Complex Formation between S100B Protein and the p90 Ribosomal S6 Kinase (RSK) in Malignant Melanoma Is Calcium-dependent and Inhibits Extracellular Signal-regulated Kinase (ERK)-mediated Phosphorylation of RSK

S100B is a prognostic marker for malignant melanoma. Increasing S100B levels are predictive of advancing disease stage, increased recurrence, and low overall survival in malignant melanoma patients. Using S100B overexpression and shRNA^S100B knockdown studies in melanoma cell lines, elevated S100B was found to enhance cell viability and modulate MAPK signaling by binding directly to the p90 ribosomal S6 kinase (RSK). S100B-RSK complex formation was shown to be Ca²⁺-dependent and to block ERK-dependent phosphorylation of RSK, at Thr-573, in its C-terminal kinase domain. Additionally, the overexpression of S100B sequesters RSK into the cytosol and prevents it from acting on nuclear targets. Thus, elevated S100B contributes to abnormal ERK/RSK signaling and increased cell survival in malignant melanoma.     

A Conserved Three-nucleotide Core Motif Defines Musashi RNA Binding Specificity

Musashi (MSI) family proteins control cell proliferation and differentiation in many biological systems. They are overexpressed in tumors of several origins, and their expression level correlates with poor prognosis. MSI proteins control gene expression by binding RNA and regulating its translation. They contain two RNA recognition motif (RRM) domains, which recognize a defined sequence element. The relative contribution of each nucleotide to the binding affinity and specificity is unknown. We analyzed the binding specificity of three MSI family RRM domains using a quantitative fluorescence anisotropy assay. We found that the core element driving recognition is the sequence UAG. Nucleotides outside of this motif have a limited contribution to binding free energy. For mouse MSI1, recognition is determined by the first of the two RRM domains. The second RRM adds affinity but does not contribute to binding specificity. In contrast, the recognition element for Drosophila MSI is more extensive than the mouse homolog, suggesting functional divergence. The short nature of the binding determinant suggests that protein-RNA affinity alone is insufficient to drive target selection by MSI family proteins.     

Insights into the Inhibitory Mechanisms of the Regulatory Protein IIAGlc on Melibiose Permease Activity

The phosphotransfer protein IIA^Glc of the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system plays a key role in the regulation of carbohydrate metabolism. Melibiose permease (MelB) is one among several permeases subject to IIA^Glc regulation. The regulatory mechanisms are poorly understood; in addition, thermodynamic features of IIA^Glc binding to other proteins are also unknown. Applying isothermal titration calorimetry and amine-specific cross-linking, we show that IIA^Glc directly binds to MelB of Salmonella typhimurium (MelB_St) and Escherichia coli MelB (MelB_Ec) at a stoichiometry of unity in the absence or presence of melibiose. The dissociation constant values are 3–10 μm for MelB_St and 25 μm for MelB_Ec. All of the binding is solely driven by favorable enthalpy forces. IIA^Glc binding to MelB_St in the absence or presence of melibiose yields a large negative heat capacity change; in addition, the conformational entropy is constrained upon the binding. We further found that the IIA^Glc-bound MelB_St exhibits a decreased binding affinity for melibiose or nitrophenyl-α-galactoside. It is believed that sugar binding to the permease is involved in an induced fit mechanism, and the transport process requires conformational cycling between different states. Thus, the thermodynamic data are consistent with the interpretation that IIA^Glc inhibits the induced fit process and restricts the conformational dynamics of MelB_St.     

A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily: PRODUCTION,FUNCTIONAL CHARACTERIZATION,AND STRUCTURE MODELING*

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.     

Unusual binding mode of scorpion toxin BmKTX onto potassium channels relies on its distribution of acidic residues

Besides classical scorpion toxin–potassium channel binding modes, novel modes remain unknown. Here, we report a novel binding mode of native toxin BmKTX towards Kv1.3 channel. The combined experimental and computational data indicated that BmKTX-D33H analog used the classical anti-parallel β-sheet domain as the channel-interacting interface together with the conserved channel pore-blocking Lys²⁶. However, the wild-type BmKTX was found to use Arg²³ rather than Lys²⁶ as the new pore-blocking residue, and mainly adopt the turn motif between the α-helix and antiparallel β-sheet domains to recognize K_v1.3 channel. Together, these findings not only reveal that scorpion toxin–potassium channel interaction modes are more diverse than thought, but also highlight the functional role of toxin acidic residues in mediating diverse toxin–potassium channel binding modes.     

Vasonatrin peptide stimulates both of the natriuretic peptide receptors,NPRA and NPRB

Vasonatrin peptide (VNP) is an active cardiovascular factor and a novel synthetic natriuretic peptide with unknown natriuretic peptide receptor (NPR) binding properties. We set out to design binding models of NPRA/VNP and NPRB/VNP, and then assessed their recognition and binding affinities using molecular dynamics. Molecular dynamics analysis indicated decreases in the values of Van der Waals, electrostatic energy and potential energy of NPRB/VNP compared to NPRA/VNP. There was a 25% increase in H-bond formation between VNP and NPRB. The cGMP stimulated by VNP in NPRB-transfected HEK-293 cells was 11-fold higher than that of NPRA. We therefore demonstrated that VNP binds with both NPRA and NPRB, but with a preference for NPRB.     

A starch-binding domain identified in α-amylase (AmyP) represents a new family of carbohydrate-binding modules that contribute to enzymatic hydrolysis of soluble starch

A novel starch-binding domain (SBD) that represents a new carbohydrate-binding module family (CBM69) was identified in the α-amylase (AmyP) of the recently established alpha-amylase subfamily GH13_37. The SBD and its homologues come mostly from marine bacteria, and phylogenetic analysis indicates that they are closely related to the CBM20 and CBM48 families. The SBD exhibited a binding preference toward raw rice starch, but the truncated mutant (AmyP_ΔSBD) still retained similar substrate preference. Kinetic analyses revealed that the SBD plays an important role in soluble starch hydrolysis because different catalytic efficiencies have been observed in AmyP and the AmyP_ΔSBD.     

The Ca-dependent interaction of calpastatin domain L with the C-terminal tail of the Cav1.2 channel

To demonstrate the interaction of calpastatin (CS) domain L (CS_L) with Cav1.2 channel, we investigated the binding of CS_L with various C-terminus-derived peptides at ≈ free, 100 nM, 10 μM, and 1 mM Ca²⁺ by using the GST pull-down assay method. Besides binding with the IQ motif, CS_L was also found to bind with the PreIQ motif. With increasing [Ca²⁺], the affinity of the CS_L–IQ interaction gradually decreased, and the affinity of the CS_L–PreIQ binding gradually increased. The results suggest that CS_L may bind with both the IQ and PreIQ motifs of the Cav1.2 channel in different Ca²⁺-dependent manners.