Pex14p phosphorylation regulates peroxisome import

Peroxisomal matrix proteins are imported into peroxisomes via membrane-bound docking/translocation machinery. One central component of this machinery is Pex14p, a peroxisomal membrane protein involved in the docking of Pex5p, the receptor for peroxisome targeting signal type 1 (PTS1). Studies in several yeast species have shown that Pex14p is phosphorylated in vivo, whereas no function has been assigned to Pex14p phosphorylation in yeast and mammalian cells. Here, we investigated peroxisomal protein import and its dynamics in mitotic mammalian cells. In mitotically arrested cells, Pex14p is phosphorylated at Ser-232, resulting in a lower import efficiency of catalase, but not the majority of proteins including canonical PTS1 proteins. Conformational change induced by the mitotic phosphorylation of Pex14p more likely increases homomeric interacting affinity and suppresses topological change of its N-terminal part, thereby giving rise to the retardation of Pex5p export in mitotic cells. Taken together, these data show that mitotic phosphorylation of Pex14p and consequent suppression of catalase import are a mechanism of protecting DNA upon nuclear envelope breakdown at mitosis

Atomistic modeling of alternating access of a mitochondrial ADP/ATP membrane transporter with molecular simulations

<div><p>The mitochondrial ADP/ATP carrier (AAC) is a membrane transporter that exchanges a cytosolic ADP for a matrix ATP. Atomic structures in an outward-facing (OF) form which binds an ADP from the intermembrane space have been solved by X-ray crystallography, and revealed their unique pseudo three-fold symmetry fold which is qualitatively different from pseudo two-fold symmetry of most transporters of which atomic structures have been solved. However, any atomic-level information on an inward-facing (IF) form, which binds an ATP from the matrix side and is fixed by binding of an inhibitor, bongkrekic acid (BA), is not available, and thus its alternating access mechanism for the transport process is unknown. Here, we report an atomic structure of the IF form predicted by atomic-level molecular dynamics (MD) simulations of the alternating access transition with a recently developed accelerating technique. We successfully obtained a significantly stable IF structure characterized by newly formed well-packed and -organized inter-domain interactions through the accelerated simulations of unprecedentedly large conformational changes of the alternating access without a prior knowledge of the target protein structure. The simulation also shed light on an atomistic mechanism of the strict transport selectivity of adenosine nucleotides over guanosine and inosine ones. Furthermore, the IF structure was shown to bind ATP and BA, and thus revealed their binding mechanisms. The present study proposes a qualitatively novel view of the alternating access of transporters having the unique three-fold symmetry in atomic details and opens the way for rational drug design targeting the transporter in the dynamic functional cycle.</p></div

Protein structure of the ADP-bound IF form.

<p>Snapshots at 3,990 ns in MD1 simulation are shown. Hydrophobic residues and charged ones participating in the cytoplasmic inter-domain hydrophobic packing and salt-bridge network in the IF form are shown in views from the cytoplasmic side in left and middle panels, respectively. A side view of the protein structure and the cytoplasmic salt-bridge network is shown in a right panel.</p

Linear Response Path Following: A Molecular Dynamics Method To Simulate Global Conformational Changes of Protein upon Ligand Binding

Molecular functions of proteins are often fulfilled by global conformational changes that couple with local events such as the binding of ligand molecules. High molecular complexity of proteins has, however, been an obstacle to obtain an atomistic view of the global conformational transitions, imposing a limitation on the mechanistic understanding of the functional processes. In this study, we developed a new method of molecular dynamics (MD) simulation called the linear response path following (LRPF) to simulate a protein’s global conformational changes upon ligand binding. The method introduces a biasing force based on a linear response theory, which determines a local reaction coordinate in the configuration space that represents linear coupling between local events of ligand binding and global conformational changes and thus provides one with fully atomistic models undergoing large conformational changes without knowledge of a target structure. The overall transition process involving nonlinear conformational changes is simulated through iterative cycles consisting of a biased MD simulation with an updated linear response force and a following unbiased MD simulation for relaxation. We applied the method to the simulation of global conformational changes of the yeast calmodulin N-terminal domain and successfully searched out the end conformation. The atomistically detailed trajectories revealed a sequence of molecular events that properly lead to the global conformational changes and identified key steps of local–global coupling that induce the conformational transitions. The LRPF method provides one with a powerful means to model conformational changes of proteins such as motors and transporters where local–global coupling plays a pivotal role in their functional processes

Opening of the matrix side observed in LRPF2 simulation.

<p>(<i>a-d</i>) Time courses of helix angles (<i>a</i>), distance between Glu29 and Arg279 (<i>b</i>), contact between Phe88 and Tyr290 (<i>c</i>), and contact between Glu29 and the amino group at the adenine ring of ADP (<i>d</i>). See Supporting Material for the definition of the helix angle and the contact. Shaded areas indicate that a time region where a hydrogen-bond between Glu29 and the amino group of the adenine ring was established. (<i>e</i>) Snapshots of LRPF2 simulation at 1900 (left) ns and 2100 ns (right) before and after formation of the hydrogen-bond between Glu29 and the adenine ring, respectively. Close up views of the ADP binding site are shown in right panels.</p

Structure of AAC in the OF form and a binding pose of ADP.

<p>(<i>a</i>) An X-ray structure of AAC in the OF form (PDB entry 1OKC). The numbering of bovine AAC isoform 1 starts after the initiating methionine (Ser1–), following the numbering in the PDB file. (<i>b</i>) Schematic illustration of the protein structure. (<i>c</i>) Water distribution in the cytoplasmic pore at the last snapshot of the ADP binding simulation drawn in surface representation and colored in cyan. (<i>d</i>) A binding pose of ADP. Residues 190–230 and 280–297 are not shown. (<i>e</i>) Charged residues participating in ADP binding from the cytoplasmic side. A view from the cytoplasmic side. ADP is not shown.</p

Conformational transition from the OF form to the IF one.

<p>(<i>a</i>) Time course of C_α-RMSD with respect to the X-ray structure along LRPF2, LRPF2+, MD1, SMD and APO1 trajectories. In LRPF2 and LRPF2+ simulations, biasing forces of and to stimulate opening and closure of the matrix and cytoplasmic sides of the protein, respectively, were applied. MD1 was an unbiased MD simulation after the LRPF simulations of the forced conformational changes. ADP substrate was then removed by forced transportation to the matrix with a steered MD method in SMD simulation. Finally, the apo protein was equilibrated by unbiased MD simulation in APO1 simulation. Details of the simulation protocols are summarized in Table 1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181489#pone.0181489.s002" target="_blank">S2 Text</a>. (<i>b</i>) Conformational changes from the ADP-bound OF form to the ADP bound IF form. The last snapshots of the ADP binding simulation (OF), LRPF2, LRPF2+ and MD1 simulations are shown. In bottom panels, water distributions contoured at 0.3 occupancy level for the corresponding simulations are drawn in cyan. The water occupancy was calculated from the last 1-ns portion of each trajectory. A red arrow indicates the cytoplasmic constriction site in the IF form.</p

Alternating access mechanism of AAC.

<p>(<i>a</i>) X-ray structures in the OF form in views from the matrix side (left) and the cytoplasmic one (right) are shown in upper panels, and the last snapshots of APO1 simulation in the IF form are shown in lower panels. Odd-numbered helices are colored in wheat and even-numbered ones in green, respectively. Residues comprising the cytoplasmic salt-bridge network in the IF form are drawn in stick representation in red for acidic residues and in blue for basic ones. Asp203 and Arg104 are colored in magenta and cyan, respectively. Residues constituting the cytoplasmic hydrophobic packing in the IF form are drawn in gray stick and vdW sphere representations. (<i>b</i>) Hydrophobic residues comprising the matrix hydrophobic core in the OF form and the cytoplasmic one in the IF form are shown in black surface representation. Basic residues participating in the binding of ADP or BA are shown in blue surface representation.</p

Typical biasing forces used in LRPF simulations.

<p>(<i>a-b</i>) Typical biasing forces, (<i>a</i>) and (<i>b</i>), applied to C_α-atoms in LRPF2 and LRPF2+ simulations, respectively, are represented by black cones. Protein backbone is drawn in ribbon representation. C_α-atoms to which the perturbative forces, and , are applied are indicated by spheres. The biasing forces were frequently updated during the LRPF simulations.</p

Examination of the regression model to quantify the degree of low back pain and lower limb symptoms in patients with lumbar disc herniation by the Japanese Orthopaedic Association Back Pain Evaluation Questionnaire (JOABPEQ).

The Japanese Orthopedic Association Back Pain Evaluation Questionnaire (JOABPEQ) was created to evaluate specific treatment outcomes in terms of physical functioning, social ability, and mental health in patients with back pain-related diseases. In this study, we investigated whether the JOABPEQ could be used to construct a regression model to quantify low back pain and lower limb symptoms in patients with lumbar disc herniation (LDH). We reviewed 114 patients with LDH scheduled to undergo surgery at our hospital. We measured the degrees of 1) lower back pain, 2) lower limb pain, and 3) lower limb numbness using the visual analog scale before the surgery. All answers and physical function data were subjected to partial least squares regression analysis. The degrees of lower back and lower limb pain could be used as a regression model from the JOABPEQ and had a significant causal relationship with them. However, the degree of lower limb numbness could not be used for the same. Based on our results, the questions of the JOABPEQ can be used to multilaterally understand the degree of lower back pain and lower limb pain in patients with LDH. However, the degree of lower limb numbness has no causal relationship, so actual measurement is essential