The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation

Abstract

BackgroundThe high intracellular salt concentration required to maintain a halophilic lifestyle poses challenges to haloarchaeal proteins that must stay soluble, stable and functional in this extreme environment. Proliferating cell nuclear antigen (PCNA) is a fundamental protein involved in maintaining genome integrity, with roles in both DNA replication and repair. To investigate the halophilic adaptation of such a key protein we have crystallised and solved the structure of Haloferax volcanii PCNA (HvPCNA) to a resolution of 2.0 Å.ResultsThe overall architecture of HvPCNA is very similar to other known PCNAs, which are highly structurally conserved. Three commonly observed adaptations in halophilic proteins are higher surface acidity, bound ions and increased numbers of intermolecular ion pairs (in oligomeric proteins). HvPCNA possesses the former two adaptations but not the latter, despite functioning as a homotrimer. Strikingly, the positive surface charge considered key to PCNA's role as a sliding clamp is dramatically reduced in the halophilic protein. Instead, bound cations within the solvation shell of HvPCNA may permit sliding along negatively charged DNA by reducing electrostatic repulsion effects.ConclusionThe extent to which individual proteins adapt to halophilic conditions varies, presumably due to their diverse characteristics and roles within the cell. The number of ion pairs observed in the HvPCNA monomer-monomer interface was unexpectedly low. This may reflect the fact that the trimer is intrinsically stable over a wide range of salt concentrations and therefore additional modifications for trimer maintenance in high salt conditions are not required. Halophilic proteins frequently bind anions and cations and in HvPCNA cation binding may compensate for the remarkable reduction in positive charge in the pore region, to facilitate functional interactions with DNA. In this way, HvPCNA may harness its environment as opposed to simply surviving in extreme halophilic conditions.

An Extremely Halophilic Proteobacterium Combines a Highly Acidic Proteome with a Low Cytoplasmic Potassium Content

“…Therefore, abundant Glu and Asp side chains on the protein surface can effectively compete with KCl for water molecules and thus recruit the hydration shell needed (31) for protein function. This argument can explain the observation that all halophilic proteins exhibit a strong preference for Glu/ Asp-K ϩ interactions at the exclusion of the Lys/Arg-Cl Ϫ interactions and is consistent with the analysis of the surface of obligately halophilic proteins as observed in crystal structures (16,32,33). It is likely that protein surface hydration and protein solubility are strongly related; recent results indicate that negative charges on the protein surface increase protein solubility (34).…”

“…Subsequent mutations that recover protein stability now occur in the protein present in a highly saline cytoplasm and can therefore recruit weakly stabilizing interactions between the already negatively charged protein surface and K ϩ ions through the introduction of additional Glu and Asp residues. Such cation-binding sites have indeed been observed both in biophysical experiments (13) and in crystal structures of obligately halophilic proteins (16,32,33). These weak K ϩ -binding sites resemble the interactions that cause protein denaturation at molar concentrations of urea (36), except that they stabilize the native conformation.…”

The archaeal PCNA proteins

“…Interestingly, out of the four positively charged residues in the eukaryotic PCNA required for DNA binding [21], only one is present in the H. volcanii protein [16,17]. These observations may suggest a different mechanism of interaction with DNA by the halophilic PCNAs.…”

“…To date, the three-dimensional structures of archaeal PCNA proteins have been determined for Pyrococcus furiosus ( Figure 1A) [14], Archaeoglobus fulgidus ( Figure 1B) [15], Haloferax volcanii ( Figure 1C) [16,17], the two PCNA homologues in T. kodakaraensis ( Figure 1D), and Sulfolobus solfataricus ( Figure 1E) [18]. Whereas PCNA from Euryarchaeota form homotrimers, those from Crenarchaeota form heterotrimers.…”

Structural and biophysical characterization of Mycobacterium tuberculosis dodecin Rv1498A

“…In general, thermostability appears to be implemented by a combination of several structural parameters, like, increased interactions of charged residues on surface (Merz et al, 2008) increased number of electrostatic interactions (Lee et al, 2005), hydrophobic-surface burial, improved core packing, greater rigidity, extended secondary structure, shorter loops, higher states of oligomerization and more prolines and less glutamines (Cambillau and Claverie, 2000;Jaenicke, 2000;Luke et al, 2007). The most commonly observed adaptations in halophilic proteins include higher surface acidity (Mevarech et al, 2000), increased number of intermolecular ion pairs in oligomeric proteins (Madern et al, 2000;Winter et al, 2009), and a decrease in hydrophobic residues (Pieper et al, 1998;Esclapez et al, 2007). However, there are exceptions from these known conserved determinants that confer thermophilic and halophilic properties (Sivakumar et al, 2006;Tadeo et al, 2009).…”