Although general trends in halophilic adaptation have been identified, there are no universal determinants, presumably a reflection on the diversity of protein structure and function in the intracellular environment. Structural analysis has found that the majority of halophilic proteins studied to date are broadly conserved architecturally in comparison to their nonhalophilic counterparts [35
]. The acidic nature of halophilic proteins is largely attributable to an increase in surface-exposed negatively charged residues and is believed to limit protein aggregation [36
]. Homology modelling suggests that HvRPA3 is typical of halophilic proteins in terms of its architectural conservation and increased acidic nature ( and Supplementary Figures
2 and 3).
Modelling suggests that HvRPA3 could bind DNA in a manner seen in other RPA proteins, given the retention of both intercalating aromatic residues and a number of positively charged residues involved in binding. Nonetheless, the binding cleft shows a marked reduction in electropositivity, and high resolution co-crystal structures are required to dissect in detail the role residues flanking these conserved residues play and particularly to identify surface ions that have been suggested by previous studies to be an important mechanism of halophilic adaption of DNA binding [16
Analysis of the P. woesei
TATA-box binding protein demonstrated a strong trend of increasing affinity for DNA binding with increasing salt concentration (0.8 to 1.2
]. However, the intracellular salt concentration of H. volcanii
is significantly higher than P. woesei,
and we wished to extend our analysis to levels more appropriate to H. volcanii
, up to 3
M KCl. Several studies of halophilic DNA binding enzymes have demonstrated increasing enzymatic activity to such levels, strongly inferring DNA binding under these conditions [21
]. Practical difficulties in adapting established protocols to quantify DNA binding in 3
M NaCl/KCl have inevitably limited detailed analysis of binding. Using a combination of SEC, FA, and agarose gel retardation, we have characterised the binding of HvRPA3.
Under physiological salt conditions, in 3
M KCl, HvRPA3 appears to bind ssDNA as a monomer under the equimolar conditions employed in SEC analysis (). In the saturating binding conditions used for FA, the estimated dissociation constant is in the nanomolar range and is only slightly reduced compared to MacRPA3 (). It is clear that HvRPA3 has adapted to function under these extreme salt conditions, with broadly comparable affinity to MacRPA3. Indeed, the slight reduction may reflect the fact that HvRPA3 possesses only a single OB-fold compared to the two identified in MacRPA3. Model fitting yields a Hill coefficient greater than one, indicating positive cooperativity of binding, supported by the binding curve in 3
M KCl, suggestive of cooperative rather than independent binding, as observed for MacRPA3 (). Positive cooperativity has previously been reported for the monomeric SSB T4 gene 32 protein and the authors ascribe this effect to distortions to the DNA and/or direct protein-protein interactions that make subsequent binding of further molecules more favourable [37
Analysis of the NTD suggests that the OB-fold domain is sufficient for DNA-binding activity, albeit with reduced binding affinity of an order of magnitude (), more pronounced than that observed in MacRPA3 (21.1 ± 0.8
]). Presumably, the effect is more profound for HvRPA3 than MacRPA3 since HvRPA3 possesses only a single OB-fold. The equivalent MacRPA3 C-terminal deletion containing a single OB-fold bound more weakly (879.0 ± 624.0
nM), presumably because this protein is optimised for two OB-fold binding.
HvRPA3 forms a dimer in 1
M KCl, seen in both the full-length protein and the NTD and appear by SEC to bind DNA as a dimer (Figures and ). ssDNA was preincubated in an equimolar ratio relative to the concentration of monomer. For full-length HvRPA3, given the lack of residual oligonucleotide peak, this suggests that the ratio of binding is 1 dimer: 2 oligonucleotides. The increased absorption of the protein, DNA complex under these conditions, when compared to the complex in 3
M KCl, suggests a variation in binding between the two protein forms. We interpret this to indicate that, in 1
M KCl, a portion of the oligonucleotide is mobile, hence the increase in UV absorption. A Hill coefficient of 0.9 suggests a degree of negative cooperativity under conditions where an excess of protein is present, suggesting that binding of a protein ligand makes the binding of a second ligand less energetically favourable. This is in contrast to the situation observed in MacRPA3 (a dimer) and probably reflects alterations in HvRPA3 as a consequence of halophilic adaptation. The OB-folds within the dimer may not be optimally oriented for sequential binding and that binding between the sites is potentially independent. This does not appear to be the case for the NTD in 1
M KCl. Fitting a two-site binding model produces a curve of better fit to the FA data (), although the large errors indicate that this model does not entirely describe the binding behaviour. The slight reduction in tryptophan fluorescence observed in the NTD under 1
M KCl conditions suggests that the tryptophan residues are less solvent exposed in the NTD dimer than the full length dimer. Taking both these factors into account, it is plausible that the NTD dimer could bind ssDNA in tandem array as found in the human RPA70-DNA co-crystal structure [27
]. In both 1 and 3
M KCl, an increase in absorption is seen on complexation of NTD and ssDNA, suggesting that a proportion of the 18mer oligonucleotide is mobile. This is consistent with the predicted binding footprint of the OB-fold relative to an 18mer oligonucleotide; the structure of human RPA70 contains tandem OB-folds binding an 8mer ssDNA molecule.
To examine binding in a context more relevant to cellular conditions, the effect of HvRPA3 on circular ssDNA was assessed by EMSA (). Although it is difficult to quantify the exact salt concentration the complexes experience during electrophoresis, two forms of complex are clearly visible. The slower migrating form appears only at higher protein concentrations. FRET analysis of MacRPA3 clearly demonstrated that the protein possesses two DNA binding modes, which are concentration-dependent [6
]. At low concentrations, a wrapping mode predominates. In contrast, at a critical concentration, the protein arranges itself such that the ssDNA becomes stretched and the protein molecules are presumably arranged in tandem array along the length of ssDNA. This latter form would migrate more slowly in EMSA. The presence of this slower migrating form at higher protein concentration suggests that HvRPA3 shows similar behaviour to MacRPA3, despite the variation in multimeric state and number of OB-folds complicating extrapolation.
Robbins and others proposed that the variation in wrapping and stretching modes would present alternate regions of RPA for interaction with protein partners or DNA and could affect ssDNA conformation [10
]. It seems likely that in the organisms that possess several independent SSBs, like M. acetivorans
and H. volcanii
, precise control of each protein and consequently their partners' proteins will be crucial for temporal and spatial regulation of DNA processing. Robbins and others demonstrated that the C-terminal zinc finger region was required for both binding modes in MacRPA3 and for positive cooperativity [6
]. Little difference in cooperativity is seen between the full-length HvRPA3 (1.2 ± 0.1) and NTD (1.1 ± 0.2) and this may reflect differences between the monomeric HvRPA3 and dimeric MacRPA3. The C-terminal domain of HvRPA3 binds zinc with comparable occupancy to the full-length protein and is likely to play a role in regulation of ssDNA binding via redox, as suggested for MacRPA3 [38
Multimerisation associated with decreasing salt concentration is a marked feature of the constructs analysed in this study (Figures and ). The sharp elution profiles observed are consistent with defined multimerisation rather than aggregation associated with the partial unfolding observed in some halophilic proteins at lower salt concentrations [39
]. A similar increase in multimerisation at low salt concentration has been observed with H. volcanii
DNA polymerase X and RadA in our hands (data not shown). Presumably at lower salt levels, the decoration of the protein surface with ions observed structurally [14
] is less than optimal, resulting in exposure of charged residues. In the case of DNA-binding proteins, which are more likely to retain positively charged residues in patches, multimerisation may be driven by salt bridge formation between basic patches and the largely negative surfaces of the neighbouring protein, resulting in the defined peaks observed, rather than aggregation of partially unfolded proteins due to exposure of hydrophobic core residues.
No evidence was found for ssDNA binding in 0.2
M KCl (Figures and ). Some H. volcanii
proteins have been shown to bind DNA in low salt conditions [40
], whereas other enzymes have no activity in the absence of salt, potentially due to abrogation of DNA binding [21
]. This variation likely reflects the diversity of both adaptation and protein function in the cell. Lack of DNA binding and the observed reduction in intrinsic fluorescence in both the full-length and NTD proteins under 0.2
M KCl conditions is consistent with at least partial occlusion of the DNA-binding cleft due to this defined multimerisation, although it is likely that suboptimal surface ion decoration under these conditions contributes to the lack of binding.
Such association under low salt conditions is likely distinct from the dimerisation effect seen in 1
M KCl in both the full-length and NTD proteins. Robbins and others [6
] identified the residues N-terminal to the first OB-fold as central to dimerisation and alignment supports conservation of this region in the single OB-fold RPAs. HvRPA3 is dimeric in 1
M KCl and presumably associates in a similar manner to MacRPA3. 3
M KCl conditions are likely refractory to formation of this dimer interface. Although increases in ion pairs have been commonly noted as a form of halophilic adaptation to stabilise interfaces under high salt conditions, it is not a universal effect. In this instance, the monomeric form has adapted to function in high salt concentrations, reflecting the range of adaptations and diversity of protein function in the cell. To further understand the diversity and adaptation of these proteins, it would be of interest to characterise the DNA binding and multimerisation behaviour of other single OB-fold archaeal RPAs, such as that of Archaeoglobus
from a thermophilic, rather than halophilic source (Supplementary Figure
1). Clustering of the halophilic RPA3 proteins and the equivalent Archaeoglobus
protein is consistent with phylogenetic analysis of other DNA replication proteins, such as the MCM complex [41
As has been noted, the archaea present a melting pot for differing arrangements of SSBs and RPAs and are an excellent model to study the evolution of such a widespread fold as the OB-fold [6
]. The two OB-fold/zinc finger arrangement in the well-characterised MacRPA3 appears to be the most common. This study represents the first characterisation of a single OB-fold-containing-RPA coupled with a zinc finger. It is also the first quantitative study of DNA binding under such extreme salt conditions and represents a significant step forward in the understanding of halophilic adaptation of this most classically salt-sensitive interaction, including the applicability of standard assays to characterise DNA binding under extreme salt conditions. Work is under way to exploit this information in structural studies, to provide detailed characterisation of a DNA protein co-crystal and fully dissect halophilic adaptation for DNA binding.