The final crystallographic model consist of 1686 protein non-H atoms from 224 amino-acid residues of two acostatin heterodimers, 293 water molecules, two sulfate ions and additional residual electron densities tentatively modeled as ten water molecules and another sulfate ion at a lower occupancy. The final refinement statistics are summarized in Table 1. The model includes amino-acid residues 5–63 for subunit A
and 5–62 for subunit C
of the Ile-lacking 62 amino-acid residues (2–63) of the α-chain of acostatin. The model also includes amino-acid residues 4–62 for subunit B
and 4–59 for subunit D
of the 64 amino-acid residues of the β-chain of acostatin. Electron densities are connected for all backbone atoms at the 1σ level except for residues Arg43D
and the tentatively assigned Lys61C
C-terminal residues. Residual electron densities are visible and could potentially be explained on the basis of disorder in the amino-terminal and carboxy-terminal residues and potential alternative conformations including the side chains of Met33B
. The model has been refined to crystallographic R
values of 18.6% and 21.5%, respectively, using all data in the 20.0–1.7 Å resolution range, with root-mean-square deviations (r.m.s.d.s) in bond lengths and bond angles of 0.013 Å and 1.3°, respectively. The geometry of the model was analyzed with MOLPROBITY
(Davis et al.
) and showed 100% of the residues to be in the core region of the Ramachandran plot. Additional stereochemistry was analyzed using Coot
and was found to be in agreement with expected values. One outlier is found in the rotamer conformation of Cys13 from all subunits. Fig. 1 shows representative electron-density fit including Cys13 and a carboxy-terminal group at residue Phe63 from the α-type subunit A
Figure 1 Electron-density fit of the model showing (a) observed differences in the amino-acid sequence of the α- and β-chains of acostatin represented by subunits A and B, respectively, (b) all Cys13 residues identified as rotamer outliers and (more ...)
3.1. Acostatin subunit structures
The overall fold of all acostatin subunits (A, B, C, D) is similar and is depicted for the heterodimer AB in Fig. 2(a). Each subunit structure can be divided into two distinct clusters: an amino-terminal cluster (up to residue 19) and a carboxy-terminal cluster (residue 20 and beyond). In subunits A, B and D, the structure contains three β-sheets each formed by a pair of antiparallel β-strands consisting of residues 8–9 with 14–15, residues 27–28 with 31–32 and residues 38–40 with 49–50; in subunit C only the latter two β-sheets are found. The β-strands are connected by β-turns and flexible loops of different lengths consisting of 4–10 residues. The typical intra-chain disulfide bridges found in the disintegrin family are also observed in the acostatin structure. For all subunits, the distances calculated between the S atoms of the pairs of Cys residues 7–30, 21–27, 26–51 and 39–58 are all within expected disulfide-bond distances. The high content of disulfide bridges in these polypeptides is likely to contribute to the formation of a stable and well defined three-dimensional structure.
Figure 2 (a) Overall structure of the acostatin heterodimer represented by a Cα tracing of subunits A (in blue) and B (in magenta) with disulfide bridges in yellow and the side chains of the RGD binding loops. (b) Superimposition of the Cα tracing (more ...)
Comparison of the structure of the α- and β-chains of acostatin does not reveal major structural changes (Fig. 2
b). The r.m.s.d. for the superimposition of the Cα atoms (residues 5–59) of the α-chains (A/C) and the β-chains (B/D) are 0.88 and 1.02 Å, respectively. In comparison, superimposition of mixed chain types gives r.m.s.d.s of 1.03 Å (A/B), 1.04 Å (A/D), 1.12 Å (C/D) and 1.57 Å (B/C). For all overlays, the deviations are mainly located in the region of residues 38–50, designated as the Arg-Gly-Asp-containing (RGD) loop, with the largest deviation of 4.3 Å at Asp45 when comparing subunits B and C. We also observed that the C-terminal residues 60–62 visible in subunits A, B and C and located adjacent to the RGD loops are found in different orientations. Most of the observed differences can be accounted for by crystal contacts. The comparison of the acostatin fold with the previously determined disintegrin structures of the monomeric trimestatin, the schistatin homodimer and the heterodimer from Echis carinatus does not indicate any major structural rearrangements, as expected from their homologous sequences (Fig. 3). The calculated r.m.s.d. of 1.2–1.5 Å in the superimposition of acostatin with other disintegrin structures is comparable to the overlay of the different chain types of acostatin. Additional conformational differences are also observed in the N-terminal residues.
Sequence alignment of acostatin with trimestatin, schistatin and the E. carinatus heterodimer.
3.2. The αβ acostatin dimer
Specific interactions are found between the α- and β-chains in both the AB
dimers. The N-terminal clusters of each pair of subunits are responsible for dimer formation (Fig. 2
). In both heterodimers, we observed that the distances calculated between the S atoms of Cys residue 8 in one chain and Cys residue 13 in the other chain are all within expected disulfide-bond distances. This pattern of disulfide bridges is identical to the pattern of intermolecular disulfide bonds observed in the homodimer of schistatin (Bilgrami et al.
) and the heterodimer from the E. carinatus
disintegrin (Bilgrami et al.
). These two intermolecular disulfide bridges per heterodimer certainly contribute to the stability and rigidity of the dimer. In addition, two hydrogen-bond distances are observed in the heterodimer AB
between the side-chain N atoms of Asn5 and the carbonyl O atoms of Ala10. In heterodimer CD
the side chains of Asn5 adopt a different rotamer conformation and the carbonyl O atoms of Ala10 and the N atoms of Cys7 are found to interact with the same water molecules. Overall, 8.3% and 9.7% of the accessible surface area of the subunits is buried in the formation of the AB
The overall fold of the acostatin AB and CD dimers is essentially similar. Superimposition of the dimers gives a calculated r.m.s.d. on Cα atoms of 1.82 Å. We observed that dimerization through the N-terminal domains takes place such that the C-terminal domains are facing away from each other. The C-terminal domains in the heterodimeric acostatin are widely separated from each other: the distances between the tips of the C-terminal domains at the Cα atoms of Asp45 are 69.5 and 69.8 Å for the AB and CD dimers, respectively. With such an orientation of the N- and C-terminal regions, it is not surprising to find that the slight differences between the heterodimers are located in this RGD-containing segment. Comparison of the acostatin dimers with previously reported dimeric disintegrins reveals a major overall difference. We observed that the dimerization through the N-terminal clusters generated a different hinge region between the C-terminal domains (Fig. 2
c), with a larger angle in the acostatin dimers. This larger angular hinge moves the tips of the C-terminal domains in acostatin further apart. In comparison, the calculated distances between the Cα atoms of Asp45 in schistatin and the E. carinatus heterodimeric disintegrin are 57.7 and 59.1 Å, respectively.
3.3. An αββα acostatin tetramer
We observed considerable interactions between the acostatin AB and CD heterodimers, as shown in Figs. 2(d) and 2(e). A substantial part (8.2%) of the accessible surface area of the subunits, mostly spread over the N- and C-terminal clusters of the β chains (B and D), is buried in this heterodimer–heterodimer interaction. We identified two regions of hydrophobic interaction involving residue Leu15 in subunit B with Phe32 and Ile54 in subunit D and vice versa. Two hydrogen bonds are also formed, with the carbonyl O atom of Ser19 interacting with the side-chain N atom of Gln20. Two additional hydrogen bonds are formed between subunits B and C; one involved the side-chain carboxyl group of Glu35 (subunit B) and the N atom of Leu15 (subunit C) and the other is made between the carbonyl O atom of Asn52 (subunit B) and the side-chain amino group of Lys14 (subunit C). These residues adopt a different conformation in the A/D subunits and the interactions found between them are mediated through a network of water molecules. The surface complementarities of the AB and CD dimers suggest the possibility of a tetrameric form of acostatin that is best represented by an αββα acostatin tetramer. In the tetrameric form the RGD loops are all pointing in different and almost orthogonal directions. The distance between the Cα atoms of Asp45 of the β-chains is 40.9 Å and that between the α-chains is 80.6 Å. The equivalent distances measured between the AD and BC subunits are 40.5 and 38.7 Å, respectively. This tetrameric arrangement is new among known disintegrin structures but could be an artifact of crystallization. Further experiments are required to identify whether this αββα acostatin complex plays a functional role in vivo.