Specific determinants of conantokins that dictate their selectivity for the NR2B subunit of N-methyl-D-aspartate receptors.

Conantokins are naturally-occurring small peptide antagonists of ion flow through NMDA/glycine activated-N-methyl-d-aspartate receptor (NMDAR) ion channels. One member of the conantokin family, conantokin (con)-G, a 17-residue peptide, is selective for NMDARs containing the N-methyl-d-aspartate receptor subunit 2 B (NR2B), whereas the homologous peptides, con-T and con-R, show broader selectivity for NR2 subunits. In this study, con-G, con-R, and con-T variants were chemically synthesized and employed to investigate their subunit selectivities as inhibitors of agonist-evoked ion currents in human embryonic kidney-293 (HEK-293) cells expressing various combinations of NMDAR subunits that contain NR1a or NR1b combined with NR2A or NR2B. Using truncation and point mutants, as well as chimeric conantokins, we determined that the N-terminus of con-G contains all the determinants for NR2B selectivity. With this information, a large number of (con) variants were synthesized and used to establish minimal sequence determinants for selectivity. Tyr at position 5 broadens the NR2 selectivity, and recovery of NR2B selectivity in Tyr5 peptides was achieved by incorporating Ala or Gly at position 8. NR2B selectivity in con-R can be conferred through deletion of the Ala at position 10, thereby shifting the γ-carboxyglutamate (Gla) from position 11 to position 10, where a Gla naturally occurs in con-G and con-T. The nature of the amino acid at position 6 is also linked to subunit selectivity. Our studies suggest that the molecular determinants of conantokins that dictate NMDAR subunit selectivity are housed in specific residues of the N-termini of these peptides. Thus, it is possible to engineer desired NMDAR functional properties into conantokin-based peptides.

Direct binding properties of conantokins to native N-methyl-d-aspartate receptors.

Conantokin-G (con-G) is a small, gamma-carboxyglutamic acid (Gla)-containing peptide that functions neurophysiologically by inhibiting the N-methyl-d-aspartate receptor (NMDAR). In the current study, the receptor binding properties of an alanine-rich, Gla-deficient con-G variant, Ala-con-G, were assessed following tracer radioiodination with 125I. Direct binding experiments with [125I]Ala-con-G yielded a single site defined by a Kd value of 516 +/- 120 nm. Displacement of [125I]Ala-con-G binding by Ala-con-G resulted in 100% displacement with an IC50 value of 564 +/- 33 nm, while heterologous displacement by con-G[S16Y], con-G, con-T, and con-R[1-17] yielded IC50 values in the range of 15-45 microm. No displacement was observed with d-gamma-con-G or con-G[L5A], analogs that are inactive at NMDARs. Specific [125I]Ala-con-G binding was displaced by NMDA and 2-amino-5-phosphopentanoic acid in a dose-dependent manner, suggesting an interaction at the glutamate binding site. The direct binding of [125I]Ala-con-G to adult rat brain sections revealed an anatomical distribution of binding sites in all regions known to contain the NR2B subunit of the NMDAR. These results constitute the only known demonstration of the direct binding of a radiolabeled conantokin to the NMDARs present in rat brain membrane preparations and rat brain sections, and suggest that radiolabeled Ala-con-G, and similar conantokin derivatives, may find utility as probes of NMDARs in a variety of systems.

Kinetic and mechanistic characterization of NMDA receptor antagonism by replacement and truncation variants of the conantokin peptides.

The characterization of conantokin-T (con-T), conantokin-R (con-R), and variants thereof, using the whole-cell patch clamp technique, was undertaken to evaluate the contribution of various residues towards the onset and recovery of N-methyl-D-aspartate (NMDA) receptor inhibition in cultured embryonic murine hippocampal neurons. The results obtained indicate that the two most C-terminal gamma-carboxyglutamic acid (Gla) residues of the conantokins, while not essential for activity, provided for more tenacious binding to the receptor. Specifically, con-T[gamma10K/gamma14K] and con-R[gamma11A/gamma15A] displayed 5.6- and 8.4-fold decreases in tau(off), respectively, compared to the parent peptides. For the truncated con-T variants, con-T[1-9/Q6G], and a sarcosine (Src)-containing species, con-T[1-9/G1Src/Q6G], the tau(off) was over 80- and 40-fold faster, respectively, compared to con-T. For the latter peptide, the coapplication of 300 microM spermine enhanced the onset rate constant from 3.1x10(3)M(-1) x s(-1) to 12.6x10(3)M(-1) x s(-1). From analysis of equilibrium dose-inhibition curves using the Cheng-Prusoff equation, a K(i) value of 1.1 microM for the peptide was obtained. Con-T[1-9/G1Src/Q6G] demonstrated an apparent competitive mode of inhibition relative to NMDA. Schild analysis of the data yielded an equilibrium dissociation constant of 2.4 microM for the interaction of con-T[1-9/G1Src/Q6G] with the receptor.

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.

The amino acid residue at sequence position 5 in the conantokin peptides partially governs subunit-selective antagonism of recombinant N-methyl-D-aspartate receptors.

Whole cell voltage clamp recordings were performed to assess the ability of conantokin-G (con-G), conantokin-T (con-T), and a 17-residue truncated form of conantokin-R (con-R[1-17]) to inhibit N-methyl-d-aspartate (NMDA)-evoked currents in human embryonic kidney 293 cells transiently expressing various combinations of NR1a, NR1b, NR2A, and NR2B receptor subunits. Con-T and con-R[1-17] attenuated ion currents in cells expressing NR1a/NR2A or NR1a/NR2B. Con-G did not affect NMDA-evoked ionic currents in cells expressing NR1a/NR2A, but it showed inhibitory activity in cells expressing NR1a/NR2B receptors and the triheteromeric combination of NR1a/NR2A/NR2B. An Ala-rich con-G analog, con-G[Q6G/gamma7K/N8A/gamma10A/gamma14A/K15A/S16A/N17A] (Ala/con-G, where gamma is Gla), in which all nonessential amino acids were altered to Ala residues, manifested subunit specificity similar to that of con-G, suggesting that the replaced residues are not responsible for selectivity in the con-G framework. A sarcosine-containing con-T truncation analog, con-T[1-9/G1Src/Q6G], inhibited currents in NR1a/NR2A and NR1a/NR2B receptors, eliminating residues 10-21 as mediators of the broad subunit selectivity of con-T. In contrast to the null effects of con-G and Ala/con-G at a NR1a/NR2A-containing receptor, some inhibition ( approximately 40%) of NMDA-evoked currents was effected by these peptides in cells expressing NR1b/NR2A. This finding suggests that the presence of exon 5 in NR1b plays a role in the activity of the conantokins. Analysis of various conantokin analogs demonstrated that Leu(5) of con-G is an important determinant of conantokin selectivity. Taken as a whole, these results suggest that the important molecular determinants on conantokins responsible for NMDA receptor activity and specificity are discretely housed in specific residues of these peptides, thus allowing molecular manipulation of the NMDA receptor inhibitory properties of the conantokins.

Amino acid determinants for NMDA receptor inhibition by conantokin-T.

Several derivatives of conantokin-T (con-T), a naturally occurring, gamma-carboxyglutamate (Gla)-containing peptide with NMDA receptor (NMDAR) antagonist properties, were synthesized and evaluated for their ability to displace [(3)H]MK-801 from adult rat forebrain membranes. Analyses of progressive C-terminal truncation analogs of the parent 21-mer revealed gradual losses in activity with decreased chain length. In this series, con-T[1-8] was identified as the shortest variant capable of manifesting inhibitory activity (< 1% of the parent peptide). Ala substitution studies of individual residues identified Gly1, Gla3, Met8 and Leu12 as important for activity, while Glu2, Gla4 and Tyr5 were shown to be essential in this regard. The effect of side-chain length and charge in the N-terminal region was probed by single amino acid replacements. No correlation was observed between potencies and circular dichroism-derived helical contents of the con-T derivatives. Further elaboration of structure-function relationships in con-T was effected through the design and synthesis of helically constrained and destabilized analogs. The results of the current study were compared with those of a previous investigation on con-G, a related conantokin. Substantial differences in activity requirements were noted between the peptides, particularly in the C-terminal regions. Chimeras of con-T and con-G were generated and revealed virtually no interchangeability of residues between these two peptides. Finally, single amino acid substitutions that resulted in analogs with enhanced inhibitory properties were combined to yield superior conantokin-based NMDAR inhibitors.

Sequence requirements for the N-methyl-D-aspartate receptor antagonist activity of conantokin-R.

Conantokin-R (con-R), a gamma-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, [(3)H]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 Leu(12)-Pro(27) and Met(8)-Pro(27) respectively. A second set of analogs featured single Ala substitutions in the fully active con-R[1-17] fragment. The replacement of Met(8) and Leu(12) 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 alpha-helix content were observed in most analogs. However, analogs with replacement of Gla(11) and Gla(15), 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 Gla(11) and Gla(15). Additional interactions were indicated by the reduced alpha-helix stability in the Ala analogs of Gla(4), Lys(7), and Arg(14).

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

Conantokin-R (con-R) is a gamma-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, [(3)H]MK-801, to rat brain membranes, and yielded an IC(50) 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 alpha-helical content. There is an increase in alpha-helicity upon the addition of divalent cations, such as Ca(2+), Mg(2+), or Zn(2+). Isothermal titration calorimetry experiments showed one detectable Mg(2+) binding site with a K(d) of 6.5 microM, and two binding sites for Zn(2+), with K(d) values of 150 nM and 170 microM. Residue-specific information of the conformational state of con-R was obtained by two-dimensional (1)H-NMR. Analyses of the alpha-proton chemical shifts, NOE patterns, and hydrogen exchange rates of the peptide indicated an alpha-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 alpha-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.

An internal histidine residue from the bacterial surface protein, PAM, mediates its binding to the kringle-2 domain of human plasminogen.

The determinants of binding of a peptide lacking C-termini-exposed lysine residues to a kringle domain were investigated using an up-regulated lysine binding kringle (K2Pg[C4G/E56D/K72Y]) of plasminogen and a peptide (a1-PAM) with a sequence derived from a surface-exposed M-like streptococcal protein. Significant kringle-induced chemical shifts in a His side-chain of a1-PAM were revealed by two-dimensional NMR. Further studies using isothermal titration calorimetry (ITC) provided support for the involvement of His12 in the peptide/ protein complex. In an effort to screen a1-PAM-derived truncation peptides, a combinatorial mixture, a1deltaa2-PAM[H12X] (where X=Pro, Arg, His, Trp, Lys, Ala, Phe, Asp and Gly), was analyzed using the surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI) platform. The major peptide that remained bound to the surface of the K2Pg[C4G/ E56D/K72Y]-containing chip was that containing His12, corresponding to the wild-type sequence. Minor peaks, representing binding, were obtained for Lys12-, Arg12- and Trp12-containing peptides. Individual peptides containing these amino acids were then examined using ITC and the binding constants obtained correlated with the relative strengths of binding estimated from the SELDI-based screen.

Conantokins: inhibitors of ion flow through the N-methyl-D-aspartate receptor channels.

Calcium flow through the ion channel of the N-methyl-D-aspartate receptor (NMDAR) has been implicated as contributing to a variety of neuropathologies. This receptor is a complex heteromeric oligomer consisting of different types of subunits, the nature of which governs its properties, as well as its response to a variety of agonists, antagonists, and other types of inhibitors. A new natural series of NMDAR inhibitors, the conantokins, have been shown to be present in the venoms of snails within the genus, Conus. These agents appear to function by inhibition of the spermine/spermidine stimulation of ion flow through the NMDAR channel. These small peptides (17-27 amino acid residues) are highly processed post-translationally. One such processing event is the vitamin K-dependent gamma-carboxylation of glutamate, resulting in placement of gamma-carboxyglutamic acid residues in these peptides. As a result, these peptides then possess the ability to interact with divalent metal ions and concomitantly undergo a conformational alteration. Rational drug design based on the characteristics of these promising peptides requires knowledge of their properties and the manner in which they target the NMDAR. This review summarizes current knowledge in this area.

Enhancement through mutagenesis of the binding of the isolated kringle 2 domain of human plasminogen to omega-amino acid ligands and to an internal sequence of a Streptococcal surface protein.

In the background of the recombinant K2 module of human plasminogen (K2(Pg)), a triple mutant, K2(Pg)[C4G/E56D/L72Y], was generated and expressed in Pichia pastoris cells in yields exceeding 100 mg/liter. The binding affinities of a series of lysine analogs, viz. 4-aminobutyric acid, 5-aminopentanoic acid, epsilon-aminocaproic acid, 7-aminoheptanoic acid, and t-4-aminomethylcyclohexane-1-carboxylic acid, to this mutant were measured and showed up to a 15-fold tighter interaction, as compared with wild-type K2(Pg) (K2(Pg)[C4G]). The variant, K2(Pg)[C4G/E56D], afforded up to a 4-fold increase in the binding affinity to these same ligands, whereas the K2(Pg)[C4G/L72Y] mutant decreased the same affinities up to 5-fold, as compared with K2(Pg)[C4G]. The thermal stability of K2(Pg)[C4G/E56D/L72Y] was increased by approximately 13 degrees C, as compared with K2(Pg)[C4G]. The functional consequence of up-regulating the lysine binding property of K2(Pg) was explored, as reflected by its ability to interact with an internal sequence of a plasminogen-binding protein (PAM) on the surface of group A streptococci. A 30-mer peptide of PAM, containing its K2(Pg)-specific binding region, was synthesized, and its binding to each mutant of K2(Pg) was assessed. Only a slight enhancement in peptide binding was observed for K2(Pg)[C4G/E56D], compared with K2(Pg)[C4G] (K(d) = 460 nM). A 5-fold decrease in binding affinity was observed for K2(Pg)[C4G/L72Y] (K(d) = 2200 nM). However, a 12-fold enhancement in binding to this peptide was observed for K2(Pg)[C4G/E56D/L72Y] (K(d) = 37 nM). Results of these PAM peptide binding studies parallel results of omega-amino acid binding to these K2(Pg) mutants, indicating that the high affinity PAM binding by plasminogen, mediated exclusively through K2(Pg), occurs through its lysine-binding site. This conclusion is supported by the 100-fold decrease in PAM peptide binding to K2(Pg)[C4G/E56D/L72Y] in the presence of 50 mM 6-aminohexanoic acid. Finally, a thermodynamic analysis of PAM peptide binding to each of these mutants reveals that the positions Asp(56) and Tyr(72) in the K2(Pg)[C4G/E56D/L72Y] mutant are synergistically coupled in terms of their contribution to the enhancement of PAM peptide binding.

Binding of cations to individual gamma-carboxyglutamate residues of conantokin-G and conantokin-T.

Conantokin-G (con-G) and conantokin-T (con-T) are naturally occurring gamma-carboxyglutamate (Gla)-containing peptides that interact with multivalent cations in functionally relevant manners. Selective 13C-enrichment of Cgamma and Cdelta in each of the Gla residues has allowed metal binding affinities to be measured at individual side chains. Con-T possesses two metal binding sites, one with high affinity at Gla10/Gla14 and another with weak binding at Gla3/Gla4. Con-G contains two sites of comparable low affinity for Ca2+. Analysis of the 13C line-widths of con-G in the presence of Mg2+ allowed the order of metal binding to be determined, with Gla10/Gla14 loading before the Gla3/Gla4/Gla7 cluster. While the variant peptide, apo-con-T[Lys7Gla], was shown to have a very low alpha-helical content, this peptide binds a second metal with much greater affinity than wild-type con-T. This provides additional evidence that Gla7 in con-G is primarily responsible for destabilizing the apo-form, but is an important ligand for metal chelation. The residue-specific alpha-helical stabilities of con-G and con-T in their metal-free and metal-loaded states were estimated by determining rates of proton exchange from backbone peptide bond amides with deuterium atoms from 2H20-containing solvents. For both peptides, the lifetimes of protons on several peptide bond amides increased as metals of higher affinity were bound to the peptides, with the longest half-lives found in the region of the alpha-helical turn stabilized by the Gla10/Gla14 metal coordination site. We propose that Gla10 and Gla14 constitute the primary tight metal ion binding site in both peptides. This detailed analysis with physiologically relevant metal cations is crucial for deciphering the roles of critical amino acids in the bioactivity of the conantokin peptides.

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 Man8GlcNAc2 to 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.

NMDA-receptor antagonist requirements in conantokin-G.

A series of variants of the neuroactive 17-residue gamma-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 alpha-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 microM), 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 alpha-helices. Peptides with notably low alpha-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.

Thermodynamics of binding of calcium, magnesium, and zinc to the N-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 gamma-carboxyglutamic acid-containing neuroactive peptides, conantokin-G (con-G) and conantokin-T (con-T), have been determined by isothermal titration calorimetry (ITC) at 25 degreesC and pH 6.5. We have previously shown by potentiometric measurements that con-G contains 2-3 equivalent Ca2+ sites with an average Kd value of 2800 microM. With Mg2+ as the ligand, two separate exothermic sites are obtained by ITC, one of Kd = 46 microM and another of Kd = 311 microM. Much tighter binding of Zn2+ is observed for these latter two sites (Kd values = 0.2 microM and 1.1 microM), and a third considerably weaker binding site is observed, characterized by a Kd value of 286 microM and an endothermic enthalpy of binding. con-T possesses a single exothermic tight binding site for Ca2+, Mg2+, and Zn2+, with Kd values of 428 microM, 10.2 microM, and 0.5 microM, respectively. Again, in the case of con-T, a weak (Kd = 410 microM) endothermic binding site is observed for Zn2+. The binding of these cations to con-G and con-T result in an increase in the alpha-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 Kd 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 Kd 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.

Conformational changes in conantokin-G induced upon binding of calcium and magnesium as revealed by NMR structural analysis.

The apo- and metal-bound solution conformations of synthetic conantokin-G (con-G, G1Egamma gammaL5Q gamma NQgamma 10LIRgamma K15SN-CONH2, gamma = gamma-carboxyglutamic acid), an antagonist of N-methyl-D-aspartate receptor-derived neuronal ion channels, have been examined by one- and two-dimensional 1H NMR at neutral pH. A complete structure for the Mg2+-loaded peptide was defined by use of distance geometry calculations and was found to exist as an alpha-helix that spans the entire peptide. The alpha-helical nature of Mg2+/con-G was also supported by the small values (<5.5 Hz) of the 3JHNalpha coupling constants measured for amino acid residues 3-5, 8, 9, and 11-16, and the small values (<4 ppb/K) of the temperature coefficients observed for the alphaNH protons of residues 5-17. This conformation contrasted with that obtained for apo-con-G, which was nearly structureless in solution. Docking of Mg2+ into con-G was accomplished by use of the genetic algorithm/molecular dynamics simulation method, employing the NMR-derived Mg2+-loaded structure for initial coordinates in the midpoint calculations. For the 3 Mg2+/con-G model, it was found that binding of one Mg2+ ion is stabilized by oxygen atoms from three gamma-carboxylates of Gla3, Gla4, and Gla7; another Mg2+ is coordinated by two oxygen atoms, one from each of the gamma-carboxylates of Gla7; and a third metal ion through three donor oxygen atoms of gamma-carboxylates from Gla10 and Gla14. As shown from direct metal binding measurements to mutant con-G peptides, these latter two Gla residues probably stabilized the tightest binding Mg2+ ion. Circular dichroism studies of these same peptide variants demonstrated that all Gla residues contribute to the adoption of the Mg2+-dependent alpha-helical conformation in con-G. The data obtained in this investigation provide a molecular basis for the large conformational alteration observed in apo-con-G as a result of divalent cation binding and allow assessment of the roles of individual Gla residues in defining certain of the structure-function properties of con-G.

The roles of individual gamma-carboxyglutamate residues in the solution structure and cation-dependent properties of conantokin-T.

The solution structure of the Ca2+-loaded conantokin-T (con-T), a gamma-carboxyglutamate (Gla)-containing 21-residue peptide (NH2-G1EgammagammaY5QKMLgamma10NLRgammaA15EVKKN20A-CONH2,gam ma = Gla), has been elucidated by use of distance geometry calculations with experimental distances derived from two-dimensional 1H NMR spectroscopy. An end-to-end alpha-helix was the dominant conformation in solution, similar to that of apo-con-T, except that reorientation of several side chains occurred in the Ca2+-coordinated complex. The most notable examples of this were those of Gla10 and Gla14, which were more optimally positioned for complexation with Ca2+. In addition to the stabilization offered to the alpha-helix by Ca2+ binding, hydrophobic clustering of the side chains of Tyr5, Met8, Leu9, and Leu12, and ionic interactions between Lys7 and Gla3/Gla10 and between Arg13 and Gla14, along with hydrogen bonding between Gln6 and Gla10, were among the side chain interactions likely playing a significant role in maintenance of the alpha-helical conformation. Docking of Ca2+ in the con-T structure was accomplished using genetic algorithm-molecular dynamics simulation approaches. The results showed that one Ca2+ ion is most likely coordinated by four side chain oxygen atoms, two each from Gla10 and Gla14. Another bound Ca2+ ion has as its donor sites three oxygen atoms, two from Gla3 and one from Gln6. To examine the functional roles of the individual Gla residues, a series of variant peptides have been synthesized with Ala substituted for each Gla residue, and several properties of the resulting variants have been examined. The data obtained demonstrated the importance of Gla10 and Gla14 in stabilizing binding of the highest affinity Ca2+ site and in governing the conformational change induced by Ca2+. The critical nature of Gla3 and Gla4 in inhibition of the spermine-induced potentiation of the binding of MK-801 to open ion channels of the N-methyl-D-aspartate receptor was established, as well as the role of Gla4 in stabilizing the apo-con-T alpha-helical conformation.

Serine-578 is a major phosphorylation locus in human plasma plasminogen.

It has been reported that human plasminogen (HPg) exists in plasma in a phosphorylated form. We now document that both major glycoforms of plasma HPg contain a phosphoserine residue in their latent protease chains, as revealed by quantitative protein phosphate determinations and 31P-NMR analysis. The sequence location of the phosphoserine residue was established by time-of-flight matrix-assisted laser desorption ionization with delayed extraction mass spectrometric analysis of peptides resulting from complete tryptic and cyanogen bromide digests of the latent protease chain of HPg. Confirmation of the presence of organic phosphate in the identified peptide was obtained by determination of the resulting mass shift after treatment of the peptide with alkaline phosphatase. The data show that Ser578 is a major phosphorylation site in HPg.

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, (G1 EgammagammaY5QKMLgamma10NLRgammaA15EVKKN20A-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 alpha-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 alpha-helix, having RMSD values for the backbone and all heavy atoms of 0.40 A and 0.77 A, 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 alpha-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.

High-level secretion in Pichia pastoris and biochemical characterization of the recombinant kringle 2 domain of tissue-type plasminogen activator.

The kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) has been expressed in Pichia pastoris cell lines GSI 15 and KM71. This construct contained a hexahistidine sequence at the C-terminus of the kringle to aid in purification by immobilized metalion-affinity chromatography. The exact amino acid sequence of the isolated kringle was EAEAYV-[K2tPA]SR(H)6, where [K2tPA] represents amino acid sequence residues C1-C82 of the kringle domain (residues 180-261 of tPA). The clones of the yeast transformants provided large amounts of the recombinant (r)-[K2tPA]-containing polypeptide at levels that allowed ready purification of several hundred mg from shake flasks and near-gram levels from a high-biomass fermenter. Purification of the kringle domain directly from cell-conditioned media was accomplished in a single step by either immobilized Ni(+)-affinity chromatography or lysine-Sepharose affinity chromatography. N-linked glycans were present on approx. 30% of this yeast-expressed material, at N5 of the kringle (corresponds to N11 of the particular construct, N184 of full-length tPA). The expressed recombinant kringle recognized a conformation-specific monoclonal antibody generated against tPA that is directed to the K2 domain of the protein, interacted properly with various omega-amino acid ligands, and showed signature conformational properties when studied by differential scanning calorimetry and high-resolution 1H-NMR. The results demonstrate that the P. pastoris system can be employed to obtain large amounts of secreted and properly folded kringle domains.