Analogues of the natural product noscapine were synthesized and their potential as antitumor agents evaluated. The discovery of a novel regioselective O-demethylation facilitated the synthesis of the potent aniline 6, which arrests mammalian cells in the G2/M phase of the cell cycle at 0.1 microM and also affects tubulin polymerization. Aniline 6 is orally bioavailable and is 250-fold more potent than noscapine in reducing cell proliferation in rapidly dividing cells.
Calmodulin is an EF-hand calcium-binding protein (148 a.a.) essential in intracellular signal transduction. Its homologous N- and C-terminal domains are separated by a linker that appears disordered in NMR studies. In a study of an N-domain fragment of Paramecium CaM (PCaM1-75), the addition of linker residues 76 to 80 (MKEQD) raised the Tm by 9 degrees C and lowered calcium binding by 0.54 kcal/mol (Sorensen et al., [Biochemistry 2002;41:15-20]), showing that these tether residues affect energetics as well as being a barrier to diffusion. To determine the individual contributions of residues 74 through 80 (RKMKEQD) to stability and calcium affinity, we compared a nested series of 7 fragments (PCaM1-74 to PCaM1-80). For the first 4, PCaM1-74 through PCaM1-77, single amino acid additions at the C-terminus corresponded to stepwise increases in thermostability and decreases in calcium affinity with a net change of 13.5 degrees C in Tm and 0.55 kcal/mol in free energy. The thermodynamic properties of fragments PCaM1-77 through PCaM1-80 were nearly identical. We concluded that the 3 basic residues in the sequence from 74 to 77 (RKMK) are critical to the increased stability and decreased calcium affinity of the longer N-domain fragments. Comparisons of NMR (HSQC) spectra of 15N-PCaM1-74 and 15N-PCaM1-80 and analysis of high-resolution structural models suggest these residues are latched to amino acids in helix A of CaM. The addition of residues E78, Q79, and D80 had a minimal effect on sites I and II, but they may contribute to the mechanism of energetic communication between the domains.
A hydrophobic core is a widely accepted determinant of protein stability. However, regulatory proteins undergoing ligand-induced conformational switching may expose interior residues to solvent and cannot afford to be extremely rigid. Optimizing the energetic balance between stability and binding is challenging. The addition of five interdomain residues to rat and Paramecium calmodulin N-domain fragments (residues 1-75) increased their thermostability by 9 degrees C and lowered their calcium affinity by a factor of 4. This demonstrates that the flexible linker regulates functional properties as well as tethering the neighboring domains and that protein stability may be increased markedly by minor modifications of the C-terminus. The sensitivity of this domain to few and conservative variations in helices A and D (D2E, S17A, T70S and M71L) is demonstrated by the rat CaM fragments having lower stability and higher calcium affinity than fragments of the same length derived from Paramecium CaM.
Calmodulin (CaM) is the major Ca2؉ sensor in eukaryotic cells. It consists of four EF-hand Ca 2؉ binding motifs, two in its N-terminal domain and two in its C-terminal domain. Through a negative feedback loop, CaM inhibits Ca 2؉ influx through N-methyl-D-aspartate-type glutamate receptors in neurons by binding to the C0 region in the cytosolic tail of the NR1 subunit. Ca 2؉ -depleted (apo)CaM is pre-associated with a variety of ion channels for fast and effective regulation of channel activities upon Ca 2؉ influx. Using the NR1 C0 region for fluorescence and circular dichroism spectroscopy studies we found that not only Ca 2؉ -saturated CaM but also apoCaM bound to NR1 C0. In vitro interaction assays showed that apoCaM also binds specifically to fulllength NR1 solubilized from rat brain and to the complete C terminus of the NR1 splice form that contains the C0 plus C2 domain. The Ca 2؉ -independent interaction of CaM was also observed with the isolated C-but not N-terminal fragment of calmodulin in the independent spectroscopic assays. Fluorescence polarization studies indicated that apoCaM associated via its C-terminal domain with NR1 C0 in an extended conformation and collapsed to adopt a more compact conformation of faster rotational mobility in its complex with NR1 C0 upon addition of Ca 2؉ . Our results indicate that apoCaM is associated with NR1 and that the complex of CaM bound to NR1 C0 undergoes a dramatic conformational change when Ca 2؉ binds to CaM. Glutamate is the prevailing excitatory neurotransmitter in the mammalian brain. The ionotropic N-methyl-D-aspartate (NMDA)1 -type glutamate receptor is a major source of Ca 2ϩ influx into neurons in the central nervous system. NMDA receptors in the cerebral cortex largely consist of two NR1 and two or three NR2A and 2B subunits, which are homologous to each other (1-4). Each subunit has an extracellular N terminus, four membrane regions (M1-4), and an intracellular C terminus. M1, M3, and M4 are transmembrane regions but M2 loops into and back out of the plasma membrane at its cytoplasmic face. The C-terminal domain of NR1 contains the 30 residue-long membrane-proximal C0 region common to all NR1 isoforms, followed in some NR1 isoforms by the C1 region (37 residues), which can be spliced in or out, and by either the C2 (38 residues) or the C2Ј (22 residues) region, which is downstream of C2 in the genome. The C2 region can be spliced out but, when present, C2Ј will not be translated because C2 carries its own stop codon. In a negative feedback loop calmodulin (CaM) inhibits Ca 2ϩ
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