Crystallization and preliminary crystallographic characterization of the origin-binding domain of the bacteriophage lambda O replication initiator.

The bacteriophage lambda O protein binds to the lambda replication origin (orilambda) and serves as the primary replication initiator for the viral genome. The binding energy derived from the binding of O to orilambda is thought to help drive DNA opening to facilitate initiation of DNA replication. Detailed understanding of this process is severely limited by the lack of high-resolution structures of O protein or of any lambdoid phage-encoded paralogs either with or without DNA. The production of crystals of the origin-binding domain of lambda O that diffract to 2.5 A is reported. Anomalous dispersion methods will be used to solve this structure.

High apparent dielectric constants in the interior of a protein reflect water penetration.

A glutamic acid was buried in the hydrophobic core of staphylococcal nuclease by replacement of Val-66. Its pK(a) was measured with equilibrium thermodynamic methods. It was 4.3 units higher than the pK(a) of Glu in water. This increase was comparable to the DeltapK(a) of 4.9 units measured previously for a lysine buried at the same location. According to the Born formalism these DeltapK(a) are energetically equivalent to the transfer of a charged group from water to a medium of dielectric constant of 12. In contrast, the static dielectric constants of dry protein powders range from 2 to 4. In the crystallographic structure of the V66E mutant, a chain of water molecules was seen that hydrates the buried Glu-66 and links it with bulk solvent. The buried water molecules have never previously been detected in >20 structures of nuclease. The structure and the measured energetics constitute compelling and unprecedented experimental evidence that solvent penetration can contribute significantly to the high apparent polarizability inside proteins. To improve structure-based calculations of electrostatic effects with continuum methods, it will be necessary to learn to account quantitatively for the contributions by solvent penetration to dielectric effects in the protein interior.

Protein crystallization by design: chymotrypsinogen without precipitants.

Protein crystals are usually obtained by an empirical approach based on extensive screening to identify suitable crystallization conditions. In contrast, we have used a systematic predictive procedure to produce data-quality crystals of bovine chymotrypsinogen A and used them to obtain a refined X-ray structure to 3 A resolution. Measurements of the osmotic second virial coefficient of chymotrypsinogen solutions were used to identify suitable solvent conditions, following which crystals were grown for approximately 30 hours by ultracentrifugal crystallization, without the use of any precipitants. Existing structures of chymotrypsinogen were obtained in solutions including 10-30 % ethanol, whereas simple buffered NaCl solutions were used here. The protein crystallized in the tetragonal space group P4(1)2(1)2, with one molecule per asymmetric unit. The quality of the refined map was very high throughout, with the main-chain atoms of all but four residues clearly defined and with nearly all side-chains also defined. Although only minor differences are seen compared to the structures previously reported, they indicate the possibility of structural changes due to the crystallization conditions used in those studies. Our results show that more systematic crystallization of proteins is possible, and that the procedure can expand the range of conditions under which crystals can be grown successfully and can make new crystal forms available.

The duplication of an eight-residue helical stretch in Staphylococcal nuclease is not helical: a model for evolutionary change.

A common method of evolutionary change is gene duplication, followed by other events that lead to new function, decoration of folds, oligomerization, or other changes. As part of a study on the potential for evolutionary change created by duplicated sequences, we have carried out a crystallographic study on a mutant of Staphylococcal nuclease in which residues 55-62 have been duplicated in a wild-type variant termed PHS. In the parental protein (PHS) these residues form the first two turns of a helix running from residue 54 to 68 (hereafter designated as helix I). The crystal structure of the mutant is very similar to that of the parental, with helix I being unaltered. The duplicated residues are accommodated by expanding an existing loop N-terminal to helix I. In addition, circular dichroism (CD) studies have been carried out on a parental peptide containing helix I with six flanking residues at each terminus (residues 48-74) and on the same peptide expanded by the duplication, as a function of 2,2,2-trifluoroethanol (TFE) concentration. Each peptide possesses only modest helical propensity in solution. Our data, which is different from what was observed in T4 lysozyme, show that the conformation of the duplicated sequence is determined by a balance of sequential and longer-range effects. Thus duplicating sequence need not mean duplicating structure. Proteins 2000;40:465-472.

Crystal structure of a conserved ribosomal protein-RNA complex.

The structure of a highly conserved complex between a 58-nucleotide domain of large subunit ribosomal RNA and the RNA-binding domain of ribosomal protein L11 has been solved at 2.8 angstrom resolution. It reveals a precisely folded RNA structure that is stabilized by extensive tertiary contacts and contains an unusually large core of stacked bases. A bulge loop base from one hairpin of the RNA is intercalated into the distorted major groove of another helix; the protein locks this tertiary interaction into place by binding to the intercalated base from the minor groove side. This direct interaction with a key ribosomal RNA tertiary interaction suggests that part of the role of L11 is to stabilize an unusual RNA fold within the ribosome.

Solution structure of Delta 5-3-ketosteroid isomerase complexed with the steroid 19-nortestosterone hemisuccinate.

The solution structure of the ketosteroid isomerase homodimer complexed with the product analogue 19-nortestosterone hemisuccinate (19-NTHS) was solved by heteronuclear multidimensional NMR methods using 1647 distance restraints, 77 dihedral angle (phi) restraints, and 67 hydrogen bond restraints per monomer. The refined secondary structure of each subunit consists of three alpha-helices, eight beta-strands, four turns, and two beta-bulges. The beta-strands form a mixed beta-sheet. One of the five proline residues, Pro-39, is cis and begins a nonclassical turn. A self-consistent ensemble of 15 tertiary/quaternary structures of the enzyme dimer-steroid complex, with no distance violations greater than 0.35 A, was generated by simulated annealing and energy minimization with the program X-PLOR. The mean pairwise RMSD of the secondary structural elements was 0.63 A for the average subunit and 1.25 A for the dimer. Within each subunit, the three alpha-helices are packed onto the concave surface of the beta-sheet with a groove between them into which the steroid binds at a site defined by 14 intermolecular distances. In the productive complex, Tyr-14, from alpha-helix 1, approaches both Asp-99 and the 3-keto group of 19-NTHS while, from beta-strand 1, the carboxylate of Asp-38 approaches the beta-face of the steroid near C4 and C6, between which it transfers a proton during catalysis. Thus the solution structure of the isomerase-steroid complex can accommodate the catalytic diad mechanism in which Asp-99 donates a hydrogen bond to Tyr-14 which in turn is hydrogen bonded to the 3-oxygen of the steroid. While direct hydrogen bonding of Asp-99 to the steroid oxygen is less likely, it cannot be excluded. All other interactions of the steroid with the enzyme are hydrophobic. The dimer interface, which is between the convex surfaces of the beta-sheets, is defined by 28 intersubunit NOEs between hydrophobic residues in the 13C-filtered NOESY-HSQC spectrum of a 13C/12C-heterolabeled dimer. Both hydrophobic and polar interactions occur at the dimer interface which contains no space that would permit additional steroid binding. Comparison of the complexed enzyme with the solution structure of the free enzyme [Wu et al. (1997) Science 276, 415-418] reveals that the three helices change position in the steroid complex, becoming more closely packed onto the concave surface of the beta-sheet, thus bringing Tyr-14 closer to Asp-99 and the substrate. Comparison of the enzyme-steroid complex in solution with the free enzyme in the crystalline state reveals similar differences between the positions of the helices.

Hydrogen bonding at the active site of delta 5-3-ketosteroid isomerase.

The solution secondary structure of the highly active Y55F/Y88F "Tyr-14-only" mutant of delta 5-3-ketosteroid isomerase complexed with 19-nortestosterone hemisuccinate has been shown to consist of three helices, a six-stranded mixed beta-sheet, and five turns. The steroid binds near the general acid, Tyr-14, on helix 1, near the general base, Asp-38, on the first strand of the beta-sheet, and on the hydrophobic face of the beta-sheet [Zhao, Q., Abeygunawardana, C., & Mildvan, A. S. (1997) Biochemistry 36, 3458-3472]. On this hydrophobic face, Asp-99 is the only polar residue. Free isomerase shows a deshielded exchangeable proton resonance at 13.1 ppm assigned to the N epsilon H of neutral His-100. Its fractionation factor (phi = 0.79) and slow exchange with solvent suggest it to be buried or involved in an H-bond. The binding of dihydroequilenin or estradiol to isomerase induces the appearance of two additional deshielded proton resonances, one at 18.2 ppm assigned to the gamma-carboxyl proton of Asp-99, and the other, at 11.6 ppm, assigned to the zeta-OH proton of Tyr-14. While mutation of Asp-99 to Ala results in the disappearance of only the resonance near 18 ppm [Wu, R. W., Ebrahemian, S., Zwrotny, M. E., Thornberg, L. D., Perez-Alverado, G. C., Brothers, P., Pollack, R. M., & Summers, M. F. (1997) Science 276, 415-418], both of these resonances disappear in mutants lacking Tyr-14, suggesting an H-bonded catalytic diad, Asp-99-COOH--Tyr14-OH--O-steroid enolate. The catalytic diad is further supported by NOEs from the beta 1 and beta 2 protons of Asp-99 to the epsilon protons of Tyr-14, and from the zeta-OH proton of Tyr-14 to the gamma-carboxyl proton of Asp-99, indicating close proximity of these two residues, and by other data from the literature. A strong, low-barrier H-bond between Asp-99 and Tyr-14 is indicated by the 6.2 ppm deshielding, low fractionation factor (phi = 0.34) and slow exchange of the resonance at 18.2 ppm. A normal H-bond between Tyr-14 and the steroid is indicated by the 1.8 ppm deshielding, fractionation factor of 0.97 and the slow exchange of the resonance at 11.6 ppm. It is suggested that the 10(4.7)-fold contribution of Tyr-14 to catalysis is made possible by strong H-bonding from Asp-99 in the catalytic diad which strengthens general acid catalysis by Tyr-14. It is also noted that highly deshielded proton resonance on enzymes between 15 and 20 ppm, assigned to low-barrier H-bonds, generally involve carboxyl groups.

Crystal structure of the actin-binding protein actophorin from Acanthamoeba.

Actophorin is a member of the actin-depolymerizing factor/cofilin family. It severs actin filaments and sequesters actin monomers. The crystal structure of actophorin will help to elucidate actin-ADF/cofilin interactions.

Experimental measurement of the effective dielectric in the hydrophobic core of a protein.

The dielectric inside a protein is a key physical determinant of the magnitude of electrostatic interactions in proteins. We have measured this dielectric phenomenologically, in terms of the dielectric that needs to be used with the Born equation in order to reproduce the observed pKa shifts induced by burial of an ionizable group in the hydrophobic core of a protein. Mutants of staphylococcal nuclease with a buried lysine residue at position 66 were engineered for this purpose. The pKa values of buried lysines were measured by difference potentiometry. The extent of coupling between the pKa and the global stability of the protein was evaluated by measuring pKa values in hyperstable forms of nuclease engineered to be 3.3 or 6.5 kcal mol-1 more stable than the wild type. The crystallographic structure of one mutant was determined to describe the environment of the buried lysine. The dielectrics that were measured range from 10 to 12. Published pKa values of buried ionizable residues in other proteins were analyzed in a similar fashion and the dielectrics obtained from these values are consistent with the ones measured in nuclease. These results argue strongly against the prevalent use of dielectrics of 4 or lower to describe the dielectric effect inside a protein in structure-based calculations of electrostatic energies with continuum dielectric models.

Solution structure of the MutT enzyme, a nucleoside triphosphate pyrophosphohydrolase.

The MutT enzyme (129 residues) catalyzes the hydrolysis of normal and mutagenic nucleoside triphosphates, such as 8-oxo-dGTP, by substitution at the rarely attacked beta-P, to yield NMP and pyrophosphate. Previous heteronuclear NMR studies of MutT have shown the secondary structure to consist of a five-stranded mixed beta-sheet connected by the loop I-alpha-helix I--loop II motif, by two tight turns, and by loop III, and terminated by loop IV--alpha-helix II [Abeygunawardana et al. (1993) Biochemistry 32, 13071-13080; Weber et al. (1993) Biochemistry 32, 13081-13087). Complete side-chain assignments of 1H and 13C resonances have now been made by 3D C(CO)NH and HCCH-TOCSY experiments. A total of 1461 interproton proximities (11 per residue), obtained by 3D 15N-resolved NOESY-HSQC and 3D 13C-resolved NOESY-HSQC spectra, including 372 long-range NOEs, as well as 65 dihedral angle (phi) restraints and 34 backbone hydrogen bond restraints were used to determine the tertiary structure of MutT by distance geometry, simulated annealing, and energy minimization with the program X-PLOR. The structure is globular and compact with the parallel portion of the beta-sheet sandwiched between the two alpha-helices, forming an alpha+beta fold. The essential divalent cation has previously been shown to bind near residues Gly-37, Gly-38, Lys-39, and Glu-57, and nucleotides have been shown to bind near residues Leu-54 and Val-58 by NMR relaxation methods [Frick et al. (1995) Biochemistry 34, 5577-5586].(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal structure of the haemopexin-like C-terminal domain of gelatinase A.

The crystal structure of the haemopexin-like C-terminal domain of gelatinase A reveals that it is a four-bladed beta-propeller protein. The four blades are arranged around a channel-like opening in which Ca2+ and a Na-Cl+ ion pair are bound.

One-step evolution of a dimer from a monomeric protein.

Deletion of six amino acids in a surface loop transforms staphylococcal nuclease from a monomeric protein into a very stable dimer (Kd < 1 x 10(-8)M). A 2 A X-ray crystal structure of the dimer (R = 0.176) shows that the carboxy-terminal alpha-helix has been stripped from its normal position in one monomer and is now incorporated into the equivalent position on the adjoining monomer. This swapping creates an association interface of 2900 A 2. A second, smaller interface of 460 A 2 is also formed. The spontaneous exchange or swapping of secondary structural elements provides a simple pathway for the formation of large, stable protein/protein interfaces and may play an important role in the evolution of oligomeric proteins.

NMR structure of a stable "OB-fold" sub-domain isolated from staphylococcal nuclease.

Similar folds often occur in proteins with dissimilar sequences. The OB-fold forms a part of the structures of at least seven non-homologous proteins that share either oligonucleotide or oligosaccharide binding functions. A 1-103 fragment corresponding to the OB-fold of the 149 amino acid residue staphylococcal nuclease gives NMR spectra characteristic of an unfolded protein, i.e. the wild-type nuclease sequence is insufficient to maintain a stable tertiary structure in the absence of the C-terminal one-third of this single-domain protein. By contrast, the 1-103 fragment of nuclease with the mutations Val66Leu and Gly88Val adopts a stable tertiary structure. The NMR solution structure of this latter fragment is a close variation of the OB-fold found in the X-ray structure of the parent protein. The Val66Leu and Gly88Val mutations appear to stabilize tertiary structure by consolidating the hydrophobic core of the nuclease OB-fold sub-domain. Taken together, these results suggest that recurrent structural motifs such as the OB-fold may in some cases represent vestiges of autonomous folding units that, during evolution, have become integrated into more complex cooperative folding domains.

Solution structure and function in trifluoroethanol of PP-50, an ATP-binding peptide from F1ATPase.

PP-50, a synthetic peptide, based on residues 141-190 of the beta-subunit of mitochondrial F1ATPase, containing the GX4GKT consensus sequence for nucleoside triphosphate binding, binds ATP tightly (Kd = 17.5 microM) as found by fluorescence titration at pH 4.0. CD and 2D proton NMR studies at pH 4.0 revealed two beta-turns, regions of extended secondary structure, transient tertiary structure, and flexibility in the GX4GKT region (W.J. Chuang, C. Abeygunawardana, P. L. Pedersen, and A. S. Mildvan, 1992, Biochemistry 31, 7915-7921). CD titration of PP-50 with trifluoroethanol (TFE) reveals a decrease in ellipticity at 208 and 222 nm, saturating at 25% TFE. Computer analysis indicates that 25% TFE increases the helix content from 5.8 to 28.6%, decreases the beta-structure from 30.2 to 20.2% and decreases the coil content from 64 to 51.2%. Fluorescence titrations of H2ATP2- with PP-50 in 25% TFE yields a Kd of 7.3 microM, 2.4-fold tighter than in H2O, probably due to TFE increasing the activity of H2ATP2- . PP-50 completely quenches the fluorescence of H2ATP2- in 25% TFE, while in H2O the fluorescence quenching is only 62%. In H2O the binding of H2ATP2- increases the structure of PP-50 as detected by CD, but in 25% TFE no significant change in CD is found on binding either H2ATP2- or Mg2+ HATP (Kd = 14 microM). The complete proton NMR spectrum of PP-50 in 25% TFE has been assigned. The solution structure, determined by distance geometry, molecular dynamics with simulated annealing, and energy minimization, consists of a coil (residues 1-8), a strand (residues 9-12), a loop (residues 13-22) containing the GX4GKT consensus sequence (residues 16-23), an alpha-helix (residues 23-36), a turn (residues 38-41), and a coil (residues 42-50), similar to that of the corresponding region of the X-ray structure of F1ATPase (J.P. Abrahams, A.G.W. Leslie, R. Lutter, and J. E. Walker, 1994 Nature 370, 621-628) and to the structure of a homologous peptide from the ATP-binding site of adenylate kinase (D. C. Fry, D. M. Byler, H. Susi, E. M. Brown, S. A. Kuby, and A. S. Mildvan, 1988 Biochemistry 27, 3588-3598), beta, gamma-Bidentate Cr3+ ATP binds to PP-50 with the Cr3+ pyrophosphate moiety approaching the epsilon-methylene group of K22 in the GX4GKT consensus sequence, in agreement with the X-ray structure of the Mg2+ AMPPNP complex of F1ATPase.

NMR studies of the conformations and location of nucleotides bound to the Escherichia coli MutT enzyme.

The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates to nucleoside monophosphates and pyrophosphate by substitution at the rarely attacked beta-phosphorus. Nucleotides containing bulky substituents at the 8 position of the purine ring are preferentially hydrolyzed. The conformation of the MutT-bound nonhydrolyzable substrate analog Mg(2+)-AMPCPP, determined by 10 intramolecular NOEs and molecular dynamics refinement using a full relaxation matrix analysis with back-calculation of the NOESY intensities, is high anti (chi = 53 +/- 9 degrees), with a C2'-exo, O1'-endo sugar pucker. Similarly, the product of dGTP hydrolysis, dGMP, also binds MutT in a high anti (chi = 73 +/- 9 degrees) C1'-endo conformation based on seven intramolecular NOEs. Such high anti rotations of the base would allow MutT to accommodate nucleotides substituted at the C-8 position with no intramolecular clashes. Changes in chemical shifts in the 1H-15N spectra of the enzyme induced by Mg2+ and Mg2+ AMPCPP suggest that the metal activator and nucleotide interact with residues in loop I, at the carboxyl end of helix I, loop II, loop III, and beta-strands A and B of the secondary structure of MutT. The displacement of Mg2+ by Mn2+ causes the selective disappearance due to paramagnetic broadening of 1H-15N cross peaks from G37, G38, and K39 in loop I and E57 in helix I. Eleven intermolecular NOEs between Mg2+AMPCPP and hydrophobic residues of MutT are found, three of which are tentatively assigned to L67 in loop II and three to L54 in helix I. Similarly, seven intermolecular NOEs between dGMP and hydrophobic residues of the enzyme are found, four of which are tentatively assigned to L54 and two to V58, both in helix I. These interactions indicate that the loop I-helix I-loop II motif contributes significantly to the active site of MutT in accord with mutagenesis studies and with sequence homologies among MutT-like NTP pyrophosphohydrolases.

Crystal structures of the binary Ca2+ and pdTp complexes and the ternary complex of the Asp21-->Glu mutant of staphylococcal nuclease. Implications for catalysis and ligand binding.

The crystal structure of the Asp21-->Glu mutant (D21E) of staphylococcal nuclease (SNase) has been determined in three different complex forms. The structure of the D21E ternary complex in which D21E is bound to both Ca2+ and the transition-state analogue, thymidine 3',5'-diphosphate (pdTp), was determined to 1.95-A resolution. The structures of both binary complexes, D21E bound either to Ca2+ or pdTp, were determined to 2.15- and 2.05-A resolution, respectively. In the ternary structure, we find a 1.5-A movement of the Ca2+ in the active site, evidence of bidentate coordination of Ca2+ by Glu21 and inner-sphere coordination of the Ca2+ by Glu43. Comparison of the D21E binary structures with the ternary model shows large movements of active site side chains expected to play a direct role in catalysis. Glu43 moves in the binary nucleotide complex, whereas Arg35 is oriented differently in the binary metal complex. From these changes, we seek to explain the basis for the 1500-fold decrease in Vmax of D21E relative to wild-type SNase (WT). Furthermore, we describe direct structural evidence which explains the cooperativity of Ca2+ and pdTp binding in the ternary complex relative to that of the binary complexes.

NMR docking of a substrate into the X-ray structure of the Asp-21-->Glu mutant of staphylococcal nuclease.

To understand the structural basis of the 1500-fold decrease in catalytic activity of the D21E mutant of staphylococcal nuclease in which an aspartate ligand of the essential Ca2+ has been enlarged to glutamate, the conformation of the enzyme-bound substrate dTdA has been determined by NMR methods and has been docked into the X-ray structure of the D21E mutant (Libson, A. M., Gittis, A.G., & Lattman, E. E. Biochemistry, preceding paper in this issue) based on distances from the bound metal ion to dTdA and on intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of dTdA, using energy minimization to relieve small overlaps. Like the wild-type enzyme, the D21E mutant forms binary E-M and E-S and ternary E-M-S complexes with Ca2+, Mn2+, Co2+, and La3+. D21E enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 of the phosphorus and on 1/T1 of four proton resonances of dTdA, and these effects are abolished by the binding of the competitive inhibitor 3',5'-pdTp. From the paramagnetic effects of enzyme-bound Co2+ on 1/T1 of phosphorus and protons, with the use of a correlation time of 1.1 ps based on 1/T1 values at 250 and 600 MHz, five metal-nucleus distances and 11 lower limit metal-nucleus distances have been calculated. The Co2+ to 31P distance of 4.1 +/- 0.9 A agrees with that found on the wild-type enzyme (Weber, D. J., Mullen, G. P., & Mildvan, A. S. (1991) Biochemistry 30, 7425-7437) and indicates at least 18% inner sphere phosphate coordination. Fourteen interproton distances and 109 lower limit interproton distances in dTdA in the ternary D21E-La(3+)-dTdA complex were determined by NOESY spectra at 50-, 100-, and 200-ms mixing times. Both the metal-nucleus and interproton distances were necessary to compute a narrow range of conformations for enzyme-bound dTdA. As on the wild-type enzyme, the conformation of dTdA on the D21E mutant is highly extended, with high-anti C-2' endo conformations for the individual nucleosides. However, significant conformational differences are found in the torsional angles chi of dA (delta chi = 49 +/- 3 degrees), in gamma of dT (delta gamma = 108 +/- 30 degrees) and in zeta of dT (delta zeta = 124 +/- 38 degrees).(ABSTRACT TRUNCATED AT 400 WORDS)

Magnetic resonance studies of the binding of oligonucleotide substrates to mutants of staphylococcal nuclease.

By a combination of NMR docking and model building, the substrate binding site on staphylococcal nuclease was found to accommodate a trinucleotide and to consist of three subsites, each interacting with a single nucleotidyl unit of DNA. Binding of the essential Ca2+ activator and substrate cleavage occur between subsites 1 and 2. Hence, catalytically productive binding would span subsites 1 and 2 while nonproductive binding would span subsites 2 and 3. Lys-49 is near subsite 1, and Lys-84 and Tyr-115 interact with substrates at subsite 3 [Weber, D.J., Gittis, A.G., Mullen, G.P., Abeygunawardana, C., Lattman, E.E., Mildvan, A.S. Proteins 13:275-287, 1992]. The proposed locations of these subsites were independently tested by the effects of the K49A, K84A, and Y115A mutations of staphylococcal nuclease on the binding of Mn2+, Ca2+, and the dinucleotide and trinucleotide substrates, 5'-pdTdA, dTdA, and dTdAdG. These three mutants have previously been shown to be fully active and to have CD and 2D NMR spectra very similar to those of the wild-type enzyme (Chuang, W.-J., Weber, D.J., Gittis, A.G., Mildvan, A.S. Proteins 17:36-48, 1993). All three mutant enzymes and their pdTdA and dTdA complexes (but not their dTdAdG complex) bind Mn2+ and Ca2+ more weakly than the wild-type enzyme by factors ranging from 2 to 11. The presence of a terminal phosphate as in 5'-pdTdA raises the affinity of the substrate for staphylococcal nuclease and its three mutants by two orders of magnitude and for the corresponding enzyme-metal complexes by three to four orders of magnitude, suggesting that the terminal phosphate is coordinated by the enzyme-bound divalent cation. Such complexation would result in the nonproductive binding of 5'-pdTdA at subsites 2 and 3. Accordingly, the K84A and Y115A mutations significantly weaken the binding of 5'-pdTdA and its metal to staphylococcal nuclease by factors of 2.2 to 37.8, while the K49A mutation has much smaller or no effect. Such nonproductive binding explains the low activity of staphylococcal nuclease with small substrates, especially those with a terminal phosphate. Similarly, the K84A and Y115A mutations weaken the binding of dTdA and its metal complexes to the enzyme by factors of 3.4 to 13.1 while the K49A mutation has smaller effects indicating significant nonproductive binding of dTdA. The trinucleotide dTdAdG binds more tightly to wild-type and mutant staphylococcal nuclease and to its metal complexes than does the dinucleotide dTdA by factors of 2.4 to 12.2, reflecting the occupancy of an additional subsite.(ABSTRACT TRUNCATED AT 400 WORDS)

NMR docking of the competitive inhibitor thymidine 3',5'-diphosphate into the X-ray structure of staphylococcal nuclease.

In the X-ray structure of the ternary staphylococcal nuclease-Ca(2+)-3',5'-pdTp complex, the conformation of the bound inhibitor 3',5'-pdTp is distorted by Lys-70* and Lys-71* from an adjacent molecule of the enzyme in the crystal lattice (Loll, P. J. and Lattman, E. E. Proteins 5:183-201, 1989; Serpersu, E. H., Hibler, D. W., Gerlt, J. A., and Mildvan, A. S. Biochemistry 28:1539-1548, 1989). Since this interaction does not occur in solution, the NMR docking procedure has been used to correct this problem. Based on 8 Co(2+)-nucleus distances measured by paramagnetic effects on T1, and 9 measured and 45 lower limit interproton distances determined by 1D and 2D NOE studies of the ternary Ca2+ complex, the conformation of enzyme-bound 3',5'-pdTp is high-anti (chi = 58 +/- 10 degrees) with a C2' endo/O1' endo sugar pucker (delta = 143 +/- 2 degrees), (-) synclinal about the C3'-O3' bond (epsilon = 273 +/- 4 degrees), trans, gauche about the C4'-C5' bond (gamma = 301 +/- 29 degrees) and either (-) or (+) clinal about the C5'-O5' bond (beta = 92 +/- 8 degrees or 274 +/- 3 degrees). The structure of 3',5'-pdTp in the crystalline complex differs due to rotations about the C4'-C5' bond (gamma = 186 +/- 12 degrees, gauche, trans) and the C5'-O5' bond [beta = 136 +/- 10 degrees, (+) anticlinal]. The undistorted conformation of enzyme-bound metal-3',5'-pdTp determined by NMR was docked into the X-ray structure of the enzyme, using 19 intermolecular NOEs from ring proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of the inhibitor. van der Waals overlaps were then removed by energy minimization. Subsequent molecular dynamics and energy minimization produced no significant changes, indicating the structure to be in a global rather than in a local minimum. While the metal-coordinated 5'-phosphate of the NMR-docked structure of 3',5'-pdTp overlaps with that in the X-ray structure, and similarly receives bifunctional hydrogen bonds from both Arg-35 and Arg-87, the thymine, deoxyribose, and 3'-phosphate are significantly displaced from their positions in the X-ray structure, with the 3'-phosphate receiving hydrogen bonds from Lys-49 rather than from Lys-84 and Tyr-85. The repositioned thymine ring permits hydrogen bonding to the phenolic hydroxyl of Tyr-115.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutational tests of the NMR-docked structure of the staphylococcal nuclease-metal-3',5'-pdTp complex.

In the X-ray structure of the staphylococcal nuclease-Ca(2+)-3',5'-pdTp complex, the conformation of the inhibitor 3',5'-pdTp is distorted by Lys-70* and Lys-71* from an adjacent molecule of staphylococcal nuclease (Loll, P.J., Lattman, E.E. Proteins 5:183-201, 1989). In order to correct this crystal packing problem, the solution conformation of enzyme-bound 3',5'-pdTp in the staphylococcal nuclease-metal-pdTp complex determined by NMR methods was docked into the X-ray structure of the enzyme [Weber, D.J., Serpersu, E.H., Gittis, A.G., Lattman, E.E., Mildvan, A.S. (preceding paper)]. In the NMR-docked structure, the 5'-phosphate of 3',5'-pdTp overlaps with that in the X-ray structure. However, the 3'-phosphate accepts a hydrogen bond from Lys-49 (2.89 A) rather than from Lys-84 (8.63 A), and N3 of thymine donates a hydrogen bond to the OH of Tyr-115 (3.16 A) which does not occur in the X-ray structure (5.28 A). These interactions have been tested by binding studies of 3',5'-pdTp, Ca2+, and Mn2+ to the K49A, K84A, and Y115A mutants of staphylococcal nuclease using water proton relaxation rate and EPR methods. Each mutant was fully active and structurally intact, as found by CD and two-dimensional NMR spectroscopy, but bound Ca2+ 9.1- to 9.9-fold more weakly than the wild-type enzyme. While the K84A mutation did not significantly weaken 3',5'-pdTp binding to the enzyme (1.5 +/- 0.7 fold), the K49A mutation weakened 3',5'-pdTp binding to the enzyme by the factor of 4.4 +/- 1.8-fold. Similarly, the Y115A mutation weakened 3',5'-pdTp binding to the enzyme 3.6 +/- 1.6-fold. Comparable weakening effects of these mutations were found on the binding of Ca(2+)-3',5'-pdTp. These results are more readily explained by the NMR-docked structure of staphylococcal nuclease-metal-3',5'-pdTp than by the X-ray structure.