Investigation of Ionization Pattern of the Adjacent Acidic Residues in the DXDXE Motif of GH-18 Chitinases Using Theoretical p<i>K</i>_a Calculations

GH-18 chitinases are chitinolytic enzymes, primarily responsible for the recycling of insoluble chitin biomaterials. These enzymes contain three invariant acidic active-site residues within a DXDXE motif, which play a synergistic role in the catalytic cycle of chitin degradation. We employed a p<i>K</i>_a calculation approach to approximate the protonation states of residues D1, D2, and E in the DXDXE motif of 75 GH-18 chitinases. Theoretical pH-activity profiles of these enzymes were subsequently constructed and compared with the experimentally determined pH-activity profiles. Theoretical p<i>K</i>_a data indicate that in the majority of chitinases the D1 side-chain is in the “up” and the E side-chain in the “down” position, while the position of the D2 side-chain is versatile and depends on the state of the enzyme. The p<i>K</i>_a values in 75 GH-18 chitinases were predicted to be <0 for D1, 8–13 for D2, and 6–9 for E, indicating that the D1–D2 pair holds exactly one net negative charge. On the other hand, the catalytic acid E is protonated over the active pH-range, agreeing with the pH-activity curves reported previously for most chitinases. The results obtained from this study help to elucidate the mechanistic details of the concerted participation of D1, D2, and E in the catalytic cycle of chitin hydrolysis by GH-18 chitinases

Probing the Residual Structure of the Low Populated Denatured State of ADA2h under Folding Conditions by Relaxation Dispersion Nuclear Magnetic Resonance Spectroscopy

The structural characterization of low populated states of proteins with accuracy comparable to that achievable for native states is important for understanding the mechanisms of protein folding and function, as well as misfolding and aggregation. Because of the transient nature of these low populated states, they are seldom detected directly under conditions that favor folding. The activation domain of human procarboxypeptidase A2 (ADA2h) is an α/β-protein that forms amyloid fibrils at low pH, presumably initiated from a denatured state with a considerable amount of residual structure. Here we used Carr–Parcell–Meiboom–Gill relaxation dispersion (CPMG RD) nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the denatured state of the ADA2h I71V mutant under conditions that favor folding. Under these conditions, the lifetime of the denatured state of I71V ADA2h is on the order of milliseconds and its population is approximately several percent, which makes this mutant amenable to studies by CPMG RD methods. The nearly complete set of CPMG RD-derived backbone ¹⁵N, ¹³C, and ¹H NMR chemical shifts in the I71V ADA2h denatured state reveals that it retains a significant fraction (up to 50–60%) of nativelike α-helical structure, while the regions encompassing native β-strands are structured to a much lesser extent. The nativelike α-helical structure of the denatured state can bring together hydrophobic residues on the same sides of α-helices, making them available for intra- or intermolecular interactions. CPMG RD data analysis thus allowed a detailed structural characterization of the ADA2h denatured state under folding conditions not previously achieved for this protein

Structural Insights into the Calcium-Mediated Allosteric Transition in the C‑Terminal Domain of Calmodulin from Nuclear Magnetic Resonance Measurements

Calmodulin is a two-domain signaling protein that becomes activated upon binding cooperatively two pairs of calcium ions, leading to large-scale conformational changes that expose its binding site. Despite significant advances in understanding the structural biology of calmodulin functions, the mechanistic details of the conformational transition between closed and open states have remained unclear. To investigate this transition, we used a combination of molecular dynamics simulations and nuclear magnetic resonance (NMR) experiments on the Ca²⁺-saturated E140Q C-terminal domain variant. Using chemical shift restraints in replica-averaged metadynamics simulations, we obtained a high-resolution structural ensemble consisting of two conformational states and validated such an ensemble against three independent experimental data sets, namely, interproton nuclear Overhauser enhancements, ¹⁵N order parameters, and chemical shift differences between the exchanging states. Through a detailed analysis of this structural ensemble and of the corresponding statistical weights, we characterized a calcium-mediated conformational transition whereby the coordination of Ca²⁺ by just one oxygen of the bidentate ligand E140 triggers a concerted movement of the two EF-hands that exposes the target binding site. This analysis provides atomistic insights into a possible Ca²⁺-mediated activation mechanism of calmodulin that cannot be achieved from static structures alone or from ensemble NMR measurements of the transition between conformations

A repertoire of representative Val60 structures generated using the CamTube force field.

<p>A selection of 135 structures whose TM-score from respective CATH structures is larger than 0.4; a-c) examples of three CATH structures with their equivalent Val60 structures. CATH codes are given bellow the respective figures.</p

Atom pair self-avoiding sphere distances, <i>d</i>, from Eq 1.

<p>Atom pair self-avoiding sphere distances, <i>d</i>, from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004435#pcbi.1004435.e001" target="_blank">Eq 1</a>.</p

Input parameters used in simulations with the CamTube force field.

<p>Input parameters used in simulations with the CamTube force field.</p

Illustration of the repulsion between C' and H atoms introduced by the curvature term in the CamTube model.

<p>The C' and H atoms belong to the α-helix and are 2 and 3 residues apart in the sequence.</p

Parameters for Cβ-Cα-N-C' and Cβ-Cα-C'-N dihedral angles used in the CamTube force field that encode the propensity of the amino acids for different regions in the Ramachandran map.

<p>Parameters for Cβ-Cα-N-C' and Cβ-Cα-C'-N dihedral angles used in the CamTube force field that encode the propensity of the amino acids for different regions in the Ramachandran map.</p

Schematic representation of a segment of a polypeptide chain in the CamTube model.

<p>The tube-like implementation is carried out by self-avoiding spheres, which for clarity of illustration are shown here only for Cα atoms. Bond lengths (apart from the Cα-Cβ bond) and angles are taken from the Amber force field. The length of the CA-Cβ bond of Val, Pro, Thr, Ser and Cys is scaled 1.5 times; Asp, Ile, Leu and Asn 2 times; Phe 2.25 times; Glu, Gln, Met and His 2.5 times; Tyr and Trp 3 times; Lys and Arg 4 times the length of the Cα-Cβ bond in the Amber force field.</p

Steric map in the CamTube model.

<p>The map shows main steric restrictions (dashed black line) imposed by H_i-H_i+1, O_i–1-O_i and O_i–1-N_i+1 distances. Allowed regions are represented by light blue colour and they contain the range of dihedral angles present in right-handed α-helices, left-handed α-helices and β-sheets.</p