Extensive counter-ion interactions seen at the surface of subtilisin in an aqueous medium †

The extent of protein and counter-ioninteractions in solution is still far from being fully described and understood. In low dielectric media there is documented evidence that counter-ions do bind and a ﬀ ect enzymatic activity. However, published crystal structures of macro-molecules of biological interest in aqueous solution often do not report the presence of any counter-ions on the surface. The extent of counter-ion interactions within subtilisin in an aqueous medium has been investigated crystallographically using CsCl soak and X-ray wavelength optimised anomalous di ﬀ raction at the Cs K-edge. Ten Cs + , as well as six Cl (cid:1) sites, have been clearly identi ﬁ ed, revealing that in aqueous salt solutions ions can bind at de ﬁ ned points around the protein surface. The counter-ions do not generally interact with formal charges on the protein; formally neutral oxygens, mostly backbone carbonyls, mostly coordinate the Cs + ions. The Cl (cid:1) ion sites are also found likely to be near positive charges on the protein surface. The presence of counter-ions substantially changes the protein surface electrical charge. The surface charge distribution on a protein is commonly discussed in relation to enzyme function. The correct identi ﬁ cation of counter-ions associated with a protein surface is necessary for a proper understanding of an enzyme's function.


Introduction
Ions can interact at many places on a protein molecule. The mechanism of interaction between ions and a protein is far from being fully understood. It is also a matter of debate whether the nature of such interactions is site-specic, or nonspecic i.e. depending on interaction with nearby groups. 1,2 Some interactions take place at relatively strong and specic binding sites, and have been well studied. 3 At higher salt concentrations, more ions become associated, with important and signicant effects on function associated with Hofmeister effects. [4][5][6] Properties following the Hofmeister series include enzyme activity, protein stability, protein-protein interactions, protein crystallization, optical rotation of sugars and amino acids and bacterial growth, as reviewed by Zhang and Cremer. 7 Further elucidation of the sites of ion interactions on protein surfaces is warranted so as to better rationalize ion-specic, Hofmeister, effects.
Molecular dynamics simulations have been used to elucidate the affinity of cations like K + and Na + on protein surfaces in aqueous solutions. These cations show a strong preference for aspartic and glutamic acid side chains, with minor contributions from other side chains or from carbonyl oxygens. 8 The "law of matching water affinity" 9 suggests that K + ,a chaotrope, clusters water molecules only weakly, and, as such, it is mismatched in its water affinity to the strongly hydrated phosphates and carboxylates. A K + ion would form weak, labile interactions with phosphate and carboxylate. 10 Molecular dynamics simulations have also shown that large so anions such as SCN À and I À interact with the backbone of a folded protein via a hybrid binding site that consists of the amide nitrogen and an alpha-carbon, with a Cl À ion binding far more weakly to the site. 11 Protein X-ray crystallography represents a source of information on these interactions with ions. If their identication is specically targeted, condence in ion assignments can be improved. The most common cations used in crystallisation, Na + and NH 4 + , are iso-electronic with water and hence will usually be falsely modelled in a protein X-ray crystal structure as waters, unless distinctive features of their coordination environment are clearly seen. Replacing these common counterions with ones based on heavier elements, but as similar as possible in their size and their chemistry, combined with anomalous diffraction measurements with soer X-rays 12,13 can be used to unambiguously identify their binding sites. 14, 15 We have been exploring such methods to give denitive information about such counter-ion interactions when investigating proteins that had been transferred to a predominantly nonaqueous environment (acetonitrile) and where interactions with counter-ions might be expected to become stronger. The enzyme subtilisin Carlsberg, in acetonitrile, was shown to have no fewer than eleven dened binding sites for Cs + cations and eight for Cl À anions, with clear evidence from an enzyme assay that their binding affected function. 14 We report here that the same enzyme, shown to retain activity in 3.5 M aqueous NaCl, 16 has a substantial number of clear binding sites for these counter-ions. Using the same approach of CsCl soak and optimised anomalous diffraction measurements with soer X-rays, ten Cs + and six Cl À sites are unambiguously identied. This nding is rather unexpected, because the improved solvation of ions in water might be expected to reduce their binding to the protein in comparison with their behaviour in non-aqueous media. The electrical potential surface of the protein will be substantially affected by the presence of these counter-ions.

Experimental section
Crystallization, cryo-protection and Cs soaking of crystals Subtilisin Carlsberg was purchased from Sigma (product code: P5380) and used for crystallization without further purication. The protein powder was dissolved in 330 mM Na cacodylate buffer at pH 5.6 to a concentration of 10 mg mL À1 and crystallized by the batch method from a buffer solution saturated with Na 2 SO 4 $13% (w/v) as precipitant. 14 Needle morphology crystals grew over a period of two weeks to typical dimensions 50 Â 50 Â 400 mm 3 . These 'native' crystals were cryo-protected using a solution of 25% glycerol and 75% buffer reservoir. The caesium derivative was prepared by soaking a single crystal with the above cryoprotectant containing 2 M CsCl for 1 minute.
X-ray diffraction data collection and processing and subsequent protein structure renement Data collection was performed on SRS BL100 17 Daresbury Laboratory. Diffraction data were collected using soer X-rays 13 This journal is © The Royal Society of Chemistry 2014 at an X-ray wavelength of 2.070Å and these data processed using the HKL2000 soware package. 18 Initial crystallographic phases were calculated from the PDB model code 2WUW 14 for the aqueous subtilisin model and 2WUV 14 for the Cs derivative subtilisin model. These protein molecular models were rened with the CCP4 Refmac 5.0 19 and the protein structure regions displaying different conformations were rebuilt with COOT. 20 Anomalous difference Fourier electron density maps were calculated and those peaks contoured at a 4s level or higher were considered signicant. Ion assignments were made on the basis of coordination geometry at a peak, the map peak height, and nally the B-factor and occupancy of each site. The individual Cl À and Cs + ion occupancies were rened with a B-factor set equal to the average of their surrounding protein atoms within a radius of 7Å of each ion. This adjustment was performed using the soware program ION_GRINDER, a Python script which uses the Python interfaces of the Computational Crystallography Toolbox (CCTBX) 21 to work on PDB models. Final R factor (R free ) were 15.9 (22.6) and 15.6 (23.4) for the aqueous subtilisin model and for the Cs derivative subtilisin model respectively. Overall X-ray diffraction data and protein model renement statistics for both are reported in Table 1. The gures presented in this paper were made using CCP4MG. 22 Fig. 1 Crystallographically detected counter-ion sites for subtilisin in water: Cs in grey, Cl in green. Anomalous difference electron density Fourier map contour level is at 4s.

Results and discussion
The clear anomalous signal shows very well dened sites (>4s level Fig. 1 and Table 2). All the caesium sites, and most of the chloride ones, had partial occupancies. The total occupancies for Cs + and Cl À sum to +3.3 and À3.5 respectively, indicating almost zero (À0.2) net charge from the counter-ions. In contrast, the caesium and chloride counter-ions in the previously determined subtilisin crystal structure in acetonitrile had a net charge of À1.7.
In protein crystallography ion's partial occupancies and ion's B factor are a measure of ion mobility likelihood. It is reasonable to think that the protein molecule has only some of the sites fully occupied, and perhaps some are mutually exclusive.
The distribution of cesium ions on the subtilisin surface in an aqueous medium was predicted by molecular dynamics simulations in a preliminary study and showed good agreement with the cation sites observed here crystallographically. 23 The electrical potential surface of subtilisin is altered by the protein-counter-ion interactions at high salt concentrations (Fig. 2). Patches of positive electric potential appear to increase in area on the protein surface overall and in close proximity of the catalytic triad (Fig. 2). Molecular dynamics simulations show a poor agreement of the distribution of chloride ions on the subtilisin surface in an aqueous medium compared with the anion sites observed crystallographically. 23 It was calculated that the surface potential of subtilisin is more negative in aqueous solution than in the crystal, limiting the binding of anions. 23 In fact, patches of negative electric potential do not show an extension of the areas in a high salt condition (Fig. 2).
Protein electrical potential surfaces are commonly calculated and discussed in structure-function relationships. However, such calculations normally do not consider any counter-ions, as well as the intrinsic difficulty of predicting ionisable amino acid side chain pKas, 24 and electric potentials are very different and even opposite in sign if counter-ions are included. 14,23 Clearly, an experimental determination of the cation and anion substructures should be sought for any of those systems where electrical potential surfaces are studied, especially at high salt concentrations.
Previous suggestions are that cations are expected to be coordinated mainly by hydrated protein side-chain carboxylates, with a minor contribution from carbonyl oxygens and other side chains. 8,10 In contrast, we observe that, most of the Cs + ions are predominantly coordinated by formally neutral oxygens, mostly backbone carbonyls: Fig. 3a shows Cs site 2 as an example. Two of the Cs + ions appear to show coordination by the aromatic rings of Tyr residues, presumably in a charge-p interaction (Fig. 3b). Only Cs site 5 is closely associated with a charged protein group, and since it interacts with two different protein molecules, the site is probably an artefact of the crystal packing. The "law of matching water affinity" 9 would suggest relatively weak association of large cations like Cs + with protein carboxylates.
Most of the Cl À ion sites have relatively few of the potential H-bond donors and they are also not near likely protein +ve charges. All the sites show coordinating interactions with an atom in an adjacent protein molecule in the crystal. Cl site 5, for instance, is placed between a Lys amino group and a Cs + ion associated with the symmetry related molecule and it might be a crystal packing site. In these cases we have to accept the possibility that Cl-sites are a result of crystal packing, as conrmed by the results of the molecular dynamics simulations, 23 and would not have particular affinity for counter-ions when the enzyme is in solution. This possibility is also in agreement with the observation that Cl À binds far more weakly to the protein surface. 11 We can also compare the counter-ion sites found in this study with those in previously reported studies, and also with one for our aqueous crystals not soaked with CsCl ( Table 2). The tightly bound Ca-1 is conserved across all reported subtilisin structures.
In the Cs-1 site, three of the previously determined crystal structures reported a Ca 2+ (Table 3). In our aqueous structure that has not had a CsCl soak, we conrm a sodium atom (Na-1)   in this position distinguishing it from a water or from bivalent cation site. In site Cs-2, a water molecule is modelled in all ve previous structures. Four carbonyl oxygens, a side chain hydroxyl and a water molecule coordinate the site. This appears more like octahedral coordination rather than a tetrahedron of waters. For all but one of the other Cs + sites in our structure, a water molecule has been modelled in at least one of the other aqueous structures. In many of these sites the coordination geometry does not obviously indicate a counter-ion rather than a water molecule, so without the optimized anomalous difference electron density data it would not be easily recognized. Our structure also contains a site clearly assigned to a Na + (Na-2).
The same site has been reported as containing Na + in some other structures, but as a second Ca 2+ in others (Table 3). This site showed no anomalous difference Fourier map peak at 2.070 A wavelength, which excluded a Ca 2+ (Ca 2+ f" at this wavelength is 2.95 e À ). The assignment as a calcium is not supported by our ndings.
Turning to the Cl À sites we identify, four of the six sites have been modelled as containing waters in one or more of the previously reported crystal structures.
We can also compare the counter-ion sites with those found in our previous subtilisin crystal structure soaked with CsCl and acetonitrile (2WUV). 14 All but one Cs + site, and all the Cl À sites, were also found in this acetonitrile structure ( Table 2). In addition, the acetonitrile based crystal structure had further counter-ion sites, 2 Cs + and 2 Cl À , which are not found in the aqueous crystal structure. The favoured sites for ion interactions are not very different in acetonitrile and aqueous media in subtilisin.

Conclusions
The present study extends the understanding association of counter-ions with the surface of subtilisin and of proteins in general. Subtilisin shows signicant numbers of dened counter-ion sites even in aqueous conditions i.e. with its large dielectric constant. Most counter-ion sites did not involve close interaction with formal protein charges. The presence of these counter-ions substantially changes the protein electrical potential surface, and which is an effect commonly discussed in relation to function.
The interactions between proteins and counter-ions may have important inuences on enzyme function, especially in systems where quite high salt concentrations are encountered. To understand these effects, it is important to have information about the sites of interaction, and which we provide in this case study.