Steroid and G protein binding characteristics of the seatrout and human progestin membrane receptor alpha subtypes and their evolutionary origins.

A novel progestin receptor (mPR) with seven-transmembrane domains was recently discovered in spotted seatrout and homologous genes were identified in other vertebrates. We show that cDNAs for the mPR alpha subtypes from spotted seatrout (st-mPRalpha) and humans (hu-mPRalpha) encode progestin receptors that display many functional characteristics of G protein-coupled receptors. Flow cytometry and immunocytochemical staining of whole MDA-MB-231 cells stably transfected with the mPRalphas using antibodies directed against their N-terminal regions show the receptors are localized on the plasma membrane and suggest the N-terminal domain is extracellular. Both recombinant st-mPRalpha and hu-mPRalpha display high affinity (Kd 4.2-7.8 nm), limited capacity (Bmax 0.03-0.32 nm), and displaceable membrane binding specific for progestins. Progestins activate a pertussis toxin-sensitive inhibitory G protein (G(i)) to down-regulate membrane-bound adenylyl cyclase activity in both st-mPRalpha- and hu-mPRalpha-transfected cells. Coimmunoprecipitation experiments demonstrate the receptors are directly coupled to the G(i) protein. Similar to G protein-coupled receptors, dissociation of the receptor/G protein complex results in a decrease in ligand binding to the mPRalphas and mutation of the C-terminal, and third intracellular loop of st-mPRalpha causes loss of ligand-dependent G protein activation. Phylogenetic analysis indicates the mPRs are members of a progesterone and adipoQ receptor (PAQR) subfamily that is only present in chordates, whereas other PAQRs also occur in invertebrates and plants. Progesterone and adipoQ receptors are related to the hemolysin3 family and have origins in the Eubacteria. Thus, mPRs arose from Eubacteria independently from members of the GPCR superfamily, which arose from Archeabacteria, suggesting convergent evolution of seven-transmembrane hormone receptors coupled to G proteins.

Fusarium solani pisi cutinase.

Cutinase from Fusarium solani pisi has been studied extensively with respect to its structural and functional properties. The crystal structure of the enzyme was solved to high atomic resolution (1 angstrom), while data on structural dynamics have been obtained from detailed NMR studies. Functional data were mainly derived from kinetic studies using substrate analogues that simplify the kinetic behaviour. The properties of wild-type cutinase are reviewed and discussed in relation with the effects brought about by site-directed variants of the enzyme.

Identification of functional and unfolding motions of cutinase as obtained from molecular dynamics computer simulations.

The implementation of cutinase from Fusarium solani pisi as a fat-stain removing ingredient in laundry washing is hampered by its unfolding in the presence of anionic surfactants. In this work we present molecular dynamics (MD) computer simulations on cutinase and analysis procedures to distinguish the movements related to its functional behavior (e.g., substrate binding) from those related to the unfolding of the enzyme. Two kinds of MD-simulations were performed: a simulation mimicking the thermal motion at room temperature, and several simulations in which unfolding is induced either by high temperature or by using a modified water-protein interaction potential. Essential dynamics analyses (A. Amadei et al., Proteins 17:412-425, 1993) on the simulations identify distinct regions in the molecular structure of cutinase in which the motions occur for function and initial unfolding. The unfolding in various simulations starts in a similar way, suggesting that mutations in the regions involved might stabilize the enzyme without affecting its functionality.

Dynamics of Fusarium solani cutinase investigated through structural comparison among different crystal forms of its variants.

In characterizing mutants and covalently inhibited complexes of Fusarium solani cutinase, which is a 197-residue lipolytic enzyme, 34 variant structures, crystallizing in 8 different crystal forms, have been determined, mostly at high resolution. Taking advantage of this considerable body of information, a structural comparative analysis was carried out to investigate the dynamics of cutinase. Surface loops were identified as the major flexible protein regions, particularly those forming the active-site groove, whereas the elements constituting the protein scaffold were found to retain the same conformation in all the cutinase variants studied. Flexibility turned out to be correlated with thermal motion. With a given crystal packing environment, a high flexibility turned out to be correlated with a low involvement in crystal packing contacts. The high degree of crystal polymorphism, which allowed different conformations with similar energy to be detected, made it possible to identify motions which would have remained unidentified if only a single crystal form had been available. Fairly good agreement was found to exist between the data obtained from the structural comparison and those from a molecular dynamics (MD) simulation carried out on the native enzyme. The crystallographic approach used in this study turned out to be a suitable tool for investigating cutinase dynamics. Because of the availability of a set of closely related proteins in different crystal environments, the intrinsic drawback of a crystallographic approach was bypassed. By combining several static pictures, the dynamics of the protein could be monitored much more realistically than what can be achieved on the basis of static pictures alone.

Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state.

Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.

Engineering surface charges in a subtilisin.

Introduction of multiple charged amino acid residues in the subtilisin Savinase by genetic engineering allowed us to modify the electrostatic properties of this enzyme in a systematic way. The effects of these charge changes were investigated theoretically with the calculated electrostatic potential at the enzyme surface and experimentally using ion exchange chromatography. Our results indicate that the effect of introducing charged residues at the enzyme surface depends on the local electrostatic potential. The effects are purely additive for residues that are not too closely packed at the enzyme surface. Although it is generally accepted that polarization effects are relatively small, our data show that substantial charge shifts arise when the dominating effect of the overall charge is taken away. These shifts are not well quantified using current methods to calculate the electrostatic potential at the enzyme surface. Our work focuses [correction of focusses] on methods that will provide a better description of this surface potential.

Cutinase from Fusarium solani pisi hydrolyzing triglyceride analogues. Effect of acyl chain length and position in the substrate molecule on activity and enantioselectivity.

Triglyceride analogues were synthesized in which one of the primary acyl ester functions has been replaced by an alkyl group and the secondary acyl ester bond has been replaced by an acyl amino bond. The chain length at either position was varied, and both (R)- and (S)-enantiomers of each compound were synthesized. These pseudo triglycerides contain only one hydrolyzable ester bond, and they are ideally suited to studying the influence of the chain length at the 1-, 2-, and 3-position on lipase activity and on stereopreference. These substrates were used to characterize cutinase from Fusarium solani pisi. Our results show that the activity of cutinase is very sensitive to the length and distribution of the acyl chains and that the highest activities are found when the chains at positions 1 and 3 contain three or four carbon atoms. The enzyme preferentially hydrolyzes the (R)-enantiomers, but this preference is strongly dependent on the acyl chain length distribution, with (R) over (S) activity ratios varying from about 30 to 1. This enantioselectivity was found in three different assay systems: a mixed micellar, a reverse micellar, and a monolayer study. Our data suggest that at least two alkyl chains of the pseudo triglycerides must be fixed during hydrolysis. Therefore, these substrates were used to characterize mutants of cutinase with mutations in putative lipid binding domains. Two mutants (A85F and A85W) have increased activities. The results obtained with these mutants suggest an interaction of the acyl chain of the scissile ester bond with a surface loop, comprising residues 80-90, in the enzyme-substrate complex.

Increasing thermal stability of subtilisin from mutations suggested by strongly interacting side-chain clusters.

In this paper we present for seven subtilisin structures a systematic comparison of densely packed side-group clusters (defined as an ensemble of side chains with extensive internal atomic contacts as compared with those made with the surrounding protein environment and measured relative to the maximum possible for each residue type). Spatially consistent clusters are observed at structurally equivalent positions in the proteins, as revealed by careful multiple superpositioning of the respective backbone atoms. The clusters are positioned at strategic loop-connecting sites near the protein surfaces. The residues within consistent clusters displaying extensive association show varying conservation at structurally equivalent alignment sites. Suggestions for residue substitutions, as observed over the seven tertiary structures, were taken from the cluster positions and were shown to be consistent with a number of point mutations in one of the seven structures (savinase) that result in increased thermal stability.

Engineering surface charges in a subtilisin: the effects on electrophoretic and ion-exchange behaviour.

The introduction or removal of multiple charged amino acid residues in the subtilisin Savinase by genetic engineering allowed us to modify the electrostatic properties of this enzyme in a systematic way. The effects of these charge changes were investigated experimentally using ion-exchange chromatography and electrophoretic mobility in native gels all under identical conditions. The experiments clearly demonstrated that the overall charge of a given protein is not the only factor determining electrophoretic mobility at low or moderate ionic strengths. For a series of variants having identical overall positive charge a linear relation was observed between mobility towards the cathode and the total number of charged residues present. This effect was found to depend on the type of (chloride) salt used: calcium ions give rise to complete screening of all negative charges, whereas only partial screening is found for magnesium and sodium ions. In contrast, in the presence of sodium phosphate the overall charge of the enzyme becomes slightly negative. These data indicate that cations as well as anions may strongly perturb the overall charge of proteins depending on the type of salt and on the number of charged amino acid residues present. The ion-exchange behaviour demonstrated similar results, i.e. showing stronger enzyme adsorption with increasing numbers of surface charges on a cation-exchange column run below the isoelectric point of the proteins. However, the apparent sign reversal noted above for electrophoresis with sodium phosphate did not appear in the ion-exchange experiments. This work provides further insight into the adsorption of proteins to surface and the role played by small ions, particularly when electrostatic forces dominate the adsorption process.

Tertiary structure of two-electron reduced Megasphaera elsdenii flavodoxin and some implications, as determined by two-dimensional 1H-NMR and restrained molecular dynamics.

The tertiary structure of the non-crystallizable two-electron-reduced Megasphaera elsdenii flavodoxin (15 kDa, 137 amino acid residues) has been determined using nuclear Overhauser enhancement restraints extracted from two-dimensional 1H-NMR spectra. A tertiary structure satisfying the experimental restraints very well (maximum NOE violation of 66 pm) was obtained with use of restrained molecular dynamics, using 509 distance restraints (including one non-NOE) on a starting structure modeled from the crystal structure of one-electron-reduced Clostridium MP flavodoxin. The protein consists of a central parallel beta-sheet surrounded on both sides by two alpha-helices. The flavin is positioned at the periphery of the molecule. The tertiary structure of the protein is highly defined with the exception of the flavin. The latter is expected to result from performing the restrained molecular dynamics simulation without water molecules and without proper charges on the flavin. The flavin, including the phosphate, the ribityl side chain and the isoalloxazine ring, is solvent accessible under the experimental conditions used and evidenced by a two-dimensional amide exchange experiment. This accessibility is expected to be important in the redox potential regulation of the semiquinone/hydroquinone couple of the protein. The amide exchange against deuterons and several typical line shapes in the two-dimensional NMR spectra are consistent with the structure generated. The structure is discussed in detail.

An NMR-based molecular dynamics simulation of the interaction of the lac repressor headpiece and its operator in aqueous solution.

The results of a 125 psec molecular dynamics simulation of a lac headpiece-operator complex in aqueous solution are reported. The complex satisfies essentially all experimental distance information derived from two-dimensional nuclear magnetic resonance (2-D-NMR) studies. The interaction between lac repressor headpiece and its operator is based on many direct- and water-mediated hydrogen bonds and nonpolar contacts which allow the formation of a tight complex. No stable hydrogen bonds between side chains and bases are found, while specific contacts occur between both nonpolar groups and, to a lesser extent, through water-mediated hydrogen bonds. The simulated complex structure in water is intrinsically stable without application of nuclear Overhauser effect (NOE) distance restraints, while being compatible with most of the available biochemical, genetic, and chemically induced dynamic nuclear polarization (CIDNP) data.

Combined procedure of distance geometry and restrained molecular dynamics techniques for protein structure determination from nuclear magnetic resonance data: application to the DNA binding domain of lac repressor from Escherichia coli.

The technique of two-dimensional nuclear magnetic resonance (2D-NMR) has recently assumed an active role in obtaining information on structures of polypeptides, small proteins, sugars, and DNA fragments in solution. In order to generate spatial structures from the atom-atom distance information obtained by the NMR method, different procedures have been developed. Here we introduce a combined procedure of distance geometry (DG) and molecular dynamics (MD) calculations for generating 3D structures that are consistent with the NMR data set and have reasonable internal energies. We report the application of the combined procedure on the lac repressor DNA binding domain (headpiece) using a set of 169 NOE and 17 "hydrogen bond" distance constraints. Eight of ten structures generated by the distance geometry algorithm were refined within 10 ps MD simulation time to structures with low internal energies that satisfied the distance constraints. Although the combination of DG and MD was designed to combine the good sampling properties of the DG algorithm with an efficient method of lowering the internal energy of the molecule, we found that the MD algorithm contributes significantly to the sampling as well.