5 research outputs found
Peptides as Inhibitors of the First Phosphorylation Step of the <i>Streptomyces coelicolor</i> Phosphoenolpyruvate: Sugar Phosphotransferase System
The phosphotransferase system (PTS) controls the use
of sugars
in bacteria. The PTS is ubiquitous in bacteria, but it does not occur
in plants and animals; it modulates catabolite repression, intermediate
metabolism, gene expression, and chemotaxis. Its uniqueness and pleiotropic
function make the PTS an attractive target for new antibacterial drugs.
The PTS is constituted of two general proteins, namely, enzyme I (EI)
and the histidine phosphocarrier (HPr), and various sugar-specific
permeases. EI has two domains: the N-terminal domain (EIN), which
binds to HPr, and the C-terminal domain (EIC), which contains the
dimerization interface. In this work, we determined the binding affinities
of peptides derived from EIN of <i>Streptomyces coelicolor</i> (EIN<sup>sc</sup>) against HPr of the same organism (HPr<sup>sc</sup>), by using nuclear magnetic resonance and isothermal titration calorimetry
techniques. Furthermore, we measured the affinity of EIN<sup>sc</sup> for (i) a peptide derived from HPr<sup>sc</sup>, containing the
active-site histidine, and (ii) other peptides identified previously
by phage display and combinatorial chemistry in <i>Escherichia
coli</i> [Mukhija, S. L., et al (1998) <i>Eur. J. Biochem</i>. <i>254</i>, 433ā438; Mukhija, S., and Erni, B.
(1997) <i>Mol. Microbiol. 25</i>, 1159ā1166]. The
affinities were in the range of ā¼10 Ī¼M, being slightly
higher for the binding of EIN<sup>sc</sup> with peptides derived from
HPr<sup>sc</sup>, phage display, or combinatorial chemistry (<i>K</i><sub>D</sub> ā¼ 5 Ī¼M). Because the affinity
of intact EIN<sup>sc</sup> for the whole HPr<sup>sc</sup> is 12 Ī¼M,
we suggest that the assayed peptides might be considered as good hit
compounds for inhibiting the interaction between HPr<sup>sc</sup> and
EIN<sup>sc</sup>
The Monomeric Species of the Regulatory Domain of Tyrosine Hydroxylase Has a Low Conformational Stability
Tyrosine
hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine
to form 3,4-dihydroxyphenylalanine, the first step in the synthesis
of catecholamine neurotransmitters. The protein contains a 159-residue
regulatory domain (RD) at its N-terminus that forms dimers in solution;
the N-terminal region of RDTyrH (residues 1ā71) is absent in
the solution structure of the domain. We have characterized the conformational
stability of two species of RDTyrH (one containing the N-terminal
region and another lacking the first 64 residues) to clarify how that
N-terminal region modulates the conformational stability of RD. Under
the conditions used in this study, the RD species lacking the first
64 residues is a monomer at pH 7.0, with a small conformational stability
at 25 Ā°C (4.7 Ā± 0.8 kcal mol<sup>ā1</sup>). On the
other hand, the entire RDTyrH is dimeric at physiological pH, with
an estimated dissociation constant of 1.6 Ī¼M, as determined
by zonal gel filtration chromatography; dimer dissociation was spectroscopically
silent to circular dichroism but not to fluoresecence. Both RD species
were disordered below physiological pH, but the acquisition of secondary
native-like structure occurs at pHs lower than those measured for
the attainment of tertiary native- and compactness-like arrangements
The PipX Protein, When Not Bound to Its Targets, Has Its Signaling CāTerminal Helix in a Flexed Conformation
PipX,
an 89-residue protein, acts as a coactivator of the global
nitrogen regulator NtcA in cyanobacteria. NtcAāPipX interactions
are regulated by 2-oxoglutarate (2-OG), an inverse indicator of the
ammonia abundance, and by P<sub>II</sub>, a protein that binds to
PipX at low 2-OG concentrations. The structure of PipX, when bound
to NtcA or P<sub>II</sub>, consists of an N-terminal, five-stranded
Ī²-sheet (conforming a Tudor-like domain), and two long Ī±-helices.
These helices adopt either a <i>flexed conformation</i>,
where they are in close contact and in an antiparallel mutual orientation,
also packing against the Ī²-sheet, or an <i>open conformation</i> (observed only in the P<sub>II</sub>āPipX complex) where
the last Ī±-helix moves apart from the rest of the protein. The
aim of this work was to study the structure and dynamics of isolated
PipX in solution by NMR. The backbone chemical shifts, the hydrogen-exchange,
and the NOE patterns indicated that the isolated, monomeric PipX structure
was formed by an N-terminal five-stranded Ī²-sheet and two C-terminal
Ī±-helices. Furthermore, the observed NOEs between the two helices,
and of Ī±-helix2 with Ī²-strand2 suggested that PipX adopted
a <i>flexed conformation</i>. The Ī²-strands 1 and
5 were highly flexible, as shown by the lack of interstrand backboneābackbone
NOEs; in addition, the <sup>15</sup>N-dynamics indicated that the
C terminus of Ī²-strand4 and the following Ī²-turn (Phe42-Thr47),
and the C-cap of Ī±-helix1 (Arg70-Asn71) were particularly mobile.
These two regions could act as hinges, allowing PipX to interact with
its partners, including PlmA in the newly recognized P<sub>II</sub>āPipXāPlmA ternary complex
Human COA3 Is an Oligomeric Highly Flexible Protein in Solution
The
assembly of the protein complex of cytochrome <i>c</i> oxidase
(COX), which participates in the mitochondrial respiratory
chain, requires a large number of accessory proteins (the so-called
assembly factors). Human COX assembly factor 3 (hCOA3), also known
as MITRAC12 or coiled-coil domain-containing protein 56 (CCDC56),
interacts with the first subunit protein of COX to form its catalytic
core and promotes its assemblage with the other units. Therefore,
hCOA3 is involved in COX biogenesis in humans and can be exploited
as a drug target in patients with mitochondrial dysfunctions. However,
to be considered a molecular target, its structure and conformational
stability must first be elucidated. We have embarked on the description
of such features by using spectroscopic and hydrodynamic techniques,
in aqueous solution and in the presence of detergents, together with
computational methods. Our results show that hCOA3 is an oligomeric
protein, forming aggregates of different molecular masses in aqueous
solution. Moreover, on the basis of fluorescence and circular dichroism
results, the protein has (i) its unique tryptophan partially shielded
from solvent and (ii) a relatively high percentage of secondary structure.
However, this structure is highly flexible and does not involve hydrogen
bonding. Experiments in the presence of detergents suggest a slightly
higher content of nonrigid helical structure. Theoretical results,
based on studies of the primary structure of the protein, further
support the idea that hCOA3 is a disordered protein. We suggest that
the flexibility of hCOA3 is crucial for its interaction with other
proteins to favor mitochondrial protein translocation and assembly
of proteins involved in the respiratory chain
Mutation of Ser-50 and Cys-66 in Snapin Modulates Protein Structure and Stability
Snapin is a 15 kDa protein present in neuronal and non-neuronal
cells that has been implicated in the regulation of exocytosis and
endocytosis. Protein kinase A (PKA) phosphorylates Snapin at Ser-50,
modulating its function. Likewise, mutation of Cys-66, which mediates
protein dimerization, impairs its cellular activity. Here, we have
investigated the impact of mutating these two positions on protein
oligomerization, structure, and thermal stability, along with the
interaction with SNARE proteins. We found that recombinant purified
Snapin in solution appears mainly as dimers in equilibrium with tetramers.
The protein exhibits modest secondary structure elements and notable
thermal stability. Mutation of Cys-66 to Ser abolished subunit dimerization,
but not higher-order oligomers. This mutant augmented the presence
of Ī±-helical structure and slightly increased the protein thermal
stability. Similarly, the S50A mutant, mimicking the unphosphorylated
protein, also exhibited a higher helical secondary structure content
than the wild type, along with greater thermal stability. In contrast,
replacement of Ser-50 with Asp (S50D), emulating the protein-phosphorylated
state, produced a loss of Ī±-helical structure, concomitant with
a decrease in protein thermal stability. In vitro, the wild type and
mutants weakly interacted with SNAP-25 and the reconstituted SNARE
complex, although S50D exhibited the strongest binding to the SNARE
complex, consistent with the observed higher cellular activity of
PKA-phosphorylated Snapin. Our observations suggest that the stronger
binding of S50D to SNAREs might be due to a destabilization of tetrameric
assemblies of Snapin that favor the interaction of protein dimers
with the SNARE proteins. Therefore, phosphorylation of Ser-50 has
an important impact on the protein structure and stability that appears
to underlie its functional modulation