Structure and dynamics of Penetratin’s association and translocation to a lipid bilayer

Penetratin belongs to the important class of small and positively charged peptides, capable of entering cells. The determination of the optimal peptidic structure for translocation is challenging; results obtained so far are varied and dependent on several factors. In this work, we review the dynamics of association of Penetratin with a modeled dioleoyl-phosphatidylcholine (DOPC) lipid membrane using molecular dynamics simulations with last generation force fields. Penetratin’s structural preferences are determined using a Markov state model. It is observed that the peptide retains a helical form in the membrane associated state, just as in water, with the exception of both termini which lose helicity, facilitating the interaction of terminal residues with the phosphate groups on the membrane’s outer layer. The optimal orientation for insertion is found to be with the peptide’s axis forming a small angle with the interface, and with R1 stretching toward the bilayer. The interaction between arginine side-chains and phosphate groups is found to be greater than the corresponding to lysine, mainly due to a higher number of hydrogen bonds between them. The free energy profile of translocation is qualitatively studied using Umbrella Sampling. It is found that there are different paths of penetration, that greatly differ in size of free energy barrier. The lowest path is compatible with residues R10 to K13 leading the way through the membrane and pulling the rest of the peptide. When the other side is reached, the C-terminus overtakes those residues, and finally breaks out of the membrane. The peptide’s secondary structure during this traversal suffers some changes with respect to the association structure but, overall, conserves its helicity, with both termini in a more disordered state

Structural and Dynamic Implications of an Effector-induced Backbone Amide cis-trans Isomerization in Cytochrome P450cam

Experimental evidence has been provided for a functionally relevant cis-trans isomerization of the Ile88-Pro89 peptide bond in cytochrome P450cam (CYP101). The isomerization is proposed to be a key element of the structural reorganization leading to the catalytically competent form of CYP101 upon binding of the effector protein putidaredoxin (Pdx). A detailed comparison of the results of molecular dynamics simulations on the cis and trans conformations of substrate- and carbonmonoxy-bound ferrous CYP101 with sequence-specific Pdx-induced structural perturbations identified by nuclear magnetic resonance is presented, providing insight into the structural and dynamic consequences of the isomerization. The mechanical coupling between the Pdx binding site on the proximal face of CYP101 and the site of isomerization is described. © 2009 Elsevier Ltd. All rights reserved

Structure and Dynamics Study of LeuT Using the Markov State Model and Perturbation Response Scanning Reveals Distinct Ion Induced Conformational States

The bacterial leucine transporter (LeuT), a close homologue of the eukaryote monoamine transporters (MATs), currently serves as a powerful template for computer simulations of MATs. Transport of the amino acid leucine through the membrane is made possible by the sodium electrochemical potential. Recent reports indicate that the substrate transport mechanism is based on structural changes such as hinge movements of key transmembrane domains. In order to further investigate the role of sodium ions in the uptake of leucine, here we present a Markov state model analysis of atomistic simulations of lipid embedded LeuT in different environments, generated by varying the presence of binding pocket sodium ions and substrate. Six metastable conformations are found, and structural differences between them along with transition probabilities are determined. We complete the analysis with the implementation of perturbation response scanning on our system, determining the most sensitive and influential regions of LeuT, in each environment. Our results show that the occupation of sites Na1 and Na2, along with the presence of the substrate, selectively influences the geometry of LeuT. In particular, the occupation of each site Na1/Na2 has strong effects (in terms of changes in influence and/or sensitivity, as compared to the case without ions) in specific regions of LeuT, and the effects are different for simultaneous occupation. Our results strengthen the rationale and provide a conformational mechanism for a putative transport mechanism in which Na2 is necessary, but may not be sufficient, to initiate and stabilize extracellular substrate access to the binding pocket

Mechanisms of Activation and Subunit Release in Ca 2+ /Calmodulin-Dependent Protein Kinase II

Calcium/calmodulin-dependent protein kinase II is an enzyme involved in many different functions, including the so-called long-term potentiation, a mechanism that strengthens synapses in a persistent mode and is believed to be a basic cellular mechanism for memory formation. Here we study the conformational changes of the enzyme due to phosphorylation of some key residues that are believed to drive the transition from an inhibited to an active state; it is this active state the one associated with long-term potentiation. We found that the conformational changes could be explained in terms of three charged regions in the three main subdomains of the enzyme: the hub, linker, and kinase. The role of phosphorylation is to change the charge relation between them, turning on and off their interactions and switching between an attractive state (nonphosphorylated or inhibited) and a not attractive one (phosphorylated or active). We also show that phosphorylated subunits become less stable, and this could favor their release from the multimer, as has been already observed experimentally.Fil: Pullara, Filippo. University of Pittsburgh; Estados UnidosFil: Asciutto, Eliana Karina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: General, Ignacio. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Phage-display-derived peptide binds to human CD206 and modeling reveals a new binding site on the receptor

We recently identified a tumor-homing peptide (mUNO, sequence: “CSPGAK”) that specifically interacts with mouse CD206 to target CD206/MRC1-expressing tumor-associated macrophages in mice. Here, we report studies on the binding of mUNO to human recombinant CD206 (hCD206) and on modeling the mUNO/hCD206 interaction by computational analysis. Fluorescence anisotropy analysis demonstrated that fluorophore-labeled mUNO interacts with hCD206. Microsecond time-scale molecular dynamics simulations and docking predictions showed that mUNO binds to a newly identified epitope between C-type lectin domains 1 and 2. The physical mechanisms that contribute to the docking interactions of mUNO include electrostatic interactions, aromatic interactions, and hydrogen bonds. We also demonstrate the selectivity of FAM-mUNO for CD206+-cultured human macrophages. The peptide mUNO appears to be the first ligand capable of interacting with this epitope of hCD206, for which no ligands have been reported. Our study has implications for targeting human M2-like tumor-associated macrophages, a subpopulation of immune cells with a major protumoral role