Phosphoribosylformylglycinamidine (FGAM) synthetase (EC 184.108.40.206) catalyses the reaction 5′-phosphoribosylformylglycinamide (FGAR) + glutamine + ATP ↔ FGAM + glutamate + ADP + Pi, the fourth step in the de novo purine biosynthetic pathway. In eukaryotes and many bacterial systems (including Escherichia coli and Salmonella typhimurium), the FGAM synthetase is encoded by a large protein with an N-terminal ATPase domain and a C-terminal glutamine-binding domain.1 In archaeal and other bacterial systems, however, FGAM synthetase is encoded by separate genes, making it a multisubunit (rather than multidomain) enzyme. For example, in Bacillus subtilis, the purL protein is homologous to the ATPase domain, whereas the purQ protein is homologous to the glutamine-binding domain of the single-chain FGAM synthetases.2
The purL and purQ genes are part of the pur operon in B. subtilis, which encodes 11 of the 12 enzymes in the purine biosynthetic pathway.2 The genetic studies also identified an open reading frame (ORF) of 84 amino acids in this operon, now known as purS,3 which is conserved in a large group of Gram-positive bacteria and methanogenic archaea (Fig. 1). Recent studies showed that disruption of the purS gene in B. subtilis resulted in a purine-auxotrophic phenotype, due to defective FGAM synthetase activity.3 Therefore, the purS protein appears to be required for the function of the purL and purQ subunits of the FGAM synthetase, but the molecular mechanism for the functional role of purS is currently not known.
We recently initiated a prototype structural genomics effort that focused on non-membrane proteins in the proteome of the thermophilic archaeon Methanobacterium thermoautotrophicum (Mth).4 One of the proteins selected for this study is MTH169, which shares 29% amino acid sequence identity with that of purS in B. subtilis. The Mth proteome also contains proteins that are homologous to the purL and purQ subunits of B. subtilis. MTH169 is, therefore, the likely purS ortholog in Mth. We will use the names purS and MTH169 interchangeably here. In addition, for ease of discussion, all purS proteins are numbered according to the sequence of MTH169 (Fig. 1).
The crystal structure of MTH169 (purS) has been determined at 2.56 Å resolution (Table I) and deposited at the Protein Data Bank (entry 1GTD). This 84-residue protein forms a tetramer, each subunit of which contains well-defined secondary structure elements, including a three-stranded anti-parallel β-sheet (β1 through β3) and two α-helices (αA and αB) [Fig. 2(A)]. Helix αB covers part of one face of the β-sheet, forming the hydrophobic core of the structure. Residues in this core are primarily on strand β1 and helix αB [Fig. 2(A)], and are generally conserved among the family of purS proteins (Fig. 1). On the other hand, residues on the other face of the β-sheet are weakly-conserved and are mostly hydrophilic or charged in nature.
|Unit cell parameters (a, c) (Å)||53.8, 142.7|
|Maximum resolution (Å)||2.56|
|Number of observations||55,012|
|Number of reflections||12,678|
|Resolution range for refinement||20–2.56|
|R factor (%)b||23.5|
|Free R factor (%)||27.8|
|rms deviation in bond lengths (Å)||0.007|
|rms deviation in bond angles (°)||1.2|
Helix αA extends away from the β-sheet structure [Fig. 2(A)]. Such a conformation is probably unstable for the molecule in the monomeric state, but these residues are stabilized by quaternary interactions in the tetramer of MTH169. There are two molecules of MTH169 in the crystallographic asymmetric unit, which form a non-crystallographic dimer [Fig. 2(B)]. The dimer is situated near a crystallographic twofold symmetry axis, and this produces a tetramer of MTH169 in the crystal [Fig. 2(C)]. Within each monomer, about 1,100 Å2 and 650 Å2 of the surface area is buried at the dimer and tetramer interface, respectively, suggesting that the dimer interface is more extensive than the tetramer interface.
Structural searches against the Protein Data Bank, with the program Dali,5 showed that there are other structures with similar backbone folds, but none of them are identical to MTH169. All the structures identified by Dali have a four-stranded anti-parallel β-sheet, most frequently with the extra strand inserted between αA and β2 and located next to β2 of MTH169. Moreover, the αA helix in these structures runs along the β-sheet and helps cover the hydrophobic core. The conformation for helix αA in MTH169 is not observed in any of these other structures. Interestingly, this folding motif of a four-stranded anti-parallel β-sheet with two helices on one face is also observed to mediate protein oligomerization, producing dimers, trimers, and tetramers for a number of proteins.
A major part of the dimer interface in MTH169 is composed of hydrogen-bonding interactions between strand β2 of one monomer with the same strand in the other monomer. This extends the 3-stranded β-sheet in the monomer to a 6-stranded, highly-twisted anti-parallel β-sheet in the dimer [Fig. 2(B)]. In addition, helix αA and the αA-β2 loop in one monomer are packed against helix αB in the other monomer [Fig. 2(B)], with conserved, hydrophobic residues in this interface (Fig. 1).
All four helices are located on the convex face of the β-sheet in the dimer [Fig. 2(B)]. Helix αA together with the β1-αA and αB-β3 loops form a ring-like structure on the surface of this sheet [Fig. 2(D)], and residues in this structure are mostly conserved and hydrophobic among the purS proteins (Fig. 1). The tetramer of MTH169 is formed by the direct contact of the ring-like structures from the two dimers [Fig. 2(C)]. This contact leaves an open space in the center of the tetramer, with a volume of about 1,100 Å3 [Fig. 2(C)]. Two charged side chains from each monomer in MTH169 (Fig. 1), Glu19 and Asp41, are pointed towards this pocket. The concave face of the β-sheet in the dimer remains exposed to the solvent in the tetramer [Fig. 2(C)].
Roughly 7,000 Å2 of the surface areas of the four monomers are buried by the formation of the tetramer, suggesting that this tetramer may be a stable entity. This structural observation was confirmed by gel filtration studies, which demonstrated that MTH169 is a tetramer in solution (unpublished results). The conserved nature of the dimer and tetramer interfaces among the purS proteins (Fig. 1) suggests that the other purS orthologs may also exist as tetramers. This is supported by our mutagenesis studies showing that single-site mutations in the dimer or tetramer interface did not disrupt the tetramer (data not shown), demonstrating the stability of this oligomer.
Our structural analysis suggests that purS is likely a protein-protein interaction module that helps bring the purL and purQ subunits together. Overall, the molecular surface of MTH169 is highly negative electrostatically [Fig. 2(E)], a feature that may be conserved among the purS proteins. All these proteins have calculated isoelectric points between 4.5 and 6, and will therefore be negatively charged at physiological pH. This surface electro-negativity may be functionally important for these proteins. Despite this overall negatively charged surface, there are conserved hydrophobic surface patches on the tetramer as well, near the dimer and tetramer interfaces. It is likely that these surface patches may have important roles in the function of purS, for example the recruitment of the purL and purQ proteins.