Richard E. Dickerson

Structure of a B-DNA dodecamer. III. Geometry of hydration.

Abstract The dodecamer d(CpGpCpGpApApTpTpCpGpCpG) or C-G-C-G-A-A-T-T-C-G-C-G crystallizes as slightly more than one full turn of right-handed B -DNA. It is surrounded in the crystal by one bound spermine molecule and 72 ordered water molecules, most of which associate with polar N and O atoms at the exposed edges of base-pairs. Hydration within the major groove is principally confined to a monolayer of water molecules associated with exposed N and O groups on the bases, with most association being monodentate. Waters hydrating backbone phosphate oxygens tend not to be ordered, except where they are immobilized by 5-methyl groups from nearby thymines. In contrast, the minor groove is hydrated in an extensive and regular manner, with a zigzag “spine” of first- and second-shell hydration along the floor of the groove serving as a foundation for less-regular outer shells extending beyond the radius of the phosphate backbone. This spine network bridges purine N-3 and pyrimidine O-2 atoms in adjacent base-pairs. It is particularly regular in the A-A-T-T center, and is disrupted at the C-G-C-G ends, in part by the presence of the N-2 amino groups on guanine residues. The minor groove hydration spine may be responsible for the stability of the B form of polymers containing only A · T and I · C base-pairs, and its disruption may explain the ease of transition to the A form of polymers with G · C pairs.

Structure of a B-DNA dodecamer: II. Influence of base sequence on helix structure☆

Abstract Detailed examination of the structure of the B-DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G, obtained by single-crystal X-ray analysis (Drew et al., 1981), reveals that the local helix parameters, twist, tilt and roll, are much more strongly influenced by base sequence than by crystal packing or any other external forces. The central EcoRI restriction endonuclease recognition site, G-A-A-T-T-C, is a B helix with an average of 9.8 base-pairs per turn. It is flanked on either side by single-base-pair steps having aspects of an A-like helix character. The dodecamer structure suggests several general principles, whose validity must be tested by other B-DNA analyses. (1) When an external bending moment is applied to a B-DNA double helix, it bends smoothly, without kinks or breaks, and with relatively little effect on local helix parameters. (2) Purine-3′,5′-pyrimidine steps open their base planes towards the major groove, pyrimidine-purine steps open toward the minor groove, and homopolymer (Pur-Pur, Pyr-Pyr) steps resist rolling in either direction. This behavior is related to the preference of pyrimidines for more negative glycosyl torsion angles. (3) CpG steps have smaller helical twist angles than do GpC, as though in compensation for their smaller intrinsic base overlap. Data on A-T steps are insufficient for generalization. (4) G.C base-pairs have smaller propellor twist than A · T, and this arises mainly from interstrand base overlap rather than the presence of the third hydrogen bond. (5) DNAase I cuts preferentially at positions of high helical twist, perhaps because of increased exposure of the backbone to attack. The correlation of the digestion patterns in solution and helical twist in the crystal argues for the essential identity of the helix structure in the two environments. (6) In the two places where the sequence TpCpG occurs, the C slips from under T in order to stack more efficiently over G. At the paired bases of this CpG step, the G and C are tilted so the angle between base planes is splayed out to the outside of the helix. This TpC is the most favored cutting site for DNAase I by a factor of 4.5 (Lomonossoff et al., 1981). (7) The EcoRI restriction endonuclease and methylase both appear to prefer a cutting site of the type purine-purine-A-T-T-pyrimidine, involving two adjacent homopolymer triplets, and this may be a consequence of the relative stiffness of homopolymer base-stacking observed in the dodecamer.

Conformation change of cytochrome c: I. Ferrocytochrome c structure refined at 1·5 Å resolution☆

Abstract Tuna ferrocytochrome c has been crystallographically refined at a resolution of 1.5 A using the Diamond real-space method followed by Jack-Levitt restrained energy and reciprocal space refinement, monitoring progress continuously with superimposed Fourier and difference Fourier maps: The final R factor for cytochrome plus 53 solvent molecules, using 13,840 reflections with intensities greater than 2 σ, is 17·3%. The overall structure remains as described earlier (Takano et al., 1977), but structural details have been clarified to the point where meaningful comparison can be made with the oxidized molecule (following paper). Main and side-chain flexibility as judged by isotropic temperature parameters correlate with position in the molecule, with greatest flexibility at external chain loops. The haem group is held tightly in place by its attachments and neighbours, and is deformed slightly into a saddle shape. The iron does not deviate significantly from the best mean plane of the haem, and bond lengths to ligands are as expected from model compounds. A water molecule buried in the haem crevice is bonded to Asn52, Tyr67 and Thr78, the latter two being bonded also to Met80 and the outer haem propionate. It is proposed that this buried water molecule is involved in the reduction of ferricytochrome c by chromous ion, and the reactions of Tyr67 with KI3 and tetranitromethane. Two other buried water molecules occur beneath the 20s loop at the right, and within the 40s loop at the bottom. Reasonable if tentative functional assignments can be made for all 24 of the evolutionarily invariant residues in the cytochrome molecule.

7 Cytochromes c

Publisher Summary The term cytochrome c is both a spectral and a structural classification, related more to the heme and its attachment to the polypeptide chain than to the protein which surrounds it. The most familiar member of the class is cytochrome c from the mitochondria1 respiratory chain, which has a single heme group per chain of 103–113 amino acids, and a reduction potential of +260 mV. Cytochromes all have a characteristic three-banded absorption spectrum in the reduced state. One of the most striking features of the heme group is the delocalization of electrons among the π orbitals of the porphyrin ring. The Soret band in the absorption spectrum represents the excitation of delocalized π electrons to unoccupied levels of the porphyrin ring of similar angular momentum. The absorption spectrum of cytochrome c, which is the basis for classification, depends on the side groups around the porphyrin ring and the way that they are connected to the protein chain. All cytochromes c are oxidation-reduction proteins involved in either respiration or photosynthesis.

Conformation change of cytochrome c. II. Ferricytochrome c refinement at 1.8 A and comparison with the ferrocytochrome structure.

Tuna ferricytochrome c has been crystallographically refined at a resolution of 1·8 A using Diamond real-space methods followed by Jack Levitt restrained energy and reciprocal space refinement, with Fourier and difference Fourier map monitoring. The final R factor for two independent cytochromes plus 49 solvent molecules, using 16,831 reflections to 1.8 A is 20·8%. The structure is essentially the same as described at 2.0 A (Swanson et al., 1977), but the increased accuracy now permits detailed comparison with the reduced cytochrome c molecule (preceding paper). Twelve water molecules are found in identical positions in both oxidized molecules and also in the reduced. Three of these are buried: one between Asn52, Tyr67 and Thr78 to the lower left of the haem, a second inside the 20s loop, and a third below the buried haem propionate. The first of these three buried water molecules is involved in a concerted shift of side-chains and even main-chain at the bottom of the molecule, that accompanies the change in redox state of the haem. The shift of this water molecule and a slight outward movement of the haem both give the haem a more hydrophilic environment in ferricytochrome c. This same buried water molecule may be involved in the alkaline conformation transition that ferricytochrome c undergoes at pH 9·4, assisting in the replacement of Met80 by a different low-spin ligand. The absence of such transition in ferrocytochrome c until pH 12 is reached may be ascribed to the shift in water position observed in the reduced state. The water molecule buried in the 20s loop, in turn, may be involved in the acid transformation that ferricytochrome c undergoes at pH 2·5. The conformations observed by X-rays for both ferricytochrome c and ferrocytochrome c appear to be post-transfer states, i.e. states that are most compatible with the particular redox state of the haem, rather than being poised to give up or receive electrons. These conformations have been interpreted as being effects of changes in haem redox state rather than causes, but if binding to another macromolecule were to induce either conformation, then transfer of an electron might thereby be facilitated.

The structure of bovine trypsin : Electron density maps of the inhibited enzyme at 5 Å and at 2·7 Å resolution☆

The crystal structure of diisopropyl phosphoryl-trypsin has been obtained by the method of isomorphous heavy-atom replacement (R. M. Stroud, L. M. Kay, A. M. Cooper & R. E. Dickerson, Abstr. 8th Int. Congr. Biochem. 1970; Stroud et al., 1971). DIP†-trypsin crystallizes in two forms depending on the pH of the crystallization medium. The orthorhombic form obtained at pH 7 to 8 was used for the crystallographic study. The space group is P212121 and the cell dimensions are a = 54·8 A, b = 58·7 A and c = 67·6 A. One asymmetric unit of the crystal structure contains one molecule of molecular weight 24,000. Analysis of the crystalline material shows that crystals contain α- and β-DIP-trypsin in approximately equal proportions. The single-chain molecule, β-trypsin, contains 223 amino acid residues linked together by six disulfide bridges. This paper reports the progress of analysis from the low-resolution (5 A) model, which shows the general molecular architecture, through high-resolution (2·7 A) density maps, from which a model of the enzyme was built. The map shows approximately 70% of the carbonyl groups, and allows interpretation of intramolecular hydrogen bonding and solvation of the protein. The tertiary structure of trypsin shows extensive homology to the related enzymes α-chymotrypsin and elastase.

Structure of cytochrome c551 from Pseudomonas aeruginosa refined at 1.6 Å resolution and comparison of the two redox forms

Abstract The molecular structures of ferri- and ferrocytochrome c 551 from Pseudomonas aeruginosa have been refined at a resolution of 1.6 A, to an R factor of 19.5% for the oxidized molecule and 18.7% for the reduced. Reduction of oxidized crystals with ascorbate produced little change in cell dimensions, a 10% mean change in F obs , and no damage to the crystals. The heme iron is not significantly displaced from the porphyrin plane. Bond lengths from axial ligands to the heme iron are as expected in a low-spin iron compound. A total of 67 solvent molecules were incorporated in the oxidized structure, and 73 in the reduced, of which four are found inside the protein molecule. The oxidized and reduced forms have virtually identical tertiary structures with 2 ° root-mean-square differences in main-chain torsion angles φ and ψ, but with larger differences along the two edges of the heme crevice. The difference map and pyrrole ring tilt suggest that a partially buried water molecule (no. 23) in the heme crevice moves upon change of oxidation state. Pseudomonas cytochrome c 551 differs from tuna cytochrome c in having: (1) a water molecule (no. 23) at the upper left of the heme crevice; that is, between Pro62 and the heme pyrrol 3 ring on the sixth ligand Met61 side, where tuna cytochrome c has an evolutionary invariant Phe82 ring; (2) a string of hydrophobic side-chains along the left side of the heme crevice, and fewer positively charged lysines in the vicinity; and (3) a more exposed and presumably more easily ionizable heme propionate group at the bottom of the molecule. A network of hydrogen bonds in the heme crevice is reminiscent of that inside the heme crevice of tuna cytochrome c . As in tuna, a slight motion of the water molecule toward the heme is observed in the oxidized state, helping to give the heme a more polar microenvironment. The continuity of solvent environment between the heme crevice and the outer medium could explain the greater dependence of redox potential on pH in cytochrome c 551 than in cytochrome c .

The cytochrome fold and the evolution of bacterial energy metabolism

Respiratory cytochrome c from eukaryotes, photosynthetic c 2 from purple nonsulfur bacteria, and respiratory c 550 from Paracoccus (formerly Micrococcus) denitrificans , have been shown by X-ray structure analysis and amino acid sequence comparisons to form a unified subfamily of evolutionarily homologous proteins (cytochromes c 2 ) in which the differences between respiratory and photosynthetic cytochromes are no greater than the variation among photosynthetic cytochromes c 2 . From sequence and structure considerations alone, these cytochromes are indistinguishable as to origin or function. Low resolution (4 A) X-ray analysis of Pseudomonas cytochrome c 551 shows it to have the same overall folding pattern as cytochrome c , with a massive deletion at the bottom of the molecule. This information enables amino acid sequence homologies to be extended with reasonable probability of correctness to all cytochromes c 551 , to Chlorobium c 555 , Pseudomonas c 5 , algal cytochromes f , and even c 553 from Desulfovibrio . All of these proteins appear to comprise a broader family of evolutionarily related electron carriers. It is proposed that bacterial and eukaryotic oxygen respiration arose from the dual-function photosynthetic and respiratory electron chain in purple non-sulfur bacteria, by the loss of photosynthetic capabilities. A detailed outline for the evolution of photosynthesis and respiration in bacteria is presented, involving first a symbiotic sulfur cycle, then the development of the Krebs cycle machinery and finally the evolution of the present-day symbiotic oxygen cycle between plants and animals.

Conformation and dynamics in a Z-DNA tetramer☆

Abstract Two kinds of conformational variability have been reported for left-handed Z -DNA: the Z to Z ′ transition, which involves a change in guanine sugar pucker from C-3′- endo to C-1′- exo , and the Z I to Z II transition, which corresponds to a simple three-atom phosphate-group rotation. Neither of these motions substantially affects base stacking or helical twist, and this is because the degree of independent motion of phosphate groups, sugar molecules and base-pairs is greater in the left-handed Z helix than in right-handed B -DNA. Detailed considerations of Z helix geometry suggest that Z I , Z II and Z ′ are not separate species, but only samplings of the full range of conformation open to Z -DNA.

Sequence and structure homologies in bacterial and mammalian-type cytochromes

Abstract A three-dimensional structure is proposed for Pseudomonas aeruginosa cytochrome c 551 , based on the X-ray analysis of horse and bonito oxidized cytochromes c , and the amino-acid sequences of these and cytochrome c 2 from Rhodospirillum rubrum . Most of the changes between Pseudomonas and the other cytochromes can be accounted for by the deletion of one 16-residue hairpin loop in the horse cytochrome c structure. The agreement between the three cytochromes in terms of the number of identical amino acids is greater for this sequence alignment than for earlier ones proposed. No evidence is found in the X-ray analysis of cytochrome c for repeated polypeptide sequences such as have been reported from sequence comparisons using the criterion of minimum mutational distance in DNA. It is demonstrated that the conservative nature of the nucleic acid code is such that functional similarity in two unrelated chains can simulate evolutionary homology, and that homologies based on minimum mutational distance in DNA must be treated with caution.