Induced fit, drug design, and dUTPase.
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Animals↗
Biomedical subjects
C David Stout
Publications and source records attributed to C David Stout.
Structure of human microsomal cytochrome P450 2C8. Evidence for a peripheral fatty acid binding site.
A 2.7-Angstrom molecular structure of human microsomal cytochrome P450 2C8 (CYP2C8) was determined by x-ray crystallography. The membrane protein was modified for crystallization by replacement of the hydrophobic N-terminal transmembrane domain with a short hydrophilic sequence before residue 28. The structure of the native sequence is complete from residue 28 to the beginning of a C-terminal histidine tag used for purification. CYP2C8 is one of the principal hepatic drug-metabolizing enzymes that oxidizes therapeutic drugs such as taxol and cerivastatin and endobiotics such as retinoic acid and arachidonic acid. Consistent with the relatively large size of its preferred substrates, the active site volume is twice that observed for the structure of CYP2C5. The extended active site cavity is bounded by the beta1 sheet and helix F' that have not previously been implicated in substrate recognition by mammalian P450s. CYP2C8 crystallized as a symmetric dimer formed by the interaction of helices F, F', G', and G. Two molecules of palmitic acid are bound in the dimer interface. The dimer is observed in solution, and mass spectrometry confirmed the association of palmitic acid with the enzyme. This novel finding identifies a peripheral binding site in P450s that may contribute to drug-drug interactions in P450 metabolism.
Aryl Hydrocarbon Hydroxylases↗
Conformational change in the NADP(H) binding domain of transhydrogenase defines four states.
Proton-translocating transhydrogenase (TH) couples direct and stereospecific hydride transfer between NAD(H) and NADP(H), bound to soluble domains dI and dIII, respectively, to proton translocation across a membrane bound domain, dII. The reaction occurs with proton-gradient coupled conformational changes, which affect the energetics of substrate binding and interdomain interactions. The crystal structure of TH dIII from Rhodospirillum rubrum has been determined in the presence of NADPH (2.4 A) and NADP (2.1 A) (space group P6(1)22). Each structure has two molecules in the asymmetric unit, differing in the conformation of the NADP(H) binding loop D. In one molecule, loop D has an open conformation, with the B face of (dihydro)nicotinamide exposed to solvent. In the other molecule, loop D adopts a hitherto unobserved closed conformation, resulting in close interactions between NADP(H) and side chains of the highly conserved residues, betaSer405, betaPro406, and betaIle407. The conformational change shields the B face of (dihydro)nicotinamide from solvent, which would block hydride transfer in the intact enzyme. It also alters the environments of invariant residues betaHis346 and betaAsp393. However, there is little difference in either the open or the closed conformation upon change in oxidation state of nicotinamide, i.e., for NADP vs. NADPH. Consequently, the occurrence of two loop D conformations for both substrate oxidation states gives rise to four states: NADP-open, NADP-closed, NADPH-open, and NADPH-closed. Because these states are distinguished by protein conformation and by net charge they may be important in the proton translocating mechanism of intact TH.
Binding Sites↗
An open conformation of mammalian cytochrome P450 2B4 at 1.6-A resolution.
The xenobiotic metabolizing cytochromes P450 (P450s) are among the most versatile biological catalysts known, but knowledge of the structural basis for their broad substrate specificity has been limited. P450 2B4 has been frequently used as an experimental model for biochemical and biophysical studies of these membrane proteins. A 1.6-A crystal structure of P450 2B4 reveals a large open cleft that extends from the protein surface directly to the heme iron between the alpha-helical and beta-sheet domains without perturbing the overall P450 fold. This cleft is primarily formed by helices B' to C and F to G. The conformation of these regions is dramatically different from that of the other structurally defined mammalian P450, 2C5/3LVdH, in which the F to G and B' to C regions encapsulate one side of the active site to produce a closed form of the enzyme. The open conformation of 2B4 is trapped by reversible formation of a homodimer in which the residues between helices F and G of one molecule partially fill the open cleft of a symmetry-related molecule, and an intermolecular coordinate bond occurs between H226 and the heme iron. This dimer is observed both in solution and in the crystal. Differences between the structures of 2C5 and 2B4 suggest that defined regions of xenobiotic metabolizing P450s may adopt a substantial range of energetically accessible conformations without perturbing the overall fold. This conformational flexibility is likely to facilitate substrate access, metabolic versatility, and product egress.
Animals↗
Mechanisms of redox-coupled proton transfer in proteins: role of the proximal proline in reactions of the [3Fe-4S] cluster in Azotobacter vinelandii ferredoxin I.
The 7Fe ferredoxin from Azotobacter vinelandii (AvFdI) contains a [3Fe-4S](+/0) cluster that binds a single proton in its reduced level. Although the cluster is buried, and therefore inaccessible to solvent, proton transfer from solvent to the cluster is fast. The kinetics and energetics of the coupled electron-proton transfer reaction at the cluster have been analyzed in detail by protein-film voltammetry, to reveal that proton transfer is mediated by the mobile carboxylate of an adjacent surface residue, aspartate-15, the pK of which is sensitive to the charge on the cluster. This paper examines the role of a nearby proline residue, proline-50, in proton transfer and its coupling to electron transfer. In the P50A and P50G mutants, a water molecule has entered the cluster binding region; it is hydrogen bonded to the backbone amide of residue-50 and to the Asp-15 carboxylate, and it is approximately 4 A from the closest sulfur atom of the cluster. Despite the water molecule linking the cluster more directly to the solvent, proton transfer is not accelerated. A detailed analysis reveals that Asp-15 remains a central part of the mechanism. However, the electrostatic coupling between cluster and carboxylate is almost completely quenched, so that cluster reduction no longer induces such a favorable shift in the carboxylate pK, and protonation of the base no longer induces a significant shift in the pK of the cluster. The electrostatic coupling is crucial for maintaining the efficiency of proton transfer both to and from the cluster, over a range of pH values.
Amino Acid Substitution↗
Essential glycine in the proton channel of Escherichia coli transhydrogenase.
The nicotinamide nucleotide transhydrogenases of mitochondria and bacteria are proton pumps that couple hydride ion transfer between NAD(H) and NADP(H) bound, respectively, to extramembranous domains I and III, to proton translocation by the membrane-intercalated domain II. Previous experiments have established the involvement of three conserved domain II residues in the proton pumping function of the enzyme: His91, Ser139, and Asn222, located on helices 9, 10, and 13, respectively. Eight highly conserved domain II glycines in helices 9, 10, 13, and 14 were mutated to alanine, and the mutant enzymes were assayed for hydride transfer between domains I and III and for proton translocation by domain II. One of the glycines on helix 14, Gly252, was further mutated to Cys, Ser, Thr, and Val, expression levels of the mutant enzymes were evaluated, and each was purified and assayed. The results show that Gly252 is essential for function and support a model for the proton channel composed of helices 9, 10, 13, and 14. Gly252 would allow spatial proximity of His91, Ser139, and Asn222 for proton conductance within the channel. Gly252 mutants are distinguished by high levels of cyclic transhydrogenation activity in the absence of added NADP(H) and by complete loss of proton pumping activity. The purified G252A mutant has <1% proton translocation and reverse transhydrogenation activity, retains 0.9 mol of NADP(H) per domain III, and has 96% intrinsic cyclic transhydrogenation activity, which does not exceed 100% upon the addition of NADP(H). These properties imply that Gly252 mutants exhibit a native-like domain II conformation while blocking proton translocation and coupled exchange of NADP(H) in domain III.
Alanine↗
Structure of mammalian cytochrome P450 2C5 complexed with diclofenac at 2.1 A resolution: evidence for an induced fit model of substrate binding.
The structure of the anti-inflammatory drug diclofenac bound in the active site of rabbit microsomal cytochrome P450 2C5/3LVdH was determined by X-ray crystallography to 2.1 A resolution. P450 2C5/3LVdH and the related enzyme 2C5dH catalyze the 4'-hydroxylation of diclofenac with apparent K(m) values of 80 and 57 microM and k(cat) values of 13 and 16 min(-1), respectively. Spectrally determined binding constants are similar to the K(m) values. The structure indicates that the pi-electron system of the dichlorophenyl moiety faces the heme Fe with the 3'- and 4'-carbons located 4.4 and 4.7 A, respectively, from the Fe. The carboxyl moiety of the substrate is hydrogen bonded to a cluster of waters that are also hydrogen bonded to the side chains of N204, K241, S289, and D290 as well as the backbone of the protein. The proximity of the diclofenac carboxylate to the side chain of D290 together with an increased binding affinity at lower pH suggests that diclofenac is protonated when bound to the enzyme. The structure exhibits conformational changes indicative of an adaptive fit to the substrate reflecting both the hydration and size of the substrate. These results indicate how structurally diverse substrates are recognized by drug-metabolizing P450 enzymes.
Animals↗
A heterocyclic peptide nanotube.
An open-ended hollow tubular structure is designed based on hydrogen-bond-directed self-assembly of a chimeric cyclic peptide subunit comprised of alternating alpha- and epsilon-amino acids. The design features a novel 1,4-disubstituted-1,2,3-triazole epsilon-amino acid and its utility as a peptide backbone substitute. The N-Fmoc-protected epsilon-amino acid was synthesized in high yield and optical purity in three steps from readily available starting materials and was employed in solid-phase peptide synthesis to afford the desired cyclic peptide structure. The cyclic peptide self-assembly has been studied in solution by (1)H NMR and mass spectrometry and the resulting tubular ensemble characterized in the solid state by X-ray crystallography.
Amino Acids↗
High-resolution structure of the soluble, respiratory-type Rieske protein from Thermus thermophilus: analysis and comparison.
The structure of the soluble Rieske protein from Thermus thermophilus has been determined at a resolution of 1.3 A at pH 8.5 using multiwavelength anomalous dispersion (MAD) techniques. This is the first report of a Rieske protein from a menaquinone-utilizing organism. The structure shows an overall fold similar to previously reported Rieske proteins. A novel feature of this crystal form appears to be a shared hydrogen between the His-134 imidazole ring ligated to Fe2 of the [2Fe-2S] cluster and its symmetry partner, His-134', one being formally an imidazolate anion, Fe2-(His-134)N(epsilon)(-)...H-N(epsilon')(His-134')-Fe2', in which crystallographic C(2) axes pass equidistant between N(epsilon)...N(epsilon') and normal to the line defined by N(epsilon)...N(epsilon'). This provides evidence for a stable, oxidized cluster with a His(-) ligand and lends support to a previously proposed mechanism of coupled proton and electron transfer. A detailed comparison of the Thermus Rieske protein with six other Rieske and Rieske-type proteins indicates: (a) The cluster binding domain is tightly conserved. (b) The 3-D structure of the 10 beta-strand fold is conserved, even among the most divergent proteins. (c) There is an approximately linear relation between acid-pH redox potential and number of H-bonds to the cluster. (d) These proteins have two faces, one points into the larger complex (bc(1), b(6)f, or other), is involved in the proton coupled electron transfer function, and is highly conserved. The second is oriented toward the solvent and shows wide variation in charge, sequence, length, hydrophobicity, and secondary elements in the loops that connect the beta-sheets.
Amino Acid Sequence↗
Structure of a substrate complex of mammalian cytochrome P450 2C5 at 2.3 A resolution: evidence for multiple substrate binding modes.
The structure of rabbit microsomal cytochrome P450 2C5/3LVdH complexed with a substrate, 4-methyl-N-methyl-N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide (DMZ), was determined by X-ray crystallography to 2.3 A resolution. Substrate docking studies and electron density maps indicate that DMZ binds to the enzyme in two antiparallel orientations of the long axis of the substrate. One orientation places the principal site of hydroxylation, the 4-methyl group, 4.4 A from the heme Fe, whereas the alternate conformation positions the second, infrequent site of hydroxylation at >5.9 A from the heme Fe. Comparison of this structure to that obtained previously for the enzyme indicates that the protein closes around the substrate and prevents open access of water from bulk solvent to the heme Fe. This reflects a approximately 1.5 A movement of the F and G helices relative to helix I. The present structure provides a complete model for the protein from residues 27-488 and defines two new helices F' and G'. The G' helix is likely to contribute to interactions of the enzyme with membranes. The relatively large active site, as compared to the volume occupied by the substrate, and the flexibility of the enzyme are likely to underlie the capacity of drug-metabolizing enzymes to metabolize structurally diverse substrates of different sizes.
Crystallography, X-Ray↗
The circumsphere as a tool to assess distortion in [4Fe-4S] atom clusters.
The geometry proposition that "four points not in a plane describe one and only one sphere" provides a novel tool for analyzing protein-induced distortions in [4Fe-4S] clusters. A geometrically perfect reference structure comprises interlaced, regular tetrahedra of Fe, S, and S gamma atoms having T(d) symmetry. Three circumspheres are defined by the three sets of four atoms, the circumcenters of which are unique points within the cluster. The structure is thus re-defined by the positions of the circumcenters in xyz space and the r, theta, phi of each atom on its respective sphere. Analysis of 12 high-resolution structures of protein-bound and small molecule [4Fe-4S](SR)(4) clusters revealed: (a) the circumcenters are generally non-coincident by approximately 0.01 to approximately 0.06 A; (b) the Fe radius, r(Fe), is nominally independent of core oxidation state, having values between 1.66 to 1.69 A, whereas r(S) and r(SG), which have ranges of 2.18-2.24 A and 3.87-3.94 A, respectively, both increase by as much as approximately 3% upon reduction from the 3+ to the 1+ core valence; (c) deviation of some atoms from the theta, phi of a perfect tetrahedron can be large, approximately 10 degrees, and sets of atoms can show patterns of motion on their spheres that result from changes in Fe-S bond lengths. Density functional theory calculations suggest that the [4Fe-4S] core itself requires rather little energy to distort (approximately 2 kcal/mol), whereas significantly more energy is required to distort the Sgamma shell (~4 kcal/mol) to that of cluster I in Clostridium acidurici ferredoxin.
Algorithms↗
Inherent protein structural flexibility at the RNA-binding interface of L30e.
The Saccharomyces cerevisiae ribosomal protein L30 autoregulates its own expression by binding to a purine-rich internal loop in its pre-mRNA and mRNA. NMR studies of L30 and its RNA complex showed that both the internal loop of the RNA as well as a region of the protein become substantially more ordered upon binding. A crystal structure of a maltose binding protein (MBP)-L30 fusion protein with two copies in the asymmetric unit has been determined. The flexible RNA-binding region in the L30 copies has two distinct conformations, one resembles the RNA bound form solved by NMR and the other is unique. Structure prediction algorithms also had difficulty accurately predicting this region, which is consistent with conformational flexibility seen in the NMR and X-ray crystallography studies. Inherent conformational flexibility may be a hallmark of regions involved in intermolecular interactions.
Amino Acid Sequence↗
Combinatorial crystallization of an RNA-protein complex.
One of the most difficult steps in X-ray crystallography of a ribonucleoprotein (RNP) complex is obtaining crystals that diffract to high resolution. This paper describes a procedure for identifying the optimal lengths of the nucleic acid components that provide high-quality crystals of the RNP. Both strands of an RNA duplex were varied in a systematic manner to generate a large number of unique RNPs that were screened for crystallization behavior. As observed in the crystallization of other nucleic acids and their complexes, the exact length of the RNA chains was found to be critical in obtaining diffraction-quality crystals, even though the relative molecular weights of the protein and RNA components were approximately 50 and approximately 10 kDa, respectively. In particular, the helix-loop-helix structure in the mRNA for the Saccharomyces cerevisiae ribosomal protein L30, which functions as an autoregulatory element for L30 expression, was synthesized as two separate RNA chains of variable length (12-14 and 15-17 nucletides). Duplex formation of these RNAs formed the asymmetric, internal loop-binding site for L30. 16 such RNA duplexes, varying by +/-1 residue at the 5' or 3' end of either chain, were used to prepare 16 unique complexes with a maltose-binding protein-L30 fusion protein. The complexes were screened against 48 standard crystallization conditions in 2304 experiments, yielding 30 conditions with single crystals in the initial screen. The most promising of these is being used for structure determination.
Carrier Proteins↗
Crystal structures of transhydrogenase domain I with and without bound NADH.
Transhydrogenase (TH) is a dimeric integral membrane enzyme in mitochondria and prokaryotes that couples proton translocation across a membrane with hydride transfer between NAD(H) and NADP(H) in soluble domains. Crystal structures of the NAD(H) binding alpha1 subunit (domain I) of Rhodospirillum rubrum TH have been determined at 1.8 A resolution in the absence of dinucleotide and at 1.9 A resolution with NADH bound. Each structure contains two domain I dimers in the asymmetric unit (AB and CD); the dimers are intimately associated and related by noncrystallographic 2-fold axes. NADH binds to subunits A and D, consistent with the half-of-the-sites reactivity of the enzyme. The conformation of NADH in subunits A and D is very similar; the nicotinamide is in the anti conformation, the A-face is exposed to solvent, and both N7 and O7 participate in hydrogen bonds. Comparison of subunits A and D to six independent copies of the subunit without bound NADH reveals multiple conformations for residues and loops surrounding the NADH site, indicating flexibility for binding and release of the substrate (product). The NADH-bound structure is also compared to the structures of R. rubrum domain I with NAD bound (PDB code 1F8G) and with NAD bound in complex with domain III of TH (PDB code 1HZZ). The NADH- vs NAD-bound domain I structures reveal conformational differences in conserved residues in the NAD(H) binding site and in dinucleotide conformation that are correlated with the net charge, i.e., oxidation state, of the nicotinamides. The comparisons illustrate how nicotinamide oxidation state can affect the domain I conformation, which is relevant to the hydride transfer step of the overall reaction.
Animals↗
Fluorescent probes for cytochrome p450 structural characterization and inhibitor screening.
We have synthesized two luminescent probes (D-4-Ad and D-8-Ad) that target cytochrome P450cam. D-4-Ad luminescence is quenched by Förster energy transfer upon binding (Kd = 0.83 muM) but is restored when the probe is displaced from the active site by camphor. In contrast, D-8-Ad (Kd approximately 0.02 muM) is not displaced from the enzyme, even in the presence of a large excess of camphor. The 2.2 A resolution crystal structure of the D-8-Ad:P450cam complex reveals extensive hydrophobic contacts between the probe and the enzyme, which result from the conformational flexibility of the B', F, and G helices. Probes with properties similar to those of D-4-Ad potentially could be useful for screening P450 inhibitors.
Adamantane↗
The proton channel of the energy-transducing nicotinamide nucleotide transhydrogenase of Escherichia coli.
The nicotinamide nucleotide transhydrogenases of mitochondria and bacteria are proton pumps that couple direct hydride ion transfer between NAD(H) and NADP(H) bound, respectively, to extramembranous domains I and III to proton translocation by the membrane-intercalated domain II. To delineate the proton channel of the enzyme, 25 conserved and semiconserved prototropic amino acid residues of domain II of the Escherichia coli transhydrogenase were mutated, and the mutant enzymes were assayed for transhydrogenation from NADPH to an NAD analogue and for the coupled outward proton translocation. The results confirmed the previous findings of others and ourselves on the essential roles of three amino acid residues and identified another essential residue. Three of these amino acids, His-91, Ser-139, and Asn-222, occur in three separate membrane-spanning alpha helices of domain II of the beta subunit of the enzyme. Another residue, Asp-213, is probably located in a cytosolic-side loop that connects to the alpha helix bearing Asn-222. It is proposed that the three helices bearing His-91, Ser-139, and Asn-222 come together, possibly with another highly conserved alpha helix to form a four-helix bundle proton channel and that Asp-213 serves to conduct protons between the channel and domain III where NADPH binding energy is used via protein conformation change to initiate outward proton translocation.
Amino Acid Sequence↗
Purification and crystallization of N-terminally truncated forms of microsomal cytochrome P450 2C5.
Engineering more soluble forms of P450 2C5 has contributed to the crystallization of the enzyme. When detergents are used in both crystallization and purification of the protein, the ability to control the content and identity of the detergent is dependent on the protein exhibiting a sufficient degree of solubility to permit its concentration in the absence of detergents. The production of concentrated solutions of the protein containing little or no detergent provides a means for screening crystallization conditions and the selection of detergents that facilitate crystallization. These detergents can then be used not only to improve the purification of the protein, but also to solublize substrates for the cocrystallization of enzyme-substrate complexes.
Animals↗
Crystal structures of ferredoxin variants exhibiting large changes in [Fe-S] reduction potential.
Elucidating how proteins control the reduction potentials (E0') of [Fe--S] clusters is a longstanding fundamental problem in bioinorganic chemistry. Two site-directed variants of Azotobacter vinelandii ferredoxin I (FdI) that show large shifts in [Fe--S] cluster E0' (100--200 mV versus standard hydrogen electrode (SHE)) have been characterized. High resolution X-ray structures of F2H and F25H variants in their oxidized forms, and circular dichroism (CD) and electron paramagnetic resonance (EPR) of the reduced forms indicate that the overall structure is not affected by the mutations and reveal that there is no increase in solvent accessibility nor any reorientation of backbone amide dipoles or NH--S bonds. The structures, combined with detailed investigation of the variation of E0' with pH and temperature, show that the largest increases in E0' result from the introduction of positive charge due to protonation of the introduced His residues. The smaller (50--100 mV) increases observed for the neutral form are proposed to occur by directing a Hdelta+--Ndelta- dipole toward the reduced form of the cluster.
Azotobacter vinelandii↗