Conformational dynamics and energetics of viral RNA recognition by lab-evolved proteins.
Ontology highlight
ABSTRACT:
SUBMITTER: Kumar A
PROVIDER: S-EPMC8579469 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Calmodulin and other members of the EF-hand protein family are known to undergo major changes in conformation upon binding Ca(2+). However, some EF-hand proteins, such as calbindin D9k, bind Ca(2+) without a significant change in conformation. Here, we show the importance of a precise balance of solvation energetics to conformational change, using mutational analysis of partially buried polar groups in the N-terminal domain of calmodulin (N-cam). Several variants were characterized using fluorescence, circular dichroism, and NMR spectroscopy. Strikingly, the replacement of polar side chains glutamine and lysine at positions 41 and 75 with nonpolar side chains leads to dramatic enhancement of the stability of the Ca(2+)-free state, a corresponding decrease in Ca(2+)-binding affinity, and an apparent loss of ability to change conformation to the open form. The results suggest a paradigm for conformational change in which energetic strain is accumulated in one state in order to modulate the energetics of change to the alternative state.
| S-EPMC2373930 | biostudies-literature
Project description:Interferon-induced proteins with tetratricopeptide repeats (IFITs) are innate immune effector molecules that are thought to confer antiviral defence through disruption of protein-protein interactions in the host translation-initiation machinery. However, it was recently discovered that IFITs can directly recognize viral RNA bearing a 5'-triphosphate group (PPP-RNA), which is a molecular signature that distinguishes it from host RNA. Here we report crystal structures of human IFIT5, its complex with PPP-RNAs, and an amino-terminal fragment of IFIT1. The structures reveal a new helical domain that houses a positively charged cavity designed to specifically engage only single-stranded PPP-RNA, thus distinguishing it from the canonical cytosolic sensor of double-stranded viral PPP-RNA, retinoic acid-inducible gene I (RIG-I, also known as DDX58). Mutational analysis, proteolysis and gel-shift assays reveal that PPP-RNA is bound in a non-sequence-specific manner and requires a 5'-overhang of approximately three nucleotides. Abrogation of PPP-RNA binding in IFIT1 and IFIT5 was found to cause a defect in the antiviral response by human embryonic kidney cells. These results demonstrate the mechanism by which IFIT proteins selectively recognize viral RNA, and lend insight into their downstream effector function.
| S-EPMC4931921 | biostudies-literature
Project description:Molecular recognition by proteins is fundamental to almost every biological process, particularly the protein associations underlying cellular signal transduction. Understanding the basis for protein-protein interactions requires the full characterization of the thermodynamics of their association. Historically it has been virtually impossible to experimentally estimate changes in protein conformational entropy, a potentially important component of the free energy of protein association. However, nuclear magnetic resonance spectroscopy has emerged as a powerful tool for characterizing the dynamics of proteins. Here we employ changes in conformational dynamics as a proxy for corresponding changes in conformational entropy. We find that the change in internal dynamics of the protein calmodulin varies significantly on binding a variety of target domains. Surprisingly, the apparent change in the corresponding conformational entropy is linearly related to the change in the overall binding entropy. This indicates that changes in protein conformational entropy can contribute significantly to the free energy of protein-ligand association.
| S-EPMC4156320 | biostudies-literature
Project description:Trans-activation response (TAR) RNA-binding protein (TRBP) has emerged as a key player in the RNA interference pathway, wherein it binds to different pre-microRNAs (miRNAs) and small interfering RNAs (siRNAs), each varying in sequence and/or structure. We hypothesize that TRBP displays dynamic adaptability to accommodate heterogeneity in target RNA structures. Thus, it is crucial to ascertain the role of intrinsic and RNA-induced protein dynamics in RNA recognition and binding. We have previously elucidated the role of intrinsic and RNA-induced conformational exchange in the double-stranded RNA-binding domain 1 (dsRBD1) of TRBP in shape-dependent RNA recognition. The current study delves into the intrinsic and RNA-induced conformational dynamics of the TRBP-dsRBD2 and then compares it with the dsRBD1 study carried out previously. Remarkably, the two domains exhibit differential binding affinity to a 12-bp dsRNA owing to the presence of critical residues and structural plasticity. Furthermore, we report that dsRBD2 depicts constrained conformational plasticity when compared to dsRBD1. Although, in the presence of RNA, dsRBD2 undergoes induced conformational exchange within the designated RNA-binding regions and other residues, the amplitude of the motions remains modest when compared to those observed in dsRBD1. We propose a dynamics-driven model of the two tandem domains of TRBP, substantiating their contributions to the versatility of dsRNA recognition and binding.
| S-EPMC11309768 | biostudies-literature
Project description:Natural and lab-evolved proteins often recognize their RNA partners with exquisite affinity. Structural analysis of such complexes can offer valuable insight into sequence-selective recognition that can be exploited to alter biological function. Here, we describe the structure of a lab-evolved RNA recognition motif (RRM) bound to the HIV-1 trans-activation response (TAR) RNA element at 1.80 Å-resolution. The complex reveals a trio of arginines in an evolved β2-β3 loop penetrating deeply into the major groove to read conserved guanines while simultaneously forming cation-π and salt-bridge contacts. The observation that the evolved RRM engages TAR within a double-stranded stem is atypical compared to most RRMs. Mutagenesis, thermodynamic analysis and molecular dynamics validate the atypical binding mode and quantify molecular contributions that support the exceptionally tight binding of the TAR-protein complex (KD,App of 2.5 ± 0.1 nM). These findings led to the hypothesis that the β2-β3 loop can function as a standalone TAR-recognition module. Indeed, short constrained peptides comprising the β2-β3 loop still bind TAR (KD,App of 1.8 ± 0.5 μM) and significantly weaken TAR-dependent transcription. Our results provide a detailed understanding of TAR molecular recognition and reveal that a lab-evolved protein can be reduced to a minimal RNA-binding peptide.
| S-EPMC6061845 | biostudies-literature
Project description:Cytoplasmic RIG-I-like receptor (RLR) proteins in mammalian cells recognize viral RNA and initiate an antiviral response that results in IFN-β induction. Melanoma differentiation-associated protein 5 (MDA5) forms fibers along viral dsRNA and propagates an antiviral response via a signaling domain, the tandem CARD. The most enigmatic RLR, laboratory of genetics and physiology (LGP2), lacks the signaling domain but functions in viral sensing through cooperation with MDA5. However, it remains unclear how LGP2 coordinates fiber formation and subsequent MDA5 activation. We utilized biochemical and biophysical approaches to observe fiber formation and the conformation of MDA5. LGP2 facilitated MDA5 fiber assembly. LGP2 was incorporated into the fibers with an average inter-molecular distance of 32 nm, suggesting the formation of hetero-oligomers with MDA5. Furthermore, limited protease digestion revealed that LGP2 induces significant conformational changes on MDA5, promoting exposure of its CARDs. Although the fibers were efficiently dissociated by ATP hydrolysis, MDA5 maintained its active conformation to participate in downstream signaling. Our study demonstrated the coordinated actions of LGP2 and MDA5, where LGP2 acts as an MDA5 nucleator and requisite partner in the conversion of MDA5 to an active conformation. We revealed a mechanistic basis for LGP2-mediated regulation of MDA5 antiviral innate immune responses.
| S-EPMC7672446 | biostudies-literature
Project description:The HIV-1 Tat protein and several small molecules bind to HIV-1 transactivation responsive RNA (TAR) by selecting sparsely populated but pre-existing conformations. Thus, a complete characterization of TAR conformational ensemble and dynamics is crucial to understand this paradigmatic system and could facilitate the discovery of new antivirals targeting this essential regulatory element. We show here that molecular dynamics simulations can be effectively used toward this goal by bridging the gap between functionally relevant time scales that are inaccessible to current experimental techniques. Specifically, we have performed several independent microsecond long molecular simulations of TAR based on one of the most advanced force fields available for RNA, the parmbsc0 AMBER. Our simulations are first validated against available experimental data, yielding an excellent agreement with measured residual dipolar couplings and order parameter S(2). This contrast with previous molecular dynamics simulations (Salmon et al., J. Am. Chem. Soc. 2013 135, 5457-5466) based on the CHARMM36 force field, which could achieve only modest accord with the experimental RDC values. Next, we direct the computation toward characterizing the internal dynamics of TAR over the microsecond time scale. We show that the conformational fluctuations observed over this previously elusive time scale have a strong functionally oriented character in that they are primed to sustain and assist ligand binding.
| S-EPMC5521259 | biostudies-literature
Project description:All-atom molecular dynamics simulations are widely used to study the flexibility of protein conformations. However, enhanced sampling techniques are required for simulating protein dynamics that occur on the millisecond timescale. In this work, we show that torsional molecular dynamics simulations enhance protein conformational sampling by performing conformational search in the low-frequency torsional degrees of freedom. In this article, we use our recently developed torsional-dynamics method called Generalized Newton-Euler Inverse Mass Operator (GNEIMO) to study the conformational dynamics of four proteins. We investigate the use of the GNEIMO method in simulations of the conformationally flexible proteins fasciculin and calmodulin, as well as the less flexible crambin and bovine pancreatic trypsin inhibitor. For the latter two proteins, the GNEIMO simulations with an implicit-solvent model reproduced the average protein structural fluctuations and sample conformations similar to those from Cartesian simulations with explicit solvent. The application of GNEIMO with replica exchange to the study of fasciculin conformational dynamics produced sampling of two of this protein's experimentally established conformational substates. Conformational transition of calmodulin from the Ca(2+)-bound to the Ca(2+)-free conformation occurred readily with GNEIMO simulations. Moreover, the GNEIMO method generated an ensemble of conformations that satisfy about half of both short- and long-range interresidue distances obtained from NMR structures of holo to apo transitions in calmodulin. Although unconstrained all-atom Cartesian simulations have failed to sample transitions between the substates of fasciculin and calmodulin, GNEIMO simulations show the transitions in both systems. The relatively short simulation times required to capture these long-timescale conformational dynamics indicate that GNEIMO is a promising molecular-dynamics technique for studying domain motion in proteins.
| S-EPMC3647154 | biostudies-literature
Project description:The p19 protein (p19) encoded from Tombusvirus is involved in various activities such as pathogenicity and virus transport. Recent studies have found that p19 is a plant suppressor of RNA silencing, which binds to short interfering RNAs (siRNAs) with high affinity. We use molecular dynamics (MD) simulations of the wild-type and mutant p19 protein (W39 and W42G) binding with a 21-nt siRNA duplex to study the p19-siRNA recognition mechanism and mutation effects. Our simulations with standard MD and steered molecular dynamics have shown that the double mutant structure is indeed much less stable than the wild-type, consistent with the recent experimental findings. Comprehensive structural analysis also shows that the W39/42G mutations first induce the loss of stacking interactions between p19 and siRNA, Trp(42)-Cyt1 (Cyt1 from the 5' to 3' strand) and Trp(39)-Gua'19 (Gua19 from the 3' to 5' strand), and then breaks the hydrophobic core formed by W39-W42 with nucleotide basepairs in the wild-type. The steered molecular dynamics simulations also show that the mutant p19 complex is "decompounded" very fast under a constant separation force, whereas the wild-type remains largely intact under the same steering force. Moreover, we have used the free energy perturbation to predict a binding affinity loss of 6.98 +/- 0.95 kcal/mol for the single mutation W39G, and 12.8 +/- 1.0 kcal/mol loss for the double mutation W39/42G, with the van der Waals interactions dominating the contribution ( approximately 90%). These results indicate that the W39/42G mutations essentially destroy the important p19-siRNA recognition by breaking the strong stacking interaction between Cyt1 and Gua'19 with end-capping tryptophans. These large scale simulations might provide new insights to the interactions and co-evolution relationship between RNA virus proteins and their hosts.
| S-EPMC2717360 | biostudies-literature
Project description:Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) F/H family are multipurpose RNA binding proteins that participate in most stages of RNA metabolism. Despite having similar RNA sequence preferences, hnRNP F/H proteins function in overlapping and, in some cases, distinct cellular processes. The domain organization of hnRNP F/H proteins is modular, consisting of N-terminal tandem quasi-RNA recognition motifs (F/HqRRM1,2) and a third C-terminal qRRM3 embedded between glycine-rich repeats. The tandem qRRMs are connected through a 10-residue linker, with several amino acids strictly conserved between hnRNP H and F. A significant difference occurs at position 105 of the linker, where hnRNP H contains a proline and hnRNP F an alanine. To investigate the influence of P105 on the conformational properties of hnRNP H, we probed the structural dynamics of its HqRRM1,2 domain with X-ray crystallography, NMR spectroscopy, and small-angle X-ray scattering. The collective results best describe that HqRRM1,2 exists in a conformational equilibrium between compact and extended structures. The compact structure displays an electropositive surface formed at the qRRM1-qRRM2 interface. Comparison of NMR relaxation parameters, including Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion, between HqRRM1,2 and FqRRM1,2 indicates that FqRRM1,2 primarily adopts a more extended and flexible conformation. Introducing the P105A mutation into HqRRM1,2 alters its conformational dynamics to favor an extended structure. Thus, our work demonstrates that the linker compositions confer different structural properties between hnRNP F/H family members that might contribute to their functional diversity.
| S-EPMC6648666 | biostudies-literature