Biomedical subjects

M Sundaralingam

Publications and source records attributed to M Sundaralingam.

At least 73 records · Page 4Linked to original sources

PubMed1994

Crystal structure of the highly distorted chimeric decamer r(C)d(CGGCGCCG)r(G).spermine complex--spermine binding to phosphate only and minor groove tertiary base-pairing.

The crystal structure of the self-complementary chimeric decamer duplex r(C)d(CGGCGCCG)r(G), with RNA base pairs at both termini, has been solved at 1.9 A resolution by the molecular replacement method and refined to an R value of 0.145 for 2,314 reflections. The C3'-endo sugar puckers of the terminal riboses apparently drive the entire chimeric duplex into an A-DNA conformation, in contrast to the B-DNA conformation adopted by the all-deoxy decamer of the same sequence. Five symmetry related duplexes encapsulate a spermine molecule which interacts with ten phosphate groups, both directly and through water molecules to form multiple ionic and hydrogen bonding interactions. The spermine interaction severely bends the duplexes by 31 degrees into the major groove at the fourth base pair G(4).C(17), jolts it and slides the 'base plate' into the minor groove. This base pair, together with the adjacent base pair in the top half and the corresponding pseudo two-fold related base pairs in the bottom half, form four minor groove base-paired multiples with the terminal base pairs of two neighboring duplexes.

Base Composition↗

PubMed1994

A single 2'-hydroxyl group converts B-DNA to A-DNA. Crystal structure of the DNA-RNA chimeric decamer duplex d(CCGGC)r(G)d(CCGG) with a novel intermolecular G-C base-paired quadruplet.

We have found that the introduction of a single 2'-hydroxyl group on the sugar-phosphate backbone of the B-DNA decamer d(CCGGCGCCGG) transforms it to A-DNA. Thus, for the first time the X-ray structures of the same sequence have been observed in both the A and B-DNA conformations, permitting a comparison. Crystals of the DNA-RNA chimeric decamer d(CCGGC)r(G)d(CCGG) belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 25.63 A, b = 45.24 A and c = 47.99 A, and one decamer duplex in the asymmetric unit. The structure was solved by a rigid body search using the coordinates of the isomorphous structure d(CCCGGCCGGG) and refined to an R value of 0.136 using 2753 unique reflections at 1.9 A resolution. The final model contains 406 nucleotide atoms and 61 water molecules. The chimeric duplex exhibits typical A-DNA geometry, with all the sugars in the C(3')-endo puckering and the base-pairs inclined and displaced from the helix axis. The 2'-hydroxyl groups on rG6 and rG16 protrude into the minor groove surface and form different types of hydrogen bonds; that on strand 1 forms an intermolecular hydrogen bond with the furanose ring O(4') of a symmetry-related C1 residue, while that on strand 2 is involved in two water bridges. Crystal packing forces the G4-G17 base-pair in the top half of the duplex to slide significantly into the minor groove compared to the corresponding G7-G14 base-pair in the bottom half, resulting in these base-pairs exhibiting different base stacking and intermolecular interactions. The base G4 of the G4-G17 base-pair forms an unorthodox base "triple", G4*(G10-C11), hydrogen-bonding through its minor groove sites N(2) and N(3) to the minor groove atoms N(2) and O(2) of both bases of the G10-C11 base-pair of a symmetry-related molecule. The base G10 of this triple in turn forms a second similar unorthodox base triple, G10*(G3*C18), with the adjacent base-pair G3-C18 of the duplex, thus G10 is involved in a double triple. On the other hand, in the bottom half of the duplex, the C7-G14 base-pair is involved only in a single similar unorthodox base triple with G20, (C7-G14)*G20, while the adjacent base-pair rG6-C15 is involved in a novel quadruple with C1-G20, (rG6-C15) *(C1-G20), where the latter base-pairs are hydrogen-bonded to each other via the minor groove sites G(N(2))...C(O(2)).(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗

PubMed1994

Structure of chicken skeletal muscle troponin C at 1.78 A resolution.

The structure of chicken skeletal muscle troponin C (TnC) has been refined to an R value of 0.168, using 14 788 reflections, in the resolution range 8.0-1.78 A. Our earlier 2 A resolution structure [Satyshur, Rao, Pyzalska, Drendel, Greaser & Sundaralingam (1988). J. Biol. Chem. 263, 1628-1647] served as the starting model. The refined model includes atoms for all protein residues (1-162), 2 Ca(2+) ions, 169 water molecules and one sulfate ion. The high-resolution refinement shows more clearly the details of the protein and water structure. The side chains Glu63, Cysl01, Arg123, Aspl40 and Asp152 adopt two discretely ordered conformations. The long central helix is only slightly curved/bent (7.9 degrees ) and all the central helix NH.O=C hydrogen bonds are intact. Seven of the nine carbonyl O atoms of the mid segment of this helix, including the D/E linker region, are hydrogen bonded to water molecules which weakens the helix hydrogen bonds. In contrast, in each of the protected upper and lower thirds of the long central helix, only two carbonyl O atoms are hydrogen bonded to water molecules. The hydrogen-bonding patterns displayed by some of the carbonyl O atoms of NT and A helices of the N-terminal domain and the F and H helices of the C-terminal domain, which are on the exposed surface of the protein, are similar. The B helix of the calcium-free site I is kinked, with the local helix axes at either end making an angle of 39 degrees, by two inserted water molecules between N-H and O=C groups, breaking the adjacent helix hydrogen bonds. A sulfate ion from the crystallization buffer is also trapped in the B helix between the guanidinium group of Arg47 and these two inserted water molecules. The C helix of site II is devoid of similar hydration and is probably responsible for the different interhelical angles A/B at site I (134 degrees ) and C/D at site II (149 degrees ). Extensive interhelix hydrogen bonds occur between the side chains of the C and D helices of the 'apo' site II: Gln51-Asp89, Asn52-Asp89, Glu57-Gln85, Glu57-Glu88 and Glu64-Arg84, which apparently are disrupted upon Ca uptake and the resulting rearrangement of the helices expose the side chains, lining the palm of the N-(and C-) terminal domains, for interaction with specific peptide fragment of troponin I (Tnl) during muscle contraction. The dominant crystal packing motif involves a head-to-tail interaction between the N-terminal domain A helix of one molecule and the palm of the C-terminal domain of the 3(2)-related molecule, in a manner similar to that which can be expected for the TnC-TnI complex. Similar interactions may also be responsible for the dimerization of TnC at low pH.

Journal Article↗

PubMed1994

Structure of the recombinant Paramecium tetraurelia calmodulin at 1.68 A resolution.

The crystal structure of the recombinant calmodulin from Paramecium tetraurelia (rPCaM, M(r) = 16 700, 148 residues) has been determined at 1.68 A resolution. X-ray intensity data were collected at 263 K using a Siemens-Nicolet area detector and Cu Kalpha radiation from a rotating-anode source. A total of 35 936 observations were processed with XENGEN1.3 and scaled to yield 16 255 unique reflections with R(symm)(I) of 4.1%. The crystals are triclinic, with unit-cell dimensions a = 29.89, b = 53.42, c = 25.35 A, alpha = 93.67, beta = 96.88, gamma = 89.24 degrees, space group P1, with one molecule in the unit cell. The atomic coordinates of the wild-type Paramecium calmodulin (PCaM) studied in our laboratory provided the starting model. Refinement of the structure by X-PLOR and refitting it into omit maps yielded an R value of 0.194 for 15 965 reflections greater than 3sigma(F) in the 6.0-1.68 A resolution range. The final model contained 1165 protein atoms for all of the 148 residues, four Ca(2+) ions, and 172 water molecules. The dumbbell structure has seven alpha-helices including a long 7.8 turn central helix connecting the two terminal domains each containing two EF-hand (helix-loop-helix motif) calcium-binding sites. The loops within each pair of EF-hand motifs in the N- and C-terminal domains are brought into juxtaposition to form a pair of hydrogen-bonded antiparallel beta-sheets which are extended at either ends by water bridges. The four calcium-binding EF-hands are superposable with r.m.s. deviations of 0.31-0.79 A. The best agreement is between site 1 and site 3 and the worst agreement is between site 1 and 4. The largest differences are in the ninth and tenth residues of the calcium-binding loops probably because of their involvement in the mini beta-sheets. The calcium coordination distances vary between 2.04 and 2.69 A, average 2.34 A. The rPCaM and wild-type PCaM have an r.m.s. deviation of 0.36 A for equivalent C(alpha) atoms. The side chains of Lys13 and Lys115 are more extended in rPCaM compared to the wild type where the post-translational modified di- and tri-methylated lysine residues are more folded. The sequence of PCaM differs from those of mammalian (MCaM) and Drosophila calmodulin (DCaM), but the overall structures are very similar, with r.m.s,. deviations of 0.44 and 1.68 A for equivalent C(alpha) atoms, respectively. However, in rPCaM, the first four N-terminal residues stretch out and make intermolecular crystal contacts, in contrast to those in recombinant Drosophila calmodulin (rDCaM), they stretch out in the opposite direction and towards the second calcium-binding site (see note below), while in MCaM and wild-type PCaM, the N-terminal residues are not visible. The central helix in rPCaM has all its backbone hydrogen bonds intact with no unusually long separation between the carbonyl and amide groups as found in MCaM and rDCaM.

Journal Article↗

PubMed1994

Structure and function of the catalytic site mutant Asp 99 Asn of phospholipase A2: absence of the conserved structural water.

To probe the role of the Asp-99 ... His-48 pair in phospholipase A2 (PLA2) catalysis, the X-ray structure and kinetic characterization of the mutant Asp-99-->Asn-99 (D99N) of bovine pancreatic PLA2 was undertaken. Crystals of D99N belong to the trigonal space group P3(1)21 and were isomorphous to the wild type (WT) (Noel JP et al., 1991, Biochemistry 30:11801-11811). The 1.9-A X-ray structure of the mutant showed that the carbonyl group of Asn-99 side chain is hydrogen bonded to His-48 in the same way as that of Asp-99 in the WT, thus retaining the tautomeric form of His-48 and the function of the enzyme. The NH2 group of Asn-99 points away from His-48. In contrast, in the D102N mutant of the protease enzyme trypsin, the NH2 group of Asn-102 is hydrogen bonded to His-57 resulting in the inactive tautomeric form and hence the loss of enzymatic activity. Although the geometry of the catalytic triad in the PLA2 mutant remains the same as in the WT, we were surprised that the conserved structural water, linking the catalytic site with the ammonium group of Ala-1 of the interfacial site, was ejected by the proximity of the NH2 group of Asn-99. The NH2 group now forms a direct hydrogen bond with the carbonyl group of Ala-1.

Animals↗

PubMed1994

Binding of two distamycin A molecules in the minor groove of an alternating B-DNA duplex.

Here we report the 1.8A X-ray structure of a 2:1 drug-DNA complex between distamycin A and an alternating B-DNA octamer duplex d(ICICICIC)2. The two distamycin A molecules are bound side by side with dyad symmetry in an antiparallel orientation in the expanded minor groove. The amides of each drug molecule are hydrogen bonded to the minor groove base atoms of only one DNA strand. The complex not only shows binding of two drug molecules, but the DNA duplex also exhibits striking low-high alternations in the helical twist angles, the sugar puckering and the phosphate conformations, providing the basis for a new model for an alternating B-DNA with a dinucleotide repeat.

Base Sequence↗

PubMed1993

Evidence for crystal environment dominating base sequence effects on DNA conformation: crystal structures of the orthorhombic and hexagonal polymorphs of the A-DNA decamer d(GCGGGCCCGC) and comparison with their isomorphous crystal structures.

We have determined the structure of the A-DNA decamer d(GCGGGCCCGC) in two crystal forms, orthorhombic and hexagonal, at 1.7- and 1.8-A resolution, respectively. In the orthorhombic form, the fifth guanine residue has nearly trans-trans conformations for the alpha-gamma backbone torsions, as in the isomorphous orthorhombic structure d(CCCGGCCGGG) [Ramakrishnan, B., & Sundaralingam, M. (1993) J. Mol. Biol. 231, 431-444]. However, in the hexagonal form, the eighth cytosine residue adopts the trans-trans conformations for the backbone alpha-gamma torsions, as in the isomorphous hexagonal structure d(ACCGGCCGGT) [Frederick, C. A., Quigley, G. J., Teng, M.-K., Coll, M., van der Marel, G. A., van Boom, J. H., Rich, A., & Wang, A. H.-J. (1989) Eur. J. Biochem. 181, 295-307]. Even though the average helix and base-pair parameters are nearly the same in the two polymorphous crystal forms having the same sequence, many of the base-dependent local helix parameters are quite different. However, in the isomorphous crystal forms, in spite of the differing base sequences, the local helix and base-pair parameters of the duplexes are nearly the same. This indicates that, in crystals, the local conformation of a DNA structure is affected severely by the crystal packing environment rather than by the base sequence.

Base Sequence↗

PubMed1993

High resolution crystal structure of the A-DNA decamer d(CCCGGCCGGG). Novel intermolecular base-paired G*(G.C) triplets.

The DNA decamer d(CCCGGCCGGG) crystallizes in the orthorhombic space group P2(1)2(1)2(1) with a = 24.88, b = 44.60 and c = 46.97 A containing a duplex in the asymmetric unit. The structure was solved by molecular replacement and refined to an R factor of 18.5% using 6033 reflections at 1.65 A resolution. The decamer duplex adopts an A-DNA conformation. The abrupt dislocation of the duplex at the fourth base-pair G(4).C(17) by an abutting symmetry related molecule results in distortion of the backbone bonds of the fifth residue G(5), P-O(5')(alpha) and C(4')-C(5')(gamma), to the trans conformations from their favored gauche- and gauche+ conformations, respectively. In this close encounter the terminal G(10).C(11) base-pair of the symmetry related molecule hydrogen bonds to the G(4).C(17) base-pair forming a novel base-paired G(4)*(G10).C(11)) triplet, where G(4) is hydrogen bonded to both G(10) and C(11). To facilitate this hydrogen bonding the G(4).C(17) base-pair slides into the minor groove, causing a toll on the backbone conformation of the adjacent residue G(5). A similar triplet base-pairing interaction with somewhat weaker hydrogen bonds occurs at the pseudo dyad related C(7).G(14) base-pair with G(20) of another symmetry related duplex. This pseudo triplet interaction (C(7).G(14))*G(20), does not perturb the backgone alpha and gamma torsions of G(15). Both the novel base triplets are non-planar. The abrupt dislocation/bend at the G(4).C(17) base-pair jolts the global helical base-pair parameters, inclination, tilt, roll, tip, etc. quite markedly. Therefore a better description of the helix parameters is obtained by splitting the duplex and calculating the local helix axis for the top half consisting of the first three base-pairs, and the lower half consisting of the last six base-pairs, omitting the fourth base-pair. The two half duplexes are bent by only 10 degrees. This structure further demonstrates that crystal packing interactions, which can also be governed by base sequence, play a dominant role in determining DNA conformation.

Base Composition↗

PubMed1993

High-resolution refinement of the hexagonal A-DNA octamer d(GTGTACAC) at 1.4 A.

The hexagonal crystal form of the octamer d(GTGTACAC), grown in the presence of spermine, has unit-cell dimensions a = b = 32.18 and c = 78.51 A, space group P6(1)22, with one DNA strand in the asymmetric unit. The structure has been refined starting with the earlier lower resolution model and using high-resolution 1.4 A data collected on a Siemens-Xentronics area detector at 258 K. There were 4365 unique reflections greater than 2sigma(F) in the resolution range 5-1.4 A. The model was refitted into 3F(o) - 2F(c). Sim-weighted omit maps and difference maps were used to locate water molecules. The final model with 161 DNA atoms and 37 water molecules gave an R factor of 19.8%. Crystals of the same octamer were also grown in the presence of spermidine instead of spermine, and refinement using nominal 1.45 A resolution data, 3292 unique reflections, final R = 19.1%, gave virtually identical DNA parameters. No bound spermine or spermidine was detected in either of these structure analyses. The electron density was clear for the DNA and showed holes in the center of the six-membered rings of bases, and also in the center of some of the sugar rings. The high-resolution structure has provided more precise DNA parameters and confirmed the features observed in the earlier 2 A study including the packing-induced distortion in the A7 (A15) sugar pucker from C(3')-endo and C(2')-endo. This change causes the end base pairs to bend away from the helix axis while the rest of the duplex is nearly linear. The hydration patterns in the deep and shallow grooves have been characterized. Chains of water molecules were found, but no rings. The familiar intermolecular contact region between the end base pair and the minor groove of a symmetry-related duplex, involving four residues on one strand and two on the other, has been analyzed. One of these interactions is a hydrogen bond.

Journal Article↗

PubMed1993

Structure of Paramecium tetraurelia calmodulin at 1.8 A resolution.

The crystal structure of calmodulin (CaM; M(r) 16,700, 148 residues) from the ciliated protozoan Paramecium tetraurelia (PCaM) has been determined and refined using 1.8 A resolution area detector data. The crystals are triclinic, space group P1, a = 29.66, b = 53.79, c = 25.49 A, alpha = 92.84, beta = 97.02, and gamma = 88.54 degrees with one molecule in the unit cell. Crystals of the mammalian CaM (MCaM; Babu et al., 1988) and Drosophila CaM (DCaM; Taylor et al., 1991) also belong to the same space group with very similar cell dimensions. All three CaMs have 148 residues, but there are 17 sequence changes between PCaM and MCaM and 16 changes between PCaM and DCaM. The initial difference in the molecular orientation between the PCaM and MCaM crystals was approximately 7 degrees as determined by the rotation function. The reoriented Paramecium model was extensively refitted using omit maps and refined using XPLOR. The R-value for 11,458 reflections with F > 3 sigma is 0.21, and the model consists of protein atoms for residues 4-147, 4 calcium ions, and 71 solvent molecules. The root mean square (rms) deviations in the bond lengths and bond angles in the model from ideal values are 0.016 A and 3 degrees, respectively. The molecular orientation of the final PCaM model differs from MCaM by only 1.7 degrees. The overall Paramecium CaM structure is very similar to the other calmodulin structures with a seven-turn long central helix connecting the two terminal domains, each containing two Ca-binding EF-hand motifs. The rms deviation in the backbone N, Ca, C, and O atoms between PCaM and MCaM is 0.52 A and between PCaM and DCaM is 0.85 A. The long central helix regions differ, where the B-factors are also high, particularly in PCaM and MCaM. Unlike the MCaM structure, with one kink at D80 in the middle of the linker region, and the DCaM structure, with two kinks at K75 and I85, in our PCaM structure there are no kinks in the helix; the distortion appears to be more gradually distributed over the entire helical region, which is bent with an apparent radius of curvature of 74.5(2) A. The different distortions in the central helical region probably arise from its inherent mobility.

Amino Acid Sequence↗

PubMed1993

A model for the calmodulin-peptide complex based on the troponin C crystal packing and its similarity to the NMR structure of the calmodulin-myosin light chain kinase peptide complex.

In the crystal structure of troponin C, the holo C-domain is bound in a head-to-tail fashion to the A-helix of the apo N-domain of a symmetry-related molecule. Using this interaction, we have proposed a model for the calmodulin-peptide complex. We find that the interaction of the C-domain with the A-helix is similar to that observed in the NMR structure of the calmodulin-myosin light chain kinase (MLCK) peptide complex. This similarity in binding has enabled us to make a precise sequence alignment of the target peptides in the calmodulin-binding cleft and to rationalize the amino acid sequence-dependent binding strengths of various peptides. Our model differs from that proposed by Strynadka and James (Proteins Struct. Funct. Genet. 7, 234-248, 1990) in that the peptides are rotated by 100 degrees in the calmodulin binding cleft.

Amino Acid Sequence↗

PubMed1993

Crystal packing effects on A-DNA helix parameters: a comparative study of the isoforms of the tetragonal & hexagonal family of octamers with differing base sequences.

The helix and base pair parameters of A-DNA octamers have been compared having different base sequences both in the tetragonal and hexagonal crystal systems. For the eight structures in the tetragonal family, the twist, rise, slide, inclination and tilt are essentially the same, influenced by the similarity in crystal packing. The propeller twist and the base pair buckle display small sequence dependent variations. But the base pair roll appears to be changed by the specific intermolecular hydrogen bonding interactions. For four of the five octamer structures in the hexagonal family, the base pair rise, slide, inclination, tilt, as well as the propeller twist and buckle are all very similar, while the twist angle and the base pair roll are not. The intermolecular hydrogen bonding interactions seem to be primarily responsible for the differences in the roll angle in the tetragonal structures but for both the roll and twist angles in the hexagonal structures. These results demonstrate that the majority of the observed helix base pair parameters for A-DNA octamers in crystals are affected by the crystal packing environment, while a few parameters, like propeller twist and base pair buckle display some base sequence dependence.

Base Composition↗

PubMed1993

Molecular dynamics investigations of DNA triple helical models: unique features of the Watson-Crick duplex.

We have built computer models of triple helical structures with a third poly(dT) strand Hoogsteen base paired to the major groove of a poly(dA).poly(dT) Watson-Crick (WC) base-paired duplex in the canonical A-DNA as well as B-DNA. For the A-DNA form, the sugar-phosphate backbone of the third strand intertwines and clashes with the poly(dA) strand requiring a radical alteration of the duplex to access the hydrogen bonding sites in the major groove. In contrast, when the duplex was in the canonical B-DNA form, the third strand was readily accommodated in the major groove without perturbing the duplex. The triple helical model, with the duplex in the B-DNA form, was equilibrated for 400ps using molecular dynamics simulations including water molecules and counter-ions. During the entire simulations, the deoxyriboses of the adenine strand oscillate between the S-type and E-type conformations. However, 30% of the sugars of the thymine strands-II & III switch to the N-type conformation early in the simulations but return to the S-type conformation after 200ps. In the equilibrium structure, the WC duplex portion of the triplex is unique and its geometry differs from both the A- or B-DNA. the deoxyriboses of the three strands predominantly exhibit S-type conformation. Besides the sugar pucker, the major groove width and the base-tilt are analogous to B-DNA, while the X-displacement and helical twist resemble A-DNA, giving a unique structure to the triplex and the Watson & Crick and Hoogsteen duplexes.

Computer Simulation↗

PubMed1992

Crystal and molecular structure of d(GTGCGCAC): investigation of the effects of base sequence on the conformation of octamer duplexes.

The structure of the self-complementary deoxyoctanucleotide d(GTGCGCAC), which crystallized as an A-type helix in the space group P4(3)2(1)2, with one strand in the crystallographic asymmetric unit has been determined and refined to a final R-value of 0.154 using 1.64-A diffraction data collected on an area detector. In contrast to the closely related sequence d(GTGTACAC)tet, there was no evidence for an ordered spermine molecule in the major groove of this octamer. Ordered water is found associated with almost all the exposed hydrogen bonding groups of the octamer. A pentagonal ring of water molecules is hydrogen bonded to O6 and N7 of G3 and the N4 and O6 of the C4.G13 base pair. A detailed comparison of the local helical parameters of d(GTGCGCAC) and d(GTGTACAC)tet is presented. The base sequence change at the center of the octamers affects several of the local helical parameters, via both intra- and interduplex interactions within the crystal.

Base Composition↗

PubMed1992

Crystal and molecular structure of the alternating dodecamer d(GCGTACGTACGC) in the A-DNA form: comparison with the isomorphous non-alternating dodecamer d(CCGTACGTACGG).

The crystal structure of the alternating dodecamer d(GCGTACGTACGC) (5'-GC) has been determined to a resolution of 2.55A using oscillation film data. The crystals belong to space group P6(1) 22, a = b = 46.2A, c = 71.5A with one strand in the asymmetric unit, and are isomorphous with a previously described non-alternating dodecamer, d(CCGTACGTACGG) (5'-CC). Refinement by X-PLOR/NUCLSQ gave a final R factor of 14.2% for 1089 observations. The molecule adopts the A-DNA form. The interchange of the terminal base pairs in the two dodecamers results in differences in the intermolecular contacts and may account for the differences in the bending. This dodecamer shows an axial deflection of 30 degrees, in the direction of the major groove compared to 20 degrees in 5'-CC and may be a consequence of additional contacts generated in 5'-GC by the interchange of end base pairs. The high helical axis deflection appreciably influences the local helical parameters. The molecule exhibits relatively high inclination angles, and has a narrow major groove. The helical parameters when described relative to the dyad-related hexamer halves of the molecule give more reasonable values. The crystal packing, local helical parameters, torsion angles, and hydration are described and also compared with the non-alternating 5'-CC dodecamer.

Amides↗

PubMed1992

Crystal and molecular structure of the A-DNA dodecamer d(CCGTACGTACGG). Choice of fragment helical axis.

The crystal structure of the dodecamer d(CCGTACGTACGG) has been determined at 2.5 A resolution. The crystals grow in the hexagonal space group P6(1)22, a = b = 46.2 A, c = 71.5 A with one strand as the asymmetric unit. Diffraction data were collected by the oscillation film method yielding 1664 unique reflections with an Rmerge of 0.04. The structure was solved by real-space rotational translational searches with idealized helical models of A, B and Z-DNA. The best agreement was given by an A-DNA model with its dyad axis along the diagonal crystallographic dyad axis, with an R-factor 0.43 and correlation coefficient of 0.59 for data between 10 and 5 A. Iterative map fitting and restrained least-squares refinement and addition of 40 solvent molecules brought the R-factor to 0.15 and the correlation coefficient to 0.97 for all data between 8.0 and 2.5 A. The stereochemistry of the atomic model is good, with a root-mean-square deviation in bond distances of 0.006 A. This is the first example of an A-DNA containing a full helical turn. The dodecamer displays a novel packing motif. In addition to the characteristic contacts between the terminal base-pairs and the minor grooves of symmetry-related molecules, there are also minor groove to minor groove interactions not previously observed. The packing leaves an approximately 25 A diameter solvent channel around the origin, along the c-axis. The presence of a prominent 3.4 A meridional reflection and other diffuse features in the diffraction pattern provided evidence for the presence of disordered B-DNA along the c-axis, which can be accommodated in these solvent channels. The molecular conformation of the dodecamer also displays novel features. The dyad-related halves of the molecule are bent at an angle of 20 degrees, and the helical parameters are affected by this bend. Unlike the shorter A-DNA octamers, the dimensions of the major groove can be directly measured. Novel correlations between local helical parameters and global conformational features are presented. Most of the solvent molecules are associated with the major groove and the sugar-phosphate backbone.

Base Sequence↗

PubMed1992

Structure of the anti-malarial drug primaquine diphosphate.

8-(4-Amino-1-methylbutylamino)-6-methoxyquinoline bis(dihydrogenphosphate), C15H23N3O2+.2(H2PO4)-, Mr = 455.35, triclinic, P1, Z = 2, a = 7.389 (6), b = 8.862 (4), c = 16.055 (10) A, alpha = 97.57 (2), beta = 100.21 (3), gamma = 77.01 (2) degrees, V = 1003.6 (5) A3, Dm = 1.495 (by flotation), Dx = 1.507 g cm-3, lambda (Cu K alpha) = 1.5418 A, mu(Cu K alpha) = 24.48 cm-1, F(000) = 480, room temperature, R = 0.068 for 3448 observed reflections. The above working cell is related to the reduced cell with angles alpha = 82.43, beta = 79.79 and gamma = 77.01 degrees by the transformation (-100/0 -10/001). Primaquine diphosphate was crystallized in the dicationic form with protonation on the quinoline ring nitrogen atom and on the terminal amino group. One dihydrogenphosphate anion is chelated by the quinoline ring and the butylamino side chain. The other dihydrogenphosphate anion is hydrogen bonded to the terminal amino group. The C(14) atom is nearly in the plane of the quinoline ring with a C(9)-C(8)-N(13)-C(14) torsion angle of 169 degrees. The butyl-diamino side chain is kinked by rotation about the C(14)-C(16) bond with a N(13)-C(14)-C(16)-C(17) torsion angle of -59 degrees. The C(15) methyl substituent is in line with the rest of the butyl chain. The terminal amino group N(19) is hydrogen bonded to three symmetry-related phosphate groups while N(1) and N(13) are 'chelated' to a fourth phosphate group.

Antimalarials↗

PubMed1992

Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines.

The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp-99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr-52 and -73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X-ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 A resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3(1)21, with cell parameters a = b = 46.3 A and c = 102.95 A. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R-factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the alpha-carbon atoms between the double mutant and wild type was 0.56 A. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro-68. However, the hydrogen bonds of the catalytic triad, His-48, Asp-99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe-52 moves toward His-48 and Asp-99 of the catalytic diad, and Phe-73 moves toward Met-8, both by about 0.5 A. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues.

Amino Acid Sequence↗