Monomer-monomer Interface Research Articles

Modifications to glucose-6-phosphate dehydrogenase for industrial applications: predictions and tests

We have been combining computational methods with experimental tests to gently modify glucose-6-phosphate dehydrogenase (G6PDH) from Leuconostoc mesenteroides for two specific goals. Firstly, it should be immobilized on an inorganic substrate as part of a longer-term project involving microreactor methods. Secondly, the natural enzyme is found as a homodimer, but we would like to have active monomers which would be better suited to the use of small pores in an aerogel based-system. Our approach has involved consideration of available conservation information, structural properties and simple methods for estimating free energy changes. The approach relies completely on the crystal structure deposited in the protein data bank [1]. Covalent binding to a fixed surface involves introducing side-chains with appropriate reactive groups such as the thiols in cysteines, but there is some choice as to groups which can be incorporated into the inorganic surface. Here, the only considerations have been surface accessibility, distance from the active site and evolutionary conservation. One would rather substitute a naturally variable residue than a conserved site. The second aim is more ambitious and speculative. The enzyme is naturally homo-dimeric, but a monomer might be easier to incorporate in the pores of the substrate. The approach here has been to identify sites at the monomer-monomer interface. From this small set of residues, a crude, but fast free energy estimator [2] was used to check substitutions which should destabilize the dimer while not destabilizing the monomer. First results from analytical chromatography and light scattering suggest a substantial monomer population with activity reduced by perhaps two orders of magnitude.

Dynamics and Structural Transitions in Rad51-Single-Stranded DNA Complexes

Repair of DNA double-stranded breaks is essential for maintaining genome integrity and is executed by homologous recombination (HR). In humans, the core catalyst of HR is RAD51, a bacterial RecA-like ATP-dependent recombinase. It forms nucleoprotein filaments around single-stranded DNA (ssDNA) that catalyze strand exchange between regions with homologous DNA sequences. At the RAD51 monomer-monomer interface, ATP binds, is hydrolyzed and dictates the structure and conformation of the filaments. Using a combination of optical trapping and fluorescence microscopy, we have studied how modifications in the monomer-monomer interface may influence filament dynamics at the single molecule level.We observed that two naturally occurring polymorphic variants of RAD51 (RAD51-Q313 and RAD51-K313) behave dramatically different. RAD51-Q313 nuclei bind statically to ssDNA, with small nuclei bound for a shorter time than larger ones. RAD51-K313, on the other hand, can slide and hop along ssDNA. More specifically, we show that RAD51-K313 monomers predominantly move in a diffusive way, while larger nuclei are more likely to bind statically to ssDNA. These results indicate that subtle differences in the local environment of the monomer-monomer interface can have a dramatic impact on filament dynamics, making this interface a likely target for control by mediators or post-translational modifications.In addition, we have analyzed how RAD51-ssDNA filaments respond to tension. We were able to separate the effects of ATP hydrolysis from filament disassembly, revealing a force-induced structural change in RAD51-ssDNA filaments. If exposed to forces above 9 pN, ADP-bound RAD51, naturally in a compressed state, switches to an extended conformation, resembling the one active for strand exchange. We reveal that the energy needed for this transition is between 3 and 4 kBT. Thermal fluctuations can therefore supply sufficient energy to drive this molecular transition, possibly playing a vital role in strand exchange.

Computational Study of RNA Translocation in a Hexameric Helicase

Hexameric helicases are ATP-driven molecular motors that participate in important genetic processes. A particularly interesting helicase is E. coli transcription termination factor Rho, which translocates towards the 3'-end of nascent transcript. It is still an open question of how the ATP hydrolysis cycle is coupled to RNA translocation in Rho. We present results from all-atom molecular dynamics simulations that studied the conformation transitions and the corresponding energy landscape in the hydrolysis cycle based on the available crystal structure of Rho (Nathan D. Thomsen and James M. Berger. 2009, Cell, 139:523-534). We define collective variables involving the conformations of key residues at the monomer-monomer interface in different ATP binding states. The simulations reveal how interface conformational changes propagate around the circular helicase and regulate RNA translocation along the central channel in a collaborative manner. Monomers change their relative positions along the translocation direction based on the ATP binding states. The suggested allosteric inter-monomer communication in Rho is also revealed by network analysis based on cross-correlation of protein motion. Lys326 in each monomer is crucial in ratcheting the RNA and its movement is coupled to the ATP binding state. Arg269 participates directly in linking monomer-monomer communication with the ATP binding state. The simulations further demonstrate the influence of different RNA sequences (poly(U) and poly(C)). Our study, which elucidates the structure-function relationship in Rho, can be extended to other hexameric helicase systems, such as E1 and DnaB, whose crystal structures in complex with substrates are available.

A mutation in a CLC anion channel alters serotonergic neuronal activity in C. elegans

Egg‐laying in C. elegans is controlled by the HSN neurons, which release serotonin to activate egg‐laying muscles. The dominant egl‐42(n995) mutation prevents serotonin release and inhibits egg‐laying. Intracellular Ca2+ imaging demonstrated that HSN activity is reduced in egl‐42 mutants. Whole genome sequencing revealed that egl‐42(n995) is a point mutation in the clh‐3 gene, which encodes the CLC anion channel splice variants CLH‐3a and CLH‐3b. Overexpression of both wild‐type and mutant clh‐3 phenocopies the egl‐42(n995) egg‐laying defect, while clh‐3 deletion leads to hyperactive egg‐laying. The n995 allele is a valine to alanine mutation that is located in membrane helix P, which contributes to the monomer‐monomer interface in CLC homodimers. This residue is highly conserved in all human CLC proteins except CLC‐Ka and CLC‐Kb. CLH‐3a and CLH‐3b mutant channels exhibit increased current amplitude, a strongly depolarized activation voltage, and more rapid voltage dependent activation kinetics. CLH‐3a and CLH‐3b mutants also exhibit loss of pre‐potentiation behavior and greatly reduced sensitivity to inhibition by GCK‐3 kinase, respectively. Our results demonstrate 1) that CLC helix P plays an important role in controlling channel voltage sensitivity and 2) that clh‐3 encoded channels likely regulate HSN membrane potential and hence Ca2+ entry and neuronal excitability.

Crystal Structure of Archaeal Chromatin Protein Alba2-Double-stranded DNA Complex from Aeropyrum pernix K1

All thermophilic and hyperthermophilic archaea encode homologs of dimeric Alba (Sac10b) proteins that bind cooperatively at high density to DNA. Here, we report the 2.0 Å resolution crystal structure of an Alba2 (Ape10b2)-dsDNA complex from Aeropyrum pernix K1. A rectangular tube-like structure encompassing duplex DNA reveals the positively charged residues in the monomer-monomer interface of each dimer packing on either side of the bound dsDNA in successive minor grooves. The extended hairpin loop connecting strands β3 and β4 undergoes significant conformational changes upon DNA binding to accommodate the other Alba2 dimer during oligomerization. Mutational analysis of key interacting residues confirmed the specificity of Alba2-dsDNA interactions.

Correlated motion of protein subdomains and large-scale conformational flexibility of RecA protein filament

Based on X-ray crystallographic data available at Protein Data Bank, we have built molecular dynamics (MD) models of homologous recombinases RecA from E. coli and D. radiodurans. Functional form of RecA enzyme, which is known to be a long helical filament, was approximated by a trimer, simulated in periodic water box. The MD trajectories were analyzed in terms of large-scale conformational motions that could be detectable by neutron and X-ray scattering techniques. The analysis revealed that large-scale RecA monomer dynamics can be described in terms of relative motions of 7 subdomains. Motion of C-terminal domain was the major contributor to the overall dynamics of protein. Principal component analysis (PCA) of the MD trajectories in the atom coordinate space showed that rotation of C-domain is correlated with the conformational changes in the central domain and N-terminal domain, that forms the monomer-monomer interface. Thus, even though C-terminal domain is relatively far from the interface, its orientation is correlated with large-scale filament conformation. PCA of the trajectories in the main chain dihedral angle coordinate space implicates a co-existence of a several different large-scale conformations of the modeled trimer. In order to clarify the relationship of independent domain orientation with large-scale filament conformation, we have performed analysis of independent domain motion and its implications on the filament geometry.

Prediction of the Monomer-Monomer Interface Region of the ß2Ar Homodimer Receptor via Docking Experiments

Increasing body of evidence indicate that G protein-coupled receptors (GPCRs) exist as dimers and oligomers in the cell membrane. The goal of this study is to estimate the homodimeric form of beta-2 adrenergic (s2AR) receptor which is a member of the GPCR family, via multiple docking experiments.The transmembrane domain VI (TMVI) of the receptor is suggested to be a significant part of the interface both experimentally and theoretically [1,2]. The amino acid sequence motif, 75LIXXGVXXG83VXXT, which is proposed to be essential for dimerization in glycophorin A is similarly observed on TMVI of s2AR as in LKTLG276IMMG280TFTL284. A peptide is derived from TMVI consisting of residues from 276 to 296, GIIMGTFTLCWLPFFIVNIVH and blindly docked to one conformer of the monomeric structure using a rigid body approach, via AutoDock v4.0 software tool [3]. Bound conformations were then reevaluated with a knowledge-based scoring function called DSXonline v0.88 [4].Docking results shows that the peptide has the highest binding affinity for TMVI which supports the possible role of TMVI at the interface of a dimeric structure. Also, a residue interface propensity data derived from a set of 32 nonhomologous homodimers [5] is able to identify TMVI as the domain which contains the highest number of residues with the highest interface propensity values.

Evidence of the Proximity of ATP Synthase Subunits 6 (a) in the Inner Mitochondrial Membrane and in the Supramolecular Forms of Saccharomyces cerevisiae ATP Synthase

The involvement of subunit 6 (a) in the interface between yeast ATP synthase monomers has been highlighted. Based on the formation of a disulfide bond and using the unique cysteine 23 as target, we show that two subunits 6 are close in the inner mitochondrial membrane and in the solubilized supramolecular forms of the yeast ATP synthase. In a null mutant devoid of supernumerary subunits e and g that are involved in the stabilization of ATP synthase dimers, ATP synthase monomers are close enough in the inner mitochondrial membrane to make a disulfide bridge between their subunits 6, and this proximity is maintained in detergent extract containing this enzyme. The cross-linking of cysteine 23 located in the N-terminal part of the first transmembrane helix of subunit 6 suggests that this membrane-spanning segment is in contact with its counterpart belonging to the ATP synthase monomer that faces it and participates in the monomer-monomer interface.

The Non-canonical Protein Binding Site at the Monomer-Monomer Interface of Yeast Proliferating Cell Nuclear Antigen (PCNA) Regulates the Rev1-PCNA Interaction and Polζ/Rev1-dependent Translesion DNA Synthesis

Rev1 and DNA polymerase ζ (Polζ) are involved in the tolerance of DNA damage by translesion synthesis (TLS). The proliferating cell nuclear antigen (PCNA), the auxiliary factor of nuclear DNA polymerases, plays an important role in regulating the access of TLS polymerases to the primer terminus. Both Rev1 and Polζ lack the conserved hydrophobic motif that is used by many proteins for the interaction with PCNA at its interdomain connector loop. We have previously reported that the interaction of yeast Polζ with PCNA occurs at an unusual site near the monomer-monomer interface of the trimeric PCNA. Using GST pull-down assays, PCNA-coupled affinity beads pull-down and gel filtration chromatography, we show that the same region is required for the physical interaction of PCNA with the polymerase-associated domain (PAD) of Rev1. The interaction is disrupted by the pol30-113 mutation that results in a double amino acid substitution at the monomer-monomer interface of PCNA. Genetic analysis of the epistatic relationship of the pol30-113 mutation with an array of DNA repair and damage tolerance mutations indicated that PCNA-113 is specifically defective in the Rev1/Polζ-dependent TLS pathway. Taken together, the data suggest that Polζ and Rev1 are unique among PCNA-interacting proteins in using the novel binding site near the intermolecular interface of PCNA. The new mode of Rev1-PCNA binding described here suggests a mechanism by which Rev1 adopts a catalytically inactive configuration at the replication fork.

Analysis of Mammalian Histidine Decarboxylase Dimerization Interface Reveals an Electrostatic Hotspot Important for Catalytic Site Topology and Function.

Selective intervention of mammalian histidine decarboxylase (EC 4.1.1.22) could provide a useful antihistaminic strategy against many different pathologies. It is known that global conformational changes must occur during reaction that involves the monomer-monomer interface of the enzyme. Thus, the dimerization surface is a promising target for histidine decarboxylase inhibition. In this work, a rat apoenzyme structural model is used to analyze the interface of the dimeric active HDC. The dimerization surface mainly involves the fragments 1-213 and 308-371 from both subunits. Part of the overlapping surfaces conforms each catalytic site entrance and the substrate-binding sites. In addition, a cluster of charged residues is located in each overlapping surface, so that both electrostatic hotspots mediate in the interaction between the catalytic sites of the dimeric enzyme. It is experimentally demonstrated that the carboxyl group of aspartate 315 is critical for the proper conformation of the holoenzyme and the progression of the reaction. Comparison to the available information on other evolutionary related enzymes also provides new insights for characterization and intervention of homologous l-amino acid decarboxylases.

RecA K72R Filament Formation Defects Reveal an Oligomeric RecA Species Involved in Filament Extension

Using an ensemble approach, we demonstrate that an oligomeric RecA species is required for the extension phase of RecA filament formation. The RecA K72R mutant protein can bind but not hydrolyze ATP or dATP. When mixed with other RecA variants, RecA K72R causes a drop in the rate of ATP hydrolysis and has been used to study disassembly of hydrolysis-proficient RecA protein filaments. RecA K72R filaments do not form in the presence of ATP but do so when dATP is provided. We demonstrate that in the presence of ATP, RecA K72R is defective for extension of RecA filaments on DNA. This defect is partially rescued when the mutant protein is mixed with sufficient levels of wild type RecA protein. Functional extension complexes form most readily when wild type RecA is in excess of RecA K72R. Thus, RecA K72R inhibits hydrolysis-proficient RecA proteins by interacting with them in solution and preventing the extension phase of filament assembly.

Atomic Force Microscopy Studies Provide Direct Evidence for Dimerization of the HIV Restriction Factor APOBEC3G

APOBEC3G (A3G) is an antiviral protein that binds RNA and single-stranded DNA (ssDNA). The oligomerization state of A3G is likely to be influenced by these nucleic acid interactions. We applied the power of nanoimaging atomic force microscopy technology to characterize the role of ssDNA in A3G oligomerization. We used recombinant human A3G prepared from HEK-293 cells and specially designed DNA substrates that enable free A3G to be distinguished unambiguously from DNA-bound protein complexes. This DNA substrate can be likened to a molecular ruler because it consists of a 235-bp double-stranded DNA visual tag spliced to a 69-nucleotide ssDNA substrate. This hybrid substrate enabled us to use volume measurements to determine A3G stoichiometry in both free and ssDNA-bound states. We observed that free A3G is primarily monomeric, whereas ssDNA-complexed A3G is mostly dimeric. A3G stoichiometry increased slightly with the addition of Mg2+, but dimers still predominated when Mg2+ was depleted. A His-248/His-250 Zn2+-mediated intermolecular bridge was observed in a catalytic domain crystal structure (Protein Data Bank code 3IR2); however, atomic force microscopy analyses showed that the stoichiometry of the A3G-ssDNA complexes changed insignificantly when these residues were mutated to Ala. We conclude that A3G exchanges between oligomeric forms in solution with monomers predominating and that this equilibrium shifts toward dimerization upon binding ssDNA.

Sensing the dissociation of a polymeric enzyme by means of an engineered intrinsic probe

One of the most remarkable characteristics of Brucella lumazine synthase (BLS) is its versatility to undergo reversible dissociation and reassociation as a polymeric scaffold. We have proposed a mechanism of dissociation and unfolding of BLS. Using static light scattering (SLS) analysis, we were able to demonstrate that the decameric assembly dissociates into two different conditions [pH 5 or 2M guanidinium chloride (GdnHCl) pH 7] forming stable folded pentamers. The transition from folded pentamers to unfolded monomers by GdnHCl denaturation is highly cooperative and can be measured by different spectroscopic techniques. In this work, we show the successful insertion of an intrinsic probe to study in more detail the equilibria described in previous publications. For that purpose, we performed single-point mutations of Phe residues 121 and 127, located at the pentamer-pentamer and monomer-monomer interface, respectively, to Trp residues. These mutations produced only a marginal perturbation of the BLS structure. We analyzed the unfolding and stability of the mutants through different techniques: far-and near-UV CD, SLS, dynamic light scattering, and fluorescence spectroscopy. The introduced intrinsic probe could be used to gain insights into the detailed folding and assembly mechanism of this protein.

Crystal Structure of HugZ, a Novel Heme Oxygenase from Helicobacter pylori

The crystal structure of a heme oxygenase (HO) HugZ from Helicobacter pylori complexed with heme has been solved and refined at 1.8 Å resolution. HugZ is part of the iron acquisition mechanism of H. pylori, a major pathogen of human gastroenteric diseases. It is required for the adaptive colonization of H. pylori in hosts. Here, we report that HugZ is distinct from all other characterized HOs. It exists as a dimer in solution and in crystals, and the dimer adopts a split-barrel fold that is often found in FMN-binding proteins but has not been observed in hemoproteins. The heme is located at the intermonomer interface and is bound by both monomers. The heme iron is coordinated by the side chain of His(245) and an azide molecule when it is present in crystallization conditions. Experiments show that Arg(166), which is involved in azide binding, is essential for HugZ enzymatic activity, whereas His(245), surprisingly, is not, implying that HugZ has an enzymatic mechanism distinct from other HOs. The placement of the azide corroborates the observed γ-meso specificity for the heme degradation reaction, in contrast to most known HOs that have α-meso specificity. We demonstrate through sequence and structural comparisons that HugZ belongs to a new heme-binding protein family with a split-barrel fold. Members of this family are widespread in pathogenic bacteria and may play important roles in the iron acquisition of these bacteria.

Structural basis for the stability of a thermophilic methionine adenosyltransferase against guanidinium chloride

The methionine adenosyltransferase from the thermophile Methanococcus jannaschii is fully and irreversibly unfolded in the presence of guanidinium chloride. Unfolding of this dimeric protein is a three-state process in which a dimeric intermediate could be identified. The less stable secondary structural elements of the protein are the C-terminal ends of β-strands E2 and E6, as deduced from the behavior of tyrosine to tryptophan mutants at residues 72 and 170, which are located in the subunit interface. Unraveling of these elements at the monomer interface may soften intersubunit interactions, leading to the observed 85% activity loss. Accumulation of the intermediate was associated with maintenance of residual activity, an increase in the elution volume of the protein upon gel filtration and a decrease in the sedimentation coefficient. Elimination of the remaining enzymatic activity occurred in conjunction with a 50% reduction in helicity and fluorescence alterations illustrating a transient burial of tryptophans at β-strands E2, E3 and E9. The available 3D-model predicted that these β-strands are involved in the central and N-terminal domains of the monomer structure. Severe perturbation of this area of the monomer-monomer interface may destroy the remaining intermolecular interactions, thus leading to dissociation and aggregation. Finally, transition to the denatured state includes completion of the changes detected in the microenvironments around tryptophans included at α-helixes H5 and H6, the loops connecting H5-E8 and E9, β-strands E3 and E12.

Positions 94−98 of the Lactose Repressor N-Subdomain Monomer−Monomer Interface Are Critical for Allosteric Communication

The central region of the LacI N-subdomain monomer-monomer interface includes residues K84, V94, V95, V96, S97, and M98. The side chains of these residues line the β-strands at this interface and interact to create a network of hydrophobic, charged, and polar interactions that significantly rearranges in different functional states of LacI. Prior work showed that converting K84 to an apolar residue or converting V96 to an acidic residue impedes the allosteric response to inducer. Thus, we postulated that a disproportionate number of substitutions in this region of the monomer-monomer interface would alter the complex features of the LacI allosteric response. To explore this hypothesis, acidic, basic, polar, and apolar mutations were introduced at positions 94-98. Despite their varied locations along the β-strands that flank the interface, ∼70% of the mutations impact allosteric behavior, with the most significant effects found for charged substitutions. Of note, many of the LacI variants with minor functional impact exhibited altered stability to urea denaturation. The results confirm the critical role of amino acids 94-98 and indicate that this N-subdomain interface forms a primary pathway in LacI allosteric response.

Conformational Changes in BAK, a Pore-forming Proapoptotic Bcl-2 Family Member, upon Membrane Insertion and Direct Evidence for the Existence of BH3-BH3 Contact Interface in BAK Homo-oligomers

During apoptosis, the pro-apoptotic Bcl-2 family proteins BAK and BAX form large oligomeric pores in the mitochondrial outer membrane. Apoptotic factors, including cytochrome c, are released through these pores from the mitochondrial intermembrane space into the cytoplasm where they initiate the cascade of events leading to cell death. To better understand this pivotal step toward apoptosis, a method was developed to induce membrane permeabilization by BAK in the membrane without using the full-length protein. Using a soluble form of BAK with a hexahistidine tag at the C terminus and a liposomal system containing the Ni(2+)-nitrilotriacetic acid lipid analog that can bind hexahistidine-tagged proteins, BAK oligomers were formed in the presence of the activator protein p7/p15Bid. In this system, we determined the conformational changes in BAK upon membrane insertion by applying the site-directed spin labeling method of EPR to 13 different amino acid locations. Upon membrane insertion, the BH3 domains were reorganized, and the alpha5-alpha6 helical hairpin structure was partially exposed to the membrane environment. The monomer-monomer interface in the oligomeric structure was also mapped by measuring the distance-dependent spin-spin interactions for each residue location. Spin labels attached in the BH3 domain were juxtaposed within 5-10 A distance in the oligomeric form in the membrane. These results are consistent with the current hypothesis that BAK or BAX forms homodimers, and these homodimers assemble into a higher order oligomeric pore. Detailed analyses of the data provide new insights into the structure of the BAX or BAK homodimer.

Unveiling the unfolding pathway of FALS associated G37R SOD1 mutant: a computational study

Although the molecular determinants of Familial Amyotrophic Lateral Sclerosis (FALS) are still largely unknown, previous studies have demonstrated that aggregation of Cu, Zn superoxide dismutase (SOD1) mutants may play a causative role in FALS. It has been proposed that this pathogenic process occurs via a multi-step pathway involving metal loss, dimer dissociation and assembly of misfolded apo-monomers. The G37R, one of the many SOD1 mutations known to be associated to FALS, is difficult to be reconciled with this model because it is located far from the metal sites and the monomer-monomer interface. Consequently, an inspection of all the steps involved in G37R SOD1 misfolding is expected to provide hints in the understanding of the molecular basis of the disease. To this aim, an array of different computational strategies--i.e. Thermodynamic Integration (TI), implicit solvent Constant Temperature Molecular Dynamics (CTMD) and Steered Molecular Dynamics (SMD)--have been applied on the G37R SOD1 mutant. A comparison with parallel studies carried out for the Wild Type (WT) SOD1 pointed out that the mutation decreases the affinity of the protein for the Cu(ii) ion. Implicit solvents MD simulations performed on the two apo proteins revealed that in the mutant SOD1 a novel, stable H-bond network involving Arg37, Lys91, Lys36 and Leu38 is created thus confirming a pivotal role of this region in driving the biophysical properties of the entire protein. Finally, the presence of energetic "traps" in the force vs. elongation curves of G37R SOD1 is an indicator of the existence of intermediate states along the unfolding pathway which may lead to abnormal conformers. Our results support a general theory suggesting that the two major hypotheses regarding mutant SOD1 toxicity, i.e. aberrant copper redox chemistry and SOD1 misfolding are causally linked. In fact it is shown that the G37R mutation, although located far away the active site, may induce subtle modification in SOD1 leading to the loosening of metal binding and to the formation of metastable intermediate states along the unfolding pathway.

Kinetic Consequences of Mutations at an Allosteric Site in Arginase from the Thermophile Bacillus Caldovelox

Arginase is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. It is involved in ureagenesis and the control of arginine levels for production of proline, creatine, polyamines, and nitric oxide. The crystal structure of the enzyme from the extreme thermophile Bacillus caldovelox reveal a second arginine-binding site, located at the monomer-monomer interface. Binding of the guanidinum group of L-arginine by bidentate hydrogen bonds to Glu256 in one monomer and bifurcated hydrogen bond to Asp199 in the neighbor, was suggested to generate a catalytically competent conformation.

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

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.