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Molecular Basis for Neurofilament Heavy Chain Side Arm Structure Modulation by Phosphorylation† Shashishekar P. Adiga*,‡ and Donald W. Brenner* Department of Materials Science and Engineering, North Carolina State UniVersity, Raleigh, North Carolina 26095 ReceiVed: June 16, 2009; ReVised Manuscript ReceiVed: October 28, 2009
The role of phosphorylation of neurofilament side arms in neurofilament transport and in several neuromuscular diseases is a topic of active research. However, owing to the lack of a secondary structure of the side arms, little is known about the precise nature of the structural modifications caused by this important post-translational modification. Here, we probe the effect of phosphorylation on the structure of the C-terminal domain of the human neurofilament heavy chain NFH using molecular dynamics simulations. Our study indicates that the unphosphorylated NFH side arm is unstructured and characterized by several flexible loops stabilized by salt bridges. Phosphorylation of multiple SER residues is shown to destabilize these bridges due to electrostatic repulsion and thereby increase side arm size. We demonstrate that phosphorylation acts locally by modulating intramolecular electrostatic interactions to cause global changes in the otherwise disordered side arm. Our findings have implications for understanding the functioning of neurofilaments in health and disease. Introduction Neurofilaments, a type of intermediate filament, are vital structural constituents of the axonal cytoplasm. They are assembled from three subunit proteins, NFL, NFM, and NFH, with different molecular masses (L, M, and H specify light, medium, and heavy relative molecular masses, respectively). The subunit polypeptides share a homology over a 300 amino acid sequence that forms the neurofilament core. The remaining C-terminal domains in NFM and NFH extend beyond the core region to form side arms.1 The side arms extend 40-50 nm from 10 nm diameter cores2 to give the neurofilaments a cylindrical brush structure similar to polymer bottle brush molecules.3 Along with microtubules, these filaments are aligned parallel to each other along the axon length with nonrandom interfilament distances.4 Subsequent to their synthesis in the cell body (perikaryon), neurofilaments move down the axon by axonal transport, at an average rate of about 1 mm per day in a series of jumps.1,5,6 It is well established that regional accumulation of neurofilaments, a process that causes radial growth of axons, is brought about through a slowing down of axonal transport. Evidence has been provided that the slowing down of neurofilament transport down the axon is regulated by phosphorylation of site-specific LYS-SER-PRO (KSP) motifs that are present in the C-terminal domains of NFH and NFM.6-8 Presumably, phosphorylation causes a change in the structure of neurofilament side arms that slows down or stops their movement and causes radial growth of axons through local neurofilament accumulation as well as increased interfilament spacing.9,10 This remarkable architecture, while allowing for nutrients and ions to pass through, provides mechanical support to the axon. The exact mechanism that imparts neurofilaments this functionality, however, is not yet established. Two different models †
Part of the “Barbara J. Garrison Festschrift”. * To whom correspondence should be addressed. E-mail: s.p.adiga@ kodak.com (S.P.A.);
[email protected] (D.W.B.). ‡ Present address: Kodak Research Laboratories, Eastman Kodak Company, Rochester, NY 14650.
have been proposed for the mechanism(s) by which neurofilaments maintain the axon diameter. In the first, the side arms are thought to be cross-linked between different neurofilaments; this lends mechanical stability to the structure.11-13 A second model, proposed by Hoh and co-workers14,15 based on entropic repulsion due to the polymer brush-like structure of neurofilaments, has gained prominence recently. The model suggests that the largely unstructured side arms repel one another through entropic forces resulting from Brownian motion, and the Coulombic interaction due to phosphorylation is thought to affect only intramolecular interaction. An increase in the extent of phosphorylation expands the side arm and thereby increases the interfilament distance. Evidence for this comes from two atomic force microscopy (AFM) experiments.14 In the first, images were obtained that clearly show an excluded region of material coisolated with the filaments around individual neurofilaments on a mica surface. The second piece of evidence for this model comes from distance-force curves for an AFM tip over neurofilaments that indicate the onset of repulsive forces at a distance starting at about 50 nm above the filament core. This is the height at which the side arms are predicted to extend due to Brownian motion. Recently, using Monte Carlo simulations, Kumar et al.4 explained the distribution of interfilament distances in mouse sciatic nerves with interfilament interaction potentials derived from the brush theory. The model considered increased in side chain charge due to phosphorylation and predicted a considerable increase in the interfilament distance. The biological structure/function of the neurofilament assembly is believed to depend on the interaction between neurofilaments, which is thought to be regulated by the extent of phosphorylation of the side arms. However, precisely how the side arm structure is modulated by phosphorylation remains an open question. From this point of view, an understanding of the structure and the mechanism that determines the interfilament spacing of neurofilaments is very critical. Investigating the structure of neurofilaments is also important because a defect in their transport and assembly is thought to be a molecular link to several neurodegenerative disorders.16 A
10.1021/jp905671u 2010 American Chemical Society Published on Web 12/23/2009
Neurofilament Heavy Chain Side Arm Structure Modulation better understanding at a molecular level of how these structures function and under what circumstances they lose their functionality could shed light on a piece of the puzzle of these disorders. While phosphorylation-regulated neurofilament transport and accumulation is critical for healthy functioning of neurons, a disruption in neurofilament transport due to physical or chemical injuries can contribute to neurodegenerative diseases.17,18 For example, accumulation of an excess neurofilament in the axons is associated with several neuromuscular diseases, in particular, amyotrophic lateral sclerosis (ALS).19 This abnormal accumulation is thought to be triggered by hyperphosphorylation of NF side arms in the perikaryon or the proximal part of axons. Experiments on transgenic animals with neurofilament overexpression or mutations that disrupt normal filament stiochiometry or assembly have also produced abnormal neurofilament accumulation similar to that observed in ALS.20 Therefore, there appears to be a clear link between abnormal neurofilament accumulation and the neurodegenerative disease process. In this work, we investigate NFH side arms, the longest of the side arms, using molecular dynamics (MD) simulations. In particular, we investigate the effect of phosphorylation on the size and structure of human NFH side arms. Our results suggest that phosphorylation of serine residues in the NFH side arms mediate the neurofilaments’ function in controlling axonal caliber, in part, by modulating intramolecular salt bridges between acidic and basic residues. Simulation Details All molecular dynamics (MD) simulations were carried out at T ) 300 K using the AMBER721 MD program, the all-atom AMBER force field ff99, and a time step of 2 fs. The MD simulations were carried out using an implicit solvent implementation using the GB/SA22 option included in AMBER. Two different salt concentrations, cS ) 0.1 and 0.2 M, were considered. The lengths of chemical bonds to hydrogen atoms were fixed. The systems were equilibrated at a temperature of 300 K for about 50 ps. This was followed by a MD simulation of 50 ns. Sampling was begun after the polypeptide chains attained their average size and began to fluctuate around a mean value (∼6 ns). The force field parameters for the phosphorylated serine (SEP) residue, which are not available in the standard AMBER library, were obtained using the generalized Amber force field (GAFF) included in the Antechamber module of AMBER7. The partial charges on the atoms of the SEP residue were derived by following the procedure described in the AMBER documentation.21,23,24 Gaussian 9825 was used to calculate the electrostatic potential (ESP) at the molecular surfaces at the HF level of theory using the 6-31G* basis set. Atom-centered charges were obtained using the restrained electrostatic potential (RESP) fitting module of AMBER.23,24 The models of the C-terminal region of the NFH subunit were built using the LEAP module of AMBER.21 Five different chains, NFH0, NFH25, NFH50, NFH100, and NPHKSP, were constructed. The NFH0, NFH25, NFH50, and NFH100 chains correspond to 0, 25, 50, and 100% phosphorylated serine residues, respectively. While NFH100 has all residues of type SER replaced with type SEP, every fourth and every second SER residues along the sequence were replaced with SEP for NFH25 and NFH50, respectively. The chain NFHKSP has only the SER residues part of the KSP motifs phosphorylated and thus contains 42 residues of type SEP. The total number of atoms in the NFH0, NFH25, NFH50, NFH100, and NPHKSP
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Figure 1. Primary structure of the NFH side arm. (a) Amino acid sequence of the C-terminal domain of human NFH protein used in the simulation. The serine residues are highlighted in red, and KSP motifs are underlined. (b) Distribution of charge along the side arm calculated with an interval of 10 residues for unphosphorylated (NFH0) and phosphorylated (NFHKSP) side arms. In the chain NFHKSP, the 42 serine residues that are part of the KSP repeats are phosphorylated.
simulation systems were 9446, 9488, 9530, 9614, and 9572, respectively. The completely extended initial model of the chain was energy-minimized for 30000 steps to remove any unfavorable contacts between atoms. The structural analysis included the end-to-end distance, radius of gyration Rg, and distances between residues that were calculated using the CARNAL module of AMBER. The salt bridge analysis was performed with the help of the salt bridge plugin provided in the Visual Molecular Dynamics (VMD) program26 and custom written scripts. A salt bridge was defined to exist when the distance between atom N of a basic residue and atom O of an acidic residue is less than or equal to 3.2 Å. The VMD program was also used for visualization and rendering of structures. Results NFH Side Arms Are Unstructured Chains Consisting of Loops Stabilized by Salt Bridges. The amino acid (aa) sequence of NFH protein can be divided into three regions, an N-terminal domain of ∼100 residues, a central helical domain of ∼310 residues, and a C-terminal domain of 606 residues.27 The first two domains along with the N-terminal and helical domains of NFL and NFM form the filament core. The one letter aa sequence of the C-terminal domain of human NFH is given in Figure 1a. The first residue in this sequence corresponds to the position of residue number 415 of the complete NFH sequence according to Lees et al.27 One of the main features of the C-terminal domain is that it is a polyampholyte containing 24.1% GLU (-1 charged) and 25.7% LYS (+1 charged). In addition to this, there are 56 serine residues that can attain a -2 charged state when phosphorylated. A fully phosphorylated C-terminal domain of NFH can have a net charge of -113. It is reported, however, that, the most probable phosphorylation sites are the SER part of the LYS-SER-PRO (KSP) motifs.28 The NFH side arm has 42 KSP repeats including the two serine residues at positions 528 and 573 that are part of a reversed KSP motif. A majority of the KSP repeats have either a 3 or a 5 residue spacer between them, and they can be divided into two groups based on the presence or absence of K in the second amino acid position following the motif, that is, KSPXK or KSPXX (X is any residue other than LYS). The charge distributions for the unphosphorylated and phosphorylated (a
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total of 42 serine residues part of KSP motifs are phosphorylated) side arms are compared in Figure 1b. The side arm has a net negative charge in the segment consisting of the first 80 residues. The main effect of phosphorylation is to impart a negative charge to the central region. The domain is also rich in PRO (∼12%), which is known to break helical structure, which is fairly uniformly distributed along the domain, suggesting an absence of a secondary structure. The side arms are widely believed to be intrinsically unstructured, with strong evidence from AFM experiments. Secondary structure prediction by the program ProteinPredict29 has suggested a total absence of secondary structure for the human NFH side arm. Circular dichroism measurements of bovine NFH have also shown that the side arm contains about 20% R helix structure.30 Due to lack of a secondary structure, the relatively long NFH side arm (606 aa residues) is expected to be a random coil that undergoes large fluctuations around the center of mass. While experimental studies have been partially successful in probing the side arm structure, high-resolution structural information and average chain properties of such a chain are readily accessible by molecular dynamics simulations. The key challenge, however, is to arrive at the equilibrium side arm structure de novo and then follow the simulation trajectories long enough to capture all structural relaxations, both of which represent time scales very expensive for MD simulations. The first concern is somewhat diminished by the fact that the NFH side arm does not fold into a stable secondary structure, the time scale for which can be several hundred microseconds. To help overcome the problem of slow structural relaxations, we carried out our MD simulations using an implicit solvent description, that is, the solute (NFH) was modeled without explicit water molecules, but the calculations considered the effects of a high-dielectric solvent that is approximated by a continuum electrostatic model. This was implemented using the generalized Born solvent and solvent-accessible surface area (GB/SA) method that considers the effect of solute exposed to the solvent.22 The use of an implicit solvent reduces the computational time by orders of magnitude because it eliminates several thousands of water molecules and it enhances the rate at which the solute explores the configurational space due to an absence of viscous drag. To illustrate the basic structural features and differences in the unphosphorylated and phosphorylated states, we considered two chains, NFH0 and NFHKSP. The former represents the unphosphorylated NFH side arm and the latter the phosphorylated state in which all SER residues part of KSP repeats are phosphorylated. Thus, NFHKSP contains 42 residues of type SEP. We started our simulations from fully extended conformations. The time evolution of the potential energy and the radius of gyration Rg of the polypeptide chain during a MD simulation are plotted in Figure 2 for NFH0. The Rg and energy values quickly (
