Resolving pathways of interaction of mipafox and a sarin analog with human acetylcholinesterase by kinetics, mass spectrometry and molecular modeling approaches.

The hydroxyl oxygen of the catalytic triad serine in the active center of serine hydrolase acetylcholinesterase (AChE) attacks organophosphorus compounds (OPs) at the phosphorus atom to displace the primary leaving group and to form a covalent bond. Inhibited AChE can be reactivated by cleavage of the Ser-phosphorus bond either spontaneously or through a reaction with nucleophilic agents, such as oximes. At the same time, the inhibited AChE adduct can lose part of the molecule by progressive dealkylation over time in a process called aging. Reactivation of the aged enzyme has not yet been demonstrated. Here, our goal was to study oxime reactivation and aging reactions of human AChE inhibited by mipafox or a sarin analog (Flu-MPs, fluorescent methylphosphonate). Progressive reactivation was observed after Flu-MPs inhibition using oxime 2-PAM. However, no reactivation was observed after mipafox inhibition with 2-PAM or the more potent oximes used. A peptide fingerprinted mass spectrometry (MS) method, which clearly distinguished the peptide with the active serine (active center peptide, ACP) of the human AChE adducted with OPs, was developed by MALDI-TOF and MALDI-TOF/TOF. The ACP was detected with a diethyl-phosphorylated adduct after paraoxon inhibition, and with an isopropylmethyl-phosphonylated and a methyl-phosphonylated adduct after Flu-MPs inhibition and subsequent aging. Nevertheless, nonaged nonreactivated complexes were seen after mipafox inhibition and incubation with oximes, where MS data showed an ACP with an NN diisopropyl phosphoryl adduct. The kinetic experiments showed no reactivation of activity. The computational molecular model analysis of the mipafox-inhibited hAChE plots of energy versus distance between the atoms separated by dealkylation showed a high energy demand, thus little aging probability. However, with Flu-MPs and DFP, where aging was observed in our MS data and in previously published crystal structures, the energy demand calculated in modeling was lower and, consequently, aging appeared as a more likely reaction. We document here direct evidence for a phosphorylated hAChE refractory to oxime reactivation, although we observed no aging.

Conformational dimorphism and transmembrane orientation of prion protein residues 110-136 in bicelles.

A fragment corresponding to the putative membrane-associating domain of the prion protein (residues 110-136) was analyzed in phospholipid bicelles. Prion(110-136) associated with bicelles and exhibited a lipid- and pH-dependent conformational dimorphism between unstructured (pH 4.5) and alpha-helical (pH 7.5). Mutational analysis indicated that the charge state of a single histidine residue was largely responsible for the dimorphism. Amide-lipid NOEs and amide-water chemical exchange measurements revealed that the helical conformation of prion(110-136) spanned the bilayer, and were corroborated by solid-state deuterium NMR experiments indicating that the helical axis rested at a 16 degrees angle with respect to the bilayer normal.

Structural evaluation of phospholipid bicelles for solution-state studies of membrane-associated biomolecules.

Several complementary physical techniques have been used to characterize the aggregate structures formed in solutions containing dimyristoylphosphatidylcholine (DMPC)/dihexanoylphosphatidylcholine (DHPC) at ratios of < or =0.5 and to establish their morphology and lipid organization as that of bicelles. (31)P NMR studies showed that the DMPC and DHPC components were highly segregated over a wide range of DMPC/DHPC ratios (q = 0.05-0.5) and temperatures (15 degrees C and 37 degrees C). Only at phospholipid concentrations below 130 mM did the bicelles appear to undergo a change in morphology. These results were corroborated by fluorescence data, which demonstrated the inverse dependence of bicelle size on phospholipid concentration as well as a distinctive change in phospholipid arrangement at low concentrations. In addition, dynamic light scattering and electron microscopy studies supported the hypothesis that the bicellar phospholipid aggregates are disk-shaped. The radius of the planar domain of the disk was found to be directly proportional to the ratio of DMPC/DHPC and inversely proportional to the total phospholipid concentration when the DMPC/DHPC ratio was held constant at 0.5. Taken together, these results suggest that bicelles with low q retain the morphology and bilayer organization typical of their liquid-crystalline counterparts, making them useful membrane mimetics.

Phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of methylesterase CheB.

The crystal structure of the unphosphorylated state of methylesterase CheB shows that the regulatory domain blocks access of substrate to the active site of the catalytic domain. Phosphorylation of CheB at Asp56 results in a catalytically active transiently phosphorylated enzyme with a lifetime of approximately two seconds. Solvent accessibility changes in this transiently phosphorylated state were probed by MALDI-TOF-detected amide hydrogen/deuterium exchange. No changes in solvent accessibility were seen in the regulatory domain upon phosphorylation of Asp56, but two regions in the catalytic domain (199-203 and 310-317) became more solvent accessible. These two regions flank the active site and contain domain-domain contact residues. Comparison with results from the isolated catalytic domain-containing C-terminal fragment of CheB (residues 147-349) showed that the increased solvent accessibility was less than would have occurred upon detachment of the regulatory domain. Thus, phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of CheB.

Solvent accessibility of the thrombin-thrombomodulin interface.

The kinetics of solvent accessibility at the protein-protein interface between thrombin and a fragment of thrombomodulin, TMEGF45, have been monitored by amide hydrogen/deuterium (H/2H) exchange detected by MALDI-TOF mass spectrometry. The interaction is rapid and reversible, requiring development of theory and experimental methods to distinguish H/2H exchange due to solvent accessibility at the interface from H/2H exchange due to complex dissociation. Association and dissociation rate constants were measured by surface plasmon resonance and amide H/2H exchange rates were measured at different pH values and concentrations of TMEGF45. When essentially 100% of the thrombin was bound to TMEGF45, two segments of thrombin became completely solvent-inaccessible, as evidenced by the pH insensitivity of the amide H/2H exchange rates. These segments form part of anion-binding exosite I and contain the residues for which alanine substitution abolishes TM binding. Several other regions of thrombin showed slowing of amide exchange upon TMEGF45 binding, but the exchange remained pH-dependent, suggesting that these regions of thrombin were rendered only partially solvent-inaccessible by TMEGF45 binding. These partially inaccessible regions of thrombin form both surface and buried contacts into the active site of thrombin and contain residues implicated in allosteric changes in thrombin upon TM binding.

Orientation and effects of mastoparan X on phospholipid bicelles.

Mastoparan X (MPX: INWKGIAAMAKKLL-NH2) belongs to a family of ionophoric peptides found in wasp venom. Upon binding to the membrane, MPX increases the cell's permeability to cations leading to a disruption in the electrolyte balance and cell lysis. This process is thought to occur either through a membrane-thinning mechanism, where the peptide resides on the membrane surface thereby disrupting lipid packing, or through formation of an oligomeric pore. To address this issue, we have used both high-resolution and solid-state 2H NMR techniques to study the structure and orientation of MPX when associated with bicelles. NOESY and chemical shift analysis showed that in bicelles, MPX formed a well-structured amphipathic alpha-helix. In zwitterionic bicelles, the helical axis was found to rest generally perpendicular to the membrane normal, which could be consistent with the "carpet" mechanism for lytic activity. In anionic bicelles, on the other hand, the helical axis was generally parallel to the membrane normal, which is more consistent with the pore model for lytic activity. In addition, MPX caused significant disruption in lipid packing of the negatively charged phospholipids. Taken together, these results show that MPX associates differently with zwitterionic membranes, where it rests parallel to the surface, compared with negatively charged membranes, where it penetrates longitudinally.

Disulfide bond plasticity in epidermal growth factor.

Epidermal growth factor (EGF) has a (1-3,2-4,5-6) disulfide-bonding pattern. This pattern is found in nearly all EGF-like domains, despite wide variation in sequences. Biological data from EGF and at least one EGF-like domain show that disulfide bond isomers have significant bioactivity and suggests that the EGF fold can accommodate alternate disulfide-bonding patterns. The disulfide bonds in murine EGF were altered to seven different patterns and structures were calculated incorporating all the restraints from the highest resolution restraint set available (Tejero et al., 1996). Results showed that besides the native (1-3,2-4,5-6), two other disulfide-bonding patterns: (1-2,3-4,5-6) and (1-3,2-5,4-6) satisfied the restraints as well as the native. The results for these two patterns were indistinguishable from the native on the basis of distance and dihedral violations, XPLOR energies, Procheck statistics, and RMSDs of the final set of structures. Two other disulfide bond patterns, (1-2,3-5, 4-6) and (1-4,2-3,5-6) were able to satisfy all the distance restraints but had one or more cysteine dihedral violations. For all seven isomers, the final calculated structures were highly similar to EGF with all-atom RMSD's in the 1. 5-2 A range. These results suggest that the EGF backbone fold has the unique property of accommodating several different disulfide-bonding patterns.

Solution structure of the smallest cofactor-active fragment of thrombomodulin.

A glycosylated fragment of thrombomodulin containing two epidermal growth factor-like domains (TMEGF45) was analyzed by NMR. The 4th-domains structure of this two-domain fragment is similar to that of the individual domain previously determined. The 5th-domain, which has uncrossed disulfide bonds, is not as well determined in the two-domain fragment than the individual domain previously solved. The flexibility of the 5th-domain is consistent with low heteronuclear NOEs. In the individual 5th-domain, Met 388 was disordered, and key thrombin binding residues formed a hydrophobic core. By contrast, in TMEGF45, Met 388 is in the 5th-domain core, positioned by Phe 376 from the 4th-domain. As a result, key thrombin binding residues that were in the core of the individual domain are expelled. Upon thrombin binding, chemical shifts of two residues in the 4th-domain, the three interdomain linker residues, and nearly all of the 5th-domain are perturbed. Thus, TMEGF45 binds thrombin by an induced fit mechanism involving a flexible 5th-domain.

Electrostatic dependence of the thrombin-thrombomodulin interaction.

The rate constants for the binding interaction between thrombin and a fully active fragment of its anticoagulant cofactor, thrombomodulin, have been determined by surface plasmon resonance. At physiological ionic strength, the k(a) was 6.7x10(6) M(-1) s(-1 )and the dissociation rate constant was 0.033 s(-1). These extremely fast association and dissociation rates resulted in an overall binding equilibrium constant of 4.9 nM, which is similar to previously reported values. Changing the ionic strength from 100 mM to 250 mM NaCl caused a tenfold decrease in the association rate while the dissociation rate did not change significantly. A similar effect was observed with tetramethylammonium chloride. A Debye-Hückel plot of the data had a slope of -6 and an intercept at 0 ionic strength of 10(9) M(-1) s(-1). The same slope and intercept were obtained for data that was collected in the presence of glycerol to slow the association rates. These results show that the thrombin-TM456 interaction is extremely rapid and nearly completely electrostatically steered. An association model is presented in which TM456 approaches thrombin along the direction of the thrombin molecular dipole.

Preparation of insoluble transmembrane peptides: glycophorin-A, prion (110-137), and FGFR (368-397).

A general procedure for the reliable preparation of large quantities of insoluble transmembrane peptides has been developed. Optimal couplings were obtained during synthesis by using high-temperature couplings in conjunction with O-(7-azabenzotriazol-1-yl)-1,1,3, 3-tetramethyluronium hexafluorophosphate (HATU) activation. Improved purification schemes were developed that use reverse- phase HPLC on a C1 column and elution with a 2:1 mixture of 1-propanol:1-butanol. Using these methods three very different transmembrane peptides all longer than 25 amino acids have been prepared: glycophorin-A, prion (110-137), and fibroblast growth factor receptor (368-397).

The role of water in the catalytic efficiency of triosephosphate isomerase.

The structural basis for the effect of the S96P mutation in chicken triosephosphate isomerase (cTIM) has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. The X-ray structure is that of the enzyme complexed with phosphoglycolohydroxamate (PGH), an intermediate analogue, solved at a resolution of 1.9 A. The S96P mutation was identified as a second-site reverent when catalytically crippled mutants, E165D and H95N, were subjected to random mutagenesis. The presence of the second mutation leads to enhanced activity over the single mutation. However, the effect of the S96P mutation alone is to decrease the catalytic efficiency of the enzyme. The crystal structures of the S96P double mutants show that this bulky proline side chain alters the water structure within the active-site cavity (E165D; ref 1) and prevents nonproductive binding conformations of the substrate (H95N; ref 2). Comparison of the S96P single mutant structure with those of the wild-type cTIM, those of the single mutants (E165D and H95N), and those of the double mutants (E165D/S96P and H95N/S96P) begins to address the role of the conserved serine residue at this position. The results indicate that the residue positions the catalytic base E165 optimally for polarization of the substrate carbonyl, thereby aiding in proton abstraction. In addition, this residue is involved in positioning critical water molecules, thereby affecting the way in which water structure influences activity.

Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation.

Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P. pastoris occurs in simple minimal defined media making this system attractive for production of labeled proteins for NMR analysis. P. pastoris is therefore the expression system of choice for NMR of proteins that cannot be refolded from inclusion bodies or that require post-translational modifications for proper folding or function. The yield of expressed proteins from P. pastoris depends critically on growth conditions, and attainment of high cell densities by fermentation has been shown to improve protein yields by 10-100-fold. Unfortunately, the cost of the isotopically enriched fermentation media components, particularly 15NH4OH, is prohibitively high. We report fermentation methods that allow for both 15N-labeling from (15NH4)2SO4 and 13C-labeling from 13C-glucose or 13C-glycerol of proteins produced in Pichia pastoris. Expression of an 83 amino acid fragment of thrombomodulin with two N-linked glycosylation sites shows that fermentation is more cost effective than shake flask growth for isotopic enrichment.

Mechanism of thrombin clearance by human astrocytoma cells.

Astroglial cells secrete a variety of factors that contribute to the regulation of neurite initiation and continued outgrowth, among them proteases and protease inhibitors. An alteration in the balance between these proteins has been implicated in Alzheimer's disease, resulting in an accumulation of thrombin:protease nexin 1 (PN1) complexes in the brains of these patients. This report aims at providing a biochemical explanation for this phenomenon. We show that human astrocytoma cells bind and internalize thrombin and thrombin:PN1 complexes efficiently by a PN1-dependent mechanism. Binding was potently inhibited by soluble heparin and did not occur with the mutant PN1 (K7E) deficient in heparin binding. Receptor-associated protein, an antagonist of the low-density lipoprotein receptor-related protein (LRP), inhibited internalization of thrombin by the astrocytoma cells, but did not affect cell-surface binding. The results are consistent with a mechanism by which astrocytoma cells clear thrombin in a sequential manner: thrombin is first complexed with PN1, then bound to cell-surface heparins, and finally internalized by LRP. This mechanism provides a link between the neuronal growth regulators thrombin and PN1 and proteins genetically associated with Alzheimer's disease, such as LRP.

Characterization of the extracellular ligand-binding domain of the type II activin receptor.

The binding of a ligand to cell surface receptors initiates a cascade of intracellular signals that generate responses to the external stimuli. Thus, this event plays a pivotal role in the mechanism of transmembrane signaling. Activin is a member of a cytokine family that is involved in diverse biological processes. To study the structural basis that underlies the transmembrane signaling mechanism, we have overexpressed the soluble extracellular domain of the type II activin receptor from mouse (ActRII-ECD). We used the methylotrophic yeast Pichia pastoris as an expression host to produce a large quantity of ActRII-ECD. Expression was carried out in a fermentor with a typical yield of 10 mg of pure ActRII-ECD from a liter of growth media. Biological function was confirmed by the ability to decrease the activin-stimulated release of FSH from cultured rat pituitary cells in addition to several activin-binding assays, including native gel shift and chemical cross-linking. The glycosylation on ActRII-ECD was shown to be dispensable for high-affinity activin binding, and nonnatural sugars from the yeast expression host did not interfere with binding, indicating that the binding of activin is not sensitive to the environment near the two positions of N-linked glycosylation. Analytical ultracentrifugation of the complex between activin A and ActRII-ECD reveals that two receptors associate with one activin A dimer, consistent with results from chemical cross-linking experiments.

Identification of protein-protein interfaces by decreased amide proton solvent accessibility.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry was used to identify peptic fragments from protein complexes that retained deuterium under hydrogen exchange conditions due to decreased solvent accessibility at the interface of the complex. Short deuteration times allowed preferential labeling of rapidly exchanging surface amides so that primarily solvent accessibility changes and not conformational changes were detected. A single mass spectrum of the peptic digest mixture was analyzed to determine the deuterium content of all proteolytic fragments of the protein. The protein-protein interface was reliably indicated by those peptides that retained more deuterons in the complex compared with control experiments in which only one protein was present. The method was used to identify the kinase inhibitor [PKI(5-24)] and ATP-binding sites in the cyclic-AMP-dependent protein kinase. Three overlapping peptides identified the ATP-binding site, three overlapping peptides identified the glycine-rich loop, and two peptides identified the PKI(5-24)-binding site. A complex of unknown structure also was analyzed, human alpha-thrombin bound to an 83-aa fragment of human thrombomodulin [TMEGF(4-5)]. Five peptides from thrombin showed significantly decreased solvent accessibility in the complex. Three peptides identified the anion-binding exosite I, confirming ligand competition experiments. Two peptides identified a new region of thrombin near the active site providing a potential mechanism of how thrombomodulin alters thrombin substrate specificity.

2H NMR studies of a myristoylated peptide in neutral and acidic phospholipid bicelles.

Deuterium NMR spectra of Myr-d27-GNAAAAKKGSEQES (Cat14), the N-terminal 14-residue peptide from the catalytic subunit of cAMP-dependent protein kinase A (PKA), illustrate how magnetically aligned neutral and acidic phospholipid bicelles can be used to characterize the ordering and mode of binding of peptides to membranes. Since Cat14 is electrically neutral, the major interaction responsible for binding is the insertion of the myristoyl group into the hydrophobic core of the bilayer. The inclusion of 25% phosphatidylserine or phosphatidylglycerol into phosphatidylcholine bicelles results in a moderate increase in the ordering of the peptide relative to the bicelle normal, presumably because of favorable electrostatic interactions between the phospholipid headgroups and the two lysines in positions 7 and 8. Successful preparation of acidic bicelles was achieved by careful adjustment of lipid composition, pH and ionic strength.

Measurement of amide hydrogen exchange by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was used to determine amide proton/deuteron (H/D) exchange rates. The method has broad application to the study of protein conformation and folding and to the study of protein-ligand interactions and requires no modifications of the instrument. Amide protons were allowed to exchange with deuterons in buffered D2O at room temperature, pD 7.25. Exchanged deuterons were "frozen" in the exchanged state by quenching at pH 2.5, 0 degree C and analyzed by MALDI-TOF MS. The matrix mixture consisted of 5 mg/mL alpha-cyano-4-hydroxycinnamic acid, acetonitrile, ethanol, and 0.1% TFA. The matrix was adjusted to pH 2.5, and the chilled MALDI target was rapidly dried. Deuteration of amide protons on cyclic AMP-dependent protein kinase was measured after short times of incubation in deuterium by pepsin protein digestion and MALDI-TOF MS analysis. The unseparated peptic digest was analyzed in a single spectrum of the mixture. From five spectra, H/D exchange rates were determined for some 40 peptides covering 65% of the protein sequence.

Energetics of thrombin-thrombomodulin interaction.

Temperature and salt dependence studies of thrombin interaction with thrombomodulin, with and without chondroitin sulfate, and two fragments containing the EGF-like domains 4-5 and 4-5-6 reveal the energetic signatures and the mechanism of recognition of this physiologically important cofactor. Binding of thrombomodulin is affected drastically by the particular salt present in solution and is positively linked to Na+ binding to thrombin and the conversion of the enzyme from the slow to the fast form, but is opposed by Cl- binding to the fibrinogen recognition site and especially to the heparin binding site. Binding of thrombomodulin has an unusually large salt dependence (gamma(salt) = -4.8) contributed mostly by the polyelectrolyte-like nature of the chondroitin sulfate moiety that binds to the heparin binding site and increases the affinity of the cofactor by almost 10-fold. On the other hand, the chondroitin sulfate has no effect on the deltaCp of binding, which is determined predominantly by contacts made by the EGF-like domains 5 and 6 with the fibrinogen recognition site. The modest heat capacity change (-0.2 kcal mol(-1) K(-1)) observed when thrombomodulin binds to the fast form suggests a rigid-body association of the cofactor with the enzyme. In the slow form, however, the heat capacity change is significantly more pronounced (-0.5 kcal mol(-1) K(-1)) and signals the presence of a conformational transition of the enzyme linked to binding of the cofactor that mimics the slow-->fast conversion. These results demonstrate that recognition of thrombomodulin by thrombin is steered electrostatically by the highly charged regions of the fibrinogen recognition site and the heparin binding site, to which the chondroitin sulfate moiety binds and enhances the affinity of the interaction. The recognition event also involves conformational changes of the enzyme in the slow form mediated by binding of the EGF-like domains 5-6 to the fibrinogen recognition site. Consistent with this model, binding of thrombomodulin to the fast form has only a small effect on the hydrolysis of nine chromogenic substrates carrying substitutions at P1, P2, and P3 aimed at probing the environment of the specificity sites S1, S2, and S3 of the enzyme. Binding to the slow form, on the other hand, enhances the specificity toward all substrates up to 15-fold. For substrates carrying a Gly at P2, binding of thrombomodulin changes the relative specificity of the slow and fast forms and makes the slow form more specific. Interestingly, these effects are not specific of thrombomodulin and depend solely on binding to the fibrinogen recognition site of the enzyme. In fact, they are also observed with the hirudin C-terminal fragment 55-65. The characterization of the mechanism of thrombin-thrombomodulin interaction and the effects of the cofactor on the hydrolysis of chromogenic substrates probing the interior of the catalytic pocket bear on the thrombomodulin-induced enhancement of protein C cleavage by thrombin. We propose that this enhancement is due predominantly to an effect of thrombomodulin on the bound protein C in the ternary complex. Therefore, thrombomodulin would carry out its physiological function by making protein C a better substrate for thrombin, rather than making thrombin a better enzyme for protein C.

Structure of the fifth EGF-like domain of thrombomodulin: An EGF-like domain with a novel disulfide-bonding pattern.

The structure of the fifth EGF-like domain (residues Q387 to E426) of thrombomodulin (TMEGF5) has been determined by two-dimensional NMR. TMEGF5 binds to thrombin with a Ki of 1.9 microM and has been shown to have a novel disulfide bonding pattern in a fully active fragment of TM. In EGF, the disulfide bonding pattern is (1-3,2-4, 5-6), while TMEGF5 has an uncrossed (1-2,3-4,5-6) pattern. The structure of this novel domain, determined from 483 NOE-derived distance restraints, appears to have diverged from the common EGF-like structure. Superposition of the 14 lowest-energy structures of TMEGF5 gives an overall r.m.s.d. of 1.09 A for the backbone atoms. The central two-stranded beta-sheet common to all EGF-like domains is not present in TMEGF5. The A loop, residues C390 to C395, is twisted away from interacting with the B loop, residues C399 to C407, as in EGF, and is close to the C loop, residues C409 to C421. This twist causes the N and C termini to be closer together in TMEGF5 than in EGF. Most of the residues that are important for activity lie on one face of the molecule, which is likely to be the thrombin-binding surface of the domain. The structure of the C loop within the domain, which is a beta-hairpin similar to EGF, is similar to the structure of a synthetic version of the loop bound to thrombin as determined by transferred NOE experiments. Despite the similarity in the structures of the loops, the residues immediately following C421 are in different positions in the two structures suggesting that these "tail" residues may change conformation upon thrombin binding.