Structural insights into the mechanism of intramolecular proteolysis.

A variety of proteins, including glycosylasparaginase, have recently been found to activate functions by self-catalyzed peptide bond rearrangements from single-chain precursors. Here we present the 1.9 A crystal structures of glycosylasparaginase precursors that are able to autoproteolyze via an N --> O acyl shift. Several conserved residues are aligned around the scissile peptide bond that is in a highly strained trans peptide bond configuration. The structure illustrates how a nucleophilic side chain may attack the scissile peptide bond at the immediate upstream backbone carbonyl and provides an understanding of the structural basis for peptide bond cleavage via an N --> O or N --> S acyl shift that is used by various groups of intramolecular autoprocessing proteins.     

Crystallographic Snapshot of Glycosylasparaginase Precursor Poised for Autoprocessing

Glycosylasparaginase belongs to a family of N-terminal nucleophile hydrolases that autoproteolytically generate their mature enzymes from single-chain protein precursors. Previously, based on a precursor structure paused at pre-autoproteolysis stage by a reversible inhibitor (glycine), we proposed a mechanism of intramolecular autoproteolysis. A key structural feature, a highly strained conformation at the scissile peptide bond, had been identified and was hypothesized to be critical for driving autoproteolysis through an N-O acyl shift. To examine this “twist-and-break” hypothesis, we report here a 1. 9-Å-resolution structure of an autoproteolysis-active precursor (a T152C mutant) that is free of inhibitor or ligand and is poised to undergo autoproteolysis. The current crystallographic study has provided direct evidence for the natural conformation of the glycosylasparaginase autocatalytic site without influence from any inhibitor or ligand. This finding has confirmed our previous proposal that conformational strain is an intrinsic feature of an active precursor.     

Two-step dimerization for autoproteolysis to activate glycosylasparaginase

Glycosylasparaginase (GA) is an amidase and belongs to a novel family of N-terminal nucleophile hydrolases that use a similar autoproteolytic processing mechanism to generate a mature/active enzyme from a single chain protein precursor. From bacteria to eukaryotes, GAs are conserved in primary sequences, tertiary structures, and activation of amidase activity by intramolecular autoproteolysis. An evolutionarily conserved His-Asp-Thr sequence is cleaved to generate a newly exposed N-terminal threonine, which plays a central role in both autoproteolysis and in its amidase activity. We have recently determined the crystal structure of the bacterial GA precursor at 1.9-A resolution, which reveals a highly distorted and energetically unfavorable conformation at the scissile peptide bond. A mechanism of autoproteolysis via an N-O acyl shift was proposed to relieve these conformational strains. However, it is not understood how the polypeptide chain distortion was generated and preserved during the folding of GA to trigger autoproteolysis. An obstacle to our understanding of GA autoproteolysis is the uncertainty concerning its quaternary structure in solution. Here we have revisited this question and show that GA forms dimers in solution. Mutants with alterations at the dimer interface cannot form dimers and are impaired in the autoproteolytic activation. This suggests that dimerization of GA plays an essential role in autoproteolysis to activate the amidase activity. Comparison of the melting temperatures of GA dimers before and after autoproteolysis suggests two states of dimerization in the process of enzyme maturation. A two-step dimerization mechanism to trigger autoproteolysis is proposed to accommodate the data presented here as well as those in the literature.     

Structural constraints on autoprocessing of the human nucleoporin Nup98

Nucleoporin Nup98, a 98-kDa protein component of the nuclear pore complex, plays an important role in both protein and RNA transport. During its maturation process, Nup98 undergoes post-translational autoproteolysis, which is critical for targeting to the NPC. Here we present high-resolution crystal structures of the C-terminal autoproteolytic domains of Nup98 (2.3 A for the wild type and 1.9 A for the S864A precursor), and propose a detailed autoproteolysis mechanism through an N-O acyl shift. Structural constraints are found at the autocleavage site, and could thus provide a driving force for autocleavage at the scissile peptide bond. Such structural constraints appear to be generated, at least in part, by anchoring a conserved phenylalanine side chain into a highly conserved hydrophobic pocket at the catalytic site. Our high-resolution crystal structures also reveal that three highly conserved residues, Tyr866, Gly867, and Leu868, provide most of the interactions between the autoproteolytic domain and the C-terminal tail. These results suggest that Nup98 may represent a new subtype of protein that utilizes autoprocessing to control biogenesis pathways and intracellular translocation.     

Survey of conformational role of ester bonds in a cyclic depsipeptide. Study on cyclo(-L-Ala-L-Hmb-)2 by energy calculation and NMR spectroscopy.

The effect of ester bond on the conformation of peptide molecule was studied by designing and synthesizing a model tetradepsipeptide cyclo(-L-Ala-L-Hmb-)2 and by analyzing the conformation both theoretically and experimentally. Theoretical analysis showed that both ester and peptide bonds in the calculated low-energy conformations within 3 kcal/mol of the global minimum take a trans but distorted configuration. The distortion is larger in ester bonds than in peptide bonds. Further, the four carbonyls project from one side of the plane of the cyclic backbone, whereas the side chains project from the other side. These results are consistent with the experimental results obtained by NMR measurement; first, the coupling constant deduced from 1H-NMR species in DMSO-d6 is consistent with the dihedral angles of the calculated low-energy conformations; second, results of NOE measurement can reproduce the calculated configuration of the carbonyls and side chains. From the consistency between theoretical and experimental results, it is concluded that this model tetradepsipeptide takes an all-trans backbone conformation in solution and this backbone conformation is stabilized by large distortion in the ester bond, which compensates the strain resulted from the 12-membered cyclic backbone structure consisting only of L-residues.     

High-resolution structure of intramolecularly proteolyzed human mucin-1 SEA domain

SEA domains are ubiquitous in large proteins associated with highly glycosylated environments. Certain SEA domains undergo intramolecular proteolysis involving a nucleophilic attack of a serine hydroxyl group on the preceding glycine carbonyl. The mucin-1 (MUC1) SEA domain has been extensively investigated as a model of intramolecular proteolysis. Since neither a general base, a general acid, nor an oxyanion hole could be identified in MUC1 SEA, it has been suggested that proteolysis is accelerated by a non-planarity of the scissile peptide bond imposed by protein folding. A reactant distorted peptide bond has been also invoked to explain the autoproteolysis of several unrelated proteins. However, the only evidence of peptide distortion in MUC1 SEA stems from molecular dynamic simulations of the reactant modeled upon a single NMR structure of the cleaved product. We report the first high-resolution X-ray structure of cleaved MUC1 SEA. Structural comparison with uncleaved SEA domains suggests that the number of residues evolutionarily inserted in the cleaved loop of MUC1 SEA precludes the formation of a properly hydrogen-bonded beta turn. By sequence analysis, we show that this conformational frustration is shared by all known cleaved SEA domains. In addition, alternative conformations of the uncleaved precursor could be modeled in which the scissile peptide bond is planar. The implications of these structures for autoproteolysis are discussed in the light of the previous research on autoproteolysis.     

Enzymatic cyclization of a potent bowman-birk protease inhibitor,sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide

The most potent known naturally occurring Bowman-Birk inhibitor, sunflower trypsin inhibitor-1 (SFTI-1), is a bicyclic 14-amino acid peptide from sunflower seeds comprising one disulfide bond and a cyclic backbone. At present, little is known about the cyclization mechanism of SFTI-1. We show here that an acyclic permutant of SFTI-1 open at its scissile bond, SFTI-1[6,5], also functions as an inhibitor of trypsin and that it can be enzymatically backbone-cyclized by incubation with bovine beta-trypsin. The resulting ratio of cyclic SFTI-1 to SFTI-1[6,5] is approximately 9:1 regardless of whether trypsin is incubated with SFTI-1[6,5] or SFTI-1. Enzymatic resynthesis of the scissile bond to form cyclic SFTI-1 is a novel mechanism of cyclization of SFTI-1[6,5]. Such a reaction could potentially occur on a trypsin affinity column as used in the original isolation procedure of SFTI-1. We therefore extracted SFTI-1 from sunflower seeds without a trypsin purification step and confirmed that the backbone of SFTI-1 is indeed naturally cyclic. Structural studies on SFTI-1[6,5] revealed high heterogeneity, and multiple species of SFTI-1[6,5] were identified. The main species closely resembles the structure of cyclic SFTI-1 with the broken binding loop able to rotate between a cis/trans geometry of the I7-P8 bond with the cis conformer being similar to the canonical binding loop conformation. The non-reactive loop adopts a beta-hairpin structure as in cyclic wild-type SFTI-1. Another species exhibits an iso-aspartate residue at position 14 and provides implications for possible in vivo cyclization mechanisms.     

Stress and strain in staphylococcal nuclease.

Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well-refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116-117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a predominantly trans Gly 116-Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the beta-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-117 loop only allow trans conformations where the local backbone interactions associated with the phi and psi torsion angles are strained. When the 116-117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X-Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.     

Empirical rules predicting conformation of cyclic tetrapeptides from primary structure

A useful set of empirical rules is put forward to predict the conformations of cyclic tetrapeptides and cyclic tetradepsipeptides on the basis of primary structure, briefly presented as follows: A conformation allowing an intramolecular hydrogen bond (IMHB) of gamma-turn is preferred, and an ester bond always adopts a trans form. On a right-handed peptide ring, the carbonyl group acylating a D residue is oriented to the upper side of the main ring. The carbonyl group acylating a D proline or an N-methyl-D-amino acid residue is oriented to the lower side of the ring, forming a cis bond. The LDDL configurational sequence adopts a cis-trans-cis-trans backbone with Ci symmetry. A glycine residue behaves as a D residue in an L-peptide. Conformations of cyclotetrapeptides containing two glycine residues at diametric positions or containing an N-methyl-dehydroamino acid residue are predicted by use of appendices of rule 5. Almost all conformations of cyclic tetrapeptides are predicted by these rules. Energetical rationalization of the rules and prediction of possible new conformations are described. Conformations of cyclo(-L-Pro-L-Leu-D-Tyr(Me)-L-Ile-)(1) and cyclo (-L-Pro-D-Leu-D-Tyr(Me)-L-Ile)(2) are compared. Results of n.m.r. experiments showed that compound 1 adopts a unique cis-trans-trans-trans backbone with a gamma-turn IMHB, and 2 has a cis-trans-cis-trans backbone with Ci symmetry. These observations confirmed the rules described above. Peptides 1 and 2 are the first diastereomeric peptides with trans (LD) and cis (DD) secondary amide bonds.     

Vicinal disulfide turns

The formation of a disulfide bond between adjacent cysteine residues is accompanied by the formation of a tight turn of the protein backbone. In nearly 90% of the structures analyzed a type VIII turn was found. The peptide bond between the two cysteines is in a distorted trans conformation, the omega torsion angle ranges from 159 to -133 degrees, with an average value of 171 degrees. The constrained nature of the vicinal disulfide turn and the pronounced difference observed between the oxidized and reduced states, suggests that vicinal disulfides may be employed as a 'redox-activated' conformational switch.     

Resonance Raman enhancement of the Mn-N-O bending mode in nitrosyl manganese "strapped" and "open" heme complexes

Resonance Raman spectra of the MnII-NO moiety in synthetic nitrosyl manganese heme complexes with and without steric hindrance are reported. The "strapped" hemes having a hydrocarbon strap (variable length) across one face of the heme hinder the perpendicular bonding of a linear ligand. These complexes were employed to investigate the effects of ligand distortion (primarily tilting) on Mn-NO stretching, Mn-N-O bending, and N-O stretching modes. It is demonstrated that ligand distortion in the MnII-NO system is a valid mechanism for causing the resonance enhancement of the Mn-N-O bending mode, similar to that observed in the FeII-CO system (Yu, N.-T., E. A. Kerr, B. Ward, and C. K. Chang. 1983. Biochemistry. 22:4534-4540). More interesting is the observation of the delta(Mn-N-O) enhancement caused by the tilting of the trans Mn-N epsilon bond in the "open" heme complexes (e.g., heme-5 and proto-1X dimethylester) with 1,2-dimethylimidazole or piperidine as a base. The nu(Mn-NO) and nu(N-O) modes exhibit an increase and a decrease, respectively, as the strap length decreases (hence the steric hindrance increases). Both nu(Mn-NO) and nu(N-O) frequencies are insensitive to the strength of the trans base. The results from "strapped" and "open" model heme systems imply that the Mn-N-O geometry is essentially linear and perpendicular in the nitrosyl complexes of monomeric manganese insect hemoglobin CTT IV and sperm whale myoglobin. The unusually low nu(N-O) frequency in the manganese myoglobin complex may be caused by the distal histidine-NO interaction. The delta(Mn-N-O) enhancement in both nitrosyl manganese CTT IV and nitrosyl manganese myoglobin may be caused by a tilting of the Mn"-Nf (proximal histidine) bond.     

Serine-53 at the tip of the glycine-rich loop of cAMP-dependent protein kinase: role in catalysis, P-site specificity, and interaction with inhibitors

The glycine-rich loop, one of the most important motifs in the conserved protein kinase catalytic core, embraces the entire nucleotide, is very mobile, and is exquisitely sensitive to what occupies the active site cleft. Of the three conserved glycines [G(50)TG(52)SFG(55) in cAMP-dependent protein kinase (cAPK)], Gly(52) is the most important for catalysis because it allows the backbone amide of Ser(53) at the tip of the loop to hydrogen bond to the gamma-phosphate of ATP [Grant, B. D. et al. (1998) Biochemistry 37, 7708]. The structural model of the catalytic subunit:ATP:PKI((5)(-)(24)) (heat-stable protein kinase inhibitor) ternary complex in the closed conformation suggests that Ser(53) also might be essential for stabilization of the peptide substrate-enzyme complex via a hydrogen bond between the P-site carbonyl in PKI and the Ser(53) side-chain hydroxyl [Bossemeyer, D. et al. (1993) EMBO J. 12, 849]. To address the importance of the Ser(53) side chain in catalysis, inhibition, and P-site specificity, Ser(53) was replaced with threonine, glycine, and proline. Removal of the side chain (i.e., mutation to glycine) had no effect on the steady-state phosphorylation of a peptide substrate (LRRASLG) or on the interaction with physiological inhibitors, including the type-I and -II regulatory subunits and PKI. However, this mutation did affect the P-site specificity; the glycine mutant can more readily phosphorylate a P-site threonine in a peptide substrate (5-6-fold better than wild-type). The proline mutant is compromised catalytically with altered k(cat) and K(m) for both peptide and ATP and with altered sensitivity to both regulatory subunits and PKI. Steric constraints as well as restricted flexibility could account for these effects. These combined results demonstrate that while the backbone amide of Ser(53) may be required for efficient catalysis, the side chain is not.     

The crystal structure of the cis-proline to glycine variant (P114G) of ribonuclease A

Replacement of a cis-proline by glycine at position 114 in ribonuclease A leads to a large decrease in thermal stability and simplifies the refolding kinetics. A crystallographic approach was used to determine whether the decrease in thermal stability results from the presence of a cis glycine peptide bond, or from a localized structural rearrangement caused by the isomerization of the mutated cis 114 peptide bond. The structure was solved at 2.0 A resolution and refined to an R-factor of 19.5% and an R(free) of 21.9%. The overall conformation of the protein was similar to that of wild-type ribonuclease A; however, there was a large localized rearrangement of the mutated loop (residues 110-117-a 9.3 A shift of the Calpha atom of residue 114). The peptide bond before Gly114 is in the trans configuration. Interestingly, a large anomalous difference density was found near residue 114, and was attributed to a bound cesium ion present in the crystallization experiment. The trans isomeric configuration of the peptide bond in the folded state of this mutant is consistent with the refolding kinetics previously reported, and the associated protein conformational change provides an explanation for the decreased thermal stability.     

The occurence of the syn-C3' endo conformation and the distorted backbone conformations for C4'-C5' and P-O5' in oligo and polynucleotides.

The nucleoside constituents of nucleic acids prefer the anti conformation (1). When the sugar pucker is taken into account the nucleosides prefer the C2'endo-anti conformation. Of the nearly 300 nucleosides known, about 250 are in the anti conformation and 50 are in the syn-conformation, i.e., anti to syn conformation is 5:1. The nucleotide building blocks of nucleic acids show the same trend as nucleosides. Both the deoxy-guanosine and riboguanosine residues in nucleosides and nucleotides prefer the syn-C2'endo conformation with an intra-molecular hydrogen bond (for nucleosides) between the O5'-H and the N3 of the base and, a few syn-C3'endo conformations are also observed. Evidence is presented for the occurrence of the C3'endo-syn conformation for guanines in mis-paired double helical right-handed structures with the distorted sugar phosphate C4'-C5' and P-O5' bonds respectively, from g+ (gg) and g- to trans. Evidence is also provided for guanosine nucleotides in left-handed double-helical (Z-DNA) oligo and polynucleotides which has the same syn-C3'endo conformation and the distorted backbone sugar-phosphate bonds (C4'-C5' and P-O5') as in the earlier right-handed case.     

Kinetic specificity in papain-catalysed hydrolyses

The specificity of the proteolytic enzyme, papain, for the peptide bond of the substrate adjacent to that about to be cleaved and for the acyl residue of some N-acylglycine derivatives is manifest almost exclusively in the formation of the acyl-enzyme from the enzyme-substrate complex. Models for the enzyme-substrate complex and acyl-enzyme intermediate are suggested that account for these observations. In particular it is suggested that the peptide bond of the substrate adjacent to that about to be cleaved, is bound in the cleft of the enzyme between the NH group of glycine-66 and the backbone C=O group of aspartic acid-158, and provides a sensitive amplification mechanism through which the specificity of the enzyme for hydrophobic amino acids such as l-phenylalanine is relayed. It is also suggested that the distortion in the enzyme-substrate complex and the binding of the peptide bond adjacent to that about to be cleaved are also linked and behave co-operatively, the distortion of the protein facilitating binding and the stronger binding facilitating distortion. The results imply that between the enzyme-substrate complex and the acyl-enzyme a relaxation of the protein conformation must occur.     

NMR chemical shift mapping of the binding site of a protein proteinase inhibitor: changes in the (1)H, (13)C and (15)N NMR chemical shifts of turkey ovomucoid third domain upon binding to bovine chymotrypsin A(alpha)

The substrate-like inhibition of serine proteinases by avian ovomucoid domains has provided an excellent model for protein inhibitor-proteinase interactions of the standard type. 1H,15N and 13C NMR studies have been undertaken on complexes formed between turkey ovomucoid third domain (OMTKY3)2 and chymotrypsin A(alpha) (Ctr) in order to characterize structural changes occurring in the Ctr binding site of OMTKY3. 15N and 13C were incorporated uniformly into OMTKY3, allowing backbone resonances to be assigned for OMTKY3 in both its free and complex states. Chemical shift perturbation mapping indicates that the two regions, K13-P22 and N33-A40, are the primary sites in OMTKY3 involved in Ctr binding, in full agreement with the 12 consensus proteinase-contact residues of OMTKY3 defined previously on the basis of X-ray crystallographic and mutational analysis. Smaller chemical shift perturbations in selected other regions may result from minor structural changes on binding. Through-bond 15N-13C correlations between P1-13C' and P1'-15N in two-dimensional H(N)CO and HN(CO) NMR spectra of selectively labeled OMTKY3 complexed with Ctr indicate that the scissile peptide bond between L18 and E19 of the inhibitor is intact in the complex. The chemical shifts of the reactive site peptide bond indicate that it is predominantly trigonal, although the data are not inconsistent with a slight perturbation of the hybridization of the peptide bond toward the first tetrahedral state along the reaction coordinate.     

The importance of anchorage in determining a strained protein loop conformation.

We examine the role of the conformational restriction imposed by constrained ends of a protein loop on the determination of a strained loop conformation. The Lys 116-Pro 117 peptide bond of staphylococcal nuclease A exists in equilibrium between the cis and trans isomers. The folded protein favors the strained cis isomer with an occupancy of 90%. This peptide bond is contained in a solvent-exposed, flexible loop of residues 112-117 whose ends are anchored by Val 111 and Asn 118. Asn 118 is constrained by 2 side-chain hydrogen bonds. We investigate the importance of this constraint by replacing Asn 118 with aspartate, alanine, and glycine. We found that removing 1 or more of the hydrogen bonds observed in Asn 118 stabilizes the trans configuration over the cis configuration. By protonating the Asp 118 side chain of N118D through decreased pH, the hydrogen bonding character of Asp 118 approached that of Asn 118 in nuclease A, and the cis configuration was stabilized relative to the trans configuration. These data suggest that the rigid anchoring of the loop end is important in establishing the strained cis conformation. The segment of residues 112-117 in nuclease A provides a promising model system for study of the basic principles that determine polypeptide conformations. Such studies could be useful in the rational design or redesign of protein molecules.     

The occurrence of the syn-C3′ endo conformation and the distorted backbone conformations for C4′-C5′ and P-O5′ in oligo and polynucleotides

Abstract

The nucleoside constituents of nucleic acids prefer the anti conformation (1). When the sugar pucker is taken into account the nucleosides prefer the C2′endo-anti conformation. Of the nearly 300 nucleosides known, about 250 are in the anti conformation and 50 are in the syn-conformation, i.e., anti to syn conformation is 5:1. The nucleotide building blocks of nucleic acids show the same trend as nucleosides. Both the deoxy-guanosine and ribo- guanosine residues in nucleosides and nucleotides prefer the syn-C2′endo conformation with an intra-molecular hydrogen bond (for nucleosides) between the O5′- H and the N3 of the base and, a few syn-C3′endo conformations are also observed. Evidence is presented for the occurrence of the C3′endo-syn conformation for guanines in mis-paired double helical right-handed structures with the distorted sugar phosphate C4′-C5′ and P-O5′ bonds respectively, from g+ (gg) and g- to trans. Evidence is also provided for guanosine nucleotides in left-handed double-helical (Z-DNA) oligo and polynucleotides which has the same syn-C3′endo conformation and the distorted backbone sugar-phosphate bonds (C4′-C5′ and P- O5′) as in the earlier right-handed case.     

Crystal structures of glutaryl 7-aminocephalosporanic acid acylase: insight into autoproteolytic activation

Glutaryl 7-aminocephalosporanic acid acylase (GCA, EC 3.5.1.11) is a member of N-terminal nucleophile (Ntn) hydrolases. The native enzyme is an (alpha beta)(2) heterotetramer originated from an enzymatically inactive precursor of a single polypeptide. The activation of precursor GCA consists of primary and secondary autoproteolytic cleavages, generating a terminal residue with both a nucleophile and a base and releasing a nine amino acid spacer peptide. We have determined the crystal structures of the recombinant selenomethionyl native and S170A mutant precursor from Pseudomonas sp. strain GK16. Precursor activation is likely triggered by conformational constraints within the spacer peptide, probably inducing a peptide flip. Autoproteolytic site solvent molecules, which have been trapped in a hydrophobic environment by the spacer peptide, may play a role as a general base for nucleophilic attack. The activation results in building up a catalytic triad composed of Ser170/His192/Glu624. However, the triad is not linked to the usual hydroxyl but the free alpha-amino group of the N-terminal serine residue of the native GCA. Mutagenesis and structural data support the notion that the stabilization of a transient hydroxazolidine ring during autoproteolysis would be critical during the N --> O acyl shift. The autoproteolytic activation mechanism for GCA is described.     

Ion binding of cyclolinopeptide A: an NMR and CD conformational study

CD and nmr techniques have been used to study, in acetonitrile solution, the ion-complexing capability of cyclolinopeptide A (CLA), a cyclic nonapeptide of sequence cyclo-(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val) endowed with remarkable cytoprotective ability in vitro, and the conformation of the Ba(2+)/CLA complex. At room temperature, CLA in acetonitrile shows a proton nmr spectrum characteristic of the coexistence of many different conformers in intermediate exchange. The backbone contains a cis Pro-Pro bond, with all other peptide bonds in the trans conformation. CLA binds Ba2+ more tightly than the other cations studied, namely K+, Na+, Mg2+, and Ca2+; CD data are indicative of the presence of both 1:2 (sandwich) and 1:1 (equimolar) type complexes, depending on the Ba2+ ion concentration, whereas nmr data are consistent with an equimolar form. The relevant conformational features of the equimolar Ba2+/CLA complex are that the backbone contains all trans peptide bonds, a type I 6----3 beta-turn and a 3----1 gamma-turn (or a distorted 3----9 beta-turn). The global shape of the complexed peptide can be described as a bowl, with the concave (polar) side hosting Ba2+ and the convex side predominantly apolar.