Geoffrey C. Ford

Three-dimensional structure of d-glyceraldehyde-3-phosphate dehydrogenase☆

Abstract The crystal structure of lobster d -glyceraldehyde-3-phosphate dehydrogenase has been analyzed. A 3.0 A resolution electron density map of an averaged subunit showed that: 1. (1) The subunit structure consists of two domains. The first (residues 1 to 149) is essentially the coenzyme binding fold whereas the second (residues 150 to 334) provides residues for catalysis, specificity and subunit-subunit co-operativity. 2. (2) The first domain is similar to structures found in other dehydrogenases and kinases. Some of the important residues involved in binding the coenzyme, NAD+, are the same in d -glyceraldehyde-3-phosphate dehydrogenase as in lactate dehydrogenase. 3. (3) The conformation of NAD+ is essentially the same in d -glyceraldehyde-3-phosphate dehydrogenase as in lactate dehydrogenase, apart from a 180 ° rotation about the ribose-nicotinamide glycosidic bond, thus exposing the B side of the nicotinamide ring to the substrate. 4. (4) The second domain contains histidine 176 situated near the essential cysteine 149 and presumably acting as a base during catalysis. 5. (5) Lysine 183 binds to the pyrophosphate in the active site of the adjacent subunit, creating the unique situation in which the catalytic center contains residues from two different subunits. Thus the NAD binding sites are directly linked in pairs by virtue of a molecular 2-fold axis, forming a functional dimer. The co-operativity (positive in yeast and negative in muscle enzyme) and the half-of-the-sites reaction properties are probably associated with this characteristic. 6. (6) The position of the two sulfate anion sites suggests the position of binding of the substrate phosphate and the inorganic phosphate to be subsequently incorporated into the product. 7. (7) The amino acids in the active center region and involved in subunit-subunit contacts are more highly conserved than other residues.

A comparison of the structures of apo dogfish M4 lactate dehydrogenase and its ternary complexes.

Abstract Details are recorded of the X-ray diffraction data collection, heavy atom refinement and preliminary structure refinement for two different dogfish M4 lactate dehydrogenase structures. One of these is the 2.0 A resolution apoenzyme structure; the other is a 3.0 A resolution abortive ternary complex. Two other ternary substrate inhibitory complexes (LDHase † : NAD: oxalate and LDHase: NADH: oxamate), isomorphous with the abortive ternary complex (LDHase: NAD-pyruvate), have also been examined. The apo-LDHase and LDHase: NAD-pyruvate structures are systematically compared to determine significant differences in their conformation. These are related to differences in structure amongst the three studied ternary complexes. These differences all occur in regions of the protein around the active site, particularly the flexible loop covering the active center pocket and the C-terminal helix αH. The changes are suggestive of a domino effect whereby the closing of the loop on binding coenzyme and substrate triggers the critical reactive residues into assuming their catalytically active positions.

Structure determination of crystalline lobster d-glyceraldehyde-3-phosphate dehydrogenase

Abstract Single crystal X-ray data were collected on film for the holoenzyme of lobster d -glyceraldehyde-3-phosphate dehydrogenase to 3·0 A resolution. Films of potassium tetraiodomercurate, K 2 HgI 4 , comprising a complete low resolution set, with some additional high resolution terms, were given to us by Drs H. C. Watson and L. J. Banaszak. A 3·0 A high resolution data set was collected of a p -chloromercuri-phenylsulfonate derivative. All these films were processed on a computer controlled Optronics film scanner. The K 2 HgI 4 derivative difference Patterson was initially interpreted in terms of four single sites, one for each polypeptide chain, consistent with the previously determined molecular 222 symmetry. Single isomorphous replacement phases were then sufficient to identify other heavy atom sites. Least-squares refined parameters were used to give multiple isomorphous replacement phases at low resolution, and single isomorphous replacement phases at high resolution. The resultant electron density map was oriented along the molecular 2-fold axes and then averaged over all four equivalent subunits. This process produced a much improved electron density map, which could easily be interpreted in terms of a single polypeptide chain per subunit consistent with the known amino acid sequence. The use of non-crystallographic symmetry to improve the electron density map is equivalent to the molecular replacement method. A comparison is also made with other dehydrogenases.

Molecular symmetry axes and subunit interfaces in certain dehydrogenases

Abstract A right-handed orthogonal set of axes P , Q , R has been denned coincident with the three different molecular 2-fold axes of lactate dehydrogenase. The four different symmetry-related subunits can then be color-coded red, yellow, green and blue to identify the three different subunit contacts made by any one subunit to the remaining three others. This nomenclature has been extended to soluble malate dehydrogenase and will facilitate the comparison of dehydrogenases with related ternary and quaternary structures.

Molecular symmetry of glyceraldehyde-3-phosphate dehydrogenase

Abstract The rotation function has been used to determine the orientation of the molecule of lobster glyceraldehyde- 3-phosphate dehydrogenase in its crystal cell. The results have also established the 222 symmetry of the molecule. Comparison with the known structure of lactate dehydrogenase suggests structural similarities and indicates the relative orientation of the corresponding LDH molecule in the GAPDH unit cell. This resemblance has been used to determine the position of the molecular center of GAPDH with respect to the crystal axes.

Atomic co-ordinates for dogfish M4 apo-lactate dehydrogenase.

Abstract Atomic co-ordinates for one subunit of LDH are given in A with respect to a Cartesian co-ordinate system corresponding to the molecular two-fold axes.

The three-dimensional structure of mitochondrial aspartate aminotransferase at 4.5 Å resolution

Abstract An X-ray crystallographic study at 4.5 A resolution has been carried out with triclinic crystals of chicken mitochondrial aspartate aminotransferase. In the electron density map, the enzyme is clearly visible as an isologous α2-dimer (105 A × 60 A × 50 A) in which the subunits are associated about a molecular 2-fold axis. Each subunit of dimensions 70 A × 50 A × 40 A contains at least seven helices, one of which is about 50 A long. Difference maps have revealed the positions of the pyridoxyl and the phosphate moieties of the coenzyme as well as the general substrate binding area. The active sites are on opposite sides of the dimer, about 30 A apart and close to the intersubunit boundary, so that probably both subunits contribute to each active site. An isolated chain segment, passing in front of the active site and ending in contact with the neighbouring subunit is interpreted as one of the chain termini.

An application of the molecular replacement technique in direct space to a known protein structure

The molecular replacement technique in real space (successive cycles of electron density calculations, averaging among non-crystallographically equivalent subunits, and structure factor calculations based upon the improved averaged density) was applied to glyceraldehyde-3-phosphate dehydrogenase. It was shown possible (i) to extend the phases from a known 6.0 A single isomorphous replacement set to at least 4.9 A resolution; (ii) to determine a reasonable structure at low resolution given the molecular envelope and non-crystallographic symmetry; and (iii) to use the resulting low-resolution phases in the determination of heavy-atom sites. The application of such procedures to the determination of virus structures and heavy-atom derivative sites is discussed.

Molecular size and symmetry of the bacterioferritin of Escherichia coli: X-ray crystallographic characterization of four crystal forms

X-ray crystallographic data from four crystal forms of Escherichia coli bacterioferritin show that the molecule has a diameter in the range 119 to 128 A. Molecules are composed of 24 subunits arranged in 432 symmetry. In both size and symmetry the molecule resembles ferritin from eukaryotes. The four crystal forms are monoclinic, space group P2(1) with unit cell dimensions a = 118.7 A, b = 211.6 A, c = 123.3 A and beta = 119.1 degrees; orthorhombic, C222(1), a = 128.7 A, b = 197.1 A, c = 202.8 A; tetragonal, P4(2)2(1)2, a = b = 210.6 A, c = 145.0 A and cubic, I432, a = 146.9 A.

The location of exon boundaries in the multimeric iron-storage protein ferritin

SummaryThe nature of the amino acids whose codons border introns in ferritin genes is novel; the disposition of these intron boundaries within the three-dimensional structure of the 24-subunit molecule differs significantly from that of other proteins. These observations are discussed in relation to the functions of isoferritins.