3.1. General comments
PurB crystallizes in an orthorhombic system and the systematic absences indicated either space group I
222 or I
. Both space groups have eight asymmetric units per unit cell and a single polypeptide chain, labelled chain A
, constitutes the asymmetric unit, with an estimated solvent content of 60%. The space-group assignment of I
222 was confirmed using default settings in the program Phaser
(McCoy et al.
) and the structure was solved by molecular replacement and refined to a resolution of 2.50 Å. The log-likelihood gain score in I
222 was 742, compared with a score of 86 in I
. As will be described, Sa
PurB forms a tetramer in the crystal structure and this rules out space group I
. The crystallographic statistics and model geometry (Table 1) indicate that the analysis has produced an acceptable medium-resolution model. Continuous and well defined electron density was observed for the entire polypeptide except for residues 401 and 402. AMP, a product of the catalyzed reaction, was present in the crystallization conditions and well defined electron density for the ligand was observed in the active site (data not shown). Electron density compatible with PEG was located near the C-terminal segment of α7 and the C-terminal segment of α5 from a symmetry-related subunit (symmetry operation −x
+ 1/2, −y
+ 1/2, −z
+ 1/2, data not shown). In addition, a strong electron-density feature appeared in the active site which we could not explain on the basis of the known chemical components of the crystallization mixture. Different molecules were considered and refinements attempted. Our tentative interpretation is that this density represents oxalate (Fig. 2), possibly acquired as a contaminant of the PEG 200 used in crystallization. Contamination of PEG and its influence on protein crystallization has been observed previously (Jurnak, 1986
). In our case, since ethylene glycol is a precursor of PEG and like PEG itself is susceptible to contamination with aldehydes and peroxides (Ray & Puvathingal, 1985
), then breakdown of PEG 200 and/or the presence of ethylene glycol and subsequent oxidation may have produced oxalate.
Figure 2 Stereoview of the OMIT map of oxalate and interactions in the active site. The OMIT F
o − F
c difference density map is shown as green chicken wire and contoured at 3σ. The protein main chain is shown (more ...)
3.2. Structure overview
The SaPurB subunit can be divided into three domains that combine to form an elongated structure. Domains I and III are placed at either end of the long helical bundle formed by domain II (Fig. 3). Domain I comprises residues 1–93, which form six helices (α1–α6). Domain II, consisting of residues 94–349, forms an elongated helical bundle assembled by α7 through α15 as well as a short two-stranded antiparallel β-sheet (β1, β2) positioned between α7 and α8. Domain III comprises five helices (α16–α20) constructed from residues 350–431. This domain carries the only unresolved portion of the structure: residues 401 and 402 that are on a loop linking α18 with α19 are disordered.
Figure 3 Ribbon diagrams of the SaPurB subunit, the dimer and the tetramer. (a) A subunit is depicted and coloured from the N-terminus (blue) to the C-terminus (red). Residues of the subunit interact with three AMP molecules, which are shown in CPK format (more ...)
Gel filtration indicates that Sa
PurB exists as a single species with an approximate mass of 85 kDa in solution, suggesting the presence of a dimer under the conditions used during the final stage of purification. In the crystal structure, a symmetry-related subunit (B
) related by the crystallographic twofold axis along unit-cell edge a
is the likely partner for such a dimeric species. The A
interface covers an area of approximately 3380 Å2
; almost 16% of the total surface area of the subunit. As observed in orthologues, the crystal structure reveals the formation of a tetramer (Fig. 3), which in the case of Sa
PurB involves rotation of the A
pair about the crystallographic axis along unit-cell edge b
to provide the partner pair of subunits, which are labelled C
. The A
interface extends for approximately 1750 Å2
or 8% of the surface area of a subunit, whilst the smallest interface, that formed between subunits A
, covers an area of about 1600 Å2
or 7.5% of the subunit surface area. Changes in the multimeric state of PurB orthologues have been reported elsewhere. The B. subtilis
enzyme has been shown to exist largely as a dimeric species when the protein concentration is low (~0.1 mg ml−1
); however, as the protein concentration increases the tetrameric species becomes the most abundant form (Palenchar & Colman, 2003
3.3. The active site, interactions with ligands and enzyme mechanism
The tetramer contains four active sites, each formed by regions donated from three different subunits. In the following description, A, B or C after the residue numbers serves to identify which subunit they belong to. We consider distances between functional groups involved in hydrogen-bonding interactions to fall within the range 2.5–3.5 Å.
A sequence alignment of PurB proteins for which structures have been determined was carried out and a subset is presented in Fig. 4 together with the secondary structure of SaPurB. The PurB sequences of T. maritima and B. anthracis share 52 and 70% identity, respectively, to that of SaPurB. The remainder, L. pneumophila, E. coli, Pl. vivax, C. elegans and Py. aerophilum, fall within a range from 20 to 33% identity. A high level of conservation in and around the ligand-binding/active site is noted, with all of the residues implicated in the enzyme mechanism being strictly conserved (Fig. 4).
Figure 4 Primary and secondary structure of SaPurB together with sequence alignment of selected PurB sequences. Dark blue stars indicate residues that interact with ligands in the active site; a dark blue circle identifies a threonine previously thought to be (more ...)
The structure of AMP in the active site of Sa
PurB (Fig. 5) is similar to that in the E. coli
PurB–AMP complex (Tsai et al.
; PDB code 2ptq
). The AMP phosphate accepts hydrogen bonds from Arg4C
a water-mediated association with Arg326A
. The ribose O2′ donates a hydrogen bond to the Arg67A
carbonyl and O3′ accepts hydrogen bonds from the amide of Asp69A
NH2. The glycosidic O4′ is 3.2 Å from Asp69A
OD1, suggesting that the latter is protonated (data not shown). The adenine N1 accepts a hydrogen bond from Thr95A
OG1; N6 donates hydrogen bonds to His141B
NE2 and Gln212A
OE1, residues which are themselves positioned by hydrogen-bonding interactions. The loop Gly182-Ala183-Val184-Gly185, which is conserved in PurB sequences (Fig. 4), is positioned such that the main-chain carbonyl of Ala183 can accept a hydrogen bond from Gln212A
NE2. His141 in Sa
PurB corresponds to His171 in E. coli
PurB and is the catalytic acid for the mechanism (Tsai et al.
; Kozlov et al.
Figure 5 Stereoview of the active site. A molecule of AMP is bound in the active site formed by regions of three of the four subunits. There are four active sites per tetramer, with each active site formed by contributions from three of the four subunits, in this (more ...)
The oxalate binds accepting hydrogen bonds donated from Ser94A
OG and Gln259A
NE2 and a water molecule that in turn interacts with the side chain of Thr95 (Fig. 2). The distance between an oxalate oxygen and Ser94 OG is 3.6 Å and this may indicate a hydrogen bond. In contrast, the oxalate is 3.4 Å distant from adenine N6 but the geometry is not optimal for hydrogen-bond formation (Leonard et al.
). The oxalate occupies a position similar to that observed for fumarate in the active site of human PurB (PDB code 2vd6
) and to that of the succinate moiety observed in the E. coli
PurB–adenylosuccinate complex (PDB code 2ptr
A model of Sa
PurB with adenylosuccinate was produced (Fig. 6) using the human PurB complex to aid the analysis. Two threonines, Thr93 and Thr140 in Sa
PurB, are proposed to bind the succinyl moiety (Segall et al.
). In Sa
PurB, Thr93 is almost 8 Å distant from N6. There is a water-mediated link between Thr140 and Asn270 and the asparagine is the more likely residue to interact with the succinyl group. Gln212 is placed to interact with AMP N6 and a carbonyl on the modelled SAICAR–adenylosuccinate. In Sa
PurB, Gln259 binds the oxalate, is positioned on a loop that displays elevated thermal parameters (data not shown) and could undergo a small conformational change to interact with substrate/products.
Stereoview of the adenylosuccinate model in the active site of SaPurB. The colour scheme is the same as that used in Fig. 2, except that the C atoms of the ligand are shown in light blue.
Two conserved residues, Ser94 and Ser306 in Sa
PurB (Figs. 4 and 5), have previously attracted attention (Segall et al.
). Ser94 is close to His68 and might influence the pK
to favour the protonated form. In Sa
PurB His68 ND1 accepts a hydrogen bond from the Val70 amide and thus NE2 would under normal conditions carry a proton. Our model suggests that Ser94, like Ser306, binds substrate. In Sa
PurB Asp69 orients Arg310 so that it can in turn bind the substrate phosphate.
One side of the Sa
PurB active site is formed by a mobile loop that links β5 and α12. The electron density for this loop is more diffuse than in other parts of the structure and the thermal parameters are higher (data not shown). In most other PurB structures this loop is completely disordered (Fig. 4). In E. coli
PurB this loop is disordered in the apo structure but ligand binding induces a reorganization of the loop to close the ligand-binding site (Tsai et al.
PurB catalyzes a β-elimination that is thought to occur via
a general acid–base uni–bi mechanism (Bridger & Cohen, 1968
; Casey & Lowenstein, 1987
). Previous studies identified His141, His68 and Ser262 as important residues (Lee et al.
; Tsai et al.
). The conserved Ser262 of Sa
PurB is positioned on the mobile loop near the active site and may abstract the Cβ
proton of the substrate, helping to create a carbanion intermediate. His171 is the likely proton donor to N6 to support breakage of the C—N bond. The placement of the adenine in the Sa
PurB active site with respect to His141 suggests it is unlikely that protonation of N1 occurs during catalysis, which was a possibility presented by Tsai et al.
His141 has been suggested to be the catalytic base (Lee et al.
), forming a charge-relay pair with Glu275 to extract a proton from the substrate (Toth & Yeates, 2000
) or alternatively Ser262 (Tsai et al.
; corresponding to Ser295 in E. coli
). The latter assignment was made from structural information on the apo form of E. coli
PurB, in which the side chain of His171 (His141 in Sa
PurB) was rotated away from the position expected for it to play a role in catalysis. In E. coli
PurB structures the highly conserved Ser295 (Ser262 of Sa
PurB) is close to the site of catalysis. This is not the situation in Sa
PurB, where the conformation of the active-site loop holds Ser262 distant from the catalytic site (Fig. 7). Lys268 of Sa
PurB, a strictly conserved residue (Fig. 4), is positioned with its NZ atom 2.8 Å from a succinyl carbonyl. Mutagenesis of the equivalent lysine in B. subtilis
PurB decreased the enzyme activity significantly (Brosius & Colman, 2002
Stereoview of the different conformations displayed by the active-site loop. The SaPurB structure is shown in purple and the E. coli enzyme structure is shown in pink. The adenylosuccinate is depicted as in Fig. 6.
Our model supports the idea that His9 of the E. coli
enzyme, which is equivalent to Sa
PurB His68, is not the catalytic base but rather binds and orients the substrate to interact with fumarate (Tsai et al.
). In Sa
PurB the oxalate is 3.8 Å distant from His68, but our model (Fig. 6) suggests that the distance would be about 3 Å in the substrate complex.
3.4. Dimer interface and the potential for inhibitor development
We were intrigued by two observations. Firstly, a tetramer (or dimer of dimers) is required to produce a functional enzyme since three subunits are required to contribute residues to form an active site. Secondly, there is less sequence conservation amongst the PurB residues involved in the assembly of the quaternary structure than those around the active sites. We decided to consider the dimer interface as a potential target for screening and structure-based drug-discovery methods.
Targeting transient protein–protein interactions or the interface of oligomeric assemblies by screening and structure-based methods to find small-molecule drug-like inhibitors presents a significant challenge (Yin & Hamilton, 2005
). Complicating factors include the high degree of specificity and the large areas of interactions involved in complex formation. Nevertheless, PurB exists in dimeric and tetrameric forms, with enzyme activity restricted to the tetrameric form. Therefore, preventing the formation of or destabilizing the tetramer provides a route to enzyme inhibition. Our interest was furthered by the observation that there is a marked reduction in sequence conservation amongst those residues involved in the assembly of the quaternary structure.
In support of such an approach, we note that in excess of 30 point mutations have been identified that cause human adenylosuccinate lyase deficiency, several of which influence the stability of the tetramer (Spiegel et al.
). For example, the mutation of Lys246 to glutamic acid in the human enzyme is located at the A
subunit interface and results in a predominantly monomeric protein with negligible enzyme activity. In addition, the presence of an Arg194-to-cysteine mutation has been demonstrated to reduce the thermal stability of the B. subtilis
enzyme tetramer, leading to significant impairment of catalytic activity (Ariyananda et al.
Visualization of the electrostatic properties of the dimer–dimer interface involved in tetramer formation for both the human and S. aureus
enzymes was achieved using the Adaptive Poisson-Boltzman Solver
(Baker et al.
; Fig. 8). In Sa
PurB two acidic pockets are formed ~13 Å from either end of the helical coil region formed by domain II of each monomer within the A
dimer. The identical view of the human enzyme reveals a less acidic and smaller pocket. The residues forming the surface of this pocket are contributed from α10, α12 and from the loop linking α12 to α13. The symmetry of the dimer leads to a second pocket being formed 15 Å away and when the symmetry of the tetramer is considered four such pockets are created which combine to form two cavities within the core of the tetramer of approximately 13 × 10 × 10 Å in size. As mentioned perviously, targeting interface regions to disrupt oligomerization presents a significant challenge to drug discovery but this region in Sa
PurB may be more attractive owing to the symmetry of the tetramer. The effect of a small molecule targeted to the pocket on the surface of one subunit could be amplified by the proximity of four such pockets, offering the potential to exploit multivalent ligands.
Figure 8 The dimer–dimer interface of (a) the human enzyme and (b) SaPurB. The protein is depicted as a van der Waals surface coloured blue for basic properties, red for acidic and grey for neutral. For reference, the position of α12 in SaPurB (more ...)