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^sc) against HPr of the same organism (HPr^sc), by using nuclear magnetic resonance and isothermal titration calorimetry techniques. Furthermore, we measured the affinity of EIN^sc for (i) a peptide derived from HPr^sc, 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>, 433–438; Mukhija, S., and Erni, B. (1997) <i>Mol. Microbiol. 25</i>, 1159–1166]. The affinities were in the range of ∼10 μM, being slightly higher for the binding of EIN^sc with peptides derived from HPr^sc, phage display, or combinatorial chemistry (<i>K</i>_D ∼ 5 μM). Because the affinity of intact EIN^sc for the whole HPr^sc is 12 μM, we suggest that the assayed peptides might be considered as good hit compounds for inhibiting the interaction between HPr^sc and EIN^sc

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 1–71) 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 °C (4.7 ± 0.8 kcal mol^–1). On the other hand, the entire RDTyrH is dimeric at physiological pH, with an estimated dissociation constant of 1.6 μM, 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 C‑Terminal Helix in a Flexed Conformation

PipX, an 89-residue protein, acts as a coactivator of the global nitrogen regulator NtcA in cyanobacteria. NtcA–PipX interactions are regulated by 2-oxoglutarate (2-OG), an inverse indicator of the ammonia abundance, and by P_II, a protein that binds to PipX at low 2-OG concentrations. The structure of PipX, when bound to NtcA or P_II, consists of an N-terminal, five-stranded β-sheet (conforming a Tudor-like domain), and two long α-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 β-sheet, or an <i>open conformation</i> (observed only in the P_II–PipX complex) where the last α-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 β-sheet and two C-terminal α-helices. Furthermore, the observed NOEs between the two helices, and of α-helix2 with β-strand2 suggested that PipX adopted a <i>flexed conformation</i>. The β-strands 1 and 5 were highly flexible, as shown by the lack of interstrand backbone–backbone NOEs; in addition, the ¹⁵N-dynamics indicated that the C terminus of β-strand4 and the following β-turn (Phe42-Thr47), and the C-cap of α-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_II–PipX–PlmA 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 α-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 α-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