Reflections on biocatalysis involving phosphorus.

Early studies on chemical synthesis of biological molecules can be seen to progress to preparation and biological evaluation of phosphonates as analogues of biological phosphates, with emphasis on their isosteric and isopolar character. Work with such mimics progressed into structural studies with a range of nucleotide-utilising enzymes. The arrival of metal fluorides as analogues of the phosphoryl group, PO(3)(-), for transition state (TS) analysis of enzyme reactions stimulated the symbiotic deployment of (19)F NMR and protein crystallography. Characteristics of enzyme transition state analogues are reviewed for a range of reactions. From the available MF(x) species, trifluoroberyllate gives tetrahedral mimics of ground states (GS) in which phosphate is linked to carboxylate and phosphate oxyanions. Tetrafluoroaluminate is widely employed as a TS mimic, but it necessarily imposes octahedral geometry on the assembled complexes, whereas phosphoryl transfer involves trigonal bipyramidal (tbp) geometry. Trifluoromagnesate (MgF(3)(-)) provides the near-ideal solution, delivering tbp geometry and correct anionic charge. Some of the forty reported tbp structures assigned as having AlF(3)(0) cores have been redefined as trifluoromagnesate complexes. Transition state analogues for a range of kinases, mutases, and phosphatases provide a detailed description of mechanism for phosphoryl group transfer, supporting the concept of charge balance in their TS and of concerted-associative pathways for biocatalysis. Above all, superposition of GS and TS structures reveals that in associative phosphoryl transfer, the phosphorus atom migrates through a triangle of three, near-stationary, equatorial oxygens. The extension of these studies to near attack conformers further illuminates enzyme catalysis of phosphoryl transfer.

Amyloid fibril formation by human stefin B: influence of the initial pH-induced intermediate state.

The amyloid fibril field is briefly described, with some stress put on differences between various proteins and possible role for domain swapping. In the main body of the text, first, a short review is given of the folding properties of both human stefins, alpha/beta-type globular proteins of 53% identity with a known three-dimensional fold. Second, in vitro study of amyloid fibril formation by human stefin B (type I cystatin) is described. Solvents of pH 4.8 and pH 3.3 with and without 2,2,2-trifluoroethanol (TFE) were probed, as it has been shown previously that stefin B forms acid intermediates, a native-like and molten globule intermediate, respectively. The kinetics of fibrillation were measured by thioflavin T fluorescence and CD. At pH 3.3, the protein is initially in the molten globule state. The fibrillation is faster than at pH 4.8; however, there is more aggregation observed. On adding TFE at each pH, the fibril formation is further accelerated.

Three-dimensional domain swapping in the folded and molten-globule states of cystatins, an amyloid-forming structural superfamily.

Cystatins, an amyloid-forming structural superfamily, form highly stable, domain-swapped dimers at physiological protein concentrations. In chicken cystatin, the active monomer is a kinetic trap en route to dimerization, and any changes in solution conditions or mutations that destabilize the folded state shorten the lifetime of the monomeric form. In such circumstances, amyloidogenesis will start from conditions where a domain-swapped dimer is the most prevalent species. Domain swapping occurs by a rearrangement of loop I, generating the new intermonomer interface between strands 2 and 3. The transition state for dimerization has a high level of hydrophobic group exposure, indicating that gross conformational perturbation is required for domain swapping to occur. Dimerization also occurs when chicken cystatin is in its reduced, molten-globule state, implying that the organization of secondary structure in this state mirrors that in the folded state and that domain swapping is not limited to the folded states of proteins. Although the interface between cystatin-fold units is poorly defined for cystatin A, the dimers are the appropriate size to account for the electron-dense regions in amyloid protofilaments.

Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones.

The translationally controlled tumor-associated proteins (TCTPs) are a highly conserved and abundantly expressed family of eukaryotic proteins that are implicated in both cell growth and the human acute allergic response but whose intracellular biochemical function has remained elusive. We report here the solution structure of the TCTP from Schizosaccharomyces pombe, which, on the basis of sequence homology, defines the fold of the entire family. We show that TCTPs form a structural superfamily with the Mss4/Dss4 family of proteins, which bind to the GDP/GTP free form of Rab proteins (members of the Ras superfamily) and have been termed guanine nucleotide-free chaperones (GFCs). Mss4 also acts as a relatively inefficient guanine nucleotide exchange factor (GEF). We further show that the Rab protein binding site on Mss4 coincides with the region of highest sequence conservation in the TCTP family. This is the first link to any other family of proteins that has been established for the TCTP family and suggests the presence of a GFC/GEF at extremely high abundance in eukaryotic cells.

Location and properties of metal-binding sites on the human prion protein.

Although a functional role in copper binding has been suggested for the prion protein, evidence for binding at affinities characteristic of authentic metal-binding proteins has been lacking. By presentation of copper(II) ions in the presence of the weak chelator glycine, we have now characterized two high-affinity binding sites for divalent transition metals within the human prion protein. One is in the N-terminal octapeptide-repeat segment and has a K(d) for copper(II) of 10(-14) M, with other metals (Ni(2+), Zn(2+), and Mn(2+)) binding three or more orders of magnitude more weakly. However, NMR and fluorescence data reveal a previously unreported second site around histidines 96 and 111, a region of the molecule known to be crucial for prion propagation. The K(d) for copper(II) at this site is 4 x 10(-14) M, whereas nickel(II), zinc(II), and manganese(II) bind 6, 7, and 10 orders of magnitude more weakly, respectively, regardless of whether the protein is in its oxidized alpha-helical (alpha-PrP) or reduced beta-sheet (beta-PrP) conformation. A role for prion protein (PrP) in copper metabolism or transport seems likely and disturbance of this function may be involved in prion-related neurotoxicity.

Wild-type and met-65-->Leu variants of human cystatin A are functionally and structurally identical.

The solution structure of an N-terminally truncated and mutant form (M65L(2-98)) of the human cysteine protease inhibitor cystatin A has been reported that reveals extensive structural differences when compared to the previously published structure of full-length wild-type (WT) cystatin A. On the basis of the M65L(2-98) structure, a model of the inhibitory mechanism of cystatin A was proposed wherein specific interactions between the N- and C-terminal regions of cystatin A are invoked as critical determinants of protease binding. To test this model and to account for the reported differences between the two structures, we undertook additional structural and mechanistic analyses of WT and mutant forms of human cystatin A. These show that modification at the C-terminus of cystatin A by the addition of nine amino acids has no effect upon the affinity of papain inhibition (K(D) = 0.18+/-0.02 pM) and the consequences of such modification are not propagated to other parts of the structure. These findings indicate that perturbation of the C-terminus can be achieved without any measurable effect on the N-terminus or the proteinase binding loops. In addition, introduction of the methionine-65 --> leucine substitution into cystatin A that retains the N-terminal methionine (M65L(1-98)) has no significant effect upon papain binding (K(D) = 0.34+/-0.02 pM). Analyses of the structures of WT and M65L(1-98) using (1)H NMR chemical shifts and residual dipolar couplings in a partially aligning medium do not reveal any evidence of significant differences between the two inhibitors. Many of the differences between the published structures correspond to major violations by M65L(2-98) of the WT constraints list, notably in relation to the position of the N-terminal region of the inhibitor, one of three structural motifs indicated by crystallographic studies to be involved in protease binding by cystatins. In the WT structure, and consistent with the crystallographic data, this region is positioned adjacent to another inhibitory motif (the first binding loop), whereas in M65L(2-98) there is no proximity of these two motifs. As the NMR data for both WT9C and M65L(1-98) are wholly consistent with the published structure of WT cystatin A and incompatible with that of M65L(2-98), we conclude that the former represents the most reliable structural model of this protease inhibitor.

The major transition state in folding need not involve the immobilization of side chains.

During protein folding in which few, if any, definable kinetic intermediates are observable, the nature of the transition state is central to understanding the course of the reaction. Current experimental data does not distinguish the relative contributions of side chain immobilization and dehydration phenomena to the major rate-limiting transition state whereas this distinction is central to theoretical models that attempt to simulate the behavior of proteins during folding. Renaturation of the small proteinase inhibitor cystatin under oxidizing versus reducing conditions is the first experimental case in which these processes can be studied independently. Using this example, we show that sidechain immobilization occurs downstream of the major folding transition state. A consequence of this is the existence of states with disordered side chains, which are distinct from kinetic protein folding intermediates and which lie within the folded state free energy well.

Structural mobility of the human prion protein probed by backbone hydrogen exchange.

Prions, the causative agents of Creutzfeldt-Jacob Disease (CJD) in humans and bovine spongiform encephalopathy (BSE) and scrapie in animals, are principally composed of PrPSc, a conformational isomer of cellular prion protein (PrPC). The propensity of PrPC to adopt alternative folds suggests that there may be an unusually high proportion of alternative conformations in dynamic equilibrium with the native state. However, the rates of hydrogen/deuterium exchange demonstrate that the conformation of human PrPC is not abnormally plastic. The stable core of PrPC has extensive contributions from all three alpha-helices and shows protection factors equal to the equilibrium constant for the major unfolding transition. A residual, hyper-stable region is retained upon unfolding, and exchange analysis identifies this as a small nucleus of approximately 10 residues around the disulfide bond. These results show that the most likely route for the conversion of PrPC to PrPSc is through a highly unfolded state that retains, at most, only this small nucleus of structure, rather than through a highly organized folding intermediate.

Differences in the effects of TFE on the folding pathways of human stefins A and B.

Trifluoroethanol (TFE) has been used to probe differences in the stability of the native state and in the folding pathways of the homologous cysteine protein inhibitors, human stefin A and B. After complete unfolding in 4.5 mol/L GuHCl, stefin A refolded in 11% (vol/vol) TFE, 0.75 mol/L GuHCl, at pH 6.0 and 20 degrees C, with almost identical first-order rate constants of 4.1 s-1 and 5.5 s-1 for acquisition of the CD signal at 230 and 280 nm, respectively, rates that were markedly greater than the value of 0.11 s-1 observed by the same two probes when TFE was absent. The acceleration of the rates of refolding, monitored by tyrosine fluorescence, was maximal at 10% (vol/vol) TFE. Similar rates of refolding (6.2s-1 and 7.2 s-1 for ellipticity at 230 and 280 nm, respectively) were observed for stefin A denatured in 66% (vol/vol) TFE, pH 3.3, when refolding to the same final conditions. After complete unfolding in 3.45 mol/L GuHCl, stefin B refolded in 7% (vol/vol) TFE, 0.57 mol/L GuHCl, at pH 6.0 and 20 degrees C, with a rate constant for the change in ellipticity at 280 nm of 32.8 s-1; this rate was only twice that observed when TFE was absent. As a major point of distinction from stefin A, the refolding of stefin B in the presence of TFE showed an overshoot in the ellipticity at 230 nm to a value 10% greater than that in the native protein; this signal relaxed slowly (0.01 s-1) to the final native value, with little concomitant change in the near-ultraviolet CD signal; the majority of this changes in two faster phases. After denaturation in 42% (vol/vol) TFE, pH 3.3, the kinetics of refolding to the same final conditions exhibited the same rate-limiting step (0.01 s-1) but were faster initially. The results show that similarly to stefin A, stefin B forms its hydrophobic core and predominant part of the tertiary structure faster in the presence of TFE. The results imply that the alpha-helical intermediate of stefin B is highly structured. Proteins 1999;36:205-216.

Identification of amino acid residues critical for aggregation of human CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES. Characterization of active disaggregated chemokine variants.

Human CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation normal T cell expressed) self-associate to form high-molecular mass aggregates. To explore the biological significance of chemokine aggregation, nonaggregating variants were sought. The phenotypes of 105 hMIP-1alpha variants generated by systematic mutagenesis and expression in yeast were determined. hMIP-1alpha residues Asp26 and Glu66 were critical to the self-association process. Substitution at either residue resulted in the formation of essentially homogenous tetramers at 0.5 mg/ml. Substitution of identical or analogous residues in homologous positions in both hMIP-1beta and RANTES demonstrated that they were also critical to aggregation. Our analysis suggests that a single charged residue at either position 26 or 66 is insufficient to support extensive aggregation and that two charged residues must be present. Solution of the three-dimensional NMR structure of hMIP-1alpha has enabled comparison of these residues in hMIP-1beta and RANTES. Aggregated and disaggregated forms of hMIP-1alpha, hMIP-1beta, and RANTES generally have equivalent G-protein-coupled receptor-mediated biological potencies. We have therefore generated novel reagents to evaluate the role of hMIP-1alpha, hMIP-1beta, and RANTES aggregation in vitro and in vivo. The disaggregated chemokines retained their human immunodeficiency virus (HIV) inhibitory activities. Surprisingly, high concentrations of RANTES, but not disaggregated RANTES variants, enhanced infection of cells by both M- and T-tropic HIV isolates/strains. This observation has important implications for potential therapeutic uses of chemokines implying that disaggregated forms may be necessary for safe clinical investigation.

Multiple folding pathways for heterologously expressed human prion protein.

Human PrP (residues 91-231) expressed in Escherichia coli can adopt several conformations in solution depending on pH, redox conditions and denaturant concentration. Oxidised PrP at neutral pH, with the disulphide bond intact, is a soluble monomer which contains 47% alpha-helix and corresponds to PrPC. Denaturation studies show that this structure has a relatively small, solvent-excluded core and unfolds to an unstructured state in a single, co-operative transition with a DeltaG for folding of -5.6 kcal mol-1. The unfolding behaviour is sensitive to pH and at 4.0 or below the molecule unfolds via a stable folding intermediate. This equilibrium intermediate has a reduced helical content and aggregates over several hours. When the disulphide bond is reduced the protein adopts different conformations depending upon pH. At neutral pH or above, the reduced protein has an alpha-helical fold, which is identical to that observed for the oxidised protein. At pH 4 or below, the conformation rearranges to a fold that contains a high proportion of beta-sheet structure. In the reduced state the alpha- and beta-forms are slowly inter-convertible whereas when oxidised the protein can only adopt an alpha-conformation in free solution. The data we present here shows that the human prion protein can exist in multiple conformations some of which are known to be capable of forming fibrils. The precise conformation that human PrP adopts and the pathways for unfolding are dependent upon solvent conditions. The conditions we examined are within the range that a protein may encounter in sub-cellular compartments and may have implications for the mechanism of conversion of PrPC to PrPSc in vivo. Since the conversion of PrPC to PrPSc is accompanied by a switch in secondary structure from alpha to beta, this system provides a useful model for studying major structural rearrangements in the prion protein.

Reversible conversion of monomeric human prion protein between native and fibrilogenic conformations.

Prion propagation involves the conversion of cellular prion protein (PrPC) into a disease-specific isomer, PrPSc, shifting from a predominantly alpha-helical to beta-sheet structure. Here, conditions were established in which recombinant human PrP could switch between the native alpha conformation, characteristic of PrPC, and a compact, highly soluble, monomeric form rich in beta structure. The soluble beta form (beta-PrP) exhibited partial resistance to proteinase K digestion, characteristic of PrPSc, and was a direct precursor of fibrillar structures closely similar to those isolated from diseased brains. The conversion of PrPC to beta-PrP in suitable cellular compartments, and its subsequent stabilization by intermolecular association, provide a molecular mechanism for prion propagation.

Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidinone.

DNA duplexes in which the target cytosine base is replaced by 2-H pyrimidinone have previously been shown to bind with a significantly greater affinity to C5-cytosine DNA methyltransferases than unmodified DNA. Here, it is shown that 2-H pyrimidinone, when incorporated into DNA duplexes containing the recognition sites for M.HgaI-2 and M.MspI, elicits the formation of inhibitory covalent nucleoprotein complexes. We have found that although covalent complexes are formed between 2-H pyrimidinone-modified DNA and both M.HgaI-2 and M.MspI, the kinetics of complex formation are quite distinct in each case. Moreover, the formation of a covalent complex is still observed between 2-H pyrimidinone DNA and M.MspI in which the active-site cysteine residue is replaced by serine or threonine. Covalent complex formation between M.MspI and 2-H pyrimidinone occurs as a direct result of nucleophilic attack by the residue at the catalytic position, which is enhanced by the absence of the 4-amino function in the base. The substitution of the catalytic cysteine residue by tyrosine or chemical modification of the wild-type enzyme with N-ethylmaleimide, abolishes covalent interaction. Nevertheless the 2-H pyrimidinone-substituted duplex still binds to M.MspI with a greater affinity than a standard cognate duplex, since the 2-H pyrimidinone base is mis-paired with guanine.

On the mechanism of human stefin B folding: I. Comparison to homologous stefin A. Influence of pH and trifluoroethanol on the fast and slow folding phases.

The folding of human stefin B has been studied by several spectroscopic probes. Stopped-flow traces obtained by circular dichroism in the near and far UV, by tyrosine fluorescence, and by extrinsic probe ANS fluorescence are compared. Most (60+/-5%) of the native signal in the far UV circular dichroism (CD) appeared within 10 ms in an unresolved "burst" phase, which was followed by a fast phase (t = 83 ms) and a slow phase (t = 25s) with amplitudes of 30% and 10%, respectively. Similar fast and slow phases were also evident in the near UV CD, ANS fluorescence, and tyrosine fluorescence. By contrast, human stefin A, which has a very similar structure, exhibited only one kinetic phase of folding (t = 6s) detected by all the spectroscopic probes, which occurred subsequent to an initial "burst" phase observed by far UV CD. It is interesting that despite close structural similarity of both homologues they fold differently, and that the less stable human stefin B folds faster by an order of magnitude (comparing the non-proline limited phase). To gain more information on the stefin B folding mechanism, effects of pH and trifluoroethanol (TFE) on the fast and slow phases were investigated by several spectroscopic probes. If folding was performed in the presence of 7% of TFE, rate acceleration and difference in the mechanism were observed.

On the mechanism of human stefin B folding: II. Folding from GuHCl unfolded, TFE denatured, acid denatured, and acid intermediate states.

It has been shown that human stefin B exhibits molten globule intermediates when denatured by acid or GuHCl. In the presence of TFE, it transforms into a highly helical state. In our first study on its folding mechanism (Zerovnik et al., Proteins 32:296-303), the kinetics measured by circular dichroism (CD) and fluorescence were correlated. In the present work the kinetics of folding were monitored by tyrosine fluorescence, ANS fluorescence, and, for certain reactions, far ultraviolet (UV) CD. The folding was started from the unfolded state in 3.45 M GuHCl, the acid denatured state at pH 1.8+/-0.2, an acid molten globule intermediate I1 (pH 3.3+/-0.1, low salt), a more structured acid molten globule intermediate I2 (pH 3.3+/-0.1, 0.42 M NaCl), and the TFE state (pH 3.3+/-0.1, 42% TFE). It has been found that all denatured states, including GuHCl, TFE, acid denatured and acid molten globule intermediate I1, fold with the same kinetics, provided that the final conditions are identical. This does not apply to the second acid molten globule intermediate I2, which demonstrates a higher rate of folding by a factor of 270. Different energy of activation and pH dependence were found for folding from states I1 or I2.

The role of Gly-4 of human cystatin A (stefin A) in the binding of target proteinases. Characterization by kinetic and equilibrium methods of the interactions of cystatin A Gly-4 mutants with papain, cathepsin B, and cathepsin L.

The importance of the evolutionarily conserved Gly-4 residue for the affinity and kinetics of interaction of cystatin A with several cysteine proteinases was assessed by site-directed mutagenesis. Even the smallest replacement, by Ala, resulted in approximately 1000-, approximately 10- and approximately 6000-fold decreased affinities for papain, cathepsin L, and cathepsin B, respectively. Substitution by Ser gave further 3-8-fold reductions in affinity, whereas the largest decreases, >10(5)-fold, were observed for mutations to Arg and Glu. The kinetics of inhibition of papain by the mutants with small side chains, Ala and Ser, were compatible with a one-step bimolecular reaction similar to that with wild-type cystatin A. The decreased affinities of these mutants for papain and cathepsin L were due exclusively to increased dissociation rate constants, but the reduced affinities for cathepsin B were due also to decreased association rate constants. The latter finding indicates that the intact N-terminal region serves as a guide directing cystatin A to the active site of cathepsin B, as has been proposed for cystatin C. The kinetics of binding of the mutants with charged side chains, Arg and Glu, to papain were consistent with a two-step binding mechanism, in which the mutant side chains are accommodated in the complex by a conformational change. The NMR solution structure of the Ala and Trp mutants showed only minor changes compared with wild-type cystatin A, indicating that the large reductions in affinity for proteinases are not due to altered structures of the mutants. Instead, a side chain larger than a hydrogen atom at position 4 affects the interaction with the proteinase most likely by interfering with the binding of the N-terminal region.

Characterisation of low free-energy excited states of folded proteins.

It is demonstrated that the identity of residues accessing excited conformational states that are of low free energy relative to the ground state in proteins can be obtained from amide proton NMR chemical shift temperature dependences displaying significant curvature. For the N-terminal domain of phosphoglycerate kinase, hen egg-white lysozyme and BPTI, conformational heterogeneity arises from a number of independent sources, including: structural instability resulting from deletion of part of the protein; a minor conformer generated through disulphide bond isomerisation; an alternative hydrogen bond network associated with buried water molecules; alternative hydrogen bonds involving backbone amides and surface-exposed side-chain hydrogen bond acceptors; and the disruption of loops, ends of secondary structural elements and chain termini. In many of these cases, the conformational heterogeneity at these sites has previously been identified by X-ray and/or NMR studies, but conformational heterogeneity of buried water molecules has hitherto received little attention. These multiple independent low free-energy excited states each involve a small number of residues and are shown to be within 2.5 kcal mol-1 of the ground state. Their relationship with the partially unfolded forms previously characterised using amide proton exchange studies is discussed.

Structure of a kinetic protein folding intermediate by equilibrium amide exchange.

A combination of equilibrium amide exchange and kinetic folding data show that the essential features of the complex topology of the N-terminal domain of a thermophilic phosphoglycerate kinase are established on a millisecond or faster timescale, before the rate-limiting step in the folding pathway commences.