Comparative Molecular Dynamics Simulation Study of Crystal Environment Effect on Protein Structure

Crystal structures of proteins are under the influence from the crystal environment. In this study, we used molecular dynamics (MD) simulations to explore the possibility of eliminating the effect of the crystal packing and recovering the structure in solution. Ten representative proteins were chosen from the Protein Structural Change Database as the target systems, and 50 ns MD stimulations starting from two crystal structures having different domain arrangements were performed for each. The MD trajectories of the relaxation processes upon the release from the crystal environment revealed that the behaviors of the proteins were classified into three groups: “single domain linker”, “harmonic motion”, and “large barrier”. We discuss the structural features common to the proteins in each group

Narrowing of configurational space with increased <i>Q</i>.

<p>Occupancy maps of barstar C_α atoms with various <i>Q</i> ranges in unbiased replica of MSES simulation generated by VMD <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003901#pcbi.1003901-Humphrey1" target="_blank">[70]</a>. Three-dimensional grids are created using a bin width of 2 Å and the grid points occupied by C_α atoms in the unbiased MSES ensemble are shown in red. The coordinates are superimposed on the barnase molecule, which is shown in gray.</p

Probability of polar contact formation.

a<p>List was made according to polar contacts formed in the complex crystal structures <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003901#pcbi.1003901-Urakubo1" target="_blank">[54]</a>. The bold numbers indicate that <i>p</i>_MM>0.70.</p>b<p><i>p</i>_MM for probability in MM simulation starting from the complex structure.</p>c<p><i>p</i>_MSES for probability in MSES simulation where C_α RMSD<4 Å from the complex structure.</p><p>Probability of polar contact formation.</p

MSES simulation.

<p>(A) Probability distributions of <i>V</i>_MMCG, <i>P</i>(<i>V</i>_MMCG), defined in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003901#pcbi.1003901.e003" target="_blank">Eq. 1</a> for 12 replicas of MSES simulation. (B) Time course of <i>V</i>_MMCG for a representative model replica, i.e., the replica fixed not by <i>k</i>_MMCG, but by the configuration. (C–E) Quantities from the unbiased MSES ensemble (with <i>k</i>_MMCG = 0) as a function of simulation time: Root-mean-square displacement for C_α atoms (C_α RMSD) of barstar after fitting to barnase, RMSD_bs (C), center-of-mass (COM) distance between two COMs for barstar and barnase, respectively, <i>d</i>_COM (D), and number of polar contacts found in eight inter-molecular pairs, #3, 4, 6, 7, 8, 11, 12, and 13, listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003901#pcbi-1003901-t001" target="_blank">Table 1</a> (E). In (D), <i>d</i>_COM in conventional equilibrium MD simulation starting from complex structure (MM simulation) is also shown by red. In (F) and (G), arrangements of barstar observed in the unbiased MSES ensemble and in MM simulation are shown, respectively. Both coordinates were superimposed on barnase.</p

Free-Energy Landscape of Protein–Ligand Interactions Coupled with Protein Structural Changes

Protein–ligand interactions are frequently coupled with protein structural changes. Focusing on the coupling, we present the free-energy surface (FES) of the ligand-binding process for glutamine-binding protein (GlnBP) and its ligand, glutamine, in which glutamine binding accompanies large-scale domain closure. All-atom simulations were performed in explicit solvents by multiscale enhanced sampling (MSES), which adopts a multicopy and multiscale scheme to achieve enhanced sampling of systems with a large number of degrees of freedom. The structural ensemble derived from the MSES simulation yielded the FES of the coupling, described in terms of both the ligand’s and protein’s degrees of freedom at atomic resolution, and revealed the tight coupling between the two degrees of freedom. The derived FES led to the determination of definite structural states, which suggested the dominant pathways of glutamine binding to GlnBP: first, glutamine migrates via diffusion to form a dominant encounter complex with Arg75 on the large domain of GlnBP, through strong polar interactions. Subsequently, the closing motion of GlnBP occurs to form ligand interactions with the small domain, finally completing the native-specific complex structure. The formation of hydrogen bonds between glutamine and the small domain is considered to be a rate-limiting step, inducing desolvation of the protein–ligand interface to form the specific native complex. The key interactions to attain high specificity for glutamine, the “door keeper” existing between the two domains (Asp10–Lys115) and the “hydrophobic sandwich” formed between the ligand glutamine and Phe13/Phe50, have been successfully mapped on the pathway derived from the FES

Probability of occurrence of polar contacts, <i>p</i>, at interface 1 and interface 2, with various numbers of native contacts observed in MSES simulation^a.

a<p>Polar contacts formed between two atoms in either interface 1 or interface 2 are listed with probability of occurrence.</p>b<p><i>n</i>_PC is number of native polar contacts in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003901#pcbi-1003901-g004" target="_blank">Fig. 4E</a>, for interface 1 (0≤<i>n</i>_PC≤5) and interface 2 (0≤<i>n</i>_PC≤3). In <i>n</i>_PC = 5 in interface 1 and <i>n</i>_PC = 3 in interface 2, the probability is unity by definition.</p>c<p>Probability <i>p</i> has relation , where is probability for <i>i</i>-th identifier and <i>n</i>_PC.</p>d<p>Identifier is same as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003901#pcbi-1003901-t001" target="_blank">Table 1</a>.</p>e<p>Bold numbers indicate probable polar contact with probability >0.8.</p><p>Probability of occurrence of polar contacts, <i>p</i>, at interface 1 and interface 2, with various numbers of native contacts observed in MSES simulation^{<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003901#nt104" target="_blank">a</a>}.</p

Funnel landscape of barnase-barstar interaction.

<p>Distributions of centers of mass (COM) of barstar with various ranges of fraction of native inter-molecular contacts formed (<i>Q</i>) after superimposing barnase in unbiased ensemble of MSES simulation. (Top) Three-dimensional distributions at <i>Q</i><0.2 (blue), 0.2<<i>Q</i><0.4 (green), 0.4<<i>Q</i><0.7 (yellow), and <i>Q</i>>0.7 (red). (Bottom) Distributions onto <i>x-y</i> plane and <i>x-z</i> plane at depicted <i>Q</i> ranges. The <i>x</i>-<i>y</i> plane was defined to be orthogonal to the vector connecting the two COM's of barnase and barstar (<i>z</i>-axis), and <i>x</i>-axis being the direction of the vector from C_α of Arg87 to C_α of Arg83 of barnase.</p

Formations of two localized interfaces.

<p>(A) 2D representation of FES along RMSD1 (non-hydrogen-atom RMSD for interface 1) and RMSD2 (non-hydrogen-atom RMSD for interface 2). In (B), the situation is the same but when both interfaces are formed. (C) and (D) show probability distributions along RMSD1 and RMSD2, respectively, when designated number of polar contacts are formed. (E) Native polar contacts at interfaces 1 and 2 (identifier is same as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003901#pcbi-1003901-t001" target="_blank">Table 1</a>). (F) Side-chain positions of interfaces 1 (red) and 2 (blue) of barnase and barstar.</p

Free energy surfaces for two localized interfaces.

<p>2-D free energy surfaces of barstar position on <i>x-y</i> plane of barnase: Two distributions are plotted on same figure for centers of mass of barstar residues comprising interface 1 (Tyr29, Asn33, and Asp39: upper right) and that for interface 2 (Asp35 and Glu76: lower left). In A–H, the distributions are drawn for the unbiased MSES ensemble under the respective conditions that the polar contacts given at the bottom (the identifier defined in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003901#pcbi-1003901-t001" target="_blank">Table 1</a>) are formed. “Interface 1”, “interface 2”, and “interfaces 1&2” indicate the structures when all the polar contacts in interface 1 and/or 2 are formed, respectively. In I–K, the distributions obtained in the MM simulations starting from the complex structure are shown for the wild type (I) and the two mutants, bs:D35A (J) and bs:D39A (K).</p

Disorder-to-Order Transition of an Intrinsically Disordered Region of Sortase Revealed by Multiscale Enhanced Sampling

Molecular functions of intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs), such as molecular recognition and cellular signaling, are ascribed to dynamic changes in the conformational space in response to binding of target molecules. Sortase, a transpeptitase in Gram-positive bacteria, has an IDR in a loop which undergoes a disordered-to-ordered transition (called “disordered loop”), accompanying a tilt of another loop (“dynamic loop”), upon binding of a signal peptide and a calcium ion. In this study, all-atom conformational ensembles of sortase were calculated for the four different binding states (with/without the peptide and with/without a calcium ion) by the multiscale enhanced sampling (MSES) simulation to examine how the binding of the peptide and/or calcium influences the conformational ensemble. The MSES is a multiscale and multicopy simulation method that allows an enhanced sampling of the all-atom model of large proteins including explicit solvent. A 100 ns MSES simulation of the ligand-free sortase using 20 replicas (in total 2 μs) demonstrated large flexibility in both the disordered and dynamic loops; however, their distributions were not random but had a clear preference which populates the N-terminal part of the disordered loop near the bound form. The MSES simulations of the three binding states clarified the allosteric mechanism of sortase: the N- and C-terminal parts of the disordered loop undergo a disorder-to-order transition independently of each other upon binding of the peptide and a calcium ion, respectively; however, upon binding of both ligands, the two parts work cooperatively to stabilize the bound peptide