Unusual Conformational Changes in 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase as Revealed by X-ray Crystallography and NMR

Abstract

Because the folate pathway is essential for microorganisms but absent from mammals, HPPK, like other enzymes in the pathway, is an important target for developing antimicrobial agents.HPPK belongs to a class of enzymes that catalyze the pyrophosphoryl transfer reaction (2). Although the mechanisms of many kinases that catalyze monophosphoryl transfer have been extensively characterized, little is known about the mechanisms of pyrophosphokinases. As a small (158 residues, ϳ18 kDa), stable, monomeric protein, Escherichia coli HPPK is an excellent model system for studying the mechanisms of enzymatic pyrophosphoryl transfer.We have recently determined the crystal structures of ligand-free (apo-) E. coli HPPK (1) and its complex with HP, ␣,␤-methyleneadenosine 5Ј-triphosphate (AMPCPP), and two Mg 2ϩ ions (3) at 1.5 and 1.25 Å resolution, respectively. Hennig and co-workers (4) have determined the crystal structure of Haemophilus influenzae HPPK in complex with an HP analog at 2.05-Å resolution. Stammers and co-workers (5) have determined the crystal structure of E. coli HPPK in complex with an HP analog, ATP, and two Mg 2ϩ ions at 2.00-Å resolution. Comparative analysis of the crystal structures of the apo-HPPK and its ternary complex has revealed the interactions of the enzyme with the substrates at the atomic resolution and the dramatic substrate-induced conformational changes involving three catalytic loops (3). It appears that the complete active center of HPPK is assembled only after both substrates bind to the enzyme. However, how the catalytic center is assembled is not known. It appears that some catalytic residues cannot move into their catalytic positions because of steric constraints.In this paper, we present the crystal structure of HPPK⅐MgADP at 1.5-Å resolution and the NMR solution structure of HPPK⅐MgAMPPCP. The two structures reveal a dramatic, unusual movement of loop 3 and other significant changes in the conformation and dynamical property of HPPK. The dramatic substrate-induced movement of loop 3 is unusual because it moves away from the active center. Comparative structural analysis suggests that the structures reported here may represent an intermediate conformation required for both substrate binding and product release in the catalytic cycle. EXPERIMENTAL PROCEDURESCrystal Structure Determination of HPPK⅐MgADP-E. coli HPPK was purified as previously described (6). Crystals of HPPK⅐MgADP were grown in hanging drops at 19 Ϯ 1°C. The protein solution contained 4 mg/ml HPPK, 5 mM MgATP in 10 mM Tris buffer (pH 8.0), and the reservoir contained 30% polyethylene glycol 4000, 0.2 M NaAc in 0.2 M Tris buffer (pH 8.5). The 4-l hanging drops contained equal volumes (2 l) of protein solution and reservoir solution. Two rounds of seeding produced diffraction-quality crystals. * This work was supported in part by National Institutes of Health Grant GM51901 (to H. Y.). This study made use of a Varian INOVA-600 NMR spectrometer at Michigan State University funded in part by National Science Foundation Grant...

Crystal Structure of the Bifunctional Dihydroneopterin Aldolase/6-hydroxymethyl-7,8-dihydropterin Pyrophosphokinase from Streptococcus pneumoniae

“…deviation for C α atoms between SulD and the E. coli HPPK domain was 1.1 ) and the only minor deviations were observed in the loop region between the α5 helix and the β5 strand ( Figure 3). Changes in the structure of E. coli HPPK on binding of substrates are well documented; 6,12,25 in this respect, the structure of SulD HPPK is most closely related to the ''open'' or unliganded conformation of the pyrophosphokinase. Electron density was missing or poorly defined for two loop regions (160-169 and 201-210) that are known to be subject to structural changes on binding of ATP.…”

“…A variety of crystallographic and enzyme studies on Escherichia coli HPPK have identified the binding sites for the two substrates, ATP and 6-hydroxymethyl-7,8-dihydropterin (HMDP). 5,[8][9][10][11][12][13][14] Three loop regions are involved in a structural change, induced on binding of ATP, to form the binding site for HMDP: this primes the active site for the direct transfer of the pyrophosphoryl moiety from ATP to HMDP.…”

The Three-dimensional Structure of the Bifunctional 6-Hydroxymethyl-7,8-Dihydropterin Pyrophosphokinase/Dihydropteroate Synthase of Saccharomyces cerevisiae

“…While a number of three-dimensional structures are available for the monofunctional bacterial DHNA, 22,23,[25][26][27][28][29][30][31][32][33] and DHPS 24,[34][35][36] enzymes, the study of the higher-order association of the trifunctional DHNA-HPPK-DHPS protein from pathogens such as P. jirovecii has been hampered by poor protein expression of full-length trifunctional protein in heterologous expression systems. However, we have now succeeded in over-expressing and purifying a bifunctional S. cerevisiae HPPK-DHPS polypeptide, and we present its three-dimensional structure in complex with the oxidized substrate analogue 6-hydroxymethyl-pterin monophosphate (PTP).…”

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…ATP binds first to hydroxymethyldihydropterin diphosphokinase, which results in large structural changes in the three loops, in particular L2 and L3, and allows the binding of the second substrate, hydroxymethyldihydropterin (164,169,170). Furthermore, two Mg 2ϩ are bound within the active site (Fig.…”