Mary Prorok

Thermodynamics of Binding of Calcium, Magnesium, and Zinc to theN-Methyl-d-aspartate Receptor Ion Channel Peptidic Inhibitors, Conantokin-G and Conantokin-T

The binding isotherms of the divalent metal cations, Ca2+, Mg2+, and Zn2+, to the synthetic γ-carboxyglutamic acid-containing neuroactive peptides, conantokin-G (con-G) and conantokin-T (con-T), have been determined by isothermal titration calorimetry (ITC) at 25 °C and pH 6.5. We have previously shown by potentiometric measurements that con-G contains 2–3 equivalent Ca2+ sites with an average K d value of 2800 μm. With Mg2+ as the ligand, two separate exothermic sites are obtained by ITC, one of K d = 46 μmand another of K d = 311 μm. Much tighter binding of Zn2+ is observed for these latter two sites (K d values = 0.2 μm and 1.1 μm), and a third considerably weaker binding site is observed, characterized by a K d value of 286 μm and an endothermic enthalpy of binding. con-T possesses a single exothermic tight binding site for Ca2+, Mg2+, and Zn2+, with K d values of 428 μm, 10.2 μm, and 0.5 μm, respectively. Again, in the case of con-T, a weak (K d = 410 μm) endothermic binding site is observed for Zn2+. The binding of these cations to con-G and con-T result in an increase in the α-helical content of the peptides. However, this helix is somewhat destabilized in both cases by binding of Zn2+ to its weakest site. Since the differences observed in binding affinities of these three cations to the peptides are substantially greater than their comparative K d values to malonate, we conclude that the structure of the peptide and, most likely, the steric and geometric properties imposed on the cation site as a result of peptide folding greatly influence the strength of the interaction of cations with con-G and con-T. Further, since the Zn2+ concentrations released in the synaptic cleft during excitatory synaptic activity are sufficiently high relative to the K d of Zn2+ for con-G and con-T, this cation along with Mg2+, are most likely the most significant metal ion ligands of these peptides in neuronal cells.

Glycosylation of Asparagine-28 of Recombinant Staphylokinase with High-Mannose-type Oligosaccharides Results in a Protein with Highly Attenuated Plasminogen Activator Activity

The properties of recombinant staphylokinase (SakSTAR) expressed in Pichia pastoris cells have been determined. The single consensus N-linked oligosaccharide linkage site in SakSTAR (at Asn28 of the mature protein) was occupied in approximately 50% of the expressed protein with high-mannose-type oligosaccharides. The majority of these glycans ranged in polymerization state from Man8GlcNAc2to Man14GlcNAc2, with the predominant species being Man10GlcNAc2 and Man11GlcNAc2. Glycosylated SakSTAR (SakSTARg) did not differ from its aglycosyl form in its aggregation state in solution, its thermal denaturation properties, its ability to form a complex with human plasmin (hPm), the amidolytic properties of the respective SakSTAR-hPm complexes, or its ability to liberate the amino-terminal decapeptide required for formation of a functional SakSTAR-hPm plasminogen activator complex. However, this latter complex with SakSTARg showed a greatly reduced ability to activate human plasminogen (hPg) as compared with the same complex with the aglycosyl form of SakSTAR. We conclude that glycosylation at Asn28 does not affect the structural properties of SakSTAR or its ability to participate in the formation of an active enzymatic complex with hPm, but it is detrimental to the ability of the SakSTAR-hPm complex to serve as a hPg activator. This is likely due to restricted access of hPg to the active site of the SakSTARg-hPm complex.

Structure-function relationships of the NMDA receptor antagonist peptide, conantokin-R.

Conantokin‐R (con‐R) is a γ‐carboxyglutamate‐containing 27‐residue neuroactive peptide present in the venom of Conus radiatus, and acts as a non‐competitive antagonist of the N‐methyl‐D‐aspartate (NMDA) receptor. This peptide features a single disulfide bond, a type of structural element found in most classes of conotoxins, but not in other conantokins. The NMDA receptor antagonist activity of chemically synthesized con‐R was determined through an assay involving inhibition of the spermine‐enhanced binding of the NMDA receptor channel blocker, [3H]MK‐801, to rat brain membranes, and yielded an IC50 of 93 nM. This value represents a 2–5 times better potency than con‐G or con‐T, the other two characterized conantokins. Circular dichroism (CD) analysis of the metal‐free form of con‐R is indicative of a low α‐helical content. There is an increase in α‐helicity upon the addition of divalent cations, such as Ca2+, Mg2+, or Zn2+. Isothermal titration calorimetry experiments showed one detectable Mg2+ binding site with a K d of 6.5 μM, and two binding sites for Zn2+, with K d values of 150 nM and 170 μM. Residue‐specific information of the conformational state of con‐R was obtained by two‐dimensional 1H‐NMR. Analyses of the α‐proton chemical shifts, NOE patterns, and hydrogen exchange rates of the peptide indicated an α‐helical conformation for residues 1–19. Synthetic con‐R‐derived peptide variants, containing deletions of 7 and 10 amino acid residues from the carboxy‐terminus of the wild‐type peptide, displayed unaltered cation binding and NMDA receptor antagonist properties. The α‐helical secondary structures of the two truncation peptides were more stable than full‐length con‐R, as evidenced by CD measurements and reduced backbone hydrogen exchange rates. These results provide experimental evidence that the structural elements common to the three conantokins thus far identified are the primary determinants for receptor function and cation binding/secondary structure stability.

The NMR solution structure of the NMDA receptor antagonist, conantokin‐T, in the absence of divalent metal ions

The solution conformation of conantokin‐T, a Gla‐containing 21‐residue peptide, (G1EγγY5QKMLγ10NLRγA15EVKKN20A‐amide), in the absence of divalent metal ions, was studied by use of two‐dimensional proton NMR spectroscopy. The peptide is helical from the N‐terminus to the C‐terminus, as defined by upfield‐shifted α‐proton resonances and by characteristic NOE connectivities. Extensive interactions among the amino acid side‐chains were identified from the NOESY spectra of this peptide in a buffered aqueous solution. Four hydrophobic residues Tyr5, Met8, Leu9, and Leu12 contact one another in a stable cluster, even in the presence of 6 M urea. The solution structure of conantokin‐T is a well‐defined α‐helix, having RMSD values for the backbone and all heavy atoms of 0.40 Å and 0.77 Å, respectively. Potential repulsion between the negatively‐charged side chains of Gla10 and Gla14 is minimized by a Gln6‐Gla10 hydrogen bond and by an Arg13‐Gla14 ion‐pair interaction. The C‐terminal amide and the Asn20 side‐chain amide both interact with the backbone and minimize fraying at the C‐terminal end of the α‐helix. This study provides a basis to evaluate the changes in peptide conformation concomitant upon the binding of divalent metal ions. In addition, this investigation demonstrates that apo‐conantokin‐T has almost all of the favorable interactions that are known to contribute to helical stabilization in proteins and monomeric helices.

Structure-Function Relationships of the NMDA Receptor Antagonist Conantokin Peptides

The three members of the conantokin peptide family identified to date are conantokin(con)-G, -T and -R. Their defining attributes include a high relative content of gamma-carboxyglutamic acid (Gla), N-terminal sequence identity, as well as considerable overall sequence homology, and antagonism of the N-methyl-D-aspartate receptor (NMDAR). As promising templates for the design of neuroprotective agents, a thorough evaluation of structure-function relationships in these peptides will be invaluable in aiding rational drug modeling. To this end, a comprehensive assessment of the contributions of individual residues to conantokin structure and function is required. The current review summarizes recent efforts in this area, and also includes the effects of peptide length, as well as structural-stabilization and -destabilization on the structural and inhibitory profiles of an extensive panel ofconantokin derivatives.

N-methyl-d-aspartate receptor inhibition by an apolipoprotein E-derived peptide relies on low-density lipoprotein receptor-associated protein

The effects of a synthetic apoE peptide, viz., residues 133-149 (apoE[133-149]), a mimetic that comprises the apoE receptor binding domain, on N-methyl-D-aspartate (NMDA)/glycine-induced ion flow through NMDA receptor (NMDAR) channels, have been investigated. The activity of apoE[133-149] was found to depend on the low-density lipoprotein receptor-related protein (LRP). Competition experiments with receptor-associated protein (RAP) and activated alpha(2)-macroglobulin (alpha(2)M*), two proteins that compete for apoE binding to LRP, demonstrate that apoE[133-149] inhibition of NMDAR function is mediated at a locus in LRP that overlaps with the binding sites of RAP and alpha(2)M*. A coreceptor of LRP, cell surface heparin sulfate proteoglycan, did not function in this system. Additional electrophysiology experiments demonstrated that the inhibitory potency of apoE[133-149] was threefold greater for NMDAR-transfected wild-type Chinese hamster ovary (CHO) cells compared with NMDAR-transfected CHO cells deficient in LRP. Studies with truncation and replacement variants of the apoE peptide demonstrated that the NMDAR inhibitory properties of these peptides correlate with their binding affinities for LRP. These novel results indicate that apoE functions as an inhibitor of NMDAR ion channels indirectly via LRP, and are suggestive of a participatory role for LRP in NMDAR-based neuropathies.

Sequence Requirements for theN-Methyl-d-aspartate Receptor Antagonist Activity of Conantokin-R

Conantokin-R (con-R), a γ-carboxyglutamate-containing 27-residue peptide, is a natural peptide inhibitor of the N-methyl-d-aspartate (NMDA) subtype glutamate receptor. Synthetic analogs of con-R were generated to evaluate the importance of the individual structural elements of this peptide in its NMDA receptor antagonist activity, measured by inhibition of the spermine-enhanced binding of the NMDA receptor-specific channel blocker, [3H]MK-801, to rat brain membranes. Progressive C-terminal truncations of the 27-residue peptide revealed stages of severe activity loss. These occurred at con-R[1–11] and con-R[1–7], corresponding to the deletions of Leu12–Pro27 and Met8–Pro27 respectively. A second set of analogs featured single Ala substitutions in the fully active con-R[1–17] fragment. The replacement of Met8 and Leu12 by Ala resulted in approximate 20- and 55-fold decreases of inhibitor potency, respectively. In addition to these two residues, the only other positions where a single Ala substitution led to substantial losses (from 11-fold to >1000-fold) of activity were those of the first five N-terminal amino acids. Based on the above findings, the binding epitope of con-R was localized to the N-terminal turn of the helix and other residues on one face along two subsequent turns. This contribution pattern of the side chains in activity closely resembles the results obtained with another member of this peptide family, conantokin-T. The secondary structure and metal ion binding properties of the con-R variants were also evaluated using circular dichroism spectroscopy. Divalent cation-dependent increases of α-helix content were observed in most analogs. However, analogs with replacement of Gla11 and Gla15, as well as truncation fragments shorter than 15 residues, lost the ability to be stabilized by metal ions. These results confirmed the location of the primary divalent cation binding locus at Gla11 and Gla15. Additional interactions were indicated by the reduced α-helix stability in the Ala analogs of Gla4, Lys7, and Arg14.

NMDA-receptor antagonist requirements in conantokin-G

A series of variants of the neuroactive 17‐residue γ‐carboxyglutamate‐(Gla)‐containing polypeptide, conantokin‐G (con‐G), were synthesized with the intention of determining those features that were important for its N‐methyl‐d‐aspartate (NMDA) receptor‐targeted antagonist activity and for adoption of its divalent cation‐dependent α‐helical conformation. Employing the binding of [3H]dizolcipine (MK‐801) as an assay for open receptor ion channels in rat brain membranes, which displays inhibition by con‐G (IC50=0.48 μM), it was found that replacement by an Ala residue of Gla4 led to complete inactivation of the peptide, whereas a similar replacement of Gla3 resulted in a 20‐fold decreased potency. Ala substitutions for Gla10 and Gla14 did not substantially affect [3H]MK‐801 binding. This same substitution at Gla7 appeared to slightly enhance binding. Ala replacements of non‐Gla residues demonstrated that four of them, viz. Glu2, Leu5, Gln9, and Ile12, possessed at least 200‐fold decreases in inhibitory potency, whereas similar replacements at Gly1, Leu11, and Arg13 resulted in peptides with 8‐ to 12‐fold increases in the IC50 values. The remaining amino acid residues tested in the single Ala replacement series showed no significant changes in the inhibitory characteristics of wild‐type con‐G. Additional studies with carboxyl‐terminal truncated peptides revealed that the carboxyl‐terminal 4 amino acids were unimportant for this activity. There was no strict correlation of inhibition of [3H]MK‐801 binding with the ability of these peptides to form cation‐dependent α‐helices. Peptides with notably low α‐helical content in the presence of these cations were lacking at least one, or both, of Gla10 and Gla14. Con‐G[Gla3,4,7,10,14E] and con‐G[Gla7,10,14E] were the only peptides that remained in a completely random conformation upon metal ion addition.

The Molecular Basis of Conantokin Antagonism of NMDA Receptor Function

The N-methyl-D-aspartate receptor (NMDAR), a subtype of ionotropic glutamate receptor, has been implicated in a host of chronic and acute neurological disorders. Accordingly, much emphasis has been placed on the development of safe and effective therapeutic agents that specifically antagonize this target. The conantokins are a class of small, naturally occurring peptides that inhibit ion flow through the NMDAR. Some conantokins demonstrate receptor subunit selectivity, a pharmacological attribute of emerging importance in the search for suitable drug candidates. The current review summarizes the NMDAR inhibitory properties of the conantokins, including structure-function relationships and mechanism of action. This information is fundamental to the rational design of suitable agents that can effectively treat pathophysiologies linked to NMDAR dysfunction.

Subtype-selective antagonism of N-methyl-D-aspartate receptor ion channels by synthetic conantokin peptides

Conantokin-G (con-G), conantokin-T (con-T), a truncated conantokin-R (con-R[1-17]), that functions the same as wild-type con-R, and variant sequences of con-T, were chemically synthesized and employed to investigate their selectivities as antagonists of glutamate/glycine-evoked ion currents in human embryonic kidney-293 cells expressing various combinations of NMDA receptor (NMDAR) subunits (NR), viz., NR1a/2A, NR1a/2B, NR1b/2A and NR1b/2B. Con-G did not substantially affect ion flow into NR1a,b/NR2A-transfected cells, but potently inhibited cells expressing NR1a,b/NR2B, showing high NR2B selectivity. Con-T and con-R served as non-selective antagonists of all of four NMDAR subunit combinations. C-terminal truncation variants of the 21-residue con-T were synthesized and examined in this regard. While NMDAR ion channel antagonist activity, and the ability to adopt the Ca(2+)-induced alpha-helical conformation, diminished as a function of shortening the COOH-terminus of con-T, NMDAR subtype selectivity was enhanced in the con-T[1-11], con-T[1-9], and con-T[1-8] variants toward NR2A, NR1b, and NR1b/2A, respectively. Receptor subtype selectivity was also obtained with Met-8 sequence variants of con-T. Con-T[M8A] and con-T[M8Q] displayed selectivity with NR2B-containing subunits, while con-T[M8E] showed enhanced activity toward NR1b-containing NMDAR subtypes. Of those studied, the most highly selective variant was con-T[M8I], which showed maximal NMDAR ion channel antagonism activity toward the NR1a/2A subtype. These studies demonstrate that it is possible to engineer NMDAR subtype antagonist specificity into con-T. Since the subunit composition of the NMDAR varies temporally and spatially in developing brain and in various disease states, conantokins with high subtype selectivities are potentially valuable drugs that may be used at specific stages of disease and in selected regions of the brain.