Solution structure of Syrian hamster prion protein rPrP(90-231).

NMR has been used to refine the structure of Syrian hamster (SHa) prion protein rPrP(90-231), which is commensurate with the infectious protease-resistant core of the scrapie prion protein PrPSc. The structure of rPrP(90-231), refolded to resemble the normal cellular isoform PrPC spectroscopically and immunologically, has been studied using multidimensional NMR; initial results were published [James et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 10086-10091]. We now report refinement with better definition revealing important structural and dynamic features which can be related to biological observations pertinent to prion diseases. Structure refinement was based on 2778 unambiguously assigned nuclear Overhauser effect (NOE) connectivities, 297 ambiguous NOE restraints, and 63 scalar coupling constants (3JHNHa). The structure is represented by an ensemble of 25 best-scoring structures from 100 structures calculated using ARIA/X-PLOR and further refined with restrained molecular dynamics using the AMBER 4.1 force field with an explicit shell of water molecules. The rPrP(90-231) structure features a core domain (residues 125-228), with a backbone atomic root-mean-square deviation (RMSD) of 0.67 A, consisting of three alpha-helices (residues 144-154, 172-193, and 200-227) and two short antiparallel beta-strands (residues 129-131 and 161-163). The N-terminus (residues 90-119) is largely unstructured despite some sparse and weak medium-range NOEs implying the existence of bends or turns. The transition region between the core domain and flexible N-terminus, i.e., residues 113-128, consists of hydrophobic residues or glycines and does not adopt any regular secondary structure in aqueous solution. There are about 30 medium- and long-range NOEs within this hydrophobic cluster, so it clearly manifests structure. Multiple discrete conformations are evident, implying the possible existence of one or more metastable states, which may feature in conversion of PrPC to PrPSc. To obtain a more comprehensive picture of rPrP(90-231), dynamics have been studied using amide hydrogen-deuterium exchange and 15N NMR relaxation times (T1 and T2) and 15N{1H} NOE measurements. Comparison of the structure with previous reports suggests sequence-dependent features that may be reflected in a species barrier to prion disease transmission.

High Throughput Screening for Drug Discovery: Continually Transitioning into New Technology.

Those working in HTS laboratories, pressured to find increasing numbers of drug leads while containing costs, are seeking larger compound sets, more automated systems to screen them faster, and an integrated set of equipment and consumables. Enabling technologies are continually being developed and suppliers are teaming up to supply integrated equipment and consumable sets. Miniaturization, microfluidic chips, subnanoliter dispensing, fluorescence, homogeneous assays for HTS, and virtual screening are just some of the evolving tools that HTS experts are continually evaluating and incorporating into drug discovery operations.

NMR determination of the major solution conformation of a peptoid pentamer with chiral side chains.

Polymers of N-substituted glycines ("peptoids") containing chiral centers at the alpha position of their side chains can form stable structures in solution. We studied a prototypical peptoid, consisting of five para-substituted (S)-N-(1-phenylethyl)glycine residues, by NMR spectroscopy. Multiple configurational isomers were observed, but because of extensive signal overlap, only the major isomer containing all cis-amide bonds was examined in detail. The NMR data for this molecule, in conjunction with previous CD spectroscopic results, indicate that the major species in methanol is a right-handed helix with cis-amide bonds. The periodicity of the helix is three residues per turn, with a pitch of approximately 6 A. This conformation is similar to that anticipated by computational studies of a chiral peptoid octamer. The helical repeat orients the amide bond chromophores in a manner consistent with the intensity of the CD signal exhibited by this molecule. Many other chiral polypeptoids have similar CD spectra, suggesting that a whole family of peptoids containing chiral side chains is capable of adopting this secondary structure motif. Taken together, our experimental and theoretical studies of the structural properties of chiral peptoids lay the groundwork for the rational design of more complex polypeptoid molecules, with a variety of applications, ranging from nanostructures to nonviral gene delivery systems.

Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform.

The scrapie prion protein (PrPSc) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrPC) by a conformational change. N-terminal truncation of PrPSc by limited proteolysis produces a protein of approximately 142 residues designated PrP 27-30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified. After refolding rPrP into an alpha-helical form resembling PrPC, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113-125 characterize a hydrophobic cluster, which packs against an irregular beta-sheet, whereas residues 90-112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200-227) and a structured loop (residues 165-171) form a discontinuous epitope for binding of a protein that facilitates PrPSc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrPC into PrPSc, whereas the discontinuous epitope near the C terminus controls this transition.

Solution structure of omega-conotoxin MVIIC, a high affinity ligand of P-type calcium channels, using 1H NMR spectroscopy and complete relaxation matrix analysis.

We have determined the solution structure of the omega-conotoxin MVIIC from Conus magus by 1H NMR. This conopeptide preferentially blocks P and Q type Ca2+ currents by binding with high affinity to voltage-sensitive Ca2+ channels in neurons. This 26 residue peptide with three disulfide bonds was chemically synthesized and refolded for NMR structural studies. The 1H NMR NOESY spectrum of this peptide was completely assigned, with stereospecific assignments made for 12 of the beta prochiral centers. Complete relaxation matrix analysis using MARDIGRAS was used to obtain initial interproton distances from peak intensities. The correlation time necessary for these calculations was determined by measuring 13C relaxation times using inversely detected natural abundance spectra. Distances were input to DG, which provided 15 starting structures which were then subjected to restrained molecular dynamics calculations using SANDER with the AMBER 91 force field in vacuo. 1H-1H vicinal coupling constants were obtained using a combination of line fitting of both E. COSY and NOESY spectra and used to generate angle restraints that were included explicitly in the restrained molecular dynamics calculations. The final set of the 15 best structures had a backbone rmsd of 0.84 A. The ensemble R1/6 factor calculated by CORMA for the final 15 structures was 11%. The final structure consists of an anti-parallel, triple-stranded beta-sheet, with four turns. In spite of significant differences in amino acid sequence and affinities for calcium channel subtypes, the backbone structure of omega-conotoxin MVIIC is very similar to the previously reported structure of omega-conotoxin GVIA.

Converting trypsin to chymotrypsin: ground-state binding does not determine substrate specificity.

Rat trypsin II has been converted to a protease with chymotrypsin-like substrate specificity [Hedstrom, L., et al. (1994) Biochemistry (preceding paper in this issue)]. The key alteration in this conversion is the exchange of two surface loops for the analogous loops of chymotrypsin. k(inact)/Ki for the inactivation of chymotrypsin, trypsin, a trypsin mutant with poor activity (D189S), and the chymotrypsin-like mutants Tr-->Ch[S1+L1+L2] and Tr-->Ch[S1+L1+L2+Y172W] by Suc-Ala-Ala-Pro-Phe-chloromethylketone correlates with kcat/Km for hydrolysis of Suc-Ala-Ala-Pro-Phe-AMC. k(inact)'s for the inactivation of Tr-->Ch[S1+L1+L2] and Tr-->Ch[S1+L1+L2+Y172W] are comparable to that of chymotrypsin, while Ki's were much higher. Ki for the inhibition of these enzymes by the transition-state analog MeOSuc-Ala-Ala-Pro-boro-Phe also correlates with kcat/Km for hydrolysis of Suc-Ala-Ala-Pro-Phe-AMC. These results suggest that the surface loops stabilize the transition state for hydrolysis of chymotrypsin substrates by improving the orientation of bound substrates relative to the catalytic residues. Lastly, trypsin and chymotrypsin have comparable affinities for proflavin, while the Kd for the Tr-->Ch[S1+L1+L2+Y172W]-proflavin complex is 10-fold higher. No proflavin binding could be observed for either D189S or Tr-->Ch-[S1+L1+L2], which suggests that the S1 binding pockets of these two mutant enzymes are deformed. This work confirms that enzyme specificity is expressed in the chemical steps of the reaction rather than in substrate binding.

Crystal versus solution structure of enzymes: NMR spectroscopy of a peptide boronic acid-serine protease complex in the crystalline state.

The effectiveness of boronic acids as inhibitors of serine proteases has been widely ascribed to the ability of the boronyl group to form a tetrahedral adduct with the active-site serine that closely mimics the putative tetrahedral intermediate or transition state formed with substrates. However, recent 15N NMR studies of alpha-lytic protease (EC 3.4.21.12) in solution have shown that some boronic acids and peptide boronic acids form adducts with the active-site histidine instead of with the serine. Such histidine-boron adducts have not thus far been reported in x-ray diffraction studies of boronic acid-serine protease complexes. Here, we report an 15N NMR study of the MeOSuc-Ala-Ala-Pro-boroPhe complex of alpha-lytic protease in the crystalline state using magic-angle spinning. Previous 15N NMR studies have shown this complex involves the formation of a histidine-boron bond in solution. The 15N NMR spectra of the crystalline complex are essentially identical to those of the complex in solution, thereby showing that the structure of this complex is the same in solution and in the crystal and that both involve formation of a histidine-boron adduct.

Crystal versus solution structures of enzymes: NMR spectroscopy of a crystalline serine protease.

The hydrogen-bonding status of His57 in the catalytic triad (Asp-His-Ser) of serine protease has important mechanistic implications for this class of enzymes. Recent nitrogen-15 nuclear magnetic resonance (NMR) studies of alpha-lytic protease find His57 and Ser195 to be strongly hydrogen-bonded, a result that conflicts with the corresponding crystallographic studies, thereby suggesting that the crystal and solution structures may differ. This discrepancy is addressed and resolved in a nitrogen-15 NMR study of the enzyme in the crystalline state. The results show that the His-Ser and Asp-His interactions are identical in crystals and solutions, but that in crystals His57 titrates with a pKa of 7.9, nearly one pKa unit higher than in solution. This elevated pKa accounts for the absence of the His-Ser hydrogen bond in previous x-ray studies.

Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes.

15N NMR spectroscopy was used to examine the active-site histidyl residue of alpha-lytic protease in peptide boronic acid inhibitor complexes. Two distinct types of complexes were observed: (1) Boronic acids that are analogues of substrates form complexes in which the active-site imidazole ring is protonated and both imidazole N-H protons are strongly hydrogen bonded. With the better inhibitors of the class this arrangement is stable over the pH range 4.0-10.5. The results are consistent with a putative tetrahedral intermediate like complex involving a negatively charged, tetrahedral boron atom covalently bonded to O gamma of the active-site serine. (2) Boronic acids that are not substrate analogues form complexes in which N epsilon 2 of the active-site histidine is covalently bonded to the boron atom of the inhibitor. The proton bound to N delta 1 of the histidine in these histidine-boronate adducts remains strongly hydrogen bonded, presumably to the active-site aspartate. Benzeneboronic acid, which falls in this category, forms an adduct with histidine. In both types of complexes the N-H protons of His-57 exchange unusually slowly as evidenced by the room temperature visibility of the low-field 1H resonances and the 15N-H spin couplings. These results, coupled with the kinetic data of the preceding paper [Kettner, C. A., Bone, R., Agard, D. A., & Bachovchin, W. W. (1988) Biochemistry (preceding paper in this issue)], indicate that occupancy of the specificity subsites may be required to fully form the transition-state binding site. The significance of these findings for understanding inhibitor binding and the catalytic mechanism of serine proteases is discussed.