14 research outputs found
Investigation of Ionization Pattern of the Adjacent Acidic Residues in the DXDXE Motif of GH-18 Chitinases Using Theoretical p<i>K</i><sub>a</sub> 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><sub>a</sub> 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><sub>a</sub> data indicate that in the majority
of chitinases the D1 side-chain is in the āupā and the
E side-chain in the ādownā position, while the position
of the D2 side-chain is versatile and depends on the state of the
enzyme. The p<i>K</i><sub>a</sub> values in 75 GH-18 chitinases
were predicted to be <0 for D1, 8ā13 for D2, and 6ā9
for E, indicating that the D1āD2 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 Ī±/Ī²-protein that forms amyloid fibrils
at low pH, presumably initiated from a denatured state with a considerable
amount of residual structure. Here we used CarrāParcellāMeiboomāGill
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 <sup>15</sup>N, <sup>13</sup>C, and <sup>1</sup>H NMR chemical shifts in the I71V ADA2h
denatured state reveals that it retains a significant fraction (up
to 50ā60%) of nativelike Ī±-helical structure, while the
regions encompassing native Ī²-strands are structured to a much
lesser extent. The nativelike Ī±-helical structure of the denatured
state can bring together hydrophobic residues on the same sides of
Ī±-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 CāTerminal 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 Ca<sup>2+</sup>-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, <sup>15</sup>N 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 Ca<sup>2+</sup> 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 Ca<sup>2+</sup>-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 Ī±-helix and are 2 and 3 residues apart in the sequence.</p
Parameters for CĪ²-CĪ±-N-C' and CĪ²-CĪ±-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 CĪ²-CĪ±-N-C' and CĪ²-CĪ±-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 CĪ± atoms. Bond lengths (apart from the CĪ±-CĪ² bond) and angles are taken from the Amber force field. The length of the CA-CĪ² 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 CĪ±-CĪ² 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<sub>i</sub>-H<sub>i+1</sub>, O<sub>iā1</sub>-O<sub>i</sub> and O<sub>iā1</sub>-N<sub>i+1</sub> distances. Allowed regions are represented by light blue colour and they contain the range of dihedral angles present in right-handed Ī±-helices, left-handed Ī±-helices and Ī²-sheets.</p