Biological therapy of ovarian cancer: current directions

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein-ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol-1) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable.     

Specific in vivo thiol-labeling of the FhuA outer membrane ferrichrome transport protein of Escherichia coli K-12: evidence for a disulfide bridge in the predicted gating loop

The multifunctional FhuA protein of Escherichia coli K-12 forms a channel that is closed by a loop, tentatively designated the 'gating loop', which is also the principal binding site for all FhuA ligands. In this report, it is shown by in vivo labeling that the two cysteines in the gating loop form a disulfide bridge, and they react weakly after reduction with biotin-maleimide, as determined by streptavidin-beta-galactosidase bound to biotin. The two cysteines close to the C-terminus of FhuA also form a disulfide bridge and react with the thiol reagents only after heat denaturation of FhuA in SDS. Replacement of the existing cysteines by serine did not alter the sensitivity of cells to the FhuA ligands tested (T5, phi 80, T1, colicin M, and albomycin) and supported growth on ferrichrome as sole iron source. The cysteines in the gating loop play no specific functional role; they are largely buried in the interior of the loop, and the disulfide bridges are not essential for maintaining the conformation of FhuA. The C-terminal cysteines are in the interior of FhuA and are also not important for the structure of FhuA. The method used allows the identification of free cysteines and disulfides in surface exposed protein regions.     

An example of a protein ligand found by database mining: description of the docking method and its verification by a 2.3 A X-ray structure of a thrombin-ligand complex

A computer program (SANDOCK) has been developed for the automated docking of small ligands to a target protein. It uses a guided matching algorithm to fit ligand atoms into the protein binding pocket. The protein is described by a modified Lee-Richard's dotted surface with each dot coded by chemical property and accessibility. Orientations of the ligand in the active site are generated such that a chemical and a shape complementary between the ligand and the active site cavity have to be fulfilled. The generated fits are evaluated with scoring functions which account for van der Waals, hydrophobic and hydrogen bonding interactions. This newly developed docking program can efficiently screen very large databases in a reasonable time and has been used to successfully identify novel ligands. The X-ray structure of a thrombin-ligand complex predicted by SANDOCK is described. The ligand binds to thrombin with a Kd of 65 microM and has an rmsd of 0.7 A for all ligand atoms from the predicted binding mode by SANDOCK.     

Structures of the Erythrina corallodendron lectin and of its complexes with mono- and disaccharides

The structures of the Erythrina corallodendron lectin (EcorL) and of its complexes with galactose, N-acetylgalactosamine, lactose and N-acetyllactosamine were determined at a resolution of 1.9 to 1.95 A. The final R-values of the five models are in the range 0.169 to 0.181. The unusual, non-canonical, dimer interface of EcorL is made of beta-strands from the two monomers, which face one another in a "hand-shake" mode. The galactose molecule in the primary binding site is bound in an identical way in all four complexes. Features of the electrostatic potential of the galactose molecule match those of the potential in the combining site, thus probably pointing to the contribution of the electrostatic energy to determining the orientation of the ligand. No conformational change occurs in the protein upon binding the ligand. Subtle variations in the binding mode of the second monosaccharide (glucose in the complex with lactose and N-acetylglucosamine in the complex with N-acetyllactosamine) were observed. The mobility of Gln219 is lower in the complexes with the disaccharides than in the complexes with the monosaccharides, indicating further recruitment of this residue to ligand binding through more extensive hydrogen bonding in the former complexes. Water molecules that have been located in the combining sites of the five structures undergo rearrangement in response to binding of the different ligands. The new structural information is in qualitative agreement with thermodynamic data on the binding to EcorL.     

Double mutagenesis of a positive charge cluster in the ligand-binding site of the ferric enterobactin receptor, FepA

Siderophores and colicins enter bacterial cells through TonB-dependent outer membrane proteins. Using site-directed substitution mutagenesis, we studied ligand recognition by a prototypic Escherichia coli siderophore receptor, FepA, that binds the iron chelate ferric enterobactin and colicins B and D. These genetic experiments identified a common binding site for two of the three ligands, containing multiple positive charges, within cell surface residues of FepA. Elimination of single residues in this region did not impair the adsorption or transport of ferric enterobactin, but double mutagenesis in the charge cluster identified amino acids (Arg-286 and Arg-316) that participate in siderophore binding and function in FepA-mediated killing by colicins B and D. Ferric enterobactin binding, furthermore, prevented covalent modification of FepA within this domain by either a fluorescent probe or an arginine-specific reagent, corroborating the involvement of this site in ligand recognition. These results identify, for the first time, residues in a TonB-dependent outer membrane protein that participate in ligand binding. They also explain the competition between ferric enterobactin and the colicins on the bacterial cell surface: all three ligands interact with the same arginine residues within FepA during their penetration through the outer membrane.     

Identification of the second cluster of ligand-binding repeats in megalin as a site for receptor-ligand interactions

Megalin is a large cell surface receptor that mediates the binding and internalization of a number of structurally and functionally distinct ligands from the lipoprotein and protease:protease inhibitor families. To begin to address how megalin is able to bind ligands with unique structurally properties, we have mapped a binding site for apolipoprotein E (apoE)-beta very low density lipoprotein (beta VLDL), lipoprotein lipase, aprotinin, lactoferrin, and the receptor-associated protein (RAP) within the primary sequence of the receptor. RAP is known to inhibit the binding of all ligands to megalin. We identified a ligand-binding site on megalin by raising mAb against purified megalin, selected for a mAb whose binding to megalin is inhibited by RAP, and mapped the epitope for this mAb. mAb AC10 inhibited the binding of apoE-beta VLDL, lipoprotein lipase, aprotinin, and lactoferrin to megalin in a concentration-dependent manner. When cDNA fragments encoding the four cysteine-rich ligand-binding repeats in megalin were expressed in a baculovirus system and immunoblotted with AC10, it recognized only the second cluster of ligand-binding repeats. The location of the epitope recognized by mAb AC10 within this domain was pinpointed to amino acids 1111-1210. From these studies we conclude that the binding of apoE-beta VLDL, lactoferrin, aprotinin, lipoprotein lipase, and RAP to megalin is either competitively or sterically inhibited by mAb AC10 suggesting that these ligands bind to the same or closely overlapping sites within the second cluster of ligand-binding repeats.     

The carboxyl terminus of B class ephrins constitutes a PDZ domain binding motif

Ephrin B proteins function as ligands for B class Eph receptor tyrosine kinases and are postulated to possess an intrinsic signaling function. The sequence at the carboxyl terminus of B-type ephrins contains a putative PDZ binding site, providing a possible mechanism through which transmembrane ephrins might interact with cytoplasmic proteins. To test this notion, a day 10.5 mouse embryonic expression library was screened with a biotinylated peptide corresponding to the carboxyl terminus of ephrin B3. Three of the positive cDNAs encoded polypeptides with multiple PDZ domains, representing fragments of the molecule GRIP, the protein syntenin, and PHIP, a novel PDZ domain-containing protein related to Caenorhabditis elegans PAR-3. In addition, the binding specificities of PDZ domains previously predicted by an oriented library approach (Songyang, Z., Fanning, A. S., Fu, C., Xu, J., Marfatia, S. M., Chishti, A. H., Crompton, A., Chan, A. C., Anderson, J. M., and Cantley, L. C. (1997) Science 275, 73-77) identified the tyrosine phosphatase FAP-1 as a potential binding partner for B ephrins. In vitro studies demonstrated that the fifth PDZ domain of FAP-1 and full-length syntenin bound ephrin B1 via the carboxyl-terminal motif. Lastly, syntenin and ephrin B1 could be co-immunoprecipitated from transfected COS-1 cells, suggesting that PDZ domain binding of B ephrins can occur in cells. These results indicate that the carboxyl-terminal motif of B ephrins provides a binding site for specific PDZ domain-containing proteins, which might localize the transmembrane ligands for interactions with Eph receptors or participate in signaling within ephrin B-expressing cells.     

Crystal structures of substrate binding site mutants of manganese peroxidase

Manganese peroxidase (MnP), an extracellular heme enzyme from the lignin-degrading basidiomycetous fungus, Phanerochaete chrysosporium, catalyzes the oxidation of MnII to MnIII. The latter, acting as a diffusible redox mediator, is capable of oxidizing a variety of lignin model compounds. The proposed MnII binding site of MnP consists of a heme propionate, three acidic ligands (Glu-35, Glu-39, and Asp-179), and two water molecules. Using crystallographic methods, this binding site was probed by altering the amount of MnII bound to the protein. Crystals grown in the absence of MnII, or in the presence of EDTA, exhibited diminished electron density at this site. Crystals grown in excess MnII exhibited increased electron density at the proposed binding site but nowhere else in the protein. This suggests that there is only one major MnII binding site in MnP. Crystal structures of a single mutant (D179N) and a double mutant (E35Q,D179N) at this site were determined. The mutant structures lack a cation at the MnII binding site. The structure of the MnII binding site is altered significantly in both mutants, resulting in increased access to the solvent and substrate.     

A strategy for the incorporation of water molecules present in a ligand binding site into a three-dimensional quantitative structure--activity relationship analysis

Water present in a ligand binding site of a protein has been recognized to play a major role in ligand-protein interactions. To date, rational drug design techniques do not usually incorporate the effect of these water molecules into the design strategy. This work represents a new strategy for including water molecules into a three-dimensional quantitative structure-activity relationship analysis using a set of glucose analogue inhibitors of glycogen phosphorylase (GP). In this series, the structures of the ligand-enzyme complexes have been solved by X-ray crystallography, and the positions of the ligands and the water molecules at the ligand binding site are known. For the structure-activity analysis, some water molecules adjacent to the ligands were included into an assembly which encompasses both the inhibitor and the water involved in the ligand-enzyme interaction. The mobility of some water molecules at the ligand binding site of GP gives rise to differences in the ligand-water assembly which have been accounted for using a simulation study involving force-field energy calculations. The assembly of ligand plus water was used in a GRID/GOLPE analysis, and the models obtained compare favorably with equivalent models when water was excluded. Both models were analyzed in detail and compared with the crystallographic structures of the ligand-enzyme complexes in order to evaluate their ability to reproduce the experimental observations. The results demonstrate that incorporation of water molecules into the analysis improves the predictive ability of the models and makes them easier to interpret. The information obtained from interpretation of the models is in good agreement with the conclusions derived from the structural analysis of the complexes and offers valuable insights into new characteristics of the ligands which may be exploited for the design of more potent inhibitors.     

Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of cAMP-dependent protein kinase complexed with the peptide inhibitor PKI(5-24) and adenosine

The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H6-rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heat-stable protein kinase inhibitor PKI(5-24) and adenosine, was determined at 2.2 A resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final crystallographic R-factor of 18.2% with good stereochemical parameters. The "active" enzyme adopts a "closed" conformation as found in rC:PKI(5-24) [Knighton et al. (1991a,b) Science 253, 407-414, 414-420] and packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nucleotide binding site found in the protein kinase family. The adenosine occupies a mostly hydrophobic pocket at the base of the cleft between the two lobes and is completely buried. The missing triphosphate moiety of ATP is filled with a water molecule (Wtr 415) which replaces the gamma-phosphate of ATP. The glycine-rich loop between beta1 and beta2 helps to anchor the phosphates while the ribose ring is buried beneath beta-strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in beta-strand 1, Glu 127 in the linker strand between the two lobes, Tyr 330, and a third water molecule, Wtr 359. The conserved nucleotide fold can be defined as a lid comprised of beta-strand 1, the glycine-rich loop, and beta-strand 2. The adenine ring is buried beneath beta-strand 1 and the linker strand (120-127) that joins the small and large lobes. The C-terminal tail containing Tyr 330, a segment that lies outside the conserved core, covers this fold and anchors it in a closed conformation. The main-chain atoms of the flexible glycine-rich loop (residues 50-55) in the ATP binding domain have a mean B-factor of 41.4 A2. This loop is quite mobile, in striking contrast to the other conserved loops that converge at the active site cleft. The catalytic loop (residues 166-171) and the Mg2+ positioning loop (residues 184-186) are a stable part of the large lobe and have low B-factors in all structures solved to date. The stability of the glycine-rich loop is highly dependent on the ligands that occupy the active site cleft with maximum stability achieved in the ternary complex containing Mg x ATP and the peptide inhibitor. In this ternary complex the gamma-phosphate is secured between both lobes by hydrogen bonds to the backbone amide of Ser 53 in the glycine-rich loop and the amino group of Lys 168 in the catalytic loop. In the adenosine ternary complex the water molecule replacing the gamma-phosphate hydrogen bonds between Lys 168 and Asp 166 and makes no contact with the small lobe. This glycine-rich loop is thus the most mobile component of the active site cleft, with the tip of the loop being highly sensitive to what occupies the gamma-subsite.     

Further characterization of [3H]-CGS 21680 binding sites in the rat striatum and cortex

1. The putative high affinity binding site for the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N- ethylcarboxamidoadenosine (CGS 21680) in the rat cerebral cortex was characterized by use of a number of selective A1 and A2 adenosine receptor ligands, and compared to the characteristics of the more abundant striatal A2A receptor. 2. The binding of [3H]-CGS 21680 to cortical membranes was performed at pH 5.5, in order to increase the amount of specific binding. 3. Reduction of the pH from 7.4 to 5.5 increased the apparent affinity of the striatal binding side for both agonists and antagonists. The relative order of potencies of both groups of ligands were the same at both pH values, and were consistent with binding to the A2A receptor. There was no observable change in the Bmax, the values being 415 and 446 fmol mg-1 protein at pH 5.5 and 7.4 respectively. 4. The cortical binding site yielded a Bmax value of 117 fmol mg-1 protein. The relative order of potencies of the adenosine receptor ligands observed at this binding site were not the same as those observed in the striatum, exhibiting a profile with both A1 and A2 characteristics. 5. Further characterization of this cortical binding site in the presence of the A1 selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) revealed a more typical A2A profile. This indicated that under the conditions used there were two components of [3H]-CGS 21680 binding, approximately 20% of the A1 receptor and 80% to the A2A receptor. 6. It is concluded that in the cerebral cortex there is a CGS 21680 binding site showing the characteristic properties of the striatal A2A receptor, and no evidence was obtained for the existence of a novelA2A-like binding site.     

Predominant expression of monoamine oxidase B isoform in rabbit renal proximal tubule: regulation by I2 imidazoline ligands in intact cells

Previous studies have shown that a subpopulation of the catecholamine-degrading enzymes monoamine oxidase (MAO) A and B holds a previously unknown regulatory site, the I2-imidazoline binding site (I2BS). In the present work, we characterized the isoforms of monoamine oxidases expressed in the rabbit renal proximal tubule, defined their relationship with I2BS, and investigated the ability of I2BS ligands to inhibit enzyme activity in intact cells. Two findings indicate that MAO-B is the predominant isoform expressed in the renal proximal tubule cells: 1) Western blot performed with an anti-MAO-A/MAO-B polyclonal antiserum revealed a single 55-kDa band corresponding to MAO-B; 2) enzyme assays showed an elevated MAO-B activity ([14C]beta-phenylethylamine oxidation: Vmax = 1.31 +/- 0.41 nmol/min/mg protein), whereas MAO-A activity was only detectable ([14C]5-HT oxidation: Vmax = 80.3 +/- 19 pmol/min/mg protein). Photoaffinity labeling with the I2BS ligand [125I]2-(3-azido-4-iodophenoxy)-methylimidazoline revealed a single 55-kDa band, which indicates that MAO-B of the renal proximal tubule cells holds the I2 imidazoline binding site. [3H]Idazoxan binding studies and enzyme assays showed that, in intact cells, I2BS ligands bind to and inhibit MAO-B. Indeed, the increase in the accessibility of intracellular compartment by cell permeabilization did not enhance [3H]idazoxan binding, which indicates that, in intact cells, intracellular I2BS are fully occupied by imidazoline ligands. In addition, enzyme assays showed that incubation of proximal tubule cells with imidazoline ligands leads to a complete, dose-dependent inhibition of MAO activity. These data show the predominant expression of MAO-B in rabbit renal proximal tubule and its regulation by imidazoline ligands in intact cells.     

Identification of a conserved hydrophobic cluster in partially folded bovine beta-lactoglobulin at pH 2

BACKGROUND: NMR studies of denatured states, both fully unfolded and partially folded, give insight into the conformations and interactions formed during folding. Although the complete structural characterization of partially folded proteins is a very difficult task, the identification of structured subsets, such as hydrophobic clusters, is of value in understanding the structural organization of such states. Here, we report the NMR characterization, in acidic conditions (pH 2), of a well-defined hydrophobic cluster localized in the core of bovine beta-lactoglobulin. RESULTS: The existence of a small hydrophobic cluster present in the lipocalin protein family has been assessed on the basis of structural alignment and NRM data obtained for the partially folded bovine beta-lactoglobulin. The presence of the cluster had been predicted identifying those residues that are highly conserved in most members of the family. An NMR study conducted at pH 2, where the protein exhibits a very stable beta-core together with disordered regions, reveals the presence of NOEs among sidechains of 11 hydrophobic residues centered around Trp19 and pointing towards the interior of the protein. This buried cluster is found to be unusually stable at pH 2, not only at room temperature but also at 323K. Furthermore, conserved hydrophobic residues pointing towards the surface of the protein define a hydrophobic surface patch located in a groove between the strands and the helix. CONCLUSIONS: The detected buried cluster most likely plays an important role in bovine beta-lactoglobulin stability. The analysis of five structurally related proteins reveals that the same extended cluster is present in these structures. We propose that the buried cluster may represent the internal binding site as well and that the hydrophobic surface patch is involved in a second external binding site.     

A 3D model of the delta opioid receptor and ligand-receptor complexes

A model for the 3D structure of the transmembrane domain of the delta opioid receptor was predicted from the sequence divergence analysis of 42 sequences of G-protein coupled peptide hormone receptors belonging to the opioid, somatostatin and angiotensin receptor families. No template was used in the prediction steps, which include multiple sequence alignment, calculation of a variability profile of the aligned sequences, use of the variability profile to identify the boundaries of transmembrane regions, prediction of their secondary structure, optimization of the packing shape in a helix bundle, prediction of side chain conformations and structural refinement. The general shape of the model is similar to that of the low resolution rhodopsin structure in that the TM3 and TM7 helices are most buried in the bundle and the TM1 and TM4 helices are most exposed to the lipid phase. An initial assessment of this model was made by determining to what extent a binding site identified using four structurally disparate high affinity delta opioid ligands was consistent with known mutational studies. With the assumption that the protonated amine nitrogen, a feature common to all delta opioid ligands, interacts with the highly conserved Asp127 in TM3, a pocket was found that satisfied the criteria of complementarity to the requirements for receptor recognition for these four diverse ligands, two delta selective antagonists (the fused ring naltrindole and the peptide Tyr-Tic-Phe-Phe-NH2) and the two agonists lofentanil and BW373U86 deduced from previous studies of the ligands alone. These ligands could be accommodated in a similar region of the receptor. The receptor binding site identified in the optimized complexes contained many residues in positions known to affect ligand binding in G-protein coupled receptors. These results also allowed identification of key residues as candidates for point mutations for further assessment and refinement of this model as well as preliminary indications of the requirements for recognition of this receptor.     

Nonequilibrium analysis alters the mechanistic interpretation of inhibition of acetylcholinesterase by peripheral site ligands

The active site gorge of acetylcholinesterase (AChE) contains two sites of ligand binding, an acylation site near the base of the gorge with a catalytic triad characteristic of serine hydrolases, and a peripheral site at the mouth of the gorge some 10-20 A from the acylation site. Many ligands that bind exclusively to the peripheral site inhibit substrate hydrolysis at the acylation site, but the mechanistic interpretation of this inhibition has been unclear. Previous interpretations have been based on analyses of inhibition patterns obtained from steady-state kinetic models that assume equilibrium ligand binding. These analyses indicate that inhibitors bound to the peripheral site decrease acylation and deacylation rate constants and/or decrease substrate affinity at the acylation site by factors of up to 100. Conformational interactions have been proposed to account for such large inhibitory effects transmitted over the distance between the two sites, but site-specific mutagenesis has failed to reveal residues that mediate the proposed conformational linkage. Since examination of individual rate constants in the AChE catalytic pathway reveals that assumptions of equilibrium ligand binding cannot be justified, we introduce here an alternative nonequilibrium analysis of the steady-state inhibition patterns. This analysis incorporates a steric blockade hypothesis which assumes that the only effect of a bound peripheral site ligand is to decrease the association and dissociation rate constants for an acylation site ligand without altering the equilibrium constant for ligand binding to the acylation site. Simulations based on this nonequilibrium steric blockade model were in good agreement with experimental data for inhibition by the peripheral site ligands propidium and gallamine at low concentrations of either acetylthiocholine or phenyl acetate if binding of these ligands slows substrate association and dissociation rate constants by factors of 5-70. Direct measurements with the acylation site ligands huperzine A and m-(N,N, N-trimethylammonio)trifluoroacetophenone showed that bound propidium decreased the association rate constants 49- and 380-fold and the dissociation rate constants 10- and 60-fold, respectively, relative to the rate constants for these acylation site ligands with free AChE, in reasonable agreement with the nonequilibrium steric blockade model. We conclude that this model can account for the inhibition of AChE by small peripheral site ligands such as propidium without invoking any conformational interaction between the peripheral and acylation sites.     

Out-of-hospital quantitative monitoring of end-tidal carbon dioxide pressure during CPR

BACKGROUND: Human growth hormone (hGH) binds to both the hGH and human prolactin (hPRL) receptors. Binding to the hPRL receptor, however, is approximately 50-fold tighter and requires a single Zn2+ cation, unlike binding of hGH to the hGH receptor. Previous mutational studies have identified putative ligands from hGH and the hPRL receptor responsible for coordinating the interfacial Zn2+. RESULTS: One of these ligands was introduced at a structurally analogous site in the extracellular domain of the hGH receptor by mutating Asn218 to His, and the resulting mutant protein showed a 20-fold increase in hGH binding in the presence of ZnCl2. Alanine-scanning mutagenesis showed that the binding site on hGH for the Asn218-->His hGH receptor in the presence of Zn2+ resembled that for the hPRL receptor. CONCLUSIONS: It is possible to introduce the metal-binding site from the hPRL receptor into the homologous hGH receptor. More generally, these studies indicate that affinity between two proteins may be enhanced by design of an interfacial metal-binding site.     

Weak substrate binding to transport proteins studied by NMR

The weak binding of sugar substrates fails to induce any quantifiable physical changes in the L-fucose-H+ symport protein, FucP, from Escherichia coli, and this protein lacks any strongly binding ligands for competitive binding assays. Access to substrate binding behavior is however possible using NMR methods which rely on substrate immobiliza-tion for detection. Cross-polarization from proton to carbon spins could detect the portion of 13C-labeled substrate associated with 0.2 micromol of the functional transport system overexpressed in the native membranes. The detected substrate was shown to be in the FucP binding site because its signal was diminished by the unlabeled substrates L-fucose and L-galactose but was unaffected by a three- to fivefold molar excess of the non-transportable stereoisomer D-fucose. FucP appeared to bind both anomers of its substrates equally well. An NMR method, designed to measure the rate of substrate exchange, could show that substrate exchanged slowly with the carrier center (>10(-1) s), although its dynamics are not necessarily coupled strongly to this site within the protein. Relaxation measurements support this view that fluctuations in the interaction with substrate would be confined to the binding site in this transport system.     

Solution structure of nickel-peptide deformylase

In the accompanying paper, we report that zinc is unlikely to be the co-factor supporting peptide deformylase activity in vivo. In contrast, nickel binding promotes full enzyme activity. The three-dimensional structure of the resulting nickel-containing peptide deformylase (catalytic domain, residues 1 to 147) was solved by NMR using a 13C-15N-doubly labelled protein sample. A set of 2261 restraints could be collected, with an average of 15.4 per amino acid. The resolution, which shows a good definition for the position of most side-chains, is greatly improved compared to that previously reported for the zinc-containing, inactive form. A comparison of the two stuctures indicates however that both share the same 3D organization. This shows that the nature of the bound metal is the primary determinant of the hydrolytic activity of this enzyme. Site-directed mutagenesis enabled us to determine the conserved residues of PDF involved in the structure of the active site. In particular, a buried arginine appears to be critical for the positioning of Cys90, one of the metal ligands. Furthermore, the 3D structure of peptide deformylase was compared to thermolysin and metzincins. Although the structural folds are very different, they all display a common structural motif involving an alpha-helix and a three-stranded beta-sheet. These conserved structural elements build a common scaffold which includes the active site, suggesting a common hydrolytic mechanism for these proteases. Finally, an invariant glycine shared by both PDF and metzincins enables us to extend the conserved motif from HEXXH to HEXXHXXG.