Polyamine binding

Abstract: The N-methyl-D-aspartate (NMDA) receptor NR1 gene encodes RNA that is alternatively spliced to generate at least seven variants. The variants arise from splicing in or out of three exons; one encodes a 21-amino acid insert in the N-terminal domain, and two encode adjacent sequences of 37 and 38 amino acids in the C-terminal domain. Splicing out of the second C-terminal exon deletes a stop codon and results in an additional open reading frame encoding an unrelated sequence of 22 amino acids before arriving at a second stop codon. We denote the NR1 variants by the presence or absence of the three alternatively spliced exons (from 5' to 3'); thus, NR1(111) has all three exons, NR1(000) has none, and NR1(100) has only the N-terminal exon. We report here electrophysiological characterization of six splice variants of the NR1 receptor expressed in Xenopus oocytes. NR1 receptors that lacked the N-terminal exon (NR1(000), NR1(010), and NR1(011)) exhibited a relatively high affinity for NMDA (EC50 approximately 13 microM) and marked potentiation by spermine. In contrast, those receptor variants with the N-terminal insert (NR1(100), NR1(101), and NR1(111)) showed a lower agonist affinity and little or no spermine potentiation at saturating glycine. All six variants showed spermine potentiation at low glycine and inhibition by spermine at more negative potentials. Variants differing only in the C-terminal domain differed little in agonist affinity and spermine potentiation. These findings indicate that the N-terminal insert either participates in agonist and polyamine binding domains or indirectly modifies their conformations. The splice variants differed in the extent to which they could be potentiated by activators of protein kinase C (PKC) from 3- to 20-fold. Presence of the N-terminal insert and absence of the C-terminal sequences increased potentiation by PKC. These findings identify the contributions of the separate polypeptide domains to modulation by polyamines and PKC and provide further support for the concept that subunit composition determines functional properties of NMDA receptors.

Abstract: The aggregation of α-synuclein is characteristic of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. The 140-aa protein is natively unstructured; thus, ligands binding to the monomeric form are of therapeutic interest. Biogenic polyamines promote the aggregation of α-synuclein and may constitute endogenous agents modulating the pathogenesis of PD. We characterized the complexes of natural and synthetic polyamines with α-synuclein by NMR and assigned the binding site to C-terminal residues 109–140. Dissociation constants were derived from chemical shift perturbations. Greater polyamine charge (+2 → +5) correlated with increased affinity and enhancement of fibrillation, for which we propose a simple kinetic mechanism involving a dimeric nucleation center. According to the analysis, polyamines increase the extent of nucleation by ~$10^4$ and the rate of monomer addition ~40-fold. Significant secondary structure is not induced in monomeric α-synuclein by polyamines at 15°C. Instead, NMR reveals changes in a region (aa 22–93) far removed from the polyamine binding site and presumed to adopt the β-sheet conformation characteristic of fibrillar α-synuclein. We conclude that the C-terminal domain acts as a regulator of α-synuclein aggregation.

Abstract: The endogenous polyamine spermine has multiple effects on the N-methyl-D-aspartate (NMDA) receptor. These include an increase in the magnitude of NMDA-induced whole-cell currents that is seen in the presence of saturating concentrations of glycine ("glycine-independent" stimulation), an increase in the affinity of the receptor for glycine ("glycine-dependent" stimulation), and voltage-dependent inhibition. Although many of the properties of native NMDA receptors are seen with homomeric NR1 receptors expressed in Xenopus oocytes, we have found that the effects of spermine are differentially regulated by NR2 subunits in heteromeric NR1/NR2 receptors. Glycine-independent stimulation by spermine occurred at homomeric NR1A receptors, which lack the amino-terminal insert in NR1, and at heteromeric NR1A/NR2B receptors but not at heteromeric NR1A/NR2A or NR1A/NR2C receptors. Glycine-independent stimulation was not seen at homomeric NR1B receptors, which include the amino-terminal insert in NR1, or at heteromeric receptors containing NR1B. Thus, glycine-independent stimulation by polyamines requires the presence of an NR1 variant, such as NR1A, that lacks the amino-terminal insert, but the manifestation of the stimulatory effect is controlled by the type of NR2 subunit present in a heteromeric complex. Glycine-dependent stimulation was seen at NR1A/NR2A and NR1A/NR2B receptors and may therefore involve a second polyamine binding site distinct from that which produces glycine-independent stimulation. The voltage-dependent inhibitory effect of spermine, which is more pronounced at hyperpolarized membrane potentials, occurred with similar magnitudes at NR1A/NR2A and NR1A/NR2B receptors but was absent at NR1A/NR2C receptors. Thus, NR2 subunits control both the stimulatory and inhibitory effects of spermine at NMDA receptors. Stimulation but not inhibition by spermine was seen at NR1A/NR2B receptors in the presence of extracellular Mg2+. Stimulation, seen in the presence of physiological concentrations of Ca2+ and Mg2+, may be the predominant effect of polyamines at NMDA receptors in the intact nervous system.

Abstract: Binding constants and binding site sizes for the interactions of the polyamines spermine (+4), spermidine (+3), and putrecine (+2) with helical DNA have been determined as a function of ionic conditions and temperature by equilibrium dialysis using 14C-labeled polyamines. In addition, competition equilibrium dialysis has been used to determine binding parameters for the divalent cations putrescine and Mg2+ from the competitive effect of these ions on the binding of spermine or spermidine. In all cases, the logarithm of the binding constant (log Kobs) varies linearly with the logarithm of the monovalent salt concentration; the slopes d log Kobs/d log[NaCl] are proportional to the valence of the ligand, and values of the extrapolated binding constants at 1M NaCl obtained from the intercepts are small (of order 1–10M−1). In those cases examined, Kobs is insensitive to temperature; the free energy of binding is predominantly entropic. Consequently, polymines as DNA-binding ligands behave analogously to the oligolysines investigated previously [cf. Record, Lohman & de Haseth (1976) J. Mol. Biol.107, 145–158; Lohman, de Haseth & Record (1980) Biochemistry19, 3522–3530]. The interactions of these oligocations with DNA are predominantly electrostatic and are driven by the release of thermodynamically bound electrolyte ions from the vicinity of the DNA. The extent to which these oligocations are localized at individual phosphate binding sites or delocalized on the DNA molecule is currently not known.