Conformational stability of ribonuclease T1 determined by hydrogen-deuterium exchange.

The hydrogen-deuterium exchange kinetics of 37 backbone amide residues in RNase T1 have been monitored at 25, 40, 45, and 50 degrees C at pD 5.6 and at 40 and 45 degrees C at pD 6.6. The hydrogen exchange rate constants of the hydrogen-bonded residues varied over eight orders of magnitude at 25 degrees C with 13 residues showing exchange rates consistent with exchange occurring as a result of global unfolding. These residues are located in strands 2-4 of the central beta-pleated sheet. The residues located in the alpha-helix and the remaining strands of the beta-sheet exhibited exchange behaviors consistent with exchange occurring due to local structural fluctuations. For several residues at 25 degrees C, the global free energy change calculated from the hydrogen exchange data was over 2 kcal/mol greater than the free energy of unfolding determined from urea denaturation experiments. The number of residues showing this unexpected behavior was found to increase with temperature. This apparent inconsistency can be explained quantitatively if the cis-trans isomerization of the two cis prolines, Pro-39 and Pro-55, is taken into account. The cis-trans isomerization equilibrium calculated from kinetic data indicates the free energy of the unfolded state will be 2.6 kcal/mol higher at 25 degrees C when the two prolines are cis rather than trans (Mayr LM, Odefey CO, Schutkowski M, Schmid FX. 1996. Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped-flow double-mixing technique. Biochemistry 35: 5550-5561). The hydrogen exchange results are consistent with the most slowly exchanging hydrogens exchanging from a globally higher free energy unfolded state in which Pro-55 and Pro-39 are still predominantly in the cis conformation. When the conformational stabilities determined by hydrogen exchange are corrected for the proline isomerization equilibrium, the results are in excellent agreement with those from an analysis of urea denaturation curves.     

Restricted backbone conformational and motional flexibilities of loops containing peptidyl-proline bonds dominate the enzyme activity of staphylococcal nuclease

The role of cis-trans isomerizations of peptidyl-proline bonds in the enzyme activity of staphylococcal nuclease (SNase) was examined by mutation of proline residues. The proline-free SNase ([Pro-]SNase), namely, P11A/P31A/P42A/P47T/P56A/P117G-mutant SNase, was adopted for elucidating the correlation between the nuclease activity and the backbone conformational and dynamic states of SNase. The 3D solution structure of [Pro-]SNase has been determined by heteronuclear NMR experiments. Comparing the structure of [Pro-]SNase with the structure of SNase revealed the conformational differences between the two proteins. In the structure of [Pro-]SNase, conformational rearrangements were observed for the loop of residues Ala112-His121 containing a trans Lys116-Gly117 peptide bond and for the C-terminal alpha-helical loop of residues Leu137-Glu142. Mutation of proline at position 117 also caused the conformational rearrangement of the p-loop (Asp77-Leu89), which is remote from the Ala112-His121 loop. The Ala112-His121 loop and p-loop are placed closer to each other in [Pro-]SNase than in SNase. The backbone dynamic features of the omega-loop (Pro42-Pro56) of SNase are different from those of [Pro-]SNase. The backbone of the omega-loop exhibits restricted flexibility with slow conformational exchange motions in SNase, but is highly flexible in [Pro-]SNase. The analysis indicates that the restrained backbone conformation of the Ala112-His121 loop and restricted flexibility of the omega-loop are two dominant factors determining the enzyme activity of SNase. Of the two factors, the former is correlated with the strained cis Lys116-Pro117 peptide bond and the latter is correlated with the cis-trans isomerizations of the His46-Pro47 peptide bond.     

NMR structure of the alpha-hemoglobin stabilizing protein: insights into conformational heterogeneity and binding

The structure of alpha-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free alpha-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three alpha-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to alpha-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer alpha1beta1 interface in tetrameric HbA.     

Structural and functional relations among thioredoxins of different species

Three-dimensional models have been constructed of homologous thioredoxins and protein disulfide isomerases based on the high resolution x-ray crystallographic structure of the oxidized form of Escherichia coli thioredoxin. The thioredoxins, from archebacteria to humans, have 27-69% sequence identity to E. coli thioredoxin. The models indicate that all the proteins have similar three-dimensional structures despite the large variation in amino acid sequences. As expected, residues in the active site region of thioredoxins are highly conserved. These include Asp-26, Ala-29, Trp-31, Cys-32, Gly-33, Pro-34, Cys-35, Asp-61, Pro-76, and Gly-92. Similar residues occur in most protein disulfide isomerase sequences. Most of these residues form the surface around the active site that appears to facilitate interactions with other enzymes. Other structurally important residues are also conserved. A proline at position 40 causes a kink in the alpha-2 helix and thus provides the proper position of the active site residues at the amino end of this helix. Pro-76 is important in maintaining the native structure of the molecule. In addition, residues forming the internal contact surfaces between the secondary structural elements are generally unchanged such as Phe-12, Val-25, and Phe-27.     

Human VAPA and the yeast VAP Scs2p with an altered proline distribution can phenocopy amyotrophic lateral sclerosis-associated VAPB(P56S)

A human isoform of the vesicle-associated membrane protein-associated proteins (VAPs), VAPB, causes amyotrophic lateral sclerosis eight due to the missense mutation of Pro-56, whereas human VAPA and the yeast VAP Scs2p proteins are not significantly affected by similar mutations. We have found that VAPA and Scs2p have three prolines present in a conserved region however VAPB has only two prolines in this region. Consequently, this mutation in VAPB (VAPB(P56S)) leaves a single proline in this region whereas other VAPs can retain two proline residues even if the proline equivalent to the Pro-56 is substituted. When Scs2p and VAPA were mutated to be equivalent to VAPB(P56S) in terms of the distribution of proline residues in this region, Scs2p became inactive and aggregated, and VAPA localize to membranous aggregates indistinguishable from those induced by VAPB(P56S). This suggests that the appropriate distribution of three conserved prolines, not the existence of a particular proline, confers VAPA and Scs2p resistance to the Pro-56 mutation and, therefore, is critical for VAP activities.     

Proline isomerism in the protein folding of ribonuclease A

The guanidinium chloride-unfolded state of ribonuclease A was found to be an equilibrium mixture of slow- and fast-refolding forms of the protein chain, as has been suggested. Both forms appear to have the same spectroscopic observables as judged by the relative changes in fluorescence emission and polarization. The equilibrium between them is thermally dependent, with deltaHapp equal to -1.4 kcal/mol. The activation energy Ea is equal to 18 kcal/mol. These findings are consistent with the proposal that cis-trans isomerism of peptide bonds that are NH2-terminal to proline residues is responsible for the slow phase of RNase A refolding. However, the actual dependence of the magnitude of the slow reaction on initial, prefolding temperature cannot be explained by a model in which the proline configurations of the fast refolding form must be identical to those of the native protein, as has been suggested. Instead, the data reveal that, although the native structure of RNase A contains two cis prolines, cis isomers need not be present in the fast-refolding form in order for folding to occur.     

Vibrational spectroscopy of bacteriorhodopsin mutants: evidence for the interaction of proline-186 with the retinylidene chromophore

Fourier-transform infrared difference spectroscopy has been used to study the role of the three membrane-embedded proline residues, Pro-50, Pro-91, and Pro-186, in the structure and function of bacteriorhodopsin. All three prolines were replaced by alanine and glycine; in addition, Pro-186 was changed to valine. Difference spectra were recorded for the bR----K and bR----M photoreactions of each of these mutants and compared to those of wild-type bacteriorhodopsin. Only substitutions of Pro-186 caused significant perturbations in the frequency of the C = C and C - C stretching modes of the retinylidene chromophore. In addition, these substitutions reduced bands in the amide I and II region associated with secondary structural changes and altered signals assigned to the adjacent Tyr-185. Pro-186----Val caused the largest alterations, producing a second species similar to bR548 and nearly blocking chromophore isomerization at 78 K but not at 250 K. These results are consistent with a model of the retinal binding site in which Pro-186 and Tyr-185 are located in direct proximity to the chromophore and may be involved in linking chromophore isomerization to protein structural changes. Evidence is also found that Pro-50 may be structurally active during the bR----K transition and that substitution of this residue by glycine preserves the normal protein structural changes during the photocycle.     

The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

Background

Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.

Results

Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding.

Conclusions

Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data. In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding. This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl cis/trans interconversion during binding to the RHFP³⁵RIW motif of N-terminal Vpr.     

Essential role of proline isomerization in stefin B tetramer formation

Here we present the tetrameric structure of stefin B, which is the result of a process by which two domain-swapped dimers of stefin B are transformed into tetramers. The transformation involves a previously unidentified process of extensive intermolecular contacts, termed hand shaking, which occurs concurrently with trans to cis isomerization of proline 74. This proline residue is widely conserved throughout the cystatin superfamily, a member of which, human cystatin C, is the key protein in cerebral amyloid angiopathy. These results are consistent with the hypothesis that isomerization of proline residues can play a decisive role in amyloidogenesis.     

A proline-90 residue unique to SUMO-4 prevents maturation and sumoylation

Four small ubiquitin-related modifier (SUMO) genes have been identified in humans. However, little is known about the basic biology of SUMO-4. Here, we report that SUMO-4 differs from SUMO-1, -2, and -3 in that the maturation process of SUMO-4 to active form containing C-terminal di-glycine residues is inhibited by a unique proline residue located at position 90 (Pro-90). Although, both the hydrolase and isopeptidase activities of SUMO peptidases are significantly diminished by Pro-90 as compared to Gln-90 (glutamine) in mutated SUMO genes, only the defective hydrolase activity appears to be biologically relevant. Native SUMO-4, thus, appears to be unable to form covalent isopeptide bonds with substrates. A biological role of SUMO-4, through non-covalent interactions is proposed.     

Preferred proline puckerings in cis andtrans peptide groups: Implications for collagen stability

The interplay between side-chain and main-chain conformations is a distinctive characteristic of proline residues. Here we report the results of a statistical analysis of proline conformations using a large protein database. In particular, we found that proline residues with the preceding peptide bond in the cis state preferentially adopt a down puckering. Indeed, out of 178 cis proline residues, as many as 145 (81%) are down. By analyzing the 1-4 and 1-5 nonbonding distances between backbone atoms, we provide a structural explanation for the observed trend. The observed correlation between proline puckering and peptide bond conformation suggests a new mechanism to explain the reported shift of the cis-trans equilibrium in proline derivatives. The implications of these results for the current models of collagen stability are also discussed.     

Nonnative isomers of proline-93 and -114 predominate in heat-unfolded ribonuclease A

The peptide bonds preceding both Pro-93 and Pro-114, which are in the cis conformation in native RNase A, are predominantly in the trans conformation in the heat-unfolded protein. The percentages are estimated to be 60% and 63%, respectively, with a standard deviation of +/- 7% in each quantity. These ratios are close to those found for corresponding sequences in X-Pro-Y peptides. The concentration of the trans proline species was determined from the integrated intensities of resonance peaks of the C alpha H protons of Tyr-92 and Asn-113, which are well resolved in the 1D proton NMR spectrum of heat-unfolded RNase A. The assignments of the resonances were deduced from 2D NOESY and DQF-COSY spectra of unfolded RNase A in D2O. Furthermore, the C alpha H protons of both Tyr-92 and Asn-113 had an intense NOE cross-peak with the C delta H and C delta' H of the respective following prolines. For both Pro-93 and Pro-114, these NOE cross-peaks would arise only if the X-Pro peptide bond were in the trans conformation. It is generally believed that the rate of refolding of RNase A is considerably reduced by nonnative proline isomers, such as trans Pro-93. Two models for folding RNase A, that are consistent with these new results and the work of previous investigators, are presented here.     

Chemical Scale Studies of the Phe-Pro Conserved Motif in the Cys Loop of Cys Loop Receptors

The functions of two conserved residues, Phe¹³⁵ and Pro¹³⁶, located at the apex of the Cys loop of the nicotinic acetylcholine receptor are investigated. Both residues were substituted with natural and unnatural amino acids, focusing on the role of aromaticity at Phe¹³⁵, backbone conformation at Pro¹³⁶, side chain polarity and volume, and the specific interaction between the aromatic side chain and the proline. NMR spectroscopy studies of model peptides containing proline and unnatural proline analogues following a Phe show a consistent increase in the population of the cis conformer relative to peptides lacking the Phe. In the receptor, a strong interaction between the Phe and Pro residues is evident, as is a strong preference for aromaticity and hydrophobicity at the Phe site. A similar influence of hydrophobicity is observed at the proline site. In addition, across a simple homologous series of proline analogues, the results reveal a correlation between receptor function and cis bias at the proline backbone. This could suggest a significant role for the cis proline conformer at this site in receptor function.     

Direct link between cytokine activity and a catalytic site for macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a secreted protein that activates macrophages, neutrophils and T cells, and is implicated in sepsis, adult respiratory distress syndrome and rheumatoid arthritis. The mechanism of MIF function, however, is unknown. The three-dimensional structure of MIF is unlike that of any other cytokine, but bears striking resemblance to three microbial enzymes, two of which possess an N-terminal proline that serves as a catalytic base. Human MIF also possesses an N-terminal proline (Pro-1) that is invariant among all known homologues. Multiple sequence alignment of these MIF homologues reveals additional invariant residues that span the entire polypeptide but are in close proximity to the N-terminal proline in the folded protein. We find that p-hydroxyphenylpyruvate, a catalytic substrate of MIF, binds to the N-terminal region and interacts with Pro-1. Mutation of Pro-1 to a glycine substantially reduces the catalytic and cytokine activity of MIF. We suggest that the underlying biological activity of MIF may be based on an enzymatic reaction. The identification of the active site should facilitate the development of structure-based inhibitors.     

Cis proline mutants of ribonuclease A. I. Thermal stability.

A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give delta Tm approximately equal to 10 degrees C and delta delta G0 (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis<==>trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.     

Study, by an enzymatic method, of the isomers of L-proline (3H-4) and of the effects of preservation]

By use of procollagen proline hydroxylas which catalyses the substitution of the hydrogen in the trans position on carbon 4 with an hydroxyl on L-proline residues of procollagen, we have shown that the L-[3H-4] proline product of C.E.N. Saclay contained 29 per cent of the trans and 71 per cent of the cis form. It was also possible to verify the molecular stability of the isomers and that a small amount of tritium was randomly linked to the whole molecule.     

Characterization of Cg10062 from Corynebacterium glutamicum: implications for the evolution of cis-3-chloroacrylic acid dehalogenase activity in the tautomerase superfamily

A 149-amino acid protein designated Cg10062 is encoded by a gene from Corynebacterium glutamicum. The physiological function of Cg10062 is unknown, and the gene encoding this protein has no obvious genomic context. Sequence analysis links Cg10062 to the cis-3-chloroacrylic acid dehalogenase ( cis-CaaD) family, one of the five known families of the tautomerase superfamily. The characterized tautomerase superfamily members have two distinctive characteristics: a beta-alpha-beta structure motif and a catalytic amino-terminal proline. Pro-1 is present in the Cg10062 amino acid sequence along with His-28, Arg-70, Arg-73, Tyr-103, and Glu-114, all of which have been implicated as critical residues for cis-CaaD activity. The gene for Cg10062 has been cloned and the protein overproduced, purified, and subjected to kinetic and mechanistic characterization. Like cis-CaaD, Cg10062 functions as a hydratase: it converts 2-oxo-3-pentynoate to acetopyruvate and processes 3-bromopropiolate to a species that inactivates the enzyme by acylation of Pro-1. Kinetic and (1)H NMR spectroscopic studies also show that Cg10062 processes both isomers of 3-chloroacrylic acid at low levels with a clear preference for the cis isomer. Pro-1 is critical for the dehalogenase and hydratase activities because the P1A mutant no longer catalyzes either reaction. The presence of the six key catalytic residues and the hydratase activity coupled with the absence of an efficient cis-CaaD activity and the lack of isomer specificity implicate factors beyond this core set of residues in cis-CaaD catalysis and specificity. This work sets the stage for in-depth mechanistic and structural studies of Cg10062, which could identify the additional features necessary for a fully active and highly specific cis-CaaD. Such results will also shed light on how cis-CaaD emerged in the tautomerase superfamily because Cg10062 could be characteristic of an intermediate along the evolutionary pathway for this dehalogenase.     

Influence of proline residues on protein conformation

To study the influence of proline residues on three-dimensional structure, an analysis has been made of all proline residues and their local conformations extracted from the Brookhaven Protein Data bank. We have considered the conformation of the proline itself, the relative occurrence of cis and trans peptides preceding proline residues, the influence of proline on the conformation of the preceding residue and the conformations of various proline patterns (Pro-Pro, Pro-X-Pro, etc.). The results highlight the unique role of proline in determining local conformation.     

Parallel channels and rate-limiting steps in complex protein folding reactions: prolyl isomerization and the alpha subunit of Trp synthase,a TIM barrel protein

A kinetic folding mechanism for the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, involving four parallel channels with multiple native, intermediate and unfolded forms, has recently been proposed. The hypothesis that cis/trans isomerization of several Xaa-Pro peptide bonds is the source of the multiple folding channels was tested by measuring the sensitivity of the three rate-limiting phases (tau(1), tau(2), tau(3)) to catalysis by cyclophilin, a peptidyl-prolyl isomerase. Although the absence of catalysis for the tau(1) (fast) phase leaves its assignment ambiguous, our previous mutational analysis demonstrated its connection to the unique cis peptide bond preceding proline 28. The acceleration of the tau(2) (medium) and tau(3) (slow) refolding phases by cyclophilin demonstrated that cis/trans prolyl isomerization is also the source of these phases. A collection of proline mutants, which covered all of the remaining 18 trans proline residues of alphaTS, was constructed to obtain specific assignments for these phases. Almost all of the mutant proteins retained the complex equilibrium and kinetic folding properties of wild-type alphaTS; only the P217A, P217G and P261A mutations caused significant changes in the equilibrium free energy surface. Both the P78A and P96A mutations selectively eliminated the tau(1) folding phase, while the P217M and P261A mutations eliminated the tau(2) and tau(3) folding phases, respectively. The redundant assignment of the tau(1) phase to Pro28, Pro78 and Pro96 may reflect their mutual interactions in non-random structure in the unfolded state. The non-native cis isomers for Pro217 and Pro261 may destabilize an autonomous C-terminal folding unit, thereby giving rise to kinetically distinct unfolded forms. The nature of the preceding amino acid, the solvent exposure, or the participation in specific elements of secondary structure in the native state, in general, are not determinative of the proline residues whose isomerization reactions can limit folding.     

Cis peptide bonds in proteins: residues involved, their conformations, interactions and locations.

An analysis of a non-redundant set of protein structures from the Brookhaven Protein Data Bank has been carried out to find out the residue preference, local conformation, hydrogen bonding and other stabilizing interactions involving cis peptide bonds. This has led to a reclassification of turns mediated by cis peptides, and their average geometrical parameters have been evaluated. The interdependence of the side and main-chain torsion angles of proline rings provided an explanation why such rings in cis peptides are found to have the DOWN puckering. A comparison of cis peptides containing proline and non-proline residues show differences in conformation, location in the secondary structure and in relation to the centre of the molecule, and relative accessibilities of residues. Relevance of the results in mutation studies and the cis-trans isomerization during protein folding is discussed.