ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
General Strategy

ABSTRACTConjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Förster resonance energy transfer (smFRET) - can lead to substantial changes of the dye’s photophysical properties, ultimately biasing the quantitative determination of inter-dye distances. In particular the popular cyanine dyes and their derivatives, which are by far the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli was developed. Applied to human NADPH- cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated. Furthermore, the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

scholarly journals Differential Conformational Dynamics Encoded by the Linker between Quasi RNA Recognition Motifs of Heterogeneous Nuclear Ribonucleoprotein H

2018 ◽  
Vol 140 (37) ◽  
pp. 11661-11673 ◽  
Author(s):  
Srinivasa R. Penumutchu ◽  
Liang-Yuan Chiu ◽  
Jennifer L. Meagher ◽  
Alexandar L. Hansen ◽  
Jeanne A. Stuckey ◽  
...  
Keyword(s):  
Conformational Dynamics ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Recognition Motifs ◽  
Nuclear Ribonucleoprotein

scholarly journals Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Stijn van Dorp ◽  
Ruoyi Qiu ◽  
Ucheor B Choi ◽  
Minnie M Wu ◽  
Michelle Yen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bound State ◽  
Structural Basis ◽  
Resting Cells ◽  
Dual Roles ◽  
Er Calcium

The dimeric ER Ca2+ sensor STIM1 controls store-operated Ca2+ entry (SOCE) through the regulated binding of its CRAC activation domain (CAD) to Orai channels in the plasma membrane. In resting cells, the STIM1 CC1 domain interacts with CAD to suppress SOCE, but the structural basis of this interaction is unclear. Using single-molecule Förster resonance energy transfer (smFRET) and protein crosslinking approaches, we show that CC1 interacts dynamically with CAD in a domain-swapped configuration with an orientation predicted to sequester its Orai-binding region adjacent to the ER membrane. Following ER Ca2+ depletion and release from CAD, cysteine crosslinking indicates that the two CC1 domains become closely paired along their entire length in the active Orai-bound state. These findings provide a structural basis for the dual roles of CC1: sequestering CAD to suppress SOCE in resting cells and propelling it towards the plasma membrane to activate Orai and SOCE after store depletion.

scholarly journals Human U2AF65 Recognizing the Major Polypyrimidine Splice Site Signal

2014 ◽  
Vol 70 (a1) ◽  
pp. C1163-C1163
Author(s):  
Anant Agrawal ◽  
Clara Kielkopf
Keyword(s):  
Splice Site ◽  
Structural Information ◽  
Genetic Diseases ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Small Nuclear Rnas ◽  
Nucleotide Interactions ◽  
Gene Transcripts ◽  
Splicing Defects ◽  
Recognition Motifs

Disease-causing mutations often occur in the polypyrimidine (Py) tract splice site signals of human gene transcripts. The essential U2AF65 protein recognizes Py tracts near 3´ splice sites and initiates assembly of the splicing "machine", a megadalton complex comprised of approximately 100 proteins and five small nuclear RNAs. Our prior structures of a shortened U2AF65 variant reveal the basis for nucleotide interactions at subset of binding sites. How intact U2AF65 recognizes the Py tract splice site signal remains unknown to date. We determined a 2.0 Å resolution structure of intact U2AF65 recognizing an optimal, all-uridine Py tract. The new structure and complementary biochemical experiments reveal integral roles for main-chain atoms of the interdomain linker and residues surrounding two core RNA recognition motifs (RRM1 and RRM2) in recognition of the Py tract. The new U2AF65 structural information sheds light on the splicing defects caused by Py tract mutations in human genetic diseases. We test the U2AF65 structural relationship for a representative Py mutation that causes X-linked retinitis pigmentosa.

scholarly journals Conformational dynamics of Cas9 governing DNA cleavage revealed by single molecule FRET

2017 ◽  
Author(s):  
Mengyi Yang ◽  
Sijia Peng ◽  
Ruirui Sun ◽  
Jingdi Lin ◽  
Nan Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nuclease Activity ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fret ◽  
Allosteric Communication ◽  
Target Binding

SummaryOff-target binding and cleavage by Cas9 pose as major challenges in its applications. How conformational dynamics of Cas9 governs its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms all spontaneously transits between three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We furthermore uncovered a surprising long-range allosteric communication between the HNH domain and RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox.

scholarly journals Inter-domain dynamics and fibrinolytic activity of matrix metalloprotease-1

2019 ◽  
Author(s):  
Lokender Kumar ◽  
Joan Planas-Iglesias ◽  
Chase Harms ◽  
Sumaer Kamboj ◽  
Derek Wright ◽  
...  
Keyword(s):  
Single Molecule ◽  
Blood Clot ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Primary Component ◽  
Water Soluble ◽  
Matrix Metalloprotease ◽  
Allosteric Control ◽  
Domain Dynamics

AbstractThe roles of protein conformational dynamics and allostery in function are well-known. However, the roles that inter-domain dynamics have in function are not entirely understood. We used matrix metalloprotease-1 (MMP1) as a model system to study the relationship between inter-domain dynamics and activity because MMP1 has diverse substrates. Here we focus on fibrin, the primary component of a blood clot. Water-soluble fibrinogen, following cleavage by thrombin, self-polymerize to form water-insoluble fibrin. We studied the inter-domain dynamics of MMP1 on fibrin without crosslinks using single-molecule Forster Resonance Energy Transfer (smFRET). We observed that the distance between the catalytic and hemopexin domains of MMP1 increases or decreases as the MMP1 activity increases or decreases, respectively. We modulated the activity using 1) an active site mutant (E219Q) of MMP1, 2) MMP9, another member of the MMP family that increases the activity of MMP1, and 3) tetracycline, an inhibitor of MMP1. We fitted the histograms of smFRET values to a sum of two Gaussians and the autocorrelations to an exponential and power law. We modeled the dynamics as a two-state Poisson process and calculated the kinetic rates from the histograms and autocorrelations. Activity-dependent inter-domain dynamics may enable allosteric control of the MMP1 function.

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