Parameterization of the proline analogue Aze (azetidine-2-carboxylic acid) for molecular dynamics simulations and evaluation of its effect on homo-pentapeptide conformations.

peer reviewedWe have parameterized and evaluated the proline homologue Aze (azetidine-2-carboxylic acid) for the gromos56a3 force-field for use in molecular dynamics simulations using GROMACS. Using bi-phasic cyclohexane/water simulation systems and homo-pentapeptides, we measured the Aze solute interaction potential energies, ability to hydrogen bond with water, and overall compaction, for comparison to Pro, Gly, and Lys. Compared to Pro, Aze has a slightly higher H-bonding potential, and stronger electrostatic but weaker non-electrostatic interactions with water. The 20-ns simulations revealed the preferential positioning of Aze and Pro at the interface of the water and cyclohexane layers, with Aze spending more time in the aqueous layer. We also demonstrated through simulations of the homo-pentapeptides that Aze has a greater propensity than Pro to undergo trans-->cis peptide bond isomerization, which results in a severe 180 degrees bend in the polypeptide chain. The results provide evidence for the hypothesis that the misincorporation of Aze within proline-rich regions of proteins could disrupt the formation of poly-proline type II structures and compromise events such as recognition and binding by SH3-domains

Biophysical characterization of 21.5-kDa myelin basic protein (MBP) and the effects of zinc on its structure

Myelin Basic Protein (MBP) is a highly abundant protein in central nervous system (CNS) myelin that has a critical role in its proper formation and functioning. The 21.5-kDa isoform of MBP has been shown to be selectively imported into the nucleus of myelin-producing cells, oligodendrocytes, and may be involved in signaling pathways that affect the formation and recovery of CNS myelin. The first step in understanding potential nuclear binding partners of 21.5-kDa MBP is to characterize the structure of the protein. In this study, circular dichroism and fluorescence spectroscopy were used to analyze the structure of 21.5-kDa rmMBP (recombinant murine MBP) in vitro in the presence and absence of Zn2+, an abundant trace metal in CNS myelin that has been suggested to affect MBP structure. Fluorescence spectroscopy with a probe for hydrophobic protein regions showed that Zn2+ may affect the conformation of 21.5-kDa MBP in aqueous solution. Keywords: myelin basic protein (MBP); intrinsically-disordered protein; circular dichroism; fluoresence spectroscop

The proline-rich region of 18.5 kDa myelin basic protein binds to the SH3-domain of Fyn tyrosine kinase with the aid of an upstream segment to form a dynamic complex in vitro

The intrinsically disordered 18.5 kDa classic isoform of MBP (myelin basic protein) interacts with Fyn kinase during oligodendrocyte development and myelination. It does so primarily via a central proline-rich SH3 (Src homology 3) ligand (T92–R104, murine 18.5 kDa MBP sequence numbering) that is part of a molecular switch due to its high degree of conservation and modification by MAP (mitogen-activated protein) and other kinases, especially at residues T92 and T95. Here, we show using co-transfection experiments of an early developmental oligodendroglial cell line (N19) that an MBP segment upstream of the primary ligand is involved in MBP–Fyn–SH3 association in cellula. Using solution NMR spectroscopy in vitro, we define this segment to comprise MBP residues (T62–L68), and demonstrate further that residues (V83–P93) are the predominant SH3-target, assessed by the degree of chemical shift change upon titration. We show by chemical shift index analysis that there is no formation of local poly-proline type II structure in the proline-rich segment upon binding, and by NOE (nuclear Overhauser effect) and relaxation measurements that MBP remains dynamic even while complexed with Fyn–SH3. The association is a new example first of a non-canonical SH3-domain interaction and second of a fuzzy MBP complex

The Effects of Threonine Phosphorylation on the Stability and Dynamics of the Central Molecular Switch Region of 18.5-kDa Myelin Basic Protein

<div><p>The classic isoforms of myelin basic protein (MBP) are essential for the formation and maintenance of myelin in the central nervous system of higher vertebrates. The protein is involved in all facets of the development, compaction, and stabilization of the multilamellar myelin sheath, and also interacts with cytoskeletal and signaling proteins. The predominant 18.5-kDa isoform of MBP is an intrinsically-disordered protein that is a candidate auto-antigen in the human demyelinating disease multiple sclerosis. A highly-conserved central segment within classic MBP consists of a proline-rich region (murine 18.5-kDa sequence –T92-P93-R94-T95-P96-P97-P98-S99–) containing a putative SH3-ligand, adjacent to a region that forms an amphipathic α-helix (P82-I90) upon interaction with membranes, or under membrane-mimetic conditions. The T92 and T95 residues within the proline-rich region can be post-translationally modified through phosphorylation by mitogen-activated protein (MAP) kinases. Here, we have investigated the structure of the α-helical and proline-rich regions in dilute aqueous buffer, and have evaluated the effects of phosphorylation at T92 and T95 on the stability and dynamics of the α-helical region, by utilizing four 36-residue peptides (S72–S107) with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the α-helical as well as the proline-rich regions are disordered in aqueous buffer, whereas they are both structured in a lipid environment (<i>cf</i>., Ahmed <i>et al</i>., Biochemistry 51, 7475-9487, 2012). Thermodynamic analysis of trifluoroethanol-titration curves monitored by circular dichroism spectroscopy reveals that phosphorylation, especially at residue T92, impedes formation of the amphipathic α-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the α-helix upon temperature perturbation and affect the global structure of the peptides through altered electrostatic interactions. The results support the hypothesis that the central conserved segment of MBP constitutes a molecular switch in which the conformation and/or intermolecular interactions are mediated by phosphorylation/dephosphorylation at T92 and T95.</p></div

Structure of phosphorylated α₂-peptides at the end of MD experiments in water and in DMPC.

<p>The dimyristoylphosphatidylcholine (DMPC) membrane bilayer and water molecules are not shown in order to improve clarity. The images are a representative snapshot of singly-phosphorylated (PhT92, PhT95), and doubly-phosphorylated (PhT92–PhT95) α₂-peptides (S72–S107) of myelin basic protein (MBP) captured during the last 10 ns of the 160 ns molecular dynamics simulation experiments. Shown are examples of the type of electrostatic interactions between phosphorylated Thr residues and different basic residues within the α₂-peptide variants.</p

Amino acid sequence and secondary structural map of 18.5-kDa murine myelin basic protein.

<p>The protein is intrinsically-disordered in aqueous solution but has three distinct segments which have α-helical propensity as shown (α₁, α₂, α₃) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068175#pone.0068175-Libich1" target="_blank">[27]</a>. The sequence enclosed by the red rectangle represents the reference α₂-peptide investigated in this study. This region consist of residues which can form an amphipathic α-helix when associated with lipids (α₂), followed by a proline-rich region of the protein which adopts a left-handed poly-proline II structure (PPII, shown in purple) that is the probable SH3-ligand. Within this poly-proline structure are two mitogen-activated protein kinase sites at T92 and T95 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068175#pone.0068175-Harauz4" target="_blank">[22]</a>. The figure is modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068175#pone.0068175-Ahmed1" target="_blank">[49]</a>.</p

Change in tilt angle and membrane penetration depth of the α-helical segment in DMPC.

<p>Data were obtained from analysis of molecular dynamics experiments conducted in the presence of a dimyristoylphosphatidylcholine (DMPC) membrane bilayer for unmodified as well as singly- (PhT92, PhT95) and doubly- (PhT92–PhT95) phosphorylated α₂-peptides of myelin basic protein (MBP). Panel A1 illustrates how the tilt angle Θ of the α-helix (residues P82-I90, represented as a cylinder) within the α₂-peptide (S72–S107) was measured. Θ was determined as the angle between the axes passing through the geometric center of the helix and the axis that is parallel to the surface of the membrane. When Θ<0, the N-terminal portion of the helix is tilted away from the membrane surface (pointing upwards) whereas the C-terminal region is embedded (pointing downwards), and when Θ>0, the opposite is true. The evolution of the tilt angle was plotted as a function of simulation time over the entire experiment (A2). The change in penetration depth of the central α-helix into the DMPC bilayer for the unmodified α₂-peptide, as well as the phosphorylated variants was also plotted (B). The penetration depth represents the distance between the center of mass of the α-helix and the surface of the membrane.</p

Secondary structure evolution of the MBP α₂-peptides in water, and in a DMPC membrane environment.

<p>The α₂-peptides (S72–S107) of myelin basic protein (MBP) were simulated for 160 ns using GROMACS 4.5.5 and the Gromos96 ffG53a6 force-field. The evolution in secondary structure at each frame was monitored using the dictionary of protein secondary structure (DSSP) algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068175#pone.0068175-Kabsch1" target="_blank">[78]</a>. Results for the unmodified peptide, the singly-phosphorylated α₂-peptides (PhT92 and PhT95) and the doubly-phosphorylated α₂-peptide (PhT92–PhT95), both in water and in a dimyristoylphosphatidylcholine (DMPC) membrane environment are shown as indicated.</p