ExoVII was discovered 35 years ago, but its structure and molecular mechanism have remained substantially unexplored (16
). ExoVII comprises two subunits, XseA and XseB. The crystal structure of XseB from B. pertussis
has been determined (doi:10.2210/pdb1vp7/pdb). Recently, Larrea et al
) characterized experimentally the XseA/B homologs from Thermatoga maritima
, TM1768 and TM1769. They predicted that the large subunit of ExoVII is composed of two domains: the N-terminal OB-fold domain and a C-terminal domain termed ‘ExoVII_Large’. Here, we present a structural model for XseA, as well as for the XseA–XseB interactions that lead to formation of the ExoVII complex. Our results show that XseA consists of four domains: the OB-fold domain, the catalytic domain, a helical domain and a C-terminal extension.
We demonstrated that residues D155, R205, H238 and D241 are essential for the nuclease activity of E. coli
ExoVII. The substitution of these residues inactivated the enzyme, but they did not hinder DNA binding. In the structural model of the putative catalytic domain, they are located on the same face of the protein; they do not form a very tight cluster, which can be attributed to the relative precision of the model. Thus, we propose that all or some of these residues belong to the active site of XseA. Recently, it has been shown that D235 and D240 residues are essential for the nuclease activity of T. maritima
). Based on these findings and sequence analyses of XseA family, the authors showed that these residues are conserved in E. coli
(D241, D246) but also in other XseA homologs, and that the conserved region has a motif RGGG(x)nGHxxDxxxxD. The motif identified by Larrea et al
) includes residues R205, H238 and D241 of E. coli
XseA. In this study, we have shown that D155, which is located outside of the conserved motif, is also important for the activity of the E. coli
ExoVII enzyme. On the other hand, we demonstrated that a substitution of residue D246 in E. coli
XseA did not inactivate the enzyme. This residue corresponds to D240 in T. maritima
, which is essential for the activity of that enzyme. Larrea et al
. substituted both T. maritima
XseA residues (D234 and D240) simultaneously; therefore, it is difficult to ascertain the role of individual residues. It is currently unknown if residues in T. maritima
that correspond to E. coli
D155, R205, H238 are essential for its nuclease activity.
The role of Q177, D246, D250 and T255 in catalysis remains unclear. Sequence analyses revealed that these residues are highly conserved in the XseA family and substitution of these residues to alanine reduced the activity to the level of about 60–80% of the wt enzyme (data not shown). In the structural model of the XseA catalytic domain, these residues are located around the putative active site; therefore, their substitution could destabilize the active site or the interactions with the DNA, without direct interference with catalysis.
Despite the fact that magnesium ions are crucial for the activity of ExoVII from T. maritima
, the activity of ExoVII from E. coli
does not depend on the presence of metal ions. Larrea et al
. postulated that ExoVII family can be divided into two groups: E. coli
-like (resistant to EDTA) and T. maritima
-like (sensitive to EDTA) (19
). Our phylogenetic analysis indicates that T. maritima
-like proteins are outliers of the family and suggests that the majority of members are E. coli
-like. The distribution of magnesium dependence in the XseA family remains to be analyzed experimentally on members of the most divergent branches, whose selection can be aided by our phylogenetic tree.
The OB-fold domain alone (XseA variant Δ104-C-term) is capable of DNA binding, while the ExoVII variant without the OB-fold domain in XseA (Δ1–103) lost this capability. The OB-fold variants: F63A and R64E/R68E/R69E showed almost complete loss of DNA binding. This result supports the model-based prediction that F63 is indeed important for DNA binding. The predicted role of R64, R68 and/or R69 is also supported, although at this stage, the role of individual Arg residues remains unknown. Substitution Q96A decreased the binding of DNA to about 50% in comparison to that of XseA variant Δ104-C-term (which was used as a reference). This residue probably also takes part in binding DNA, as predicted with the help of the model, but clearly it is not essential for this process. Surprisingly, the full-length XseA alone (without XseB) does not form a complex with DNA, while inactive ExoVII variants (with substitutions in the XseA catalytic domain, and in the presence of XseB) are able to bind DNA. We speculate that the XseA protein without XseB does not fold properly and therefore is unable to bind DNA. We were able to isolate and purify XseA and XseB separately, but when the two subunits were mixed together, denaturated and refolded, they failed to form a catalytically active complex (data not shown), suggesting that the complex formation between the subunits may begin already at the stage of the protein synthesis.
We attempted to identify the interaction site(s) between XseA and XseB and gain information about the structural arrangement of the complex. The bioinformatic analyses showed that XseA contains a region consisting of three α-helices predicted to be involved in coiled-coil-like interactions. This has led us to a hypothesis that these helices may be involved in interactions with XseB, which has been confirmed experimentally. We did not observe XseB binding to the XseA variant that had all three helices deleted (Δ123), and XseB binding was decreased for other XseA variants that had one or two helices deleted. While deletions of individual helices in XseA only decreased the XseB binding, they all abolished the nuclease activity of ExoVII. This result is surprising, given the observation that XseA from T. maritima is active, yet natively contains only two coiled-coil helices and hence corresponds structurally to a variant of E. coli XseA with one helix deleted. It may indicate that XseA forms a functionally active complex only when it interacts with a precise number of XseB subunits, a possibly different number in the case of ExoVII enzymes in different species. Our attempts to express and purify isolated helices were not successful; therefore, we could not examine how many XseB subunits are bound by one helix.
It has been suggested, based on the results of size exclusion chromatography, sedimentation in sucrose gradient (16
), and native gel electrophoresis, that ExoVII from E. coli
and T. maritima
are pentamers, composed of one XseA subunit and four XseB subunits (10
). The results from our size exclusion experiments suggested that E. coli
ExoVII is actually a heptamer, which consists of one XseA subunit and six XseB subunits. This result agrees with the bioinformatic-based prediction that a single helical segment of XseA can bind one XseB dimer. Consequently, we predict that T. maritima
XseA, which has only two coiled-coil units, should bind only four XseB subunits.
We found that the full-length XseA, which possesses the OB-fold domain responsible for DNA binding, is not able to form a complex with DNA, unless it is also complexed with XseB. Earlier, it has been shown that ExoVII activity was reduced when the XseB protein was overexpressed (10
). It was demonstrated that the transcription of xseB
gene was induced upon interaction of Neisseria meningitidis
with host cells (41
). In this case, the up-regulation of XseB resulted in an induction of a DNA repair system and an increase of frequency of phase variation. That XseB expression is regulated, which in turn may influence the ExoVII activity, is suggested by the finding that a transcription factor SlyA that contributes to the virulence of Salmonella typhimurium
, binds upstream of the xseB
). Thus, we hypothesize that the binding of XseB to XseA is the key element that regulates the activity of ExoVII. The exact nature of XseA–XseB interactions and the structure of the ExoVII complex remain to be elucidated.