Local Fluctuations and Conformational Transitions in Proteins

The intrinsic plasticity of protein residues, along with the occurrence of transitions between distinct residue conformations, plays a pivotal role in a variety of molecular recognition events in the cell. Analysis aimed at identifying both of these features has been limited so far to protein-complex structures. We present a computationally efficient tool (T-pad), which quantitatively analyzes protein residues’ flexibility and detects backbone conformational transitions. T-pad is based on directional statistics of NMR structural ensembles or molecular dynamics trajectories. T-pad is here applied to human ubiquitin (hU), a paradigmatic cellular interactor. The calculated plasticity is compared to hU’s Debye–Waller factors from the literature as well as those from experimental work carried out for this paper. T-pad is able to identify most of the key residues involved in hU’s molecular recognition, also in the absence of its cellular partners. Indeed, T-pad identified as many as 90% of ubiquitin residues interacting with their cognate proteins. Hence, T-pad might be a useful tool for the investigation of interactions between proteins and their cellular partners at the genome-wide level

Specific cholesterol binding to hA_2AR A) Cartoon showing cholesterol-binding pose in H1/H2 cleft in system III.

<p>The receptor is shown in yellow cartoon; the cholesterol molecule is shown as green sticks surrounded by its solvent accessible surface; CFF, cholesterol-interacting residues, VAL57, LEU58, as well as CFF-interacting residues ILE66, SER67 are shown as green sticks with oxygen and nitrogen atoms colored in red and blue, respectively. <b>B) The diffusion of cholesterol into of the H1/H2 cleft enhances hydrophobic contacts between CFF and H2.</b> The minimum distances between the specific cholesterol molecule and H1 (residues 5–34), between cholesterol and H2 (residues 41–67), between C5@CFF and heavy atoms of ILE66 and SER67 side chains, are shown in black, red, blue and green, respectively.</p

Populations of CFF binding poses (%) detected across systems I-III over the last 400 ns of MD simulated time.

<p>Populations of CFF binding poses (%) detected across systems I-III over the last 400 ns of MD simulated time.</p

A Molecular Dynamics Simulation-Based Interpretation of Nuclear Magnetic Resonance Multidimensional Heteronuclear Spectra of α‑Synuclein·Dopamine Adducts

Multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy provides valuable structural information about adducts between naturally unfolded proteins and their ligands. These are often highly pharmacologically relevant. Unfortunately, the determination of the contributions to observed chemical shifts changes upon ligand binding is complicated. Here we present a tool that uses molecular dynamics (MD) trajectories to help interpret two-dimensional (2D) NMR data. We apply this tool to the naturally unfolded protein human α-synuclein interacting with dopamine, an inhibitor of fibril formation, and with its oxidation products in water solutions. By coupling 2D NMR experiments with MD simulations of the adducts in explicit water, the tool confirms with experimental data that the ligands bind preferentially to ¹²⁵YEMPS¹²⁹ residues in the C-terminal region and to a few residues of the so-called NAC region consistently. It also suggests that the ligands might cause conformational rearrangements of distal residues located at the N-terminus. Hence, the performed analysis provides a rationale for the observed changes in chemical shifts in terms of direct contacts with the ligand and conformational changes in the protein

CFF’s most populated binding poses in systems I-III (A-D).

<p>For each binding pose, the upper panel shows the protein backbone in yellow cartoon, CFF and residues interacting with CFF in thick and thin sticks, respectively. Water molecules forming H-bonds with CFF and residues are represented as red sphere; the lower panel shows the corresponding 2-d chart.</p

Composition of the three systems simulated here.

<p>Composition of the three systems simulated here.</p

The distribution of CFF center of mass within the ligand-binding cavity of hA_2AR across systems I-III.

<p>The receptor and CFF are shown as yellow cartoon and red sticks, respectively. CFF center of mass at each collected frame over the last 400 ns of MD simulated time is depicted as one black dot.</p

Membrane-sensitive folding of H8.

<p><b>A)</b> The cartoon representations of receptor’s backbone of systems <b>I</b>–<b>III</b> are shown in blue to red according to residues’ increased flexibility, as emerging from the so-called PAD index values [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126833#pone.0126833.ref032" target="_blank">32</a>]; the inserted panel shows the location of H8 (in yellow cartoon) in each membrane; POPC, POPE and cholesterol molecules are shown in red, blue, green lines respectively. The phosphorous atoms are shown as violet Van de Waals spheres. <b>B)</b> Secondary structure content of H8-including C segment (res 292 to 329) (²⁹²REFRQTFRKIIRSHVLRQQEPFKAAAAHHHHHHHHHHH³²⁹) is reported as a function of the simulated time. β sheet, α helix, coil and bend, and turn are shown in red, blue, white, green and yellow, respectively.</p

Structural features of SIRT2.

<p><b>(A)</b> Primary sequences of SIRT2 isoform 1 (SIRT2-iso1) and isoform 2 (SIRT2-iso2) aligned with that of Hst2 from <i>S</i>. <i>cerevisiae</i> (obtained using the BLAST webserver (<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a>)). Identical and structurally similar residues are indicated in red and green, respectively. Solid lines indicate the N-terminal (NT, blue) and C-terminal (CT, red) regions, and so-called NAD⁺ cofactor-binding loop (green). The catalytic center residue H150 (black diamond) and the phosphorylation sites S331 and S335 (orange diamonds) are shown. <b>(B)</b> X-ray structure of the catalytic core (CC) domain of human SIRT2 (PDB ID: 1J8F [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139095#pone.0139095.ref013" target="_blank">13</a>]). The CC consists of (i) a Rossmann fold (orange), made of six β-strands forming a parallel β–sheet and six α-helices, (ii) a small domain made up by a zinc binding pocket and a hydrophobic pocket, which contains a three-stranded antiparallel β-sheet (yellow), an α-helix (yellow) and a zinc ion (light blue) coordinated by four cysteine residues (C195, C200, C221 and C224 (yellow sticks)), and four α-helices forming a hydrophobic pocket (purple), and (iii) four connecting loops (green). NAD⁺ is absent in the crystal structure. It has been included here by superposing the CC of SIRT2 with that of Sir2-Af1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139095#pone.0139095.ref014" target="_blank">14</a>], for which a structure with NAD⁺ is available (NAD⁺ is in magenta). N and C indicate the N-terminal and C-terminal ends of the CC, respectively. <b>(C)</b> Structural comparison of the CC domain obtained from X-ray structure determinations and of the modeled SIRT2/NAD⁺ complex, the modeled SIRT2 and the modeled SIRT2-pS331. The CC crystal structure and our models are colored in gray and green, respectively. The NT and CT are colored in blue and pink, respectively. The NAD⁺ cofactor in the CC of the modeled SIRT2/NAD⁺ is represented by orange sticks. The S331/pS331 residues in the CT are represented by pink sticks.</p

Hydrogen bond and salt bridge interactions between the CC and the CT in SIRT2.

<p>Standard deviations are given in parentheses.</p><p>^<i>a</i> Residues belong to the CT.</p><p>^<i>b</i> Residues belong to the CC.</p><p>^<i>c</i> Atomic distances (h1–h12) between donor and acceptor, and center-of-mass distances (s1–s3) between negative and positive side chains are calculated on the equilibrium trajectory from MD simulations of SIRT2.</p><p>Hydrogen bond and salt bridge interactions between the CC and the CT in SIRT2.</p