Proc. Nat. Acad. Sci. USA Vol. 70, No. 11, pp. 3052-3054, November 1973 D-Glyceraldehyde-3-Phosphate Dehydrogenase: Three-Dimensional Structure and Evolutionary Significance (NAD binding/lactate dehydrogenase/x-ray crystallography) MANFRED BUEHNER*, GEOFFREY C. FORD, DINO MORAS, KENNETH W. OLSEN, AND MICHAEL G. ROSSMANNt Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Communicated by Dr. William N. Lipscomb, July 6, 1973 ABSTRACT A 3.0-A resolution electron density map orientation of the mutually perpendicular molecular 2-fold of lobster glyceraldehyde-3-phosphate dehydrogenase axes within the unit cell had been established by Rossmann (EC 1.2.1.12) was computed. The essentially single iso- morphous replacement map was very substantially im- et al. (11) using the rotation function (12). This knowledge, proved by averaging subunits. NAD binds in an open con- along with the accurate data obtained from an Optronics formation at sites close to subunit interfaces. The coen- Film Scanner (13), enabled us to solve the difference Patter- zyme binding portion of the enzyme has almost the same son function of the K2HgI4 heavy-atom derivative for its fold as the corresponding portion of lactate dehydro- major set of four sites. This in turn genase (EC 1.1.1.27). The presence of this structure in the permitted determination of five enzymes, analyzed so far, that use nucleotide co- the other heavy-atom sites in all derivatives by difference enzymes might indicate a fundamental primordial struc- Fourier methods, to give three chemically independent sites tural element. per polypeptide chain. The 3.0-A electron density map, which was essentially D-Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) phased by single isomorphous replacement (14), was re- catalyzes the NAD-mediated oxidative phosphorylation of oriented by means of a skew plane program (15) so that the its substrate to D-1,3-diphosphoglyceric acid. It is a tetramer molecular axes defined the new coordinate system. The of molecular weight 143,000. Park and coworkers (1) have electron density was then averaged over all four subunits. made extensive studies of the chemical groups involved in its Such a process is in principle equivalent to the molecular activity as a dehydrogenase and also as a transferase, phos- replacement technique (16). The assumption that the sub- phatase, and esterase. The enzyme exhibits various coopera- units are identical at 3-A resolution is justified by the heavy- tive phenomena (2). The primary structures of pig (3), lobster atom sites obeying the molecular 222 symmetry to better (4), and yeast (5) glyceraldehyde-3-phosphate dehydrogenase than 0.5 A. The averaging procedure enhanced the features have been determined. common to all subunits by eliminating much of the noise due Preliminary crystallographic studies have been reported on to single isomorphous phasing. In addition, the molecular glyceraldehyde-3-phosphate dehydrogenase from lobster (6), boundary is clearly shown, since the electron density outside crayfish (7), Bacillus stearothermophilus (8), and humans (9). the molecule does not obey the local noncrystallographic Watson et al. (10) reported a low-resolution structure de- symmetry. termination of human hologlyceraldehyde-3-phosphate de- All of the averaged electron density map was immediately hydrogenase. interpretable in terms of a single polypeptide chain. A pre- Structure determination liminary comparison with the bigger residues of the sequence Lobster showed compatibility. Helices could be seen to have right- hologlyceraldehyde-3-phosphate dehydrogenase crys- handed turns to them, and the outstanding sheet areas have tallizes in the orthorhombic space group P212121 with one mol- left-handed twists. ecule per asymmetric unit (6). Data were collected by pre- cession photography to 3-A resolution for the native protein Nucleotide-binding structure and a p-chloromercuriphenyl-sulfonate derivative. In addi- tion, a set of low-resolution precession photographs of a The most striking feature (Fig. 1) was the similarity of the K2HgI4 derivative and a partial low-resolution diffractometer coenzyme binding portion in glyceraldehyde-3-phosphate de- data set of a methyl-mercury-2-thioglycolate derivative was hydrogenase and in lactate dehydrogenase (EC 1.1.1.27) made available to us by Drs. H. C. Watson and L. J. Ban- (17, 18). The polypeptide fold between lactate dehydrogenase aszak. Anomalous dispersion was measured for both the residues 22-164 is essentially the same as in glyceraldehyde-3- K2HgI4 and p-chloromercuriphenyl-sulfonate derivatives. The phosphate dehydrogenase, albeit with some significant dif- ferences. There is an extra antiparallel sheet excursion in- serted between aC and The between (D and aD is * Present address: Fachbereich Biologie, Universitaet Konstanz, OC. "loop" BRD-775, Konstanz, Postfach 733, Germany. smaller in glyceraldehyde-3-phosphate dehydrogenase than in t Reprint requests to: Dr. Michael G. Rossmann, Dept. of lactate dehydrogenase. The helix a1F connecting the fifth Biological Sciences, Purdue University, West Lafayette, Ind. and sixth strands (OE and OF) of the parallel sheet has slipped 47907. around the edge of the sheet and is rather irregular. There are 3052 Downloaded by guest on October 1, 2021 Proc. Nat. Acad. Sci. USA 70 (1978) D-Glyceraldehyde-3-phosphate Dehydrogenase 3053 FIG. 1. Diagrammatic comparison of the conformation of the coenzvme-binding portion of glyceraldehyde-3-phosphate dehydrogenase (left) and lactate dehydrogenase (right). The lactate dehydrogenase diagram is labeled with the accepted nomenclature of the helices and sheets (21, 26). Labeling of the glyceraldehyde-3-phosphate dehydrogenase diagram is by comparison with the lactate dehydrogenase struc- ture. some structural elements in the second half of glyceralde- Evolutionary implications hyde-3-phosphate dehydrogenase that resemble features of The conservation of the basic nucleotide-binding structure in lactate dehydrogenase. For instance, the helix a2F (the various different proteins is now becoming apparent. The "essential thiol" peptide of lactate dehydrogenase) has fold was first reported for lactate dehydrogenase (17). 8- changed direction somewhat, but it does connect with the Malate dehydrogenase was shown (21) to have a similar first strand, /3G, of an antiparallel sheet, extending back to subunit structure as lactate dehydrogenase. Rao and Ross- the substrate-binding site, as in lactate dehydrogenase. The mann (22) showed that the nucleotide-binding moiety of carboxy-terminal helix in glyceraldehyde-3-phosphate dehy- these structures was made up of two roughly identical halves, drogenase takes the position of helix a3G in lactate dehydro- associated each with one nucleotide-binding area, and related genase. by an approximate 2-fold axis. They also showed the presence The coenzyme represents the highest electron density of of the mononucleotide structure to be used in the binding of the averaged map and is clearly visible. It is in an open con- FMN to flavodoxin (23). BrAnden and coworkers (24) have re- formation and is associated with the parallel pleated sheet cently demonstrated the presence of the dinucleotide binding as in lactate dehydrogenase. This is in contrast to the closed moiety in liver alcohol dehydrogenase, although the associa- structure predicted by Velick (19) on the grounds of fluores- tion of subunits is quite different from that in lactate de- ence studies. Its nicotinamide end approaches close to the hydrogenase and glyceraldehyde-3-phosphate dehydrogenase. beginning of the a2F helix. Our chain tracing places the es- The retention of an essentially identical fold, despite the sential thiol, cysteine 149t, near the nicotinamide of NAD+. wide variety of sequences, demonstrates again the stability In addition, histidine 176 can be positioned so that it could of tertiary structure even where there is no apparent con- participate as an acid-base catalyst. servation of primary structure (24). The various dehydro- The association of subunits in glyceraldehyde-3-phosphate genases could be related by an evolutionary model involving dehydrogenase is different from that in lactate dehydrogenase gene duplication as shown (LADH, s-MDH, LDH, and (Fig. 2). The subunit-subunit interactions across the molec- GAPDH are liver alcohol, s-malate, lactate, and glyceralde- ular 2-fold axis labeled Q in lactate dehydrogenase (20) have hyde-3-phosphate dehydrogenase, respectively): essentially been maintained, as in malate dehydrogenase (ref. 21; and see below). Although the direction of the P and Nucleotide-binding R axes with respect to the nucleotide-binding structure are protein roughly the same, the interactions across these axes are dif- ferent because the Q-axis dimers are associated in opposite ways in the two enzymes. Thus, while in lactate dehydro- NAD-binding protein genase the coenzyme sites are on the outside of the molecule, I Q-axis in glyceraldehyde-3-phosphate dehydrogenase they are so close formation to the subunit interfaces that direct interactions of the NAD + with the R-axis related subunit occur. Lysine 183 binds to the pyrosphosphate in the adjacent active site, N-Terminai creating the unique situation in which the catalytic center "arm" formation I ~~~~~~~~~~~~~~~~~~~I contains residues from two different subunits. Thus, the Flavodoxin LADH s-MIDH LDII (GAPDH different association of the glyceraldehyde-3-phosphate de- monomer dimer dimer tetramer tetramer