The single polypeptide of O67745_AQUAE has a ‘bowl-with-handle’ conformation, with a diameter of approximately 60 Å at its widest point and a depth of 35 Å (Fig. 1). It contains 29 secondary-structure elements consisting of 20 α-helices and nine β-strands. The protein exhibits four antiparallel β-sheets. The first antiparallel two-stranded sheet consists of strands β1 (residues 3–7) and β2 (residues 10–15) and identifies the beginning of the polypeptide chain. This structure is followed by two nearly perpendicular short α-helices α1 (residues 17–24) and α2 (residues 26–33). The third short helix α3 (residues 39–43) together with two longer helices α4 (residues 51–69) and α5 (residues 78–86) constitute the bottom of the ‘bowl’. At approximately residue 86, where helix α5 ends, the polypeptide contains residues that form the sides of the ‘bowl’. The secondary-structure elements that participate in building the ‘bowl’ include helices α6–α17 (α6, residues 95–99; α7, residues 108–115; α8, residues 117–125; α9, residues 128–139; α10, residues 145–155; α11, residues 160–173; α12, residues 183–188; α13, residues 203–222; α14, residues 224–242; α15, residues 249–254; α16, residues 257–266; α17, residues 271–278). The second consecutive antiparallel sheet is formed by strands β3 (residues 189–192) and β4 (residues 195–199). Strand β5 (residues 284–289) and helix α18 (residues 292–305) begin to form the ‘handle’, which is completed by the last helix α20 (residues 358–368). A long stretch of residues from amino acids 311–356 form the other side of the ‘bowl’ and consists of strands β6 (residues 311–318), β7 (residues 324–328) and β8 (residues 331–338), helix α19 (residues 341–346) and strand β9 (residues 349–356). Strands β7 and β8 make up the third short antiparallel sheet, while strands β5, β6 and β9 constitute the fourth and longest antiparallel sheet. Notably, helices α10 and α11 would be combined into one helix if it was not for an intervening four-amino-acid loop which does not conform to the requirements for the ϕ and ψ angles of α-helices.
Figure 1 Stereoview of the polypeptide fold of O67745_AQUAE. Approximately every 25th residue is numbered. Residues 1–285 and 305–355 constitute the ‘bowl’ and residues 286–304 and 356–369 form the ‘handle’. (more ...)
The amino-acid sequence of O67745_AQUAE is identified by Pfam
(Bateman et al.
) as belonging to the HD-domain family, a member of a superfamily of enzymes with a predicted or known phosphohydrolase activity. These enzymes appear to be involved in nucleic acid metabolism, signal transduction and possibly other functions in bacteria, archaea and eukaryotes. Since all the highly conserved residues within the HD superfamily are histidines or aspartates, the coordination of divalent cations could be essential for the activity of these proteins. A sequence alignment of several members of subfamily PF01966 is presented in Fig. 2. A metal ion is found at the bottom of the ‘bowl’ and a phosphate ion is present in the coordination sphere of the ion (Fig. 3). Based on the position of the metal and the phosphate ion, this region of the structure has been identified as the putative active site. The metal ion was modeled as Zn2+
based on the tetrahedral nature of its first coordination sphere. The occupancy was adjusted manually to 0.5 according to both the B
factors (Zn1 has a B
factor of 17 Å2
and Zn2 37 Å2
) of neighboring atoms and the presence of positive or negative (F
) electron density.
Figure 2 Amino-acid sequence alignment of several members of the HD superfamily. Conserved residues are shaded red. The fact that all the highly conserved residues in the HD superfamily are histidines or aspartates suggests that coordination of divalent cations (more ...)
Stereoview of the putative active site of O67745_AQUAE.
The asymmetric unit of O67745_AQUAE contains two polypeptides that interact through the bottom of the ‘bowl’ as shown in Fig. 4.
Stereoview of the interaction between two molecules of O67745_AQUAE related by NCS.
(Krissinel & Henrick, 2005
) server finds that the complexation score for the two molecules in the asymmetric unit is only 0.063, which implies that the interface is not significant for complexation and may solely be a result of crystal packing. Only about 11% of the residues from each polypeptide participate in the interface and these residues account for less than 7% of the surface area (the total surface area of a single polypeptide is about 17 200 Å2
). The PISA
server also suggests that the most probable complexation state for O67745_AQUAE would be a tetramer (Fig. 5), in which the buried area would account for nearly 40% of the total surface area of the tetramer (56 136 Å2
total surface area compared with 22 413 Å2
buried surface area).
Dimerization (a) and tetramerization (b) of O67745_AQUAE.
Experimental data on the oligomerization of O67745_AQUAE is limited to dynamic light scattering which, across a wide range of protein concentrations, consistently showed a monodisperse peak with a hydrodynamic radius of 35 Å corresponding to a molecular weight of about 50 kDa. Size-exclusion chromatography or sedimentation and static light-scattering experiments may have provided a more reliable and definite determination of the oligomerization state of O67745_AQUAE in solution. Although the dynamic light-scattering data suggest no complexation in solution and the PISA server predicts that the interface between the two molecules in the asymmetric unit is not significant for complexation, we still hypothesize that O67745_AQUAE is a dimer in solution. Such a hypothesis is based on the presence of two GDP molecules detected at the interface between two polypeptides of the asymmetric unit (Fig. 6).
Figure 6 (a) Fragment of the electron density along with a model of the protein with GDP and glycerol fitted. Residues in green and in red belong to different polypeptide chains of the asymmetric unit. Eight sites within the asymmetric unit were fitted with glycerol (more ...)
Upon dimerization, the two polypeptides in the asymmetric unit create two similar and highly positively charged deep clefts that accommodate the GDP molecules (Fig. 7). Amino acids from both polypeptides in the asymmetric unit coordinate the GDP molecules. Arg280 and His282 from one polypeptide unit and Lys3, Glu4, Asp24, Glu29 and Arg32 from the other polypeptide unit are within hydrogen-bonding distance of the docked GDP molecule. The second GDP molecule of the asymmetric unit has nearly identical coordination.
Figure 7 The positively charged cleft formed on the interface of two polypeptides of the asymmetric unit accommodates a GDP molecule. The surface electrostatic potential was calculated using the Adaptive Poisson-Boltzmann Solver software package (Baker et al. (more ...)
3.1. Comparison to structural homologues
In an attempt to establish the identity and subsequently assign the polypeptide fold of O67745_AQUAE, a spatial similarity search was carried out using the DALI
server (Holm & Sander, 1999
). Four structures were found with Z
scores above 6. The highest score was with the exopolyphosphatase from Escherichia coli
(PDB code 1u6z
; Hasson et al.
, unpublished work). 147 residues of the C-terminal second domain of exopolyphosphatase match the N-terminal portion of O67745_AQUAE with a root-mean-square deviation (r.m.s.d.) value of 3.2 Å. Within the aligned regions of these two polypeptides the sequence identity is only 12% owing to 17 gaps in the alignment spread over 320 residues of O67745_AQUAE.
The second highest Z
score was found with the enteropathogenic E. coli
type III secretion-system protein EscJ (PDB code 1yj7
; Yip et al.
). The N-terminal 54 residues of EscJ, equivalent to one third of the protein, very closely match (r.m.s.d. value of 1.6 Å) the fold of the ‘handle’ of O67745_AQUAE, although the sequence identity is only 13%. In the supermolecular complex of the type III secretion system, the N-termini of all the EscJ molecules are localized towards the broad face of the complex and are believed to be involved in the anchoring of the protein to the inner membrane. Whether or not the ‘handle’ in O67745_AQUAE plays a similar anchoring role is currently unknown.
The third highest Z
score of 6.7 was with the hypothetical protein AF1432 (PDB code 1ynb
; Dong et al.
, unpublished), where 133 residues of the 167-amino-acid protein match O67745_AQUAE with a sequence identity of 11%, the insertion of 15 gaps and an r.m.s.d. value of 4.6 Å.
The fourth highest Z
score of 6.6 was found by a comparison of the protein with phosphodiesterase 5 (PDE5; PDB code 1rkp
; Huai et al.
). In PDE5, 157 residues of the 311-amino-acid polypeptide match O67745_AQUAE with an r.m.s.d. value of 4.2 Å. Superimposition of the structures of O67745_AQUAE and PDE5 shows the distance between zinc ions in the two polypeptides to be only 1.5 Å, with most of the identical residues shared by the proteins occupying very close positions in the vicinity of the metal ion. Histidines 54, 84 and 88 of O67745_AQUAE align with histidines 617, 653 and 657 of PDE5, and aspartic acids 85 and 151 of O67745_AQUAE align with aspartic acids 654 and 764 of PDE5. The only mismatch of residues is Arg51 of O67745_AQUAE and His613 of PDE5. Notably, the NH1 atom of Arg51, which is within hydrogen-bonding distance of the zinc-bound phosphate ion, is only 1.2 Å away from the NE2 atom of His613. In addition, the glycerol molecule found in the putative active site of O67745_AQUAE aligns well with the inhibitor IBMX found in the active site of PDE5.
All the structures to which DALI found some degree of similarity belong to different Pfam families.
Based on the presence of two GDP molecules and the similarity to phosphodiesterase PDE5 found by DALI, we would like to hypothesize that O67745_AQUAE is a homodimeric enzyme belonging to one of the 23 known phosphoesterases/phosphodiesterase families, making the fold space of phosphodiesterases even broader.