Backbone Folding Research Articles

Nonessential role for methionines in the productive association between calmodulin and the plasma membrane Ca-ATPase.

To investigate the role of hydrophobic interactions involving methionine side chains in facilitating the productive association between calmodulin (CaM) and the plasma membrane (PM) Ca-ATPase, we have substituted the polar amino acid Gln for Met at multiple positions in both the amino- and carboxyl-terminal domains of CaM. Conformationally sensitive fluorescence signals indicate that these mutations have little effect on the backbone fold of the carboxyl-terminal domain of CaM. The insertion of multiple Gln in either globular domain results in a decrease in the apparent affinity of CaM for the PM-Ca-ATPase. However, despite the multiple substitution of Gln for four methionines at positions 36, 51, 71, and 72 in the amino-terminal domain or for three methionines at positions 124, 144, and 145 in the carboxyl-terminal domain, these mutant CaMs are able to fully activate the PM-Ca-ATPase. Thus, although these CaM mutants have a decreased affinity for the CaM-binding site on the Ca-ATPase, they retain the ability to fully activate the Ca-ATPase at saturating concentrations of CaM. The role of individual methionines in modulating the affinity between the carboxyl terminus and the PM-Ca-ATPase was further investigated through the substitution of individual Met with Gln. Upon substitution of Met(124) and Met(144) with Gln, there is a 5- and 10-fold increase in the amount of CaM necessary to obtain half-maximal activation of the PM-Ca-ATPase, indicating that these methionine side chains participate in the high-affinity association between CaM and the PM-Ca-ATPase. However, substitution of Gln for Met(145) results in no change in the apparent affinity between CaM and the PM-Ca-ATPase, indicating that in contrast to all other known CaM targets, Met(145) does not participate in the interaction between CaM and the PM-Ca-ATPase. These results emphasize differences in the binding interactions between individual methionines in CaM and different target enzymes, and suggest that hydrophobic interactions between methionines in CaM and the binding site on the PM-Ca-ATPase are not necessary for enzyme activation. Calculation of the binding affinities of individual CaM domains associated with activation of the PM-Ca-ATPase suggests that mutations of methionines located in either domain of CaM can decrease the initial high-affinity association between CaM and the PM-Ca-ATPase, but have little effect upon the subsequent binding of the opposing globular domain. These results suggest that the initial associations between CaM and the CaM-binding sequence in the PM-Ca-ATPase are guided by nonspecific hydrophobic interactions involving both domains of CaM.

NMR structure and dynamics of monomeric neutrophil-activating peptide 2

Neutrophil-activating peptide 2 (NAP-2), which demonstrates a range of proinflammatory activities, is a 72-residue protein belonging to the alpha-chemokine family. Although NAP-2, like other alpha-chemokines, is known to self-associate into dimers and tetramers, it has been shown that the monomeric form is physiologically active. Here we investigate the solution structure of monomeric NAP-2 by multi-dimensional 1H-NMR and 15N-NMR spectroscopy and computational modelling. The NAP-2 monomer consists of an amphipathic, triple-stranded, anti-parallel beta-sheet on which is folded a C-terminal alpha-helix and an aperiodic N-terminal segment. The backbone fold is essentially the same as that found in other alpha-chemokines. 15N T1, T2 and nuclear Overhauser effects (NOEs) have been measured for backbone NH groups and used in a model free approach to calculate order parameters and conformational exchange terms that map out motions of the backbone. N-terminal residues 1 to 17 and the C-terminus are relatively highly flexible, whereas the beta-sheet domain forms the most motionally restricted part of the fold. Conformational exchange occurring on the millisecond time scale is noted at the top of the C-terminal helix and at proximal residues from beta-strands 1 and 2 and the connecting loop. Dissociation to the monomeric state is apparently responsible for increased internal mobility in NAP-2 compared with dimeric and tetrameric states in other alpha-chemokines.

NMR structure and dynamics of monomeric neutrophil-activating peptide 2

Neutrophil-activating peptide 2 (NAP-2), which demonstrates a range of proinflammatory activities, is a 72-residue protein belonging to the α-chemokine family. Although NAP-2, like other α-chemokines, is known to self-associate into dimers and tetramers, it has been shown that the monomeric form is physiologically active. Here we investigate the solution structure of monomeric NAP-2 by multi-dimensional 1H-NMR and 15N-NMR spectroscopy and computational modelling. The NAP-2 monomer consists of an amphipathic, triple-stranded, anti-parallel β-sheet on which is folded a C-terminal α-helix and an aperiodic N-terminal segment. The backbone fold is essentially the same as that found in other α-chemokines. 15N T1, T2 and nuclear Overhauser effects (NOEs) have been measured for backbone NH groups and used in a model free approach to calculate order parameters and conformational exchange terms that map out motions of the backbone. N-terminal residues 1 to 17 and the C-terminus are relatively highly flexible, whereas the β-sheet domain forms the most motionally restricted part of the fold. Conformational exchange occurring on the millisecond time scale is noted at the top of the C-terminal helix and at proximal residues from β-strands 1 and 2 and the connecting loop. Dissociation to the monomeric state is apparently responsible for increased internal mobility in NAP-2 compared with dimeric and tetrameric states in other α-chemokines.

Topographically constrained aromatic α-aza-amino acids. Part 2. New azaTic-containing peptides: Synthesis, conformation, and intramolecular NH…N interaction

The new pseudodipeptide Boc-azaTic-Leu-OMe ( 1), incorporating the conformationally and topographically constrained 3,4-dihydro-2(1 H)-phthalazinecarboxylic acid (azaTic) residue, has been synthesized together with the three related models Boc-azaTic-NHMe ( 3), MeCO-azaTic-Gly-OMe ( 4), and azaTic-Leu-OMe ( 6). Both the epimers of 1, generated by opposite absolute configuration at an azaTic nitrogen stereogenic centre, have been found in the asymmetric unit of the crystal. The conformational perturbations, deriving by the introduction of the azaTic residue into a peptide backbone, are described and the nature of the NH…N interaction, which gives rise to a typical backbone folding closing a 5-membered ring, has been studied.

Role of ligand substitution in ferrocytochrome c folding.

The ligand substitutions that occur during the folding of ferrocytochrome c [Fe(II)cyt c] have been monitored by transient absorption spectroscopy. The folding reaction was triggered by photoinduced electron transfer to unfolded Fe(III)cyt c in guanidine hydrochloride (GuHCl) solutions. Assignments of ligation states were made by reference to the spectra of the imidazole and methionine adducts of N-acetylated microperoxidase 8. At pH 7, the heme in unfolded Fe(II)cyt c is ligated by native His18 and HisX (X = 26, 33) residues. The native Met80 ligand displaces HisX only in the last stages of folding. The ferroheme is predominantly five-coordinate in acidic solution; it remains five-coordinate until the native methionine binds the heme to give the folded protein (the rate of the methionine binding step is 16 +/- 5 s-1 at pH 5, 3.2 M GuHCl). The evidence suggests that the substitution of histidine by methionine is strongly coupled to backbone folding.

Solution Structure of a β-Neurotoxin from the New World ScorpionCentruroides sculpturatusEwing

We report the detailed solution structure of the 7.2 kDa protein CsE-I, a β-neurotoxin from the New World scorpionCentruroides sculpturatusEwing. This toxin binds to sodium channels, but unlike the α-neurotoxins, shifts the voltage of activation toward more negative potentials causing the membrane to fire spontaneously. Sequence-specific proton NMR assignments were made using 600 MHz 2D-NMR data. Distance geometry and dynamical simulated annealing refinements were performed using experimental distance and torsion angle constraints from NOESY and pH-COSY data. A family of 40 structures without constraint violations was generated, and an energy-minimized average structure was computed. The backbone conformation of the CsE-I toxin shows similar secondary structural features as the prototypical α-neurotoxin, CsE-v3, and is characterized by a short 212-turn α-helix and a 3-strand antiparallel β-sheet, both held together by disulfide bridges. The RMSD for the backbone atoms between CsE-I and CsE-v3 is 1.48 Å. Despite this similarity in the overall backbone folding, the these two proteins show some important differences in the primary structure (sequence) and electrostatic potential surfaces. Our studies provide a basis for unravelling the role of these differences in relation to the known differences in the receptor sites on the voltage sensitive sodium channel for the α- and β-neurotoxins.

5] Nuclear magnetic resonance spectroscopy of peptide ion channel ligands: Cloning and expression as aid to evaluation of structural and dynamic properties

As candidates for structural measurements ion channels are daunting targets. Their large size and the fact that they are anchored in biological membranes make atomic resolution information unattainable by available techniques. There is a vast array of peptide ligands whose structures are more accessible that bind tightly to the outer vestibules of different ion channels. Peptides from this class are found in a variety of animal venoms and are characterized by high cysteine content and a conserved backbone fold. Structural information about these ligands is important for understanding their function as pharmacologic tools and as therapeutic entities. The high binding affinity and exquisite selectivity of these ligands suggests that structural inferences can be drawn about the ion channel from an adequate knowledge of the structure of the ligands themselves. Additionally, more direct information may be obtained about the ion channel by studying the ligand bound to a relevant fragment of the channel. These peptides are particularly well suited to nuclear magnetic resonance (NMR) measurements. This chapter briefly discusses the aspects of NMR spectroscopy pertinent to the study of peptide toxins that bind to ion channels, and detail an expression and purification protocol that will both enable NMR-active isotope incorporation into these peptides and provide a facile system for producing mutant analogs for structure-function studies. The chapter discusses experimental considerations for carrying out NMR studies on this class of peptides.

Distinguishing between sequential and nonsequentially folded proteins: implications for folding and misfolding.

We describe here an algorithm for distinguishing sequential from nonsequentially folding proteins. Several experiments have recently suggested that most of the proteins that are synthesized in the eukaryotic cell may fold sequentially. This proposed folding mechanism in vivo is particularly advantageous to the organism. In the absence of chaperones, the probability that a sequentially folding protein will misfold is reduced significantly. The problem we address here is devising a procedure that would differentiate between the two types of folding patterns. Footprints of sequential folding may be found in structures where consecutive fragments of the chain interact with each other. In such cases, the folding complexity may be viewed as being lower. On the other hand, higher folding complexity suggests that at least a portion of the polypeptide backbone folds back upon itself to form three-dimensional (3D) interactions with noncontiguous portion(s) of the chain. Hence, we look at the mechanism of folding of the molecule via analysis of its complexity, that is, through the 3D interactions formed by contiguous segments on the polypeptide chain. To computationally splice the structure into consecutively interacting fragments, we either cut it into compact hydrophobic folding units or into a set of hypothetical, transient, highly populated, contiguous fragments ("building blocks" of the structure). In sequential folding, successive building blocks interact with each other from the amino to the carboxy terminus of the polypeptide chain. Consequently, the results of the parsing differentiate between sequentially vs. nonsequentially folded chains. The automated assessment of the folding complexity provides insight into both the likelihood of misfolding and the kinetic folding rate of the given protein. In terms of the funnel free energy landscape theory, a protein that truly follows the mechanism of sequential folding, in principle, encounters smoother free energy barriers. A simple sequentially folded protein should, therefore, be less error prone and fold faster than a protein with a complex folding pattern.

Modulation of gramicidin channel structure and function by the aliphatic "spacer" residues 10, 12, and 14 between the tryptophans.

In the linear gramicidins, the four aromatic residues at positions 9, 11, 13, and 15 are well-known to be important for the structure and function of membrane-spanning gramicidin channels. To investigate whether the "spacer" residues between the tryptophans in gramicidin A (gA) are important for channel structure and function, D-Leu-10, -12. and -14 of gA were replaced by Ala, Val, or Ile. (For practical reasons, the Ile substitutions were introduced into the enantiomeric gramicidin A-, gA-.) Circular dichroism spectra of [D-Ala10,12,14]gA, [D-Val10,12,14]gA, or [Ile10,12,14]gA- incorporated into sodium dodecyl sulfate micelles or 1, 2-dimyristoyl-sn-glycero-3-phosphocholine vesicles differ from the spectrum of the native [D-Leu10,12,14]gA. All the analogue spectra display reduced ellipticity at both 218 and 235 nm, indicating the presence of double-stranded conformers with the Ala analogue spectra showing the largest departure from the native gA spectra. Size-exclusion chromatograms of the Val and Ile analogues show both monomer and dimer peaks, accompanied by peak broadening; the chromatograms for the Ala analogue show broad, overlapping peaks and suggest the presence of higher oligomers and/or (rapidly) interconverting conformations. All three analogues form membrane-spanning channels, with the channel-forming potency of the Ala analogue being much less than that of gA or the other analogues. In 1.0 M CsCl, the conductance of each analogue channel is approximately 25% less than that of [D-Leu10,12,14]gA channels. The lifetimes of the analogue channels also are less than of [D-Leu10,12, 14]gA channels, with the largest (8-fold) reduction being for [D-Ala10,12,14]gA channels. Hybrid channel experiments show that the beta6.3-helical backbone folding pattern is retained in the channel-forming subunits and that the substitutions primarily influence ion entry. Both the bulk and the stereochemistry of the aliphatic residues between the tryptophans of gA are important for channel structure and function.

A search for the ideal type I beta-turn.

In 1968 C. Venkatachalam (Biopolymers, Vol. 6, pp. 1425-1436) predicted the ideal forms of beta-turns (type I, type II, etc.) based entirely on theoretical calculations. Subsequently, over a thousand x-ray structures of different globular proteins have been analyzed, with results suggesting that the most important form among the hairpin conformers is the type I beta-turn. For the latter type of hairpin conformation, the original computations had predicted phi i+I = -60 degrees, psi i+1 = -30 degrees, phi i+2 = -90 degrees, and psi i +2 = 0 degrees as backbone torsion angle values, and these have been used from that time as reference values for the identification of the type I beta-turn. However, it has never been clarified whether these "ideal" backbone torsion angle values exist in real structures, or whether these torsion angles are only "theoretical values." Using the most recent release of the Protein Data Bank (1994), a survey has been made to assign amino acid pairs that approach the ideal form of the type I beta-turn. The analysis resulted in four sequences where the deviation from ideal values for any main-chain torsion angles was less than 2 degrees. In order to determine whether such a backbone fold is possible only in proteins owing to fortuitous cooperation of different folding effects, or whether it occurs even in short peptides, various attempts have been made to design the optimal amino acid sequence. Such a peptide model compound adopting precisely the predicted torsion angle values [phi i+1 = -60 degrees, psi i +1 = -30 degrees, phi i +2 = -90 degrees, and psi i+2 = 0 degrees] could provide valuable information. The solid state conformation of cyclo[(delta)Ava-Gly-Pro-Thr(OtBu)-Gly] reported herein, incorporating the -Pro-Thr- subunit, yields values suggesting that the "ideal" type I beta-turn is even possible for a peptide where there are no major environmental effects present.

Accuracy of side-chain prediction upon near-native protein backbones generated by Ab initio folding methods.

The ab initio folding problem can be divided into two sequential tasks of approximately equal computational complexity: the generation of native-like backbone folds and the positioning of side chains upon these backbones. The prediction of side-chain conformation in this context is challenging, because at best only the near-native global fold of the protein is known. To test the effect of displacements in the protein backbones on side-chain prediction for folds generated ab initio, sets of near-native backbones (< or = 4 A C alpha RMS error) for four small proteins were generated by two methods. The steric environment surrounding each residue was probed by placing the side chains in the native conformation on each of these decoys, followed by torsion-space optimization to remove steric clashes on a rigid backbone. We observe that on average 40% of the chi1 angles were displaced by 40 degrees or more, effectively setting the limits in accuracy for side-chain modeling under these conditions. Three different algorithms were subsequently used for prediction of side-chain conformation. The average prediction accuracy for the three methods was remarkably similar: 49% to 51% of the chi1 angles were predicted correctly overall (33% to 36% of the chi1+2 angles). Interestingly, when the inter-side-chain interactions were disregarded, the mean accuracy increased. A consensus approach is described, in which side-chain conformations are defined based on the most frequently predicted chi angles for a given method upon each set of near-native backbones. We find that consensus modeling, which de facto includes backbone flexibility, improves side-chain prediction: chi1 accuracy improved to 51-54% (36-42% of chi1+2). Implications of a consensus method for ab initio protein structure prediction are discussed.

Conserved mode of peptidomimetic inhibition and substrate recognition of human cytomegalovirus protease.

Human cytomegalovirus (HCMV) protease belongs to a new class of serine proteases, with a unique polypeptide backbone fold. The crystal structure of the protease in complex with a peptidomimetic inhibitor (based on the natural substrates and covering the P4 to P1' positions) has been determined at 2.7 A resolution. The inhibitor is bound in an extended conformation, forming an anti-parallel beta-sheet with the protease. The P3 and P1 side chains are less accessible to solvent, whereas the P4 and P2 side chains are more exposed. The inhibitor binding mode shows significant similarity to those observed for peptidomimetic inhibitors or substrates of other classes of serine proteases (chymotrypsin and subtilisin). HCMV protease therefore represents example of convergent evolution. In addition, large conformational differences relative to the structure of the free enzyme are observed, which may be important for inhibitor binding.

The Three-Dimensional Structure of Aspergillus niger Pectin Lyase B at 1.7-Å Resolution1

The three-dimensional structure of Aspergillus niger pectin lyase B (PLB) has been determined by crystallographic techniques at a resolution of 1.7 A. The model, with all 359 amino acids and 339 water molecules, refines to a final crystallographic R factor of 16.5%. The polypeptide backbone folds into a large right-handed cylinder, termed a parallel beta helix. Loops of various sizes and conformations protrude from the central helix and probably confer function. The largest loop of 53 residues folds into a small domain consisting of three antiparallel beta strands, one turn of an alpha helix, and one turn of a 3(10) helix. By comparison with the structure of Erwinia chrysanthemi pectate lyase C (PelC), the primary sequence alignment between the pectate and pectin lyase subfamilies has been corrected and the active site region for the pectin lyases deduced. The substrate-binding site in PLB is considerably less hydrophilic than the comparable PelC region and consists of an extensive network of highly conserved Trp and His residues. The PLB structure provides an atomic explanation for the lack of a catalytic requirement for Ca2+ in the pectin lyase family, in contrast to that found in the pectate lyase enzymes. Surprisingly, however, the PLB site analogous to the Ca2+ site in PelC is filled with a positive charge provided by a conserved Arg in the pectin lyases. The significance of the finding with regard to the enzymatic mechanism is discussed.

Three-dimensional solution structure of alpha-conotoxin MII, an alpha3beta2 neuronal nicotinic acetylcholine receptor-targeted ligand.

alpha-Conotoxin MII, isolated from Conus magus, is a potent peptidic toxin which specifically targets the mammalian neuronal nicotinic acetylcholine receptor, alpha3beta2 subtype. The three-dimensional structure of alpha-conotoxin MII in aqueous solution has been determined by two-dimensional 1H NMR spectroscopy. NOE-derived distances, refined by an iterative relaxation matrix approach, as well as dihedral and chirality restraints were used in high-temperature biphasic simulated annealing calculations. Fourteen minimum energy structures out of 50 subjected to the SA simulations were chosen for evaluation; these 14 structures have a final RMS deviation of 0.76 +/- 0.31 and 1.35 +/- 0.34 A for the backbone and heavy atoms, respectively. The overall structure is unusually well-defined due to a large helical component around the two disulfide bridges. The principal backbone folding motif may be common to a subclass of alpha-conotoxins. There are two distinct surfaces on the molecule almost at right angles to one another. One entirely consists of the hydrophobic residues Gly1, Cys2, Cys3, Leu15, and Cys16. The second comprises the hydrophilic residues Glu11, His12, Ser13, and Asn14. These surfaces on the ligand could be essential for the subtype-specific recognition of the receptor.

A novel thermostable inhibitor of trypsin and subtilisin from the seeds of Brassica nigra: amino acid sequence, inhibitory and spectroscopic properties and thermostability

A novel thermostable protein inhibitor of trypsin and subtilisin, called BN, was isolated from the seeds of Brassica nigra. The purified protein gave a single band on SDS-PAGE, corresponding to a molecular mass of 15 500±1000 Da. The inhibitor is composed of two disulfide-linked polypeptide chains, consisting of 39 and 90 residues, respectively. The amino acid sequence of the two chains was determined by Edman degradation of peptides, isolated from enzyme hydrolysates with TPCK-trypsin, EndoLysC proteinase and a Glu-specific proteinase of reduced and vinylpyridinated protein samples. A segment of the `heavy' chain, between residues 65 and 81, showed homology with the reactive site loop region of the 6-kDa trypsin inhibitors from Nicotiana alata. The basic residue in position 39 ( N. alata) or 70 (napins) is conserved as arginine or lysine in all inhibitors from N. alata and in all napins hitherto sequenced. Probably, the two families of trypsin inhibitors have structurally similar reactive sites. BN exhibits an extremely high thermostability: CD measurements showed that during heating to 97°C it preserves a considerable part of the polypeptide backbone folding. Studies on the fluorescence properties of the inhibitor BN in the absence and presence of neutral or ionic quenchers demonstrated that the intrinsic emission of this protein is dominated by a tryptophyl residue, buried in the interior of the protein matrix. 20% of the light absorbed by Tyr 63 of the `heavy' chain is transferred to Trp 26 of the `light' chain.

Lipopeptides with improved properties: structure by NMR, purification by HPLC and structure-activity relationships of new isoleucyl-rich surfactins.

The biosynthesis of bacterial isoleucyl-rich surfactins was controlled by supplementation of L-isoleucine to the culture medium. Two new variants, the [Ile4,7]- and [Ile2,4,7]surfactins, were thus produced by Bacillus subtilis and their separation was achieved by reverse-phase HPLC. Amino acids of the heptapeptide moiety were analysed by chemical methods, and the lipid moiety was identified by beta-hydroxy anteiso pentadecanoic acid by combined GC/MS. Sequences were established on the basis of two-dimensional NMR data. Because conformational parameters issuing from NMR spectra suggested that the cyclic backbone fold was globally conserved in the new variants, structure-activity relationships were discussed in details on the basis of the three-dimensional model of surfactin in solution. Indeed, both variants have increased surface properties compared with that of surfactin, and this improvement is assigned to an increase of the hydrophobicity of the apolar domain favouring micellization. Furthermore, the additional Leu-to-Ile substitution at position 2 in the [Ile2,4,7]surfactin leads to a substantial increase of its affinity for calcium, when compared with that of [Ile4,7]surfactin or surfactin. This effect is assigned, from the model, to an increase in the accessibility of the acidic side chains constituting the calcium binding site. Thus, the propensities of such active lipopeptides for both hydrophobic and electrostatic interactions were improved, further substantiating that they can be rationally designed.

Solution structure of an SRYD-containing sequence (250?257) of the fibronectin-like Leishmania gp63 protein by restrained molecular dynamics

The IASRYDQL synthetic octapeptide (250–257) of the Leishmania major surface glycoprotein gp63 efficiently inhibits parasite attachment to the macrophage receptors in in vitro experiments, and the SRYD-containing tetrapeptide mimics antigenically and functionally the RGDS sequence of fibronectin. The conformational properties of the octapeptide were investigated in dimethylsulfoxide (DMSO) with the combined use of NMR data (vicinal coupling constants, nuclear Overhauser effects (NOEs) and temperature coefficient values), molecular modeling by energy minimization and molecular dynamics. The structure is characterized by the high occurrence, exceeding 95%, of the Arg-Asp side-chain-side-chain ionic interaction, which plays a key role in the backbone folding through a distorted type-I β-turn involving the Gln256-NH to Arg253-CO hydrogen bond.

A new serine-protease fold revealed by the crystal structure of human cytomegalovirus protease.

Human cytomegalovirus (hCMV), a herpesvirus, infects up to 70% of the general population in the United States and can cause morbidity and mortality in immunosuppressed individuals (organ-transplant recipients and AIDS patients) and congenitally infected newborns. hCMV protease is essential for the production of mature infectious virions, as it performs proteolytic processing near the carboxy terminus (M-site) of the viral assembly protein precursor. hCMV protease is a serine protease, although it has little homology to other clans of serine proteases. Here we report the crystal structure of hCMV protease at 2.0 angstroms resolution, and show that it possesses a new polypeptide backbone fold. Ser 132 and His 63 are found in close proximity in the active site, confirming earlier biochemical and mutagenesis studies. The structure suggests that the third member of the triad is probably His 157. A dimer of the protease with an extensive interface is found in the crystal structure. This structure information will help in the design and optimization of inhibitors against herpesvirus proteases.

A new serine protease fold revealed by the crystal structure of human cytomegalovirus protease

HUMAN cytomegalovirus (hCMV), a herpesvirus, infects up to 70% of the general population in the United States and can cause morbidity and mortality in immunosuppressed individuals (organ-transplant recipients and AIDS patients) and congenitally infected newborns1. hCMV protease is essential for the production of mature infectious virions, as it performs proteolytic processing near the carboxy terminus (M-site) of the viral assembly protein precursor (for a review, see ref. 2). hCMV protease is a serine protease2, although it has little homology to other clans of serine proteases2,3 Here we report the crystal structure of hCMV protease at 2.0A resolution, and show that it possesses a new polypeptide backbone fold. Ser 132 and His 63 are found in close proximity in the active site, confirming earlier biochemical and mutagenesis studies2. The structure suggests that the third member of the triad is probably His 157. A dimer of the protease with an extensive interface is found in the crystal structure. This structure information will help in the design and optimization of inhibitors against herpesvirus proteases.

A search for the ideal type I β‐turn

In 1968 C. Venkatachalam (Biopolymers, Vol. 6, pp. 1425–1436) predicted the ideal forms of β-turns (type I, type II, etc.) based entirely on theoretical calculations. Subsequently, over a thousand x-ray structures of different globular proteins have been analyzed, with results suggesting that the most important form among the hairpin conformers is the type I β-turn. For the latter type of hairpin conformation, the original computations had predicted ϕi+1 = −60°, ψi+1 = −30°, ϕi+2 = −90°, and ψi+2 = 0° as backbone torsion angle values, and these have been used from that time as reference values for the identification of the type I β-turn. However, it has never been clarified whether these “ideal” backbone torsion angle values exist in real structures, or whether these torsion angles are only “theoretical values.” Using the most recent release of the Protein Data Bank (1994), a survey has been made to assign amino acid pairs that approach the ideal form of the type I β-turn. The analysis resulted in four sequences where the deviation from ideal values for any main-chain torsion angles was less than 2°. In order to determine whether such a backbone fold is possible only in proteins owing to fortuitous cooperation of different folding effects, or whether it occurs even in short peptides, various attempts have been made to design the optimal amino acid sequence. Such a peptide model compound adopting precisely the predicted torsion angle values [ϕi+1 = −60°, ψi+1 = −30°, ϕi+2 = −90°, and ψi+2 = 0°] could provide valuable information. The solid state conformation of cyclo[(δ) Ava-Gly-Pro-Thr (O1Bu)-Gly] reported herein, incorporating the -Pro-Thr- subunit, yields values suggesting that the “ideal” type I β-turn is even possible for a peptide where there are no major environmental effects present. © 1996 John Wiley & Sons, Inc.