Optimization of reorganization energy drives evolution of the designed Kemp eliminase KE07.

Understanding enzymatic evolution is essential to engineer enzymes with improved activities or to generate enzymes with tailor-made activities. The computationally designed Kemp eliminase KE07 carries out an unnatural reaction by converting of 5-nitrobenzisoxazole to cyanophenol, but its catalytic efficiency is significantly lower than those of natural enzymes. Three series of designed Kemp eliminases (KE07, KE70, KE59) were shown to be evolvable with considerable improvement in catalytic efficiency. Here we use the KE07 enzyme as a model system to reveal those forces, which govern enzymatic evolution and elucidate the key factors for improving activity. We applied the Empirical Valence Bond (EVB) method to construct the free energy pathway of the reaction in the original KE07 design and the evolved R7 1/3H variant. We analyzed catalytic effect of residues and demonstrated that not all mutations in evolution are favorable for activity. In contrast to the small decrease in the activation barrier, in vitro evolution significantly reduced the reorganization energy. We developed an algorithm to evaluate group contributions to the reorganization energy and used this approach to screen for KE07 variants with potential for improvement. We aimed to identify those mutations that facilitate enzymatic evolution, but might not directly increase catalytic efficiency. Computational results in accord with experimental data show that all mutations, which appear during in vitro evolution were either neutral or favorable for the reorganization energy. These results underscore that distant mutations can also play role in optimizing efficiency via their contribution to the reorganization energy. Exploiting this principle could be a promising strategy for computer-aided enzyme design. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.

Patterns of Dynamics Comprise a Conserved Evolutionary Trait.

The importance of protein dynamics in function may suggest an evolutionary selection on large-scale protein motions. Here we systematically studied the dynamic characteristics in 2221 protein domains (58477 sequences) of the Pfam database. We defined the patterns of dynamics (PODs) based on the estimated NMR order parameters and the predicted degree of disorder, and found a significant correlation between them in families of both structured and disordered protein domains. We demonstrate that conservation of dynamic patterns frequently exceeds conservation of sequence and is comparable to the patterns of hydropathy and nonspecific interaction potential. Similarity of dynamic patterns is weakly correlated to structure similarity and to the degree of disorder. We illustrate that POD alignments could be applied to sequentially divergent or intrinsically disordered regions. We propose that patterns of dynamics comprise a conserved evolutionary trait, which could be used to infer evolutionary relationships as an alternative to sequence and structure.

Asymmetric dynamic coupling promotes alternative evolutionary pathways in an enzyme dimer.

The importance of dynamic factors in enzyme evolution is gaining recognition. Here we study how the evolution of a new enzymatic activity exploits conformational tinkering and demonstrate that conversion of a dimeric phosphotriesterase to an arylesterase in Pseudomonas diminuta is accompanied by structural divergence between the two subunits. Deviations in loop conformations increase with promiscuity, leading to functionally distinct states, while they decrease during specialisation for the new function. We show that opposite loop movements in the two subunits are due to a dynamic coupling with the dimer interface, the importance of which is also corroborated by the co-evolution of the loop and interface residues. These results illuminate how protein dynamics promotes conformational heterogeneity in a dimeric enzyme, leading to alternative evolutionary pathways for the emergence of a new function.

Altered dynamics may drift pathological fibrillization in membraneless organelles.

Protein phase transition can generate non-membrane bound cellular compartments, which can convert from liquid-like to solid-like states. While the molecular driving forces of phase separation have been largely understood, much less is known about the mechanisms of material-state conversion. We apply a recently developed algorithm to describe the weak interaction network of multivalent motifs, and simulate the effect of pathological mutations. We demonstrate that linker dynamics is critical to the material-state of biomolecular condensates. We show that linker flexibility/mobility is a major regulator of the weak, heterogeneous meshwork of multivalent motifs, which promotes phase transition and maintains a liquid-like state. Decreasing linker dynamics increases the propensity of amyloid-like fragments via hampering the motif-exchange and reorganization of the weak interaction network. In contrast, increasing linker mobility may compensate rigidifying mutations, suggesting that the meshwork of weak, variable interactions may provide a rescue mechanism from aggregation. Motif affinity, on the other hand, has a moderate impact on fibrillization. Here we demonstrate that the fuzzy framework provides an efficient approach to handle the intricate organization of membraneless organelles, and could also be applicable to screen for pathological effects of mutations.

Role of stabilization centers in 4 helix bundle proteins.

Stabilization centers (SCs) were shown to play an important role in preventing decay of three-dimensional protein structures. These residue clusters, stabilized by cooperative long-range interactions, were proposed to serve as anchoring points for arranging secondary structure elements. In all-alpha proteins, SC elements appear less frequently than in all-beta, alpha/beta, and alpha+beta proteins suggesting that tertiary structure formation of all-alpha proteins is governed by different principles than in other protein classes. Here we analyzed the relation between the formation of stabilization centers and the inter-axial angles (Omega) of alpha-helices in 4 helix bundle proteins. In the distance range, where dipoles have dominant effect on the helix pair arrangement, those helix pairs, where residues from both helices participate in SC elements, appear as parallel more frequently than those helices where no SC elements are present. For SC containing helix pairs, the energetic difference between the parallel and anti-parallel states decreases considerably from 1.1 kcal/mol to 0.4 kcal/mol. Although the observed effect is weak for more distant helices, a competition between the SC element formation and the optimal dipole-dipole interaction of alpha-helices is proposed as a mechanism for tertiary structure formation in 4 helix bundle proteins. The SC-forming potential of different arrangements as well as the pitfalls of the SC definition are also discussed.

The role of hydrophobic microenvironments in modulating pKa shifts in proteins.

The screened Coulomb potential (SCP) method, combined with a quantitative description of the microenvironments around titratable groups, based on the Hydrophobic Fragmental Constants developed by Rekker, has been applied to calculate the pK(a) values of groups embedded in extremely hydrophobic microenvironments in proteins. This type of microenvironment is not common; but constitutes a small class, where the protein's architecture has evolved to lend special properties to the embedded residue. They are of significant interest because they are frequently important in catalysis and in proton and electron transfer reactions. In the SCP treatment these special cases are treated locally and therefore do not affect the accuracy of the pK(a) values calculated for other residues in less hydrophobic environments. Here the calibration of the algorithm is extended with the help of earlier results from lysozyme and of three mutants of staphylococcal nuclease (SNase) that were specially designed to measure the energetics of ionization of titratable groups buried in extremely hydrophobic microenvironments. The calibrated algorithm was subsequently applied to a fourth mutant of SNase and then to a very large dimeric amine oxidase of 1284 residues, where 334 are titratable. The observed pK(a) shifts of the buried residues are large (up to 4.7 pK units), and all cases are well reproduced by the calculations with a root mean square error of 0.22. These results support the hypothesis that protein electrostatics can only be described correctly and self-consistently if the inherent heterogeneity of these systems is properly accounted for.

Type II restriction endonucleases: structure and mechanism.

Type II restriction endonucleases are components of restriction modification systems that protect bacteria and archaea against invading foreign DNA. Most are homodimeric or tetrameric enzymes that cleave DNA at defined sites of 4-8 bp in length and require Mg2+ ions for catalysis. They differ in the details of the recognition process and the mode of cleavage, indicators that these enzymes are more diverse than originally thought. Still, most of them have a similar structural core and seem to share a common mechanism of DNA cleavage, suggesting that they evolved from a common ancestor. Only a few restriction endonucleases discovered thus far do not belong to the PD...D/ExK family of enzymes, but rather have active sites typical of other endonuclease families. The present review deals with new developments in the field of Type II restriction endonucleases. One of the more interesting aspects is the increasing awareness of the diversity of Type II restriction enzymes. Nevertheless, structural studies summarized herein deal with the more common subtypes. A major emphasis of this review will be on target site location and the mechanism of catalysis, two problems currently being addressed in the literature.

Probing the general base catalysis in the first step of BamHI action by computer simulations.

BamHI is a type II restriction endonuclease that catalyzes the scission of the phoshodiester bond in the GAGTCC cognate sequence in the presence of two divalent metal ions. The first step of the reaction is the preparation of water for nucleophilic attack by Glu-113, which has been proposed to abstract the proton from the attacking water molecule. Alternatively, the 3'-phosphate group to the susceptible phosphodiester bond has been suggested to play a role as the general base. The two hypotheses have been tested by computer simulations using the semiempirical protein dipoles Langevin dipoles (PDLD/S) method. Deprotonation of water by Glu-113 has been found to be less favorable by 5.7 kcal/mol than metal-catalyzed deprotonation with a concomitant proton transfer to bulk solvent. The preparation of the nucleophile by the 3'-phosphate group is less favorable by 12.3 kcal/mol. These results suggest that both the general base and the substrate-assisted mechanisms in the first step of BamHI action are less likely than the metal-catalyzed reaction. The metal ions in the active site of BamHI make the largest contributions to the reduction of the free energy of hydroxide ion formation. On the basis of these findings we propose that the first step of endonuclease catalysis does not require a general base; rather, the essential attacking nucleophile in BamHI catalytic action is stabilized by the metal ions.

Role of active site residues in the glycosylase step of T4 endonuclease V. Computer simulation studies on ionization states.

T4 Endonuclease V (EndoV) is a base excision repair enzyme that removes thymine dimers (TD) from damaged DNA. To elucidate the role of the active site residues in catalysis, their pK(a)'s were evaluated using the semimicroscopic version of the protein dipoles-Langevin dipoles method (PDLD/S). Contributions of different effects to the pK(a) such as charge-charge interactions, conformational rearrangement, protein relaxation, and DNA binding were analyzed in detail. Charging of the active site residues was found to be less favorable in the complex than in the free enzyme. The pK(a) of the N-terminus decreased from 8.01 in the free enzyme to 6.52 in the complex, while the pK(a) of Glu-23 increased from 1. 52 to 7.82, which indicates that the key residues are neutral in the reactant state of the glycosylase step. These pK(a)'s are in agreement with the optimal pH range of the reaction and support the N-terminus acting as a nucleophile. The Glu-23 in its protonated form is hydrogen bonded to O4' of the sugar of 5' TD and can play a role in increasing the positive charge of C1' and, hence, accelerating the nucleophilic substitution. Furthermore, the neutral Glu-23 is a likely candidate to protonate O4' to induce ring opening required to complete the glycosylase step of EndoV. The positively charged Arg-22 and Arg-26 provide an electrostatically favorable environment for the leaving base. To distinguish between S(N)1 and S(N)2 mechanisms of the glycosylase step the energetics of protonating O2 of 5' TD was calculated. The enzyme was found to stabilize the neutral thymine by approximately 3.6 kcal/mol, whereas it destabilizes the protonated thymine by approximately 6.6 kcal/mol with respect to an aqueous environment. Consequently, the formation of a protonated thymine intermediate is unlikely, indicating an S(N)2 reaction mechanism for the glycosylase step.

Specificity of damage recognition and catalysis of DNA repair.

A common feature of DNA repair enzymes is their ability to recognize the damage independently of sequence in which they are found. The presence of a flipped out base inserted into the protein in several DNA-enzyme complexes suggests a contribution to enzyme specificity. Molecular simulations of damaged DNA indicate that the damage produces changes in DNA structure and changes the dynamics of DNA bending. The reduced bending force constant can be used by the enzyme to induce DNA bending and facilitate base flipping. We show that a thymine dimer (TD) containing DNA requires less energy to bend, lowering the barrier for base flipping. On the other hand, bending in DNA with U-G mismatch is affected only by a small amount and flipping is not enhanced significantly. T4 endonuclease V (endoV), which recognizes TD, utilizes the reduced barrier for flipping as a specific recognition element. In uracil DNA glycosylase (UDG), which recognizes U-G mismatches, base flipping is not enhanced and recognition is encoded in a highly specific binding pocket for the flipped base. Simulations of UDG and endoV in complex with damaged DNA provide insight into the essential elements of the catalytic mechanism. Calculations of pKas of active site residues in endoV and endoV-DNA complex show that the pKa, of the N-terminus is reduced from 8.01 to 6.52 while that of Glu-23 increases from 1.52 to 7.82. Thus, the key catalytic residues are in their neutral form. The simulations also show that Glu-23 is also H-bonded to O4' of the 5'-TD enhancing the nucleophilic attack on Cl and that Arg-26 enhances the hydrolysis by electrostatic stabilization but does not participate in proton transfer. In the enzyme-substrate complex of UDG, the role of electrostatic stabilization is played by His-268, whose pKa increases to 7.1 from 4.9 in the free enzyme. The pKa of Asp-145, the other important catalytic residue, remains around 4.2 in the free enzyme and in the complex. Thus, it can not act as a proton acceptor. In the complex the 3'-phosphate of uracil is stabilized next to Asp-145 by two bridging water molecules. Such a configuration activates one water molecule to act as a proton acceptor to produce a stabilizing hydronium ion and the other as a proton donor to produce the nucleophilic hydroxide. It appears that DNA glycosylases share commonalties in recognition of damage but differ in their catalytic mechanisms.

Molecular modelling of xylose isomerase catalysis: the role of electrostatics and charge transfer to metals.

The two main steps of the mechanism of xylose-xylulose conversion catalysed by D-xylose isomerase, the ring opening of xylose and the isomerization of the opened product by hydride transfer, were investigated by molecular mechanical and molecular orbital techniques. The activation energies calculated for these reactions clearly showed that hydrogen transfer is the rate-determining step of the enzymatic isomerization and that Mg2+ ions activate whereas Zn2+ ions inhibit the reaction, in agreement with the experiments. The remarkable differences between the net charges of these ions found by molecular orbital calculations and the inspection of the protein electrostatic potential around the reaction intermediates indicate that the main role of bivalent metal ions should be the electrostatic stabilization of the substrate transition states. In order to propose a more detailed mechanism, an attempt was made to clarify the effects of nearby residues (e.g. His54, Asp57, Lys183, Asp257) in the reaction. Different isomerization mechanisms, such as through an enediol intermediate, were examined and could be excluded, in addition to the charge-relay mechanism during the ring opening.

Effects of physostigmine on the excitation-contraction coupling of skeletal muscle fibres.

The effects of physostigmine on the electrophysiological properties of the surface membrane and on the different steps of excitation-contraction coupling were studied on the skeletal muscle of the frog (Rana esculenta). On analysing the phase plane trajectories of electrically evoked action potentials it was found that, similarly to the previously described pH dependence of the depolarizing effect of the alkaloid [14], the physostigmine-induced inhibition of the voltage-dependent sodium and potassium conductances responsible for the generation of the spike was increased in correlation with the decrease of external hydrogen ion concentration (pH 6.4, 7.0 and 8.4). When performing examinations on cut muscle fibres using the single vaseline gap voltage clamp technique [9], physostigmine did not exert any characteristic effect on the strength-duration curve of the contraction threshold determined by short depolarizing pulses even at higher concentrations (2 and 10 mmol/l; pH 7.0). In some cases the value of the rheobase was shifted to a variable extent towards more negative membrane potentials. On using the metallochromic indicator dye antipyrylazo III it was found that the application of 2 mmol/l physostigmine (pH 7.0) decreased the amount of calcium released during the depolarizing pulses. To reach the contraction threshold, a smaller increase in calcium concentration was necessary in the presence of the alkaloid. The relaxing phase of contractions elicited by depolarizing pulses was slowed down due to 2 mmol/l (pH 7.0) physostigmine treatment although the rate of the falling phase of the calcium transients increased simultaneously. The decrease in external hydrogen ion concentration facilitated the development of modifications in the shape of the contractions. The conclusion was drawn that the effects of physostigmine exhibit pH dependence. The alkaloid decreases calcium release from the sarcoplasmic reticulum and increases the calcium sensitivity of the contractile proteins.

How perchlorate improves excitation-contraction coupling in skeletal muscle fibers.

The effect of the "chaotropic" anion, perchlorate, on the activation of contraction has been studied in voltage clamped frog skeletal muscle fibers. It was found that the voltage dependence of either the contractile force or the intramembrane charge movement was shifted towards more negative membrane potentials. The maximum values of force or charge movement attained with large depolarizing pulses did not change significantly. It is concluded that a specific perchlorate effect on the movement of charged particles can explain the potentiating effect of perchlorate anions on contractile force, strengthening the view that these charged particles serve as voltage sensors regulating Ca2+ release from the sarcoplasmic reticulum.

Crystallization and preliminary X-ray analysis of porcine muscle prolyl oligopeptidase.

Prolyl oligopeptidase from pig muscle has been crystallized in complex with an inhibitor, using PEG 8000 and calcium acetate as precipitants. The crystals are orthorombic and the space group is P212121 with cell dimensions a = 111.8, b = 101.8, c = 72.4 A. The asymmetric unit contains a single chain of prolyl oligopeptidase, corresponding to a specific volume of 2.55 A3 Da-1 and a solvent content of 52%. The observed diffraction pattern extends to 2.3 A resolution and the native crystals are well suited for structural analysis by X-ray diffraction methods.

Role of electrostatics at the catalytic metal binding site in xylose isomerase action: Ca(2+)-inhibition and metal competence in the double mutant D254E/D256E.

The catalytic metal binding site of xylose isomerase from Arthrobacter B3728 was modified by protein engineering to diminish the inhibitory effect of Ca2+ and to study the competence of metals on catalysis. To exclude Ca2+ from Site 2 a double mutant D254E/D256E was designed with reduced space available for binding. In order to elucidate structural consequences of the mutation the binary complex of the mutant with Mg2+ as well as ternary complexes with bivalent metal ions and the open-chain inhibitor xylitol were crystallized for x-ray studies. We determined the crystal structures of the ternary complexes containing Mg2+, Mn2+, and Ca2+ at 2.2 to 2.5 A resolutions, and refined them to R factors of 16.3, 16.6, and 19.1, respectively. We found that all metals are liganded by both engineered glutamates as well as by atoms O1 and O2 of the inhibitor. The similarity of the coordination of Ca2+ to that of the cofactors as well as results with Be2+ weaken the assumption that geometry differences should account for the catalytic noncompetence of this ion. Kinetic results of the D254E/D256E mutant enzyme showed that the significant decrease in Ca2+ inhibition was accompanied by a similar reduction in the enzymatic activity. Qualitative argumentation, based on the protein electrostatic potential, indicates that the proximity of the negative side chains to the substrate significantly reduces the electrostatic stabilization of the transition state. Furthermore, due to the smaller size of the catalytic metal site, no water molecule, coordinating the metal, could be observed in ternary complexes of the double mutant. Consequently, the proton shuttle step in the overall mechanism should differ from that in the wild type. These effects can account for the observed decrease in catalytic efficiency of the D254E/D256E mutant enzyme.

Checking nucleic acid crystal structures.

The program SFCHECK [Vaguine et al. (1999), Acta Cryst. D55, 191-205] is used to survey the quality of the structure-factor data and the agreement of those data with the atomic coordinates in 105 nucleic acid crystal structures for which structure-factor amplitudes have been deposited in the Nucleic Acid Database [NDB; Berman et al. (1992), Biophys. J. 63, 751-759]. Nucleic acid structures present a particular challenge for structure-quality evaluations. The majority of these structures, and DNA molecules in particular, have been solved by molecular replacement of the double-helical motif, whose high degree of symmetry can lead to problems in positioning the molecule in the unit cell. In this paper, the overall quality of each structure was evaluated using parameters such as the R factor, the correlation coefficient and various atomic error estimates. In addition, each structure is characterized by the average values of several local quality indicators, which include the atomic displacement, the density correlation, the B factor and the density index. The latter parameter measures the relative electron-density level at the atomic position. In order to assess the quality of the model in specific regions, the same local quality indicators are also surveyed for individual groups of atoms in each structure. Several of the global quality indicators are found to vary linearly with resolution and less than a dozen structures are found to exhibit values significantly different from the mean for these indicators, showing that the quality of the nucleic acid structures tends to be rather uniform. Analysis of the mutual dependence of the values of different local quality indicators, computed for individual residues and atom groups, reveals that these indicators essentially complement each other and are not redundant with the B factor. Using several of these indicators, it was found that the atomic coordinates of the nucleic acid bases tend to be better defined than those of the backbone. One of the local indicators, the density index, is particularly useful in spotting regions of the model that fit poorly in the electron density. Using this parameter, the quality of crystallographic water positions in the analyzed structures was surveyed and it was found that a sizable fraction of these positions have poorly defined electron density and may therefore not be reliable. The possibility that cases of poorly positioned water molecules are symptomatic of more widespread problems with the structure as a whole is also raised.

Comparative redox and pKa calculations on cytochrome c3 from several Desulfovibrio species using continuum electrostatic methods.

A comparative study of the pH-dependent redox mechanisms of several members of the cytochrome c3 family has been carried out. In a previous work, the molecular determinants of this dependency (the so-called redox-Bohr effect) were investigated for one species using continuum electrostatic methods to find groups with a titrating range and strength of interaction compatible with a mediating role in the redox-Bohr effect. Here we clarify these aspects in the light of new and improved pKa calculations, our findings supporting the hypothesis of propionate D from heme I being the main effector in the pH-dependent modulation of the cytochrome c3 redox potentials in all the c3 molecules studied here. However, the weaker (but significant) role of other titrating groups cannot be excluded, their importance and identity changing with the particular molecule under study. We also calculate the relative redox potentials of the four heme centers among the selected members of the c3 family, using a continuum electrostatic method that takes into account both solvation and interaction effects. Comparison of the calculated values with available data for the microscopic redox potentials was undertaken, the quality of the agreement being dependent upon the choice of the dielectric constant for the protein interior. We find that high dielectric constants give best correlations, while low values result in better magnitudes for the calculated potentials. The possibility that the crystallographic calcium ion in c3 from Desulfovibrio gigas may be present in the solution structure was tested, and found to be likely.