Search NASA⌕ Search

SEARCH · Search NASA

Results for “protein conformational dynamics”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Identification of structural transitions in bacterial fatty acid binding proteins that permit ligand entry and exit at membranes

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein–FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a “closed” FakB1 state to an “open” state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

All-Atom Simulations Uncover Structural and Dynamical Properties of STING Proteins in the Membrane System

Recent studies have shown that the stimulator of interferon gene (STING) protein plays a central role in the immune system by facilitating the production of type I interferons in cells. The STING signaling pathway is also a prominent activator of cancer-killing T cells that initiate a powerful adaptive immune response. Since biomolecular signaling pathways are complicated and not easily identified through traditional experiments, molecular dynamics (MD) has often been used to study structural and dynamical responses of biological pathways. In this work, we carried out MD simulations for full-length chicken and human STING (chSTING and hSTING) proteins. Specifically, we investigated ligand-bound closed (holo) and ligand-unbound open (apo) forms of STING in the membrane system by comparing their conformational and dynamical differences. Our research provides clues for understanding the mechanism of the STING signaling pathway by uncovering detailed insights for the examined systems: the residues from each chain in the binding pocket are strongly correlated to one another in the open STING structure compared with those in the closed STING structure. Ligand-bound closed STING displays ~174° rotation of the ligand-binding domain (LBD) relative to the open STING structure. The dynamical analysis of residue Cys148 located in the linker region of hSTING does not support the earlier hypothesis that Cys148 can form disulfide bonds between adjacent STING dimers. We also demonstrate that using the full-length proteins is critical, since the MD simulations of the LBD portion alone cannot properly describe the global conformational properties of STING.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Structural and dynamic changes in P-Rex1 upon activation by PIP 3 and inhibition by IP 4

PIP 3 -dependent Rac exchanger 1 (P-Rex1) is abundantly expressed in neutrophils and plays central roles in chemotaxis and cancer metastasis by serving as a guanine-nucleotide exchange factor (GEF) for Rac. The enzyme is synergistically activated by PIP 3 and heterotrimeric Gβγ subunits, but mechanistic details remain poorly understood. While investigating the regulation of P-Rex1 by PIP 3 , we discovered that Ins(1,3,4,5)P 4 (IP 4 ) inhibits P-Rex1 activity and induces large decreases in backbone dynamics in diverse regions of the protein. Cryo-electron microscopy analysis of the P-Rex1·IP 4 complex revealed a conformation wherein the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain. This configuration is stabilized by interactions between the first DEP domain (DEP1) and the DH domain and between the PH domain and a 4-helix bundle (4HB) subdomain that extends from the C-terminal domain of P-Rex1. Disruption of the DH–DEP1 interface in a DH/PH-DEP1 fragment enhanced activity and led to a more extended conformation in solution, whereas mutations that constrain the occluded conformation led to decreased GEF activity. Variants of full-length P-Rex1 in which the DH–DEP1 and PH–4HB interfaces were disturbed exhibited enhanced activity during chemokineinduced cell migration, confirming that the observed structure represents the autoinhibited state in living cells. Interactions with PIP 3 -containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. Our results further suggest that inositol phosphates such as IP 4 help to inhibit basal P-Rex1 activity in neutrophils, similar to their inhibitory effects on phosphatidylinositol-3-kinase.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Reduced dynamic complexity allows structure elucidation of an excited state of KRAS G13D

Localized dynamics of RAS, including regions distal to the nucleotide-binding site, is of high interest for elucidating the mechanisms by which RAS proteins interact with effectors and regulators and for designing inhibitors. Among several oncogenic mutants, methyl relaxation dispersion experiments reveal highly synchronized conformational dynamics in the active (GMPPNP-bound) KRAS G13D , which suggests an exchange between two conformational states in solution. Methyl and 31 P NMR spectra of active KRAS G13D in solution confirm a two-state ensemble interconverting on the millisecond timescale, with a major Pγ atom peak corresponding to the dominant State 1 conformation and a secondary peak indicating an intermediate state different from the known State 2 conformation recognized by RAS effectors. High-resolution crystal structures of active KRAS G13D and KRAS G13D -RAF1 RBD complex provide snapshots of the State 1 and 2 conformations, respectively. We use residual dipolar couplings to solve and cross-validate the structure of the intermediate state of active KRAS G13D , showing a conformation distinct from those of States 1 and 2 outside the known flexible switch regions. The dynamic coupling between the conformational exchange in the effector lobe and the breathing motion in the allosteric lobe is further validated by a secondary mutation in the allosteric lobe, which affects the conformational population equilibrium.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Perturbative diffraction methods resolve a conformational switch that facilitates a two-step enzymatic mechanism

Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to poor experimental access. This shortcoming is evident with Escherichia coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we describe ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments to map the conformational dynamics of the Michaelis complex of DHFR. We resolve coupled global and local motions and find that these motions are engaged by the protonated substrate to promote efficient catalysis. This result suggests a fundamental design principle for multistep enzymes in which pre-existing dynamics enable intermediates to drive rapid electrostatic reorganization to facilitate subsequent chemical steps.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structural basis of RNA conformational switching in the transcriptional regulator 7SK RNP

7SK non-coding RNA (7SK) negatively regulates RNA polymerase II (RNA Pol II) elongation by inhibiting positive transcription elongation factor b (P-TEFb), and its ribonucleoprotein complex (RNP) is hijacked by HIV-1 for viral transcription and replication. Methylphosphate capping enzyme (MePCE) and La-related protein 7 (Larp7) constitutively associate with 7SK to form a core RNP, while P-TEFb and other proteins dynamically assemble to form different complexes. Here, we present the cryo-EM structures of 7SK core RNP formed with two 7SK conformations, circular and linear, and uncover a common RNA-dependent MePCE-Larp7 complex. Further, together with NMR, biochemical, and cellular data, these structures reveal the mechanism of MePCE catalytic inactivation in the core RNP, unexpected interactions between Larp7 and RNA that facilitate a role as an RNP chaperone, and that MePCE-7SK-Larp7 core RNP serves as a scaffold for switching between different 7SK conformations essential for RNP assembly and regulation of P-TEFb sequestration and release.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Self-assembly and regulation of protein cages from pre-organised coiled-coil modules

Coiled-coil protein origami (CCPO) is a modular strategy for the de novo design of polypeptide nanostructures. CCPO folds are defined by the sequential order of concatenated orthogonal coiled-coil (CC) dimer-forming peptides, where a single-chain protein is programmed to fold into a polyhedral cage. Self-assembly of CC-based nanostructures from several chains, similarly as in DNA nanotechnology, could facilitate the design of more complex assemblies and the introduction of functionalities. Here, we show the design of a de novo triangular bipyramid fold comprising 18 CC-forming segments and define the strategy for the two-chain self-assembly of the bipyramidal cage from asymmetric and pseudo-symmetric pre-organised structural modules. In addition, by introducing a protease cleavage site and masking the interfacial CC-forming segments in the two-chain bipyramidal cage, we devise a proteolysis-mediated conformational switch. This strategy could be extended to other modular protein folds, facilitating the construction of dynamic multi-chain CC-based complexes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Solvent organization in the ultrahigh-resolution crystal structure of crambin at room temperature

Ultrahigh-resolution structures provide unprecedented details about protein dynamics, hydrogen bonding and solvent networks. The reported 0.70 Å, room-temperature crystal structure of crambin is the highest-resolution ambient-temperature structure of a protein achieved to date. Sufficient data were collected to enable unrestrained refinement of the protein and associated solvent networks using SHELXL . Dynamic solvent networks resulting from alternative side-chain conformations and shifts in water positions are revealed, demonstrating that polypeptide flexibility and formation of clathrate-type structures at hydrophobic surfaces are the key features endowing crambin crystals with extraordinary diffraction power.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Data-guided Multi-Map variables for ensemble refinement of molecular movies

Driving molecular dynamics simulations with data-guided collective variables offer a promising strategy to recover thermodynamic information from structure-centric experiments. In this study, the three-dimensional electron density of a protein, as it would be determined by cryo-EM or x-ray crystallography, is used to achieve simultaneously free-energy costs of conformational transitions and refined atomic structures. Unlike previous density-driven molecular dynamics methodologies that determine only the best map-model fits, our work employs the recently developed Multi-Map methodology to monitor concerted movements within equilibrium, non-equilibrium, and enhanced sampling simulations. Construction of all-atom ensembles along the chosen values of the Multi-Map variable enables simultaneous estimation of average properties, as well as real-space refinement of the structures contributing to such averages. Using three proteins of increasing size, we demonstrate that biased simulation along the reaction coordinates derived from electron densities can capture conformational transitions between known intermediates. The simulated pathways appear reversible with minimal hysteresis and require only low-resolution density information to guide the transition. The induced transitions also produce estimates for free energy differences that can be directly compared to experimental observables and population distributions. The refined model quality is superior compared to those found in the Protein Data Bank. We find that the best quantitative agreement with experimental free-energy differences is obtained using medium resolution density information coupled to comparatively large structural transitions. Practical considerations for probing the transitions between multiple intermediate density states are also discussed.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Distinct binding conformations of epinephrine with α- and β-adrenergic receptors

Abstract Agonists targeting α 2 -adrenergic receptors (ARs) are used to treat diverse conditions, including hypertension, attention-deficit/hyperactivity disorder, pain, panic disorders, opioid and alcohol withdrawal symptoms, and cigarette cravings. These receptors transduce signals through heterotrimeric Gi proteins. Here, we elucidated cryo-EM structures that depict α 2A -AR in complex with Gi proteins, along with the endogenous agonist epinephrine or the synthetic agonist dexmedetomidine. Molecular dynamics simulations and functional studies reinforce the results of the structural revelations. Our investigation revealed that epinephrine exhibits different conformations when engaging with α-ARs and β-ARs. Furthermore, α 2A -AR and β 1 -AR (primarily coupled to Gs, with secondary associations to Gi) were compared and found to exhibit different interactions with Gi proteins. Notably, the stability of the epinephrine–α 2A -AR–Gi complex is greater than that of the dexmedetomidine–α 2A -AR–Gi complex. These findings substantiate and improve our knowledge on the intricate signaling mechanisms orchestrated by ARs and concurrently shed light on the regulation of α-ARs and β-ARs by epinephrine.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Design of intrinsically disordered protein variants with diverse structural properties

Intrinsically disordered proteins (IDPs) perform a broad range of functions in biology, suggesting that the ability to design IDPs could help expand the repertoire of proteins with novel functions. Computational design of IDPs with specific conformational properties has, however, been difficult because of their substantial dynamics and structural complexity. We describe a general algorithm for designing IDPs with specific structural properties. We demonstrate the power of the algorithm by generating variants of naturally occurring IDPs that differ in compaction, long-range contacts, and propensity to phase separate. We experimentally tested and validated our designs and analyzed the sequence features that determine conformations. We show how our results are captured by a machine learning model, enabling us to speed up the algorithm. Our work expands the toolbox for computational protein design and will facilitate the design of proteins whose functions exploit the many properties afforded by protein disorder.

Science & Technology - Other Topics↗
DOE OSTI2022

Exact reaction coordinates for flap opening in HIV-1 protease

The primary goal of protein science is to understand how proteins function, which requires understanding the functional dynamics responsible for transitions between different functional structures of a protein. A central concept is the exact reaction coordinates that can determine the value of committor for any protein configuration, which provide the optimal description of functional dynamics. Despite intensive efforts, identifying the exact reaction coordinates (RCs) in complex molecules remains a formidable challenge. Using the recently developed generalized work functional, we report the discovery of the exact RCs for an important functional process—the flap opening of HIV-1 protease. Our results show that this process has six RCs, each one is a linear combination of ~240 backbone dihedrals, providing the precise definition of collectivity and cooperativity in the functional dynamics of a protein. Applying bias potentials along each RC can accelerate flap opening by 10 3 to 10 4 folds. The success in identifying the RCs of a protein with 198 residues represents a significant progress beyond that of the alanine dipeptide, currently the only other complex molecule for which the exact RCs for its conformational changes are known. Our results suggest that the generalized work functional (GWF) might be the fundamental operator of mechanics that controls protein dynamics.

Science & Technology - Other Topics↗
DOE OSTI2024

Dynamic Implications of Noncovalent Interactions in Amphiphilic Single-Chain Polymer Nanoparticles

Single-chain polymer nanoparticles (SCNPs) combine the chemical diversity of synthetic polymers with the intricate structure of biopolymers, generating versatile biomimetic materials. The mobility of polymer chain segments at length scales similar to secondary structural elements in proteins is critical to SCNP structure and thus function. However, the influence of noncovalent interactions used to form SCNPs (e.g., hydrogen-bonding and biomimetic secondary-like structure) on these conformational dynamics is challenging to quantitatively assess. To isolate the effects of noncovalent interactions on SCNP structure and conformational dynamics, we synthesized a series of amphiphilic copolymers containing dimethylacrylamide and monomers capable of forming these different interactions: (1) di(phenylalanine) acrylamide that forms intramolecular β-sheet-like cross-links, (2) phenylalanine acrylamide that forms hydrogen-bonds but lacks a defined local structure, and (3) benzyl acrylamide that has the lowest propensity for hydrogen-bonding. Each SCNP formed folded structures comparable to those of intrinsically disordered proteins, as observed by size exclusion chromatography and small angle neutron scattering. The dynamics of these polymers, as characterized by a combination of dynamic light scattering and neutron spin echo spectroscopy, was well described using the Zimm with internal friction (ZIF) model, highlighting the role of each noncovalent interaction to additively restrict the internal relaxations of SCNPs. These results demonstrate the utility of local scale interactions to control SCNP polymer dynamics, guiding the design of functional biomimetic materials with refined binding sites and tunable kinetics.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Differences in the dynamics of the tandem-SH2 modules of the Syk and ZAP-70 tyrosine kinases

The catalytic activity of Syk-family tyrosine kinases is regulated by a tandem Src homology 2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation that stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to phosphorylated immunoreceptor tyrosine-based activation motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP-70, that its conformation more closely resembles that of the peptide-bound state, rather than the autoinhibited state. Hydrogen–deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP-70 differ, with most of these differences occurring in the C-terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP-70 have been tuned differently, such that the autoinhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP-70, consistent with tighter control of downstream signaling pathways in T cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The Role of Ligand–Ligand Interactions in Multimodal Ligand Conformational Equilibria and Surface Pattern Formation

Multimodal chromatography uses multiple modes of interaction such as charge, hydrophobic, or hydrogen bonding to separate proteins. Recently, we used molecular dynamics (MD) simulations to show that ligands immobilized on surfaces can interact and associate with neighboring ligands to form hydrophobic and charge patches, which may have important implications for the nature of protein–surface interactions. Here, we study interfacial systems of increasing complexity—from a single immobilized multimodal ligand to high density surfaces—to better understand how ligand behavior is affected by the presence of a surface and the presence of other ligands in the vicinity, and how this behavior scales to larger systems. Furthermore, we find that tethering a ligand to a surface restricts its conformations to a subset of those observed in free solution, yet the ligand maintains flexibility in the plane of the surface and can form contacts with neighboring ligands. We find that although the formation of a contact between two neighboring ligands is slightly unfavorable, three neighboring ligands exhibit a preference for the formation of a fully connected cluster. To explore how these trends in ligand association extend to a larger surface with high density of ligands, we performed coarse-grained Monte Carlo (MC) simulations of a 132-ligand surface using ligand interactions parametrized based on free energies obtained from the three-ligand MD simulations. Despite their simplicity, the coarse-grained simulations qualitatively capture the cluster size distribution of ligands observed in detailed MD simulations. Quantitative differences between the two suggest opportunities for improvements in the coarse-grained energy function for efficient predictions of cluster and pattern formations. Our approach presents a promising route to the engineering of multimodal patterns for future chromatographic resin design.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Activation dynamics of a water-soluble human mu-opioid receptor

The mu-opioid receptor (MOR), a class A G protein-coupled receptor mediates opioid analgesia and remains a central target for pain therapeutics. While crystal structures of MOR exist, they provide limited insight into the receptor’s dynamic conformational landscape underlying function. Here, we engineered a thermostable water-soluble MOR variant (wsMOR) that retains native-like ligand-binding and activation dynamics. This variant enables high-yield production and detailed solution-phase structural studies that are challenging with membrane-embedded MOR, providing a valuable tool for studying receptor activation and aqueous-phase drug screening. Using a combined computational and experimental approach, we performed long-timescale all-atom molecular dynamics simulations together with neutron scattering and single-molecule FRET, revealing a structurally stable receptor with a diverse ensemble of conformations at different temporal resolutions. In the ligand-free state, wsMOR displayed high conformational flexibility, which decreased upon agonist binding, particularly in transmembrane helix 6, a hallmark of G protein-coupled receptor activation. Positive allosteric modulation and G protein binding further stabilized active-like states. These findings highlight wsMOR’s conformational plasticity across picosecond to millisecond timescales and provide a foundation for structure-guided development of next-generation opioid ligands with improved efficacy and safety.

E, Agyemang [University of Tennessee Knoxville]↗
DOE OSTI2021

Molecular Dynamics Free Energy Simulations Reveal the Mechanism for the Antiviral Resistance of the M66I HIV-1 Capsid Mutation

While drug resistance mutations can often be attributed to the loss of direct or solvent-mediated protein−ligand interactions in the drug-mutant complex, in this study we show that a resistance mutation for the picomolar HIV-1 capsid (CA)-targeting antiviral (GS-6207) is mainly due to the free energy cost of the drug-induced protein side chain reorganization in the mutant protein. Among several mutations, M66I causes the most suppression of the GS-6207 antiviral activity (up to ~84,000-fold), and only 83- and 68-fold reductions for PF74 and ZW-1261, respectively. To understand the molecular basis of this drug resistance, we conducted molecular dynamics free energy simulations to study the structures, energetics, and conformational free energy landscapes involved in the inhibitors binding at the interface of two CA monomers. To minimize the protein−ligand steric clash, the I66 side chain in the M66I−GS-6207 complex switches to a higher free energy conformation from the one adopted in the apo M66I. In contrast, the binding of GS-6207 to the wild-type CA does not lead to any significant M66 conformational change. Based on an analysis that decomposes the absolute binding free energy into contributions from two receptor conformational states, it appears that it is the free energy cost of side chain reorganization rather than the reduced protein−ligand interaction that is largely responsible for the drug resistance against GS-6207.

Sun, Qinfang (ORCID:0000000173621543)↗
DOE OSTI2020

Transient Unfolding and Long-Range Interactions in Viral BCL2 M11 Enable Binding to the BECN1 BH3 Domain

Viral BCL2 proteins (vBCL2s) help to sustain chronic infection of host proteins to inhibit apoptosis and autophagy. However, details of conformational changes in vBCL2s that enable binding to BH3Ds remain unknown. Using all-atom, multiple microsecond-long molecular dynamic simulations (totaling 17 μs) of the murine γ-herpesvirus 68 vBCL2 (M11), and statistical inference techniques, we show that regions of M11 transiently unfold and refold upon binding of the BH3D. Further, we show that this partial unfolding/refolding within M11 is mediated by a network of hydrophobic interactions, which includes residues that are 10 Å away from the BH3D binding cleft. We experimentally validate the role of these hydrophobic interactions by quantifying the impact of mutating these residues on binding to the Beclin1/BECN1 BH3D, demonstrating that these mutations adversely affect both protein stability and binding. To our knowledge, this is the first study detailing the binding-associated conformational changes and presence of long-range interactions within vBCL2s.

59 BASIC BIOLOGICAL SCIENCES↗