6 research outputs found
Engineering Aromatic–Aromatic Interactions To Nucleate Folding in Intrinsically Disordered Regions of Proteins
Aromatic
interactions are an important force in protein folding
as they combine the stability of a hydrophobic interaction with the
selectivity of a hydrogen bond. Much of our understanding of aromatic
interactions comes from “bioinformatics” based analyses
of protein structures and from the contribution of these interactions
to stabilizing secondary structure motifs in model peptides. In this
study, the structural consequences of aromatic interactions on protein
folding have been explored in engineered mutants of the molten globule
protein apo-cytochrome <i>b</i><sub>5</sub>. Structural
changes from disorder to order due to aromatic interactions in two
variants of the protein, viz., WF-cytb5 and FF-cytb5,
result in significant long-range secondary and tertiary structure.
The results show that 54 and 52% of the residues in WF-cytb5 and FF-cytb5,
respectively, occupy ordered regions versus 26% in apo-cytochrome <i>b</i><sub>5</sub>. The interactions between the aromatic groups
are offset-stacked and edge-to-face for the Trp-Phe and Phe-Phe mutants,
respectively. Urea denaturation studies indicate that both mutants
have a <i>C</i><sub>m</sub> higher than that of apo-cytochrome <i>b</i><sub>5</sub> and are more stable to chaotropic agents than
apo-cytochrome <i>b</i><sub>5</sub>. The introduction of
these aromatic residues also results in “trimer” interactions
with existing aromatic groups, reaffirming the selectivity of the
aromatic interactions. These studies provide insights into the aromatic
interactions that drive disorder-to-order transitions in intrinsically
disordered regions of proteins and will aid in <i>de novo</i> protein design beyond small peptide scaffolds
The Structure of the Thioredoxin–Triosephosphate Isomerase Complex Provides Insights into the Reversible Glutathione-Mediated Regulation of Triosephosphate Isomerase
Protein–protein interactions are crucial for many
biological
functions. The redox interactome encompasses numerous weak transient
interactions in which thioredoxin plays a central role. Proteomic
studies have shown that thioredoxin binds to numerous proteins belonging
to various cellular processes, including energy metabolism. Thioredoxin
has cross talk with other redox mechanisms involving glutathionylation
and has functional overlap with glutaredoxin in deglutathionylation
reactions. In this study, we have explored the structural and biochemical
interactions of thioredoxin with the glycolytic enzyme, triosephosphate
isomerase. Nuclear magnetic resonance chemical shift mapping methods
and molecular dynamics-based docking have been applied in deriving
a structural model of the thioredoxin–triosephosphate isomerase
complex. The spatial proximity of active site cysteine residues of
thioredoxin to reactive thiol groups on triosephosphate isomerase
provides a direct link to the observed deglutathionylation of cysteine
217 in triosephosphate isomerase, thereby reversing the inhibitory
effect of S-glutathionylation of triosephosphate isomerase
Structural, Functional, and Mutational Studies of a Potent Subtilisin Inhibitor from Budgett’s Frog, Lepidobatrachus laevis
Subtilases
play a significant role in microbial pathogen
infections
by degrading the host proteins. Subtilisin inhibitors are crucial
in fighting against these harmful microorganisms. LL-TIL, from skin
secretions of Lepidobatrachus laevis, is a cysteine-rich peptide belonging to the I8 family of inhibitors.
Protease inhibitory assays demonstrated that LL-TIL acts as a slow-tight
binding inhibitor of subtilisin Carlsberg and proteinase K with inhibition
constants of 91 pM and 2.4 nM, respectively. The solution structures
of LL-TIL and a mutant peptide reveal that they adopt a typical TIL-type
fold with a canonical conformation of a reactive site loop (RSL).
The structure of the LL-TIL-subtilisin complex and molecular dynamics
(MD) simulations provided an in-depth view of the structural basis
of inhibition. NMR relaxation data and molecular dynamics simulations
indicated a rigid conformation of RSL, which does not alter significantly
upon subtilisin binding. The energy calculation for subtilisin inhibition
predicted Ile31 as the highest contributor to the binding
energy, which was confirmed experimentally by site-directed mutagenesis.
A chimeric mutant of LL-TIL broadened the inhibitory profile and attenuated
subtilisin inhibition by 2 orders of magnitude. These results provide
a template to engineer more specific and potent TIL-type subtilisin
inhibitors
Nuclear Magnetic Resonance Structure of a Major Lens Protein, Human γC-Crystallin: Role of the Dipole Moment in Protein Solubility
A hallmark
of the crystallin proteins is their exceptionally high
solubility, which is vital for maintaining the high refractive index
of the eye lens. Human γC-crystallin is a major γ-crystallin
whose mutant forms are associated with congenital cataracts but whose
three-dimensional structure is not known. An earlier study of a homology
model concluded that human γC-crystallin has low intrinsic solubility,
mainly because of the atypical magnitude and fluctuations of its dipole
moment. On the contrary, the high-resolution tertiary structure of
human γC-crystallin determined here shows unequivocally that
it is a highly soluble, monomeric molecule in solution. Notable differences
between the orientations and interactions of several side chains are
observed upon comparison to those in the model. No evidence of the
pivotal role ascribed to the effect of dipole moment on protein solubility
was found. The nuclear magnetic resonance structure should facilitate
a comprehensive understanding of the deleterious effects of cataract-associated
mutations in human γC-crystallin
Solution Nuclear Magnetic Resonance Structure of the GATase Subunit and Structural Basis of the Interaction between GATase and ATPPase Subunits in a <i>two-subunit-type</i> GMPS from <i>Methanocaldococcus jannaschii</i>
The
solution structure of the monomeric glutamine amidotransferase
(GATase) subunit of the <i>Methanocaldococcus janaschii</i> (Mj) guanosine monophosphate synthetase (GMPS) has been determined
using high-resolution nuclear magnetic resonance methods. Gel filtration
chromatography and <sup>15</sup>N backbone relaxation studies have
shown that the Mj GATase subunit is present in solution as a 21 kDa
(188-residue) monomer. The ensemble of 20 lowest-energy structures
showed root-mean-square deviations of 0.35 ± 0.06 Å for
backbone atoms and 0.8 ± 0.06 Å for all heavy atoms. Furthermore,
99.4% of the backbone dihedral angles are present in the allowed region
of the Ramachandran map, indicating the stereochemical quality of
the structure. The core of the tertiary structure of the GATase is
composed of a seven-stranded mixed β-sheet that is fenced by
five α-helices. The Mj GATase is similar in structure to the <i>Pyrococcus horikoshi</i> (Ph) GATase subunit. Nuclear magnetic
resonance (NMR) chemical shift perturbations and changes in line width
were monitored to identify residues on GATase that were responsible
for interaction with magnesium and the ATPPase subunit, respectively.
These interaction studies showed that a common surface exists for
the metal ion binding as well as for the protein–protein interaction.
The dissociation constant for the GATase–Mg<sup>2+</sup> interaction
has been found to be ∼1 mM, which implies that interaction
is very weak and falls in the fast chemical exchange regime. The GATase–ATPPase
interaction, on the other hand, falls in the intermediate chemical
exchange regime on the NMR time scale. The implication of this interaction
in terms of the regulation of the GATase activity of holo GMPS is
discussed
A Disulfide Stabilized β‑Sandwich Defines the Structure of a New Cysteine Framework M‑Superfamily Conotoxin
The structure of a new cysteine framework
(−CCCCCC−) “M”-superfamily
conotoxin, Mo3964, shows it to have a β-sandwich structure that
is stabilized by inter-sheet cross disulfide bonds. Mo3964 decreases
outward K<sup>+</sup> currents in rat dorsal root ganglion neurons
and increases the reversal potential of the Na<sub>V</sub>1.2 channels.
The structure of Mo3964 (PDB ID: 2MW7) is constructed from the disulfide connectivity
pattern, i.e., 1-3, 2-5, and 4-6, that is hitherto undescribed for
the “M”-superfamily conotoxins. The tertiary structural
fold has not been described for any of the known <i>conus</i> peptides. NOE (549), dihedral angle (84), and hydrogen bond (28)
restraints, obtained by measurement of <sup>h3</sup><i>J</i><sub>NC′</sub> scalar couplings, were used as input for structure
calculation. The ensemble of structures showed a backbone root mean
square deviation of 0.68 ± 0.18 Å, with 87% and 13% of the
backbone dihedral (ϕ, ψ) angles lying in the most favored
and additional allowed regions of the Ramachandran map. The conotoxin
Mo3964 represents a new bioactive peptide fold that is stabilized
by disulfide bonds and adds to the existing repertoire of scaffolds
that can be used to design stable bioactive peptide molecules