bims-lances 2024-02-18 papers

bims-lances

Biomed News

on Landscapes from Cryo-EM and Simulations

Issue of 2024–02–18
seven papers selected by
James M. Krieger, National Centre for Biotechnology

Regulatory sites of CaM-sensitive adenylyl cyclase AC8 revealed by cryo-EM and structural proteomics.
Probing the dynamic landscape of peptides in molecular assemblies by synergized NMR experiments and MD simulations.
Dynamics of activation in the voltage-sensing domain of Ciona intestinalis phosphatase Ci-VSP.
Log-periodic oscillations as real-time signatures of hierarchical dynamics in proteins.
Allosteric mechanism of MntR transcription factor from alkalophilic bacterium Halalkalibacterium halodurans.
Lipid bilayer membrane permeability mechanism of the K-Ras(G12D)-inhibitory bicyclic peptide KS-58 elucidated by molecular dynamics simulations.
Protracted molecular dynamics and secondary structure introspection to identify dual-target inhibitors of Nipah virus exerting approved small molecules repurposing.

EMBO Rep. 2024 Feb 13.

Regulatory sites of CaM-sensitive adenylyl cyclase AC8 revealed by cryo-EM and structural proteomics.

Basavraj Khanppnavar, Dina Schuster, Pia Lavriha, Federico Uliana, Merve Özel, Ved Mehta, Alexander Leitner, Paola Picotti, Volodymyr M Korkhov.

  Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca2+ signalling. Despite the importance of AC8 in physiology, the structural basis of its regulation by G proteins and CaM is not well defined. Here, we report the 3.5 Å resolution cryo-EM structure of the bovine AC8 bound to the stimulatory Gαs protein in the presence of Ca2+/CaM. The structure reveals the architecture of the ordered AC8 domains bound to Gαs and the small molecule activator forskolin. The extracellular surface of AC8 features a negatively charged pocket, a potential site for unknown interactors. Despite the well-resolved forskolin density, the captured state of AC8 does not favour tight nucleotide binding. The structural proteomics approaches, limited proteolysis and crosslinking mass spectrometry (LiP-MS and XL-MS), allowed us to identify the contact sites between AC8 and its regulators, CaM, Gαs, and Gβγ, as well as to infer the conformational changes induced by these interactions. Our results provide a framework for understanding the role of flexible regions in the mechanism of AC regulation.

Keywords:  Adenylyl cyclase; Calmodulin; Heterotrimeric G protein; Structural Proteomics; cryo-Electron Microscopy (cryo-EM)

DOI:  https://doi.org/10.1038/s44319-024-00076-y
Commun Chem. 2024 Feb 13. 7(1): 28

Probing the dynamic landscape of peptides in molecular assemblies by synergized NMR experiments and MD simulations.

Ricky Nencini, Morgan L G Regnier, Sofia M Backlund, Efstathia Mantzari, Cory D Dunn, O H Samuli Ollila.

Peptides or proteins containing small biomolecular aggregates, such as micelles, bicelles, droplets and nanodiscs, are pivotal in many fields ranging from structural biology to pharmaceutics. Monitoring dynamics of such systems has been limited by the lack of experimental methods that could directly detect their fast (picosecond to nanosecond) timescale dynamics. Spin relaxation times from NMR experiments are sensitive to such motions, but their interpretation for biomolecular aggregates is not straightforward. Here we show that the dynamic landscape of peptide-containing molecular assemblies can be determined by a synergistic combination of solution state NMR experiments and molecular dynamics (MD) simulations. Solution state NMR experiments are straightforward to implement without an excessive amount of sample, while direct combination of spin relaxation data to MD simulations enables interpretation of dynamic landscapes of peptides and other aggregated molecules. To demonstrate this, we interpret NMR data from transmembrane, peripheral, and tail anchored peptides embedded in micelles. Our results indicate that peptides and detergent molecules do not rotate together as a rigid body, but peptides rotate in a viscous medium composed of detergent micelle. Spin relaxation times also provide indirect information on peptide conformational ensembles. This work gives new perspectives on peptide dynamics in complex biomolecular assemblies.

DOI: https://doi.org/10.1038/s42004-024-01115-4
Nat Commun. 2024 Feb 15. 15(1): 1408

Dynamics of activation in the voltage-sensing domain of Ciona intestinalis phosphatase Ci-VSP.

Spencer C Guo, Rong Shen, Benoît Roux, Aaron R Dinner.

The Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) is a membrane protein containing a voltage-sensing domain (VSD) that is homologous to VSDs from voltage-gated ion channels responsible for cellular excitability. Previously published crystal structures of Ci-VSD in putative resting and active conformations suggested a helical-screw voltage sensing mechanism in which the S4 helix translocates and rotates to enable exchange of salt-bridge partners, but the microscopic details of the transition between the resting and active conformations remained unknown. Here, by combining extensive molecular dynamics simulations with a recently developed computational framework based on dynamical operators, we elucidate the microscopic mechanism of the resting-active transition at physiological membrane potential. Sparse regression reveals a small set of coordinates that distinguish intermediates that are hidden from electrophysiological measurements. The intermediates arise from a noncanonical helical-screw mechanism in which translocation, rotation, and side-chain movement of the S4 helix are only loosely coupled. These results provide insights into existing experimental and computational findings on voltage sensing and suggest ways of further probing its mechanism.

DOI: https://doi.org/10.1038/s41467-024-45514-6
J Chem Phys. 2024 Feb 21. pii: 074103. [Epub ahead of print]160(7):

Log-periodic oscillations as real-time signatures of hierarchical dynamics in proteins.

Emanuel Dorbath, Adnan Gulzar, Gerhard Stock.

The time-dependent relaxation of a dynamical system may exhibit a power-law behavior that is superimposed by log-periodic oscillations. D. Sornette [Phys. Rep. 297, 239 (1998)] showed that this behavior can be explained by a discrete scale invariance of the system, which is associated with discrete and equidistant timescales on a logarithmic scale. Examples include such diverse fields as financial crashes, random diffusion, and quantum topological materials. Recent time-resolved experiments and molecular dynamics simulations suggest that discrete scale invariance may also apply to hierarchical dynamics in proteins, where several fast local conformational changes are a prerequisite for a slow global transition to occur. Employing entropy-based timescale analysis and Markov state modeling to a simple one-dimensional hierarchical model and biomolecular simulation data, it is found that hierarchical systems quite generally give rise to logarithmically spaced discrete timescales. By introducing a one-dimensional reaction coordinate that collectively accounts for the hierarchically coupled degrees of freedom, the free energy landscape exhibits a characteristic staircase shape with two metastable end states, which causes the log-periodic time evolution of the system. The period of the log-oscillations reflects the effective roughness of the energy landscape and can, in simple cases, be interpreted in terms of the barriers of the staircase landscape.

DOI: https://doi.org/10.1063/5.0188220
J Biomol Struct Dyn. 2024 Feb 12. 1-15

Allosteric mechanism of MntR transcription factor from alkalophilic bacterium Halalkalibacterium halodurans.

Ana Marija Knez, Martina Manenica, Zoe Jelić Matošević, Branimir Bertoša.

  Bacterium Halalkalibacterium halodurans is an industrially important alkalophilic bacteria. It is recognized as a producer of enzymes such as β-galactosidase, xylanase, amylase and protease which are able to function at higher pH values and thus can be used in textile, food, paper industry and more. This bacterium, as any other bacterium, requires a sensitive mechanism for regulation of homeostasis of manganese ions (Mn2+) in order to survive. The key protein regulating this mechanism in H. halodurans is MntR - a transcriptional factor that binds to DNA and regulates the transcription of genes for proteins involved in manganese homeostasis. Long range all-atom molecular dynamics (MD) simulations, from 500 ns up to 1.25 µs, were used to study different forms of H. halodurans MntR in order to investigate the differences in the protein's structural and dynamical properties upon Mn2+ binding. Simulations revealed an allosteric mechanism which is activated by Mn2+ binding. The results of simulations show that Mn2+ binding alters the non-covalent interaction network of the protein structure which leads to a conformational change that primarily affects the positions of the DNA binding domains and, consequently, the DNA binding affinity of H. halodurans MntR. The key amino acid residues of the proposed mechanism were identified and their role in the proposed mechanism was computationally confirmed by MD simulations of in silico mutants.Communicated by Ramaswamy H. Sarma.

Keywords:  Halalkalibacterium halodurans; Molecular dynamics; manganese homeostasis; manganese transport regulator (MntR); non-covalent interaction network

DOI:  https://doi.org/10.1080/07391102.2024.2314265
Bioorg Med Chem Lett. 2024 Feb 09. pii: S0960-894X(24)00051-9. [Epub ahead of print]100 129649

Lipid bilayer membrane permeability mechanism of the K-Ras(G12D)-inhibitory bicyclic peptide KS-58 elucidated by molecular dynamics simulations.

Kotaro Sakamoto, Takatsugu Hirokawa.

  Peptides are mid-size molecules (700-2000 g/mol) and have attracted particular interest as therapeutic modalities as they are superior in controlling protein-protein interactions, a process that is a typical drug target category, compared with small molecules (<500 g/mol). In 2020, we identified KS-58 (1333 g/mol) as a K-Ras(G12D)-inhibitory bicyclic peptide and suggested its cell membrane permeability. However, the membrane permeability mechanism had not been elucidated. In this study, we aim to clarify the mechanism by molecular dynamics (MD) simulations. Initially, we simulated the molecular conformations of KS-58 in water (a polar solvent) and in chloroform (a non-polar solvent). The identified stable conformations were significantly different in each solvent. KS-58 behaves as a chameleon-like molecule as it alters its polar surface area (PSA) depending on the solvent environment. It was also discovered that orientation of Asp's side chain is a critical energy barrier for KS-58 altering its conformation from hydrophilic to lipophilic. Taking these properties into consideration, we simulated its lipid bilayer membrane permeability. KS-58 shifted toward the inside of the lipid bilayer membrane with altering its conformations to lipophilic. When the simulation condition was set in deionized form of that carboxy group of Asp, KS-58 traveled deeper inside the cell membrane. PSA and the depth of the membrane penetration correlated. In vitro data suggested that cell membrane permeability of KS-58 is improved in weakly acidic conditions leading to partial deionization of the carboxy group. Our data provide an example of the molecular properties of mid-size peptides with membrane accessibility and propose an effective metadynamics approach to elucidate such molecular mechanisms by MD simulations.

Keywords:  Chameleon-like molecule; Conformational changes; Cyclic peptide; KS-58; Membrane permeability; Mid-size peptide

DOI:  https://doi.org/10.1016/j.bmcl.2024.129649
Sci Rep. 2024 02 14. 14(1): 3696

Protracted molecular dynamics and secondary structure introspection to identify dual-target inhibitors of Nipah virus exerting approved small molecules repurposing.

Siyun Yang, Supratik Kar.

Nipah virus (NiV), with its significantly higher mortality rate compared to COVID-19, presents a looming threat as a potential next pandemic, particularly if constant mutations of NiV increase its transmissibility and transmission. Considering the importance of preventing the facilitation of the virus entry into host cells averting the process of assembly forming the viral envelope, and encapsulating the nucleocapsid, it is crucial to take the Nipah attachment glycoprotein-human ephrin-B2 and matrix protein as dual targets. Repurposing approved small molecules in drug development is a strategic choice, as it leverages molecules with known safety profiles, accelerating the path to finding effective treatments against NiV. The approved small molecules from DrugBank were used for repurposing and were subjected to extra precision docking followed by absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling. The 4 best molecules were selected for 500 ns molecular dynamics (MD) simulation followed by Molecular mechanics with generalized Born and surface area solvation (MM-GBSA). Further, the free energy landscape, the principal component analysis followed by the defined secondary structure of proteins analysis were introspected. The inclusive analysis proposed that Iotrolan (DB09487) and Iodixanol (DB01249) are effective dual inhibitors, while Rutin (DB01698) and Lactitol (DB12942) were found to actively target the matrix protein only.

DOI: https://doi.org/10.1038/s41598-024-54281-9