skip to main content

Attention:

The NSF Public Access Repository (NSF-PAR) system and access will be unavailable from 11:00 PM ET on Friday, September 29 until 11:59 PM ET on Saturday, September 30 due to maintenance. We apologize for the inconvenience.

Explore Research Products in the NSF-PAR

  • NSF Public Access
  • Search Results
  • An Unbound Proline-Rich Signaling Peptide Frequently Samples Cis Conformations in Gaussian Accelerated Molecular Dynamics Simulations
Title: An Unbound Proline-Rich Signaling Peptide Frequently Samples Cis Conformations in Gaussian Accelerated Molecular Dynamics Simulations
Disordered proline-rich motifs are common across the proteomes of many species and are often involved in protein-protein interactions. Proline is a unique amino acid due to the covalent bond between the backbone nitrogen and the proline side chain. The resulting five-membered ring allows proline to sample the cis state about its peptide bond, which other residues cannot do as readily. Because proline-rich disordered sequences exist as ensembles that likely include structures with the proline peptide bond in cis , a robust methodology to accurately account for these conformations in the overall ensemble is crucial. Observing the cis conformations of proline in a disordered sequence is challenging both experimentally and computationally. Nitrogen-hydrogen NMR spectroscopy cannot directly observe proline residues, which lack an amide bond, and computational methods struggle to overcome the large kinetic barrier between the cis and trans states, since isomerization usually occurs on the order of seconds. In the current work, Gaussian accelerated molecular dynamics was used to overcome this free energy barrier and simulate proline isomerization in a tetrapeptide (KPTP) and in the 12-residue proline-rich SH3 binding peptide, ArkA. We found that Gaussian accelerated molecular dynamics, when combined with a lowered peptide bond dihedral angle potential energy barrier (15 kcal/mol), allowed sufficient sampling of the proline cis and trans states on a microsecond timescale. All ArkA prolines spend a significant fraction of time in cis , leading to a more compact ensemble with less polyproline II helix structure than an ArkA ensemble with all peptide bonds in trans . The ensemble containing cis prolines also matches more closely to in vitro circular dichroism data than the all- trans ensemble. The ability of the ArkA prolines to isomerize likely affects the peptide’s ability to bind its partner SH3 domain, and should be studied further. This is the first molecular dynamics simulation study of proline isomerization in a biologically relevant proline-rich sequence that we know of, and a similar protocol could be applied to study multi-proline isomerization in other proline-containing proteins to improve conformational diversity and agreement with in vitro data.  more » « less
Award ID(s):
1852677 2018427
NSF-PAR ID:
10328300
Author(s) / Creator(s):
; ; ; ; ; ; ;
Date Published:
Journal Name:
Frontiers in Molecular Biosciences
Volume:
8
ISSN:
2296-889X
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ( , Scientific Reports)
    Abstract

    The intrinsically disordered ATHP3 was studied at native conditions and in complex with DNA using single amino acid substitutions and high-resolution ion mobility spectrometry coupled to mass spectrometry (trapped IMS-MS). Results showed that ATHP3 can exist in multiple conformations at native conditions (at least 10 conformers were separated), with a variety of prolinecis/transorientations, side chain orientations and protonation sites. When in complex with AT rich DNA hairpins, the -RGRP- core is essential for stabilizing the ATHP3: DNA complex. In particular, the arginine in the sixth position plays an important role during binding to AT-rich regions of hairpin DNA, in good agreement with previous NMR and X-ray data. Mobility based correlation matrices are proposed as a way to reveal differences in structural motifs across the peptide mutants based on the conformational space and relative conformer abundance.

     
    more » « less
  2. ; ; ( , Proceedings of the National Academy of Sciences)

    Protein-based molecular switches play critical roles in biological processes. The importance of the prolylcis−transswitch is underscored by the ubiquitous presence of peptidyl prolyl isomerases such as cyclophilins that accelerate the intrinsically slow isomerization rate. In rice, a tryptophan−proline (W-P)cis−transswitch in transcription repressor protein OsIAA11 along with its associated cyclophilin LRT2 are essential components in a negative feedback gene regulation circuit that controls lateral root initiation in response to the plant hormone auxin. Importantly, no quantitative characterizations of the individual (microscopic) thermodynamic and kinetic parameters for any cyclophilin-catalyzed W-P isomerization have been reported. Here we present NMR studies that determine and independently validate these parameters for LRT2 catalysis of the W-P motif in OsIAA11, providing predictive power for understanding the role of this switch in the auxin-responsive circuit and the resulting lateral rootless phenotype in rice. We show that the observed isomerization rate is linearly dependent on LRT2 concentration but is independent of OsIAA11 concentration over a wide range, and LRT2 is optimally tuned to maintain OsIAA11 at itscis−transequilibrium to supply the slower downstreamcis-specific proteasomal degradation with maximal OsIAA11 substrate. This indicates that accelerating the LRT2-catalyzed isomerization would not accelerate OsIAA degradation, whereas decreasing this rate via targeted mutation could reveal relationships between circuit dynamics and lateral root development. Moreover, we show that sequences flanking the highly conserved Aux/IAA W-P motif do not impact LRT2 catalysis, suggesting that the parameters determined here are broadly applicable across highly conserved cyclophilins and their Aux/IAA targets.

     
    more » « less
  3. ; ; ; ; ; ( , Proceedings of the National Academy of Sciences)

    Proline-rich domains (PRDs) are among the most prevalent signaling modules of eukaryotes but often unexplored by biophysical techniques as their heterologous recombinant expression poses significant difficulties. Using a “divide-and-conquer” approach, we present a detailed investigation of a PRD (166 residues; ∼30% prolines) belonging to a human protein ALIX, a versatile adaptor protein involved in essential cellular processes including ESCRT-mediated membrane remodeling, cell adhesion, and apoptosis. In solution, the N-terminal fragment of ALIX-PRD is dynamically disordered. It contains three tandem sequentially similar proline-rich motifs that compete for a single binding site on its signaling partner, TSG101-UEV, as evidenced by heteronuclear NMR spectroscopy. Global fitting of relaxation dispersion data, measured as a function of TSG101-UEV concentration, allowed precise quantitation of these interactions. In contrast to the soluble N-terminal portion, the C-terminal tyrosine-rich fragment of ALIX-PRD forms amyloid fibrils and viscous gels validated using dye-binding assays with amyloid-specific probes, congo red and thioflavin T (ThT), and visualized by transmission electron microscopy. Remarkably, fibrils dissolve at low temperatures (2 to 6 °C) or upon hyperphosphorylation with Src kinase. Aggregation kinetics monitored by ThT fluorescence shows that charge repulsion dictates phosphorylation-mediated fibril dissolution and that the hydrophobic effect drives fibril formation. These data illuminate the mechanistic interplay between interactions of ALIX-PRD with TSG101-UEV and polymerization of ALIX-PRD and its central role in regulating ALIX function. This study also demonstrates the broad functional repertoires of PRDs and uncovers the impact of posttranslational modifications in the modulation of reversible amyloids.

     
    more » « less
  4. ; ; ; ; ; ; ; ; ( , Advanced Functional Materials)
    Abstract

    The use of benign stimuli to control the binding and release of labile biologics for their isolation from complex feedstocks is a key goal of modern biopharmaceutical technology. This study introduces cyclic azobenzene‐peptide (CAP) ligands for the rapid and discrete photo‐responsive capture and release of blood coagulation factor VIII (FVIII). A predictive method—based on amino acid sequence and molecular architecture of CAPs—is developed to correlate the conformation ofcis/trans‐CAP photo‐isomers to FVIII binding and release. Combined in silico ‐ in vitro analysis of FVIII:peptide interactions guide the design of a rational approach to optimize isomerization kinetics and biorecognition of CAPs. A photoaffinity adsorbent, prepared by conjugating selected CAP G‐cycloAZOB[Lys‐YYKHLYN‐Lys]‐G on translucent chromatographic beads, features high binding capacity (>6 mg of FVIII per mL of resin) and rapid photo‐isomerization kinetics (τ < 30 s) when exposed to 420–450 nm light at the intensity of 0.1 W cm−2. The adsorbent purifies FVIII from a recombinant harvest using a single mobile phase, affording high product yield (>90%), purity (>95%), and blood clotting activity. The CAPs introduced in this report demonstrate a novel route integrating gentle operational conditions in a rapid and efficient bioprocess for the purification of life‐saving biotherapeutics.

     
    more » « less
  5. ( , Nature structural molecular biology)
    Sara Osman Carolina Perdigoto (Ed.)
    Gene expression in all eukaryotes depends critically on the function of transcriptional activation domains of gene activator proteins. The conventional model for activation domain (AD) function is the direct physical recruitment of specific coactivators and transcriptional machinery components. However, ADs are short and astronomically variable sequences, with up to 10^24 possible interchangeable sequence variants for a single gene activator; each variant is intrinsically disordered in structure and interacts with its targets with low specificity and affinity. How these peptides recruit their targets is becoming increasingly difficult to explain, exposing a massive knowledge gap in molecular biology. Here, we show that the single required characteristic of ADs—consistent with their extreme variability, intrinsic structural disorder, and near-stochastic interaction mode—is an amphiphilic aromatic–acidic surfactant-like property. We propose that the AD surfactant, by triggering the local gene-promoter chromatin phase transition, catalyzes the formation of “transcription factory” condensates. We demonstrate that the presence of tryptophan and aspartic acid residues in the AD sequence is sufficient for in vivo functionality, even when present only as a single pair of residues within a 20-amino-acid sequence containing nothing more than additional 18 glycine residues. We demonstrate that the amphipathic α-helix structure, suggested previously as beneficial for AD function, is actually detrimental, and breaking this helix by inserting prolines significantly increases activation domain functionality. The proposed surfactant action mechanism based on near-stochastic interactions implied by the minimalistic activation domains changes not only the paradigm for the explanation of gene activation but also the fundamental biochemistry paradigm based on the specificity of sequence-to-structure-to-functional-interaction. The mechanism of activity regulation by near-stochastic allosteric interactions could easily be applied to other biological processes. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email