The structure of HPPK has been determined by X-ray crystallography and NMR for various stages of the catalytic cycle using substrates, substrate analogues, and products (). The crystal structure of apo HPPK from E. coli
was determined by multiwavelength anomalous diffraction at 1.5-Å resolution (PDB code: 1HKA) [12
]. The structure reveals a three-layered αβα fold formed by six β-strands and four α-helices (Fig. 5 in [12
]). The fold of the HPPK molecule creates a valley that is approximately 26-Å long, 10-Å wide, and 10-Å deep. Three flexible loops, β1-α1 (loop 1), β2-β3 (loop 2), and α2-β4 (loop 3), form one wall of the valley. The other wall of the valley is relatively rigid and is constructed by the structural motif β6-loop-α3, which is part of the protein's hydrophobic core. ATP- and HP-binding sites were initially identified by NMR spectroscopy and molecular modeling [12
]. Binding of ATP causes significant changes in the chemical shifts of many backbone amide resonances, not only in ATP-binding site but also in HP-binding site, suggesting that the binding of ATP induces significant conformational changes. The side chain positions of several conserved residues such as R82 and R92 also suggest conformational changes upon substrate binding, because these side chains point away from the active center of the enzyme as observed in the crystal structure of apo HPPK.
Figure 3 Snapshots of the catalytic cycle of the HPPK-catalyzed reaction. The HPPK molecule is illustrated as a ribbon diagram (arrows, β-strands; spirals, helices; tubes, loops), the ligands in the crystal structures as stick models (substrates in red, (more ...)
The assignment of substrate-binding sites and suggested substrate-induced conformational changes were confirmed by the crystal structure of the ternary complex of HPPK with AMPCPP, HP, and two Mg2+
ions at 1.25-Å resolution (PDB code: 1Q0N) [15
]. We also determined the NMR solution structure of HPPK in complex with AMPCPP, 7,7-dimethyl-6-hydroxymethylpterin, and Mg2+
ions (PDB code: 2F63) [17
], which is very similar to the crystal structure of the ternary complex. In the crystal structure, HP is sandwiched between two aromatic rings of F123 and Y53 and forms six hydrogen bonds with residues T42, P43, L45, and N55 (Fig. 5(a) in [15
]). Two Mg2+
ions are found in the ternary complex, one between α- and β-phosphate and the other between β- and γ-phosphate, and both are six-coordinated (Fig. 6(a) in [15
]). Twelve residues are involved in the binding of AMPCPP, including Q74, E77, R84, R88, W89, R92, I98, R110, T112, H115, Y116, and R121, among which E77, R92, H115, and R121 are conserved. For I98, R110, and T112, the functional groups involved in AMPCPP binding are amides and/or carbonyls. The γ-phosphate group of AMPCPP is tethered by the side chains of H115, Y116, and R121, whereas the β- and α-phosphate groups form hydrogen bonds with the guanidinium groups of R92 and R84. The ribose forms hydrogen bonds with the side chain of Q74 and the carbonyl group of R110. The backbone amide and carbonyl groups of I98 and T112 are responsible for the recognition of the adenine moiety.
The conformational changes upon the formation of the ternary complex are illustrated in . The valley created by the fold of the enzyme has both ends and the front open to the solvent when the enzyme is ligand-free (Fig. 3(a) in [15
]). In the ternary complex, one end and the front of the valley are sealed, leaving only one end open (Fig. 3(a) in [15
]). If the apo enzyme looks like a half-closed right hand, the ternary complex appears to be a tightly closed fist. The most significant conformational differences reside in the three aforementioned flexible loops. A network of hydrogen bonds that couples the three flexible loops and helps to stabilize the complex and seal the active center where the pyrophosphoryl transfer occurs has been identified (Fig. 4 in [15
]). The HP-binding site in the free HPPK is not blocked. Rather, the hydrogen bond partners for the binding of HP are not in place for hydrogen bonding. Consequently, HP has a very low affinity for the free HPPK. The critical part of the active center of HPPK is assembled only when both substrates bind to the enzyme.
Stammer and coworkers determined the crystal structure of a ternary complex of HPPK with MgATP and an HP analogue at 2-Å resolution (PDB code: 1DY3) [18
]. In comparison with our ternary structure, the main differences are the conformations of loops 2 and 3 of the protein. The pterin moiety of the bound HP analogue is displaced somewhat, but the hydrogen bonds between the pterin and the protein are maintained. ATP and AMPCPP superimpose very well, and the coordination chemistry of the two Mg2+
ions is the same between the two structures, indicating that AMPCPP is an excellent analogue for ATP for HPPK. The differences in the conformations of loops 2 and 3 are caused by the two substituents of the HP analogue, particularly the bulky phenethyl group. As a result, the side-chains of R82 and R92 have quite different conformations and interact with the nucleotide triphosphate differently. Both guanidinium groups of R82 and R92 move inwards with R82 interacting with both α- and β-phosphate and R92 interacting with β-phosphate only. Both of the two distinct conformations for each of the two arginine residues revealed by the two structures were observed in the crystal structure of HPPK in complex with 6-hydroxymethylpterin/6-carboxylpterin, two Mg2+
ions, and AMPCPP at 0.89-Å resolution (PDB code: 1F9Y) [19
], suggesting that the roles of R82 and R92 are rather dynamic. R92 first binds to the α-phosphate group of ATP and then shifts to interact with the β-phosphate as R82, which initially does not bind to ATP, moves in and binds to α-phosphate when the pyrophosphoryl transfer is about to occur.
After the chemical step, HPPK undergoes another conformational change as revealed by the crystal structure of HPPK in complex with the products HPPP and AMP at 1.56-Å resolution (PDB code: 1RAO) () [20
]. In particular, loop 3 moves away from the active center and adopts a conformation even more open than in the apo HPPK. In comparison with the ternary complex with a substrate (HP) and a substrate analogue (AMPCPP), the Cα atom of E87 moves by ~23 Å, the guanidinium group of R82 by ~8 Å, and the guanidinium group of R84 by ~23 Å. The conformational change not only opens up the active center but also weakens the interactions between the products and the enzyme. The phosphate of AMP and the pyrophosphate of HPPP are disordered, suggesting that the two products are rather dynamic. AMP is more exposed to the solvent than HPPP, in accordance with the preferential release of AMP. After the departure of AMP, HPPK undergoes yet another conformational change and adopts a conformation similar to that of the apo HPPK. HPPP becomes less exposed to solvent and is well fixed in the binary product complex with a single conformation, suggesting that the dissociation of HPPP is the rate-liming step in the HPPK-catalyzed reaction. HPPP interacts with not only loop 2 but also loop 3. The dynamics of the loops, particularly loop 3, may facilitate the release of HPPP.
The crystallization of HPPK with MgATP has not been successful, because of the low level of ATPase activity of the enzyme [21
]. The published structures of HPPK in complex with a nucleotide include the crystal structure of HPPK in complex with MgADP (PDB code: 1EQM; 1.5-Å resolution) [21
], which was obtained because of the hydrolysis of ATP to ADP during the crystallization process, and two NMR solution structures, one in complex with MgAMPPCP (PDB code: 1EQ0) [21
] and the other with MgAMPCPP (PDB code: 2F65) [17
]. Loop 3 in both the HPPK•MgADP crystal structure and the HPPK•MgAMPPCP NMR solution structure adopts a conformation more open than in the apo HPPK, very similar to that in the ternary product structure. The fact that loop 3 can also adopt a “super open” conformation in solution indicates that the “super open” conformation observed in the crystal structures are not due to crystal packing [21
]. In comparison with the structures of the apo HPPK and the ternary complex with the substrates and substrate analogues, it appears that such an unusual conformational change is required for the assembly of the active center. In particular, the side chains of catalytically important R82 and R92 cannot move into the active center without first opening loop 3 [15
The conformational properties of HPPK in complex with MgAMPCPP are quite different from those of the other two binary nucleotide complexes described above. In particular, both loops 2 and 3 can assume multiple conformations, which are generally in-between those of the apo HPPK and the ternary substrate complex. Because AMPCPP is a better ATP analogue for HPPK than ADP or AMPPCP in terms of both binding affinity and bound conformation, the conformational properties of the binary substrate complex HPPK•MgATP may be better represented by those of HPPK•MgAMPCPP. The structures of HPPK•MgADP and HPPK•MgAMPPCP may represent intermediate conformations before the formation of the stable binary substrate complex.