Crystal structure of a bacterial signal peptidase apoenzyme: implications for signal peptide binding and the Ser-Lys dyad mechanism.

We report here the x-ray crystal structure of a soluble catalytically active fragment of the Escherichia coli type I signal peptidase (SPase-(Delta2-75)) in the absence of inhibitor or substrate (apoenzyme). The structure was solved by molecular replacement and refined to 2.4 A resolution in a different space group (P4(1)2(1)2) from that of the previously published acyl-enzyme inhibitor-bound structure (P2(1)2(1)2) (Paetzel, M., Dalbey, R.E., and Strynadka, N.C.J. (1998) Nature 396, 186-190). A comparison with the acyl-enzyme structure shows significant side-chain and main-chain differences in the binding site and active site regions, which result in a smaller S1 binding pocket in the apoenzyme. The apoenzyme structure is consistent with SPase utilizing an unusual oxyanion hole containing one side-chain hydroxyl hydrogen (Ser-88 OgammaH) and one main-chain amide hydrogen (Ser-90 NH). Analysis of the apoenzyme active site reveals a potential deacylating water that was displaced by the inhibitor. It has been proposed that SPase utilizes a Ser-Lys dyad mechanism in the cleavage reaction. A similar mechanism has been proposed for the LexA family of proteases. A structural comparison of SPase and members of the LexA family of proteases reveals a difference in the side-chain orientation for the general base lysine, both of which are stabilized by an adjacent hydroxyl group. To gain insight into how signal peptidase recognizes its substrates, we have modeled a signal peptide into the binding site of SPase. The model is built based on the recently solved crystal structure of the analogous enzyme LexA (Luo, Y., Pfuetzner, R. A., Mosimann, S., Paetzel, M., Frey, E. A., Cherney, M., Kim, B., Little, J. W., and Strynadka, N. C. J. (2001) Cell 106, 1-10) with its bound cleavage site region.     

The Thermoplasma acidophilum Lon protease has a Ser-Lys dyad active site.

A gene with significant similarity to bacterial Lon proteases was identified during the sequencing of the genome of the thermoacidophilic archaeon Thermoplasma acidophilum. Protein sequence comparison revealed that Thermoplasma Lon protease (TaLon) is more similar to the LonB proteases restricted to Gram-positive bacteria than to the widely distributed bacterial LonA. However, the active site residues of the protease and ATPase domain are highly conserved in all Lon proteases. Using site-directed mutagenesis we show here that TaLon and EcLon, and probably all other Lon proteases, contain a Ser-Lys dyad active site. The TaLon active site mutants were fully assembled and, similar to TaLon wild-type, displayed an apparent molar mass of 430 kDa upon gelfiltration. This would be consistent with a hexameric complex and indeed electron micrographs of TaLon revealed ring-shaped particles, although of unknown symmetry. Comparison of the ATPase activity of Lon wild-type from Thermoplasma or Escherichia coli with respective protease active site mutants revealed differences in Km and V values. This suggests that in the course of protein degradation by wild-type Lon the protease domain might influence the activity of the ATPase domain.     

The catalytic domain of Escherichia coli Lon protease has a unique fold and a Ser-Lys dyad in the active site

ATP-dependent Lon protease degrades specific short-lived regulatory proteins as well as defective and abnormal proteins in the cell. The crystal structure of the proteolytic domain (P domain) of the Escherichia coli Lon has been solved by single-wavelength anomalous dispersion and refined at 1.75-A resolution. The P domain was obtained by chymotrypsin digestion of the full-length, proteolytically inactive Lon mutant (S679A) or by expression of a recombinant construct encoding only this domain. The P domain has a unique fold and assembles into hexameric rings that likely mimic the oligomerization state of the holoenzyme. The hexamer is dome-shaped, with the six N termini oriented toward the narrower ring surface, which is thus identified as the interface with the ATPase domain in full-length Lon. The catalytic sites lie in a shallow concavity on the wider distal surface of the hexameric ring and are connected to the proximal surface by a narrow axial channel with a diameter of approximately 18 A. Within the active site, the proximity of Lys(722) to the side chain of the mutated Ala(679) and the absence of other potential catalytic side chains establish that Lon employs a Ser(679)-Lys(722) dyad for catalysis. Alignment of the P domain catalytic pocket with those of several Ser-Lys dyad peptide hydrolases provides a model of substrate binding, suggesting that polypeptides are oriented in the Lon active site to allow nucleophilic attack by the serine hydroxyl on the si-face of the peptide bond.     

The catalytic role of aspartate in a short strong hydrogen bond of the Asp274-His32 catalytic dyad in phosphatidylinositol-specific phospholipase C can be substituted by a chloride ion

Phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis catalyzes the cleavage of the phosphorus-oxygen bond in phosphatidylinositol. The focus of this work is to dissect the roles of the carboxylate side chain of Asp(274) in the Asp(274)-His(32) dyad, where a short strong hydrogen bond (SSHB) was shown to exist based on NMR criteria. A regular hydrogen bond (HB) was observed in D274N, and no low field proton resonance was detected for D274E and D274A. Comparison of the activity of wild type, D274N, and D274A suggested that the regular HB contributes significantly (approximately 4 kcal/mol) to catalysis, whereas the SSHB contributes only an additional 2 kcal/mol. The mutant D274E displays high activity similar to wild type, suggesting that the negative charge is sufficient for the catalytic role of Asp(274). To further support this interpretation and rule out possible contribution of regular HB or SSHB in D274E, we showed that the activity of D274G can be rescued by exogenous chloride ions to a level comparable with that of D274E. Comparison between different anions suggested that the ability of an anion to rescue the activity is due to the size and the charge of the anion not the property as a HB acceptor. In conclusion, a major fraction of the functional role of Asp(274) in the Asp(274)-His(32) dyad can be attributed to a negative charge (as in D274E and D274G-Cl(-)), and the SSHB in the wild type enzyme provides minimal contribution to catalysis. These results represent novel insight for an Asp-His catalytic dyad and for the mechanism of phosphatidylinositol-specific phospholipase C.     

Involvement of electrostatic interactions in the mechanism of peptide folding induced by sodium dodecyl sulfate binding

Sodium dodecyl sulfate (SDS) has consistently been shown to induce secondary structure, particularly alpha-helices, in polypeptides, and is commonly used to model membrane and other hydrophobic environments. However, the precise mechanism by which SDS induces these conformational changes remains unclear. To examine the role of electrostatic interactions in this mechanism, we have designed two hydrophilic, charged amphipathic alpha-helical peptides, one basic (QAPAYKKAAKKLAES) and the other acidic (QAPAYEEAAEELAKS), and their structures were studied by CD and NMR. The design of the peptides is based on the sequence of the segment of residues 56-70 of human platelet factor 4 [PF4(56-70), QAPLYKKIIKKLLES]. Both peptides were unstructured in water, and in the presence of neutral, zwitterionic, or cationic detergents. However, in SDS at neutral pH, the basic peptide folded into an alpha-helix. By contrast, the pH needed to be lowered to 1.8 before alpha-helix formation was observed for the acidic peptide. Strong, attractive electrostatic interactions, between the anionic groups of SDS and the cationic groups of the lysines, appeared to be necessary to initiate the folding of the basic peptide. NMR analysis showed that the basic peptide was fully embedded in SDS-peptide micelles, and that its three-dimensional alpha-helical structure could be superimposed on that of the native structure of PF4(56-70). These results enabled us to propose a working model of the basic peptide-SDS complex, and a mechanism for SDS-induced alpha-helical folding. This study demonstrates that, while the folding of peptides is mostly driven by hydrophobic effects, electrostatic interactions play a significant role in the formation and the stabilization of SDS-induced structure.     

The hydrolysis and resynthesis of a single reactive site peptide bond in recombinant antistasin by coagulation factor Xa.

Antistasin (ATS) is a 119-amino acid, leech-derived protein which exhibits selective, tight-binding inhibition of blood coagulation factor Xa. Prolonged incubation of ATS with factor Xa leads to the highly specific hydrolysis of the peptide bond between residues Arg34 and Val35, implicating this peptide bond as the putative reactive site. We report here the preparation of pure, cleaved (modified) recombinant ATS (rATS) and utilize this material to provide additional proof that the cleaved peptide bond is in fact the reactive site. Modified rATS retains strong inhibitory potency against factor Xa as evidenced by a dissociation constant of 166.3 +/- 9.6 pM; four-fold greater than that of native inhibitor, 43.4 +/- 1.4 pM. Incubation of pure, modified rATS with catalytic amounts of factor Xa results in resynthesis of the hydrolyzed peptide bond, achieving an equilibrium near unity between native and modified inhibitors. Specific removal of the newly formed carboxy-terminal Arg residue from modified rATS by carboxypeptidase B treatment obviates its conversion to native inhibitor coincident with the complete loss of inhibitory activity. These results establish that rATS inhibits factor Xa according to a standard mechanism of serine protease inhibitors and support the contention that the Arg34-Val35 peptide bond constitutes the reactive site.     

Phosphoregulation of sorting signal-VHS domain interactions by a direct electrostatic mechanism

Phosphorylation of the cytosolic tails of transmembrane receptors can regulate their intracellular trafficking. The structural basis for such regulation, however, has not been explained in most cases. The cytosolic tail of the cation-independent mannose 6-phosphate receptor contains a serine residue within an acidic-cluster dileucine signal that is important for the function of the receptor in the biosynthetic sorting of lysosomal hydrolases. We show here that phosphorylation of this Ser enhances interactions of the signal with its recognition module, the VHS domain of the GGA proteins. Crystallographic analyses demonstrate that the phosphoserine residue interacts electrostatically with two basic residues on the VHS domain of GGA3, thus providing an additional point of attachment of the acidic-cluster dileucine signal to its recognition module.     

The role of electrostatic interactions in calmodulin-peptide complex formation

The complex between calmodulin and the calmodulin-binding portion of smMLCKp has been studied. Electrostatic interactions have been anticipated to be important in this system where a strongly negative protein binds a peptide with high positive charge. Electrostatic interactions were probed by varying the pH in the range from 4 to 11 and by charge deletions in CaM and smMLCKp. The change in net charge of CaM from approximately -5 at pH 4.5 to -15 at pH 7.5 leaves the binding constant virtually unchanged. The affinity was also unaffected by mutations in CaM and charge substitutions in the peptide. The insensitivity of the binding constant to pH may seem surprising, but it is a consequence of the high charge on both protein and peptide. At low pH it is further attenuated by a charge regulation mechanism. That is, the protein releases a number of protons when binding the positively charged peptide. We speculate that the role of electrostatic interactions is to discriminate against unbound proteins rather than to increase the affinity for any particular target protein.     

The role of electrostatic interactions in the membrane binding of melittin

The binding of melittin and the C-terminally truncated analogue of melittin (21Q) to a range of phospholipid bilayers was studied using surface plasmon resonance (SPR). The phospholipid model membranes included zwitterionic dimyristylphosphatidylcholine (DMPC) and dimyristylphosphatidylethanolamine (DMPE), together with mixtures DMPC/dimyristylphosphatidylglycerol (DMPG), DMPC/DMPG/cholesterol and DMPE/DMPG. Melittin bound rapidly to all membrane mixtures, whereas 21Q, which has a reduced charge, bound much more slowly on the DMPC and DMPC/DMPG mixtures reflecting the role of the initial electrostatic interaction. The loss of the cationic residues also significantly decreased the binding of 21Q with DMPC/DMPG/Cholesterol, DMPE and DMPE/DMPG. The role of electrostatics was also highlighted with NaCl in the buffer, which affected the way melittin bound to the different membranes, causing a more uniform, concentration dependant increase in response. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high α-helicity was associated with high binding affinity. Overall, the results demonstrate that the positively charged residues at the C-terminus of melittin play an essential role in membrane binding, that modulation of peptide charge influences selectivity of binding to different phospholipids and that manipulation of the cationic regions of antimicrobial peptides can be used to modulate membrane selectivity.     

The optimization of protein-solvent interactions: thermostability and the role of hydrophobic and electrostatic interactions.

Protein-solvent interactions were analyzed using an optimization parameter based on the ratio of the solvent-accessible area in the native and the unfolded protein structure. The calculations were performed for a set of 183 nonhomologous proteins with known three-dimensional structure available in the Protein Data Bank. The dependence of the total solvent-accessible surface area on the protein molecular mass was analyzed. It was shown that there is no difference between the monomeric and oligomeric proteins with respect to the solvent-accessible area. The results also suggested that for proteins with molecular mass above some critical mass, which is about 28 kDa, a formation of domain structure or subunit aggregation into oligomers is preferred rather than a further enlargement of a single domain structure. An analysis of the optimization of both protein-solvent and charge-charge interactions was performed for 14 proteins from thermophilic organisms. The comparison of the optimization parameters calculated for proteins from thermophiles and mesophiles showed that the former are generally characterized by a high degree of optimization of the hydrophobic interactions or, in cases where the optimization of the hydrophobic interactions is not sufficiently high, by highly optimized charge-charge interactions.     

Stereochemical analysis of peptide bond hydrolysis catalyzed by the aspartic proteinase penicillopepsin

The X-ray crystal structures of native penicillopepsin and of its complex with a synthetic analogue of the inhibitor pepstatin have been refined recently at 1.8-A resolution. These highly refined structures permit a detailed examination of peptide hydrolysis in the aspartic proteinases. Complexes of penicillopepsin with substrate and catalytic intermediates were modeled, by using computer graphics, with minimal perturbation of the observed inhibitor complex. A thallium ion binding experiment shows that the position of solvent molecule O39, between Asp-33(32) and Asp-213(215) in the native structure, is favorable for cations, a fact that places constraints on possible mechanisms. A mechanism for hydrolysis is proposed in which Asp-213(215) acts as an electrophile by protonating the carbonyl oxygen of the substrate, thereby polarizing the carbon-oxygen bond, a water molecule bound to Asp-33(32) (O284 in the native structure) attacks the carbonyl carbon as the nucleophile in a general-base mechanism, the newly pyramidal peptide nitrogen is protonated, either from the solvent after nitrogen inversion or by an internal proton transfer via Asp-213(215) from a hydroxyl of the tetrahedral carbon, and the tetrahedral intermediate breaks down in a manner consistent with the stereoelectronic hypothesis. The models permit the rationalization of observed subsite preferences for substrates and may be useful in predicting subsite preferences of other aspartic proteinases.     

The role of electrostatic interactions in the antitumor activity of dimeric RNases

The cytotoxic action of some ribonucleases homologous to bovine pancreatic RNase A, the superfamily prototype, has interested and intrigued investigators. Their ribonucleolytic activity is essential for their cytotoxic action, and their target RNA is in the cytosol. It has been proposed that the cytosolic RNase inhibitor (cRI) plays a major role in determining the ability of an RNase to be cytotoxic. However, to interact with cRI RNases must reach the cytosol, and cross intracellular membranes. To investigate the interactions of cytotoxic RNases with membranes, cytotoxic dimeric RNases resistant, or considered to be resistant to cRI, were assayed for their effects on negatively charged membranes. Furthermore, we analyzed the electrostatic interaction energy of the RNases complexed in silico with a model membrane. The results of this study suggest that close correlations can be recognized between the cytotoxic action of a dimeric RNase and its ability to complex and destabilize negatively charged membranes.     

Optimization of the electrostatic interactions between ionized groups and peptide dipoles in proteins.

The three-dimensional optimization of the electrostatic interactions between the charged amino acid residues and the peptide partial charges was studied by Monte-Carlo simulations on a set of 127 nonhomologous protein structures with known atomic coordinates. It was shown that this type of interaction is very well optimized for all proteins in the data set, which suggests that they are a valuable driving force, at least for the native side-chain conformations. Similar to the optimization of the charge-charge interactions (Spassov VZ, Karshikoff AD, Ladenstein R, 1995, Protein Sci 4:1516-1527), the optimization effect was found more pronounced for enzymes than for proteins without enzymatic function. The asymmetry in the interactions of acidic and basic groups with the peptide dipoles was analyzed and a hypothesis was proposed that the properties of peptide dipoles are a factor contributing to the natural selection of the basic amino acids in the chemical composition of proteins.     

Isomerization of the Xaa-Pro peptide bond induced by ionic interactions of arginine

Inclusion of Arg or Pro residues in proteins and peptides has been proved to play an essential role in biochemical functions through ionic interactions, conformational transitions, and formation of turns as well. In this study we present the conformational properties of the Ac-Arg-Ala-Pro (1), Ac-Arg-Ala-Pro-NH₂ (2), Ac-Arg-Pro-Asp-NH₂ (3), and Ac-Arg-Pro-Asp (4) tripeptides, using ¹H-nmr spectroscopy and molecular dynamics. These peptides were modeled with the aim of studying the role of the Arg-guanidinium to carboxylate ionic interactions on the Xaa-Pro peptide bond isomerization. It was found with 1 and 4 that arginine preferentially interacts with the C-terminal carboxylate group, even though the β-carboxylate is also accessible. This tendency of the Arg moiety was found to induce the cis disposition of the Ala-Pro peptide bond in 1. It was also confirmed that the Arg…Asp side chain-side chain ionic interaction in 3 plays a key role in backbone folding and structural stabilization through a type I β-turn. The nmr pattern for 3 showed a remarkable similarity with that for various Arg-Tyr-Asp containing peptides, a sequence that is crucial for the adhesion properties of the Leishmania gp63 glycoprotein. © 1996 John Wiley & Sons, Inc.     

The role of electrostatic interactions in human serum albumin binding and stabilization by halothane

Electrostatic interactions have been proposed as a potentially important force for anesthetics and protein binding but have not yet been tested directly. In the present study, we used wild-type human serum albumin (HSA) and specific site-directed mutants as a native protein model to investigate the role of electrostatic interactions in halothane binding. Structural geometry analysis of the HSA-halothane complex predicted an absence of significant electrostatic interactions, and direct binding (tryptophan fluorescence and zonal elution chromatography) and stability experiments (hydrogen exchange) confirmed that loss of charge in the binding sites, by charged to uncharged mutations and by changing ionic strength of the buffer, generally increased both regional (tryptophan region) and global halothane/HSA affinity. The results indicate that electrostatic interactions (full charges) either do not contribute or diminish halothane binding to HSA, leaving only the more general hydrophobic and van der Waals forces as the major contributors to the binding interaction.     

Crystal structure of a novel viral protease with a serine/lysine catalytic dyad mechanism

The blotched snakehead virus (BSNV), an aquatic birnavirus, encodes a polyprotein (NH2-pVP2-X-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease (VP4) to liberate itself and the viral proteins pVP2, X and VP3. The protein pVP2 is further processed by VP4 to give rise to the capsid protein VP2 and four structural peptides. We report here the crystal structure of a VP4 protease from BSNV, which displays a catalytic serine/lysine dyad in its active site. This is the first crystal structure of a birnavirus protease and the first crystal structure of a viral protease that utilizes a lysine general base in its catalytic mechanism. The topology of the VP4 substrate binding site is consistent with the enzymes substrate specificity and a nucleophilic attack from the si-face of the substrates scissile bond. Despite low levels of sequence identity, VP4 shows similarities in its active site to other characterized Ser/Lys proteases such as signal peptidase, LexA protease and Lon protease. Together, the structure of VP4 provides insights into the mechanism of a recently characterized clan of serine proteases that utilize a lysine general base and reveals the structure of potential targets for antiviral therapy, especially for other related and economically important viruses, such as infectious bursal disease virus in poultry and infectious pancreatic necrosis virus in aquaculture.     

The role of electrostatic energy in prediction of obligate protein-protein interactions

Background

Prediction and analysis of protein-protein interactions (PPI) and specifically types of PPIs is an important problem in life science research because of the fundamental roles of PPIs in many biological processes in living cells. In addition, electrostatic interactions are important in understanding inter-molecular interactions, since they are long-range, and because of their influence in charged molecules. This is the main motivation for using electrostatic energy for prediction of PPI types.

Results

We propose a prediction model to analyze protein interaction types, namely obligate and non-obligate, using electrostatic energy values as properties. The prediction approach uses electrostatic energy values for pairs of atoms and amino acids present in interfaces where the interaction occurs. The main features of the complexes are found and then the prediction is performed via several state-of-the-art classification techniques, including linear dimensionality reduction (LDR), support vector machine (SVM), naive Bayes (NB) and k-nearest neighbor (k-NN). For an in-depth analysis of classification results, some other experiments were performed by varying the distance cutoffs between atom pairs of interacting chains, ranging from 5Å to 13Å. Moreover, several feature selection algorithms including gain ratio (GR), information gain (IG), chi-square (Chi2) and minimum redundancy maximum relevance (mRMR) are applied on the available datasets to obtain more discriminative pairs of atom types and amino acid types as features for prediction.

Conclusions

Our results on two well-known datasets of obligate and non-obligate complexes confirm that electrostatic energy is an important property to predict obligate and non-obligate protein interaction types on the basis of all the experimental results, achieving accuracies of over 98%. Furthermore, a comparison performed by changing the distance cutoff demonstrates that the best values for prediction of PPI types using electrostatic energy range from 9Å to 12Å, which show that electrostatic interactions are long-range and cover a broader area in the interface. In addition, the results on using feature selection before prediction confirm that (a) a few pairs of atoms and amino acids are appropriate for prediction, and (b) prediction performance can be improved by eliminating irrelevant and noisy features and selecting the most discriminative ones.

     

A theoretical study of the mechanism for peptide hydrolysis by thermolysin

The catalytic mechanism for peptide hydrolysis by thermolysin has been investigated using the B3LYP hybrid density functional method. The starting structure for the calculations was based on the X-ray crystal structure of the enzyme inhibited with the ZF (p)LA phosphonamidate transition-state analogue. Besides the three Zn ligands His142, His146 and Glu166, a few additional residues were also included in the model. Following the order of importance, the outer-sphere ligands Glu143, His231 and Asp226 were shown to play significant catalytic roles, well correlated with results from site-directed mutagenesis experiments. A single-step reaction mechanism was obtained starting from the initial enzyme-substrate complex with a pentacoordinated metal center and proceeding to the enzyme-carboxylate complex as a final product, following a proposal by Matthews and co-workers. The transition state combines a nucleophilic water oxygen attack on the peptide carbon and a proton transfer from the water to the peptide nitrogen, mediated by the Glu143 carboxylate. A free activation energy of 15.2 kcal/mol was obtained, compared to the experimental 12.4-16.3 kcal/mol range for various peptide substrates. An interesting aspect of the present single-step mechanism is that the Glu143 carboxylate moves a significant distance of ~1.0 A. Different chemical models were examined, both related to the system size and proper side-chain modeling. The significance of the protein frame rigidity around the active site was estimated by fixing and subsequently releasing the edge atom positions. Finally, alternative mechanistic proposals are briefly summarized.     

On the mechanism of SPP-catalysed intramembrane proteolysis; conformational control of peptide bond hydrolysis in the plane of the membrane

Intramembrane-cleaving proteases are members of a novel type of enzyme that hydrolyse substrate proteins within transmembrane regions. The presently known proteases that catalyse such cleavage reactions are membrane proteins of high hydrophobicity and multiple predicted transmembrane regions. A key feature is the positioning of active site residues in hydrophobic segments implying that the catalytic centre is assembled within the plane of the membrane. Nevertheless, all these proteases appear to utilise catalytic mechanisms similar to classic proteases that expose their active site domains in aqueous compartments. In the present review, we will address the mechanism of intramembrane proteolysis on the example of the signal peptide peptidase, and discuss how enzyme-catalysed hydrolysis of peptide bonds within the plane of a cellular membrane might occur.