Michele Cianci*a,
Jacopo Negronia,
John R. Helliwellb and
Peter J. Hallingc
aEuropean Molecular Biology Laboratory c/o DESY, 22603 Hamburg, Germany. E-mail: m.cianci@embl-hamburg.de; Fax: +49 40 89902149; Tel: +49 40 89902118
bSchool of Chemistry, University of Manchester, Oxford road, Manchester, M13 9PL, UK. E-mail: j.r.helliwell@man.ac.uk
cWestCHEM, Department of P & A Chemistry, University of Strathclyde, Glasgow G1 1XL, UK. E-mail: p.j.halling@strath.ac.uk; Fax: +44 (0)141 548 4822; Tel: +44 (0)141 548 2683
First published on 18th July 2014
The extent of protein and counter-ion interactions 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 affect enzymatic activity. However, published crystal structures of macromolecules 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 diffraction at the Cs K-edge. Ten Cs+, as well as six Cl− sites, have been clearly identified, revealing that in aqueous salt solutions ions can bind at defined 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− 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 identification of counter-ions associated with a protein surface is necessary for a proper understanding of an enzyme's function.
Some interactions take place at relatively strong and specific binding sites, and have been well studied.3 At higher salt concentrations, more ions become associated, with important and significant effects on function associated with Hofmeister effects.4–6 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-specific, 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 soft anions such as SCN− and I− 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− 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 identification is specifically targeted, confidence in ion assignments can be improved. The most common cations used in crystallisation, Na+ and NH4+, 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 counter-ions with ones based on heavier elements, but as similar as possible in their size and their chemistry, combined with anomalous diffraction measurements with softer X-rays12,13 can be used to unambiguously identify their binding sites.14,15 We have been exploring such methods to give definitive information about such counter-ion interactions when investigating proteins that had been transferred to a predominantly non-aqueous 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 defined binding sites for Cs+ cations and eight for Cl− 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 softer X-rays, ten Cs+ and six Cl− sites are unambiguously identified. This finding 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.
Aqueous | Cs-Aqueous | |
---|---|---|
a The values in parentheses are for the highest-resolution shell.b Rmerge = ΣhklΣi |Ii(hkl) − 〈I(hkl)〉|/ΣhklΣi |Ii(hkl)|.c Data taken from PROCHECK.25 | ||
PDB deposition code | 4c3v | 4c3u |
Crystallographic data | ||
Wavelength (Å) | 2.070 | 2.070 |
Cell parameter (a, b, c) | 51.89, 55.07, 75.31 | 52.17, 55.23, 75.85 |
Space group | P212121 | P212121 |
Rmergea,b | 0.07 (0.236) | 0.124 (0.210) |
Completenessa | 100.0 (100.0) | 99.9 (99.2) |
Unique reflections | 10628 (1025) | 10346 (994) |
Redundancya | 10.1 (8.5) | 5.8 (4.6) |
Resolutiona | 50.0–2.26 (2.34–2.26) | 50.0–2.28 (2.36–2.28) |
I/σ(I) | 55.61 (14.3) | 10.6 (6.9) |
Average Bfactor from Wilson plotc (Å2) | 20.07 | 23.63 |
Matthews' coefficient (Vm) | 1.98 | 2.01 |
Solvent content (Vs) | 37.7 | 38.7 |
Refinement results | ||
Rfactor (Rfree) | 15.9 (22.6) | 15.6 (23.4) |
Bond lengths refined atoms (rms; weight) | 0.016; 0.019 | 0.014; 0.019 |
Bond angles refined atoms (rms; weight) | 1.840; 1.948 | 1.718; 1.941 |
Cruickshank's DPI for coordinate error (Å) | 0.39 | 0.41 |
DPI based on free R factor (Å) | 0.23 | 0.25 |
Number of bound waters and ions | ||
Waters | 120 | 118 |
Cesium | — | 12 |
Chlorine | — | 6 |
Calcium | 1 | 1 |
Sodium | 3 | 1 |
Sulfate | 3 | — |
Ramachandran plotc | ||
Most favoured regions (%) | 89.0 | 87.3 |
Additional allowed regions (%) | 11.0 | 12.7 |
Generously allowed regions (%) | 0.0 | 0.0 |
Disallowed regions (%) | 0.0 | 0.0 |
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 4σ. |
Atom | Occ. | B-fac. | σ | Potentially charged groups within 0.8 nm | Coordinating atoms within 0.4 nm |
---|---|---|---|---|---|
a Atoms are identified by the usual PDB codes, with O, being the alpha-carbonyl oxygen; * – the amino groups of Lys136 and Lys170 appear to be in salt bridges with the carboxylates of Asp140 and Glu195 respectively; ** – these groups are in an adjacent protein molecule in the crystal. | |||||
Cs-1 | 0.73 | 30.31 | 29 | His39 ND (0.68), Cl3** (0.76) | Leu42 O (0.30), His39 O (0.30), Ala37 O (0.30), W40 (0.34) |
Cs-2 | 0.79 | 31.37 | 29 | Cl8 (0.49), Glu195 OE (0.71) | Ala194 O (0.30), Leu196 O (0.31), Ser260 O (0.31), Gly193 O (0.33), Ser260 OG (0.39), W34 (0.35), W152 (0.37) |
Cs-3 | 0.26 | 31.86 | 11 | Cl7 (0.72), Asp60 OD (0.79) | Ser98 O (0.33), Gly61 O (0.33), W98 (0.25) |
Cs-5 | 0.36 | 33.76 | 11 | Glu54** OE (0.39) | Tyr256 ring C's (0.34–0.39), W127** (0.31), Glu54** OE (0.39), Ala52** O (0.29) |
Cs-6 | 0.24 | 31.37 | 6 | Arg247 NH (0.72) | Tyr143 O (0.33), Ser244 OG (0.37), Tyr143 ring C's (0.37–0.39), W113 (0.23) |
Cs-7 | 0.24 | 35.68 | 7 | Asp120 OD (0.57), Cl3 (0.71) | Leu241 O (0.28), Pro239 O (0.32), W46 (0.27), W62 (0.28), W119 (0.32) |
Cs-9 | 0.22 | 29.49 | 4 | Ala1 N (0.50), Asp41 OD (0.77) | Thr3 OG (0.28), Ala1 O (0.30), Gly80 O (0.33), Thr3 N (0.38), W53 (0.27), W107** (0.23) |
Cs-10 | 0.12 | 34.61 | 5 | Cl5 (0.42), Arg186 NH (0.71) | Ser156 O (0.33), Asn158 O (0.34) |
Cs-11 | 0.12 | 29.94 | 4 | None | Gly47 O (0.35), Tyr57 OH (0.40) |
Cs-12 | 0.24 | 45.47 | 5 | None | Ser159 O (0.31), Thr162 OG (0.31), Ser159 OG (0.37) |
Cl-1 | 1.00 | 16.00 | 22 | Asp181 OD (0.76) | Asn185 ND (0.30), Ser184 O (0.33), Tyr263 OH (0.38), Leu257 O (0.38), W123 (0.34), Gln2** O (0.28), W54** (0.36) |
Cl-3 | 0.74 | 39.36 | 6 | Cs7 (0.71), Cs1** (0.76) | Asn240 O (0.30), Gln245 NE (0.32) |
Cl-4 | 0.64 | 31.88 | 5 | Glu195 OE (0.41), Asp172 OD (0.51), Na2 (0.63), Lys170 NZ (0.67*), Lys136 NZ (0.73*), Tyr167 OH (0.61) | Lys170 O (0.30), W3 (0.35), W87 (0.40) |
Cl-5 | 0.48 | 44.62 | 5 | Lys15** NZ (0.38), Cs10 (0.42), Arg186 NH (0.66) | Asn158 O (0.29), Asn158 ND (0.29) |
Cl-7 | 0.39 | 32.75 | 5 | Lys265** NZ (0.69), Cs3 (0.72) | Ser99 O (0.30), W147 (0.33), Asn248** ND (0.31) |
Cl-8 | 0.27 | 25.81 | 4 | Cs2 (0.49), Glu195 OE (0.66), Glu197 OE (0.76) | Ala194 O (0.30), W22 (0.32) |
In protein crystallography ion's partial occupancies and ion's Bfactor 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).
Fig. 2 Change of electrostatic potential of the protein surface. Red areas have a negative potential, blue areas a positive potential, white areas are neutral. (a) Subtilisin soaked in a CsCl salt aqueous solution; (b) in aqueous solution; (c and d) views at 180°. Electrostatic potential of the protein surface were computed using CCP4MG.22 |
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–π 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− 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 confirmed 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− 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 Ca2+ (Table 3). In our aqueous structure that has not had a CsCl soak, we confirm 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 five 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 Ca2+ in others (Table 3). This site showed no anomalous difference Fourier map peak at 2.070 Å wavelength, which excluded a Ca2+ (Ca2+ f” at this wavelength is 2.95 e−). The assignment as a calcium is not supported by our findings.
Cs ion structure | Native structure | 1CSE | 1C3L | 1SCA | 1SCN | 2SEC | 2WUV | 2WUW | 1SCB | 1SCD | 1AF4 | 1BFU |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Water | Water | Water | Water | Water | Water | Water | Acetonitrile | Acetonitrile | Acetonitrile | Acetonitrile | Dioxane 100% | Dioxane 20% |
Cs-1 | Na-1 | — | — | Ca-403 | Ca-403 | Ca-278 | Cs-1 | Na-1277 | W371 | W598 | — | W315 |
Cs-2 | Na-3 | W57 | W398 | W578 | W582 | W382 | Cs-2 | W2114 | W357 | W539 | — | — |
Cs-3 | — | W795 | — | W681 | — | — | Cs-3 | — | — | — | — | — |
— | — | — | — | — | — | — | Cs-4 | — | — | W597 | — | — |
Cs-5 | — | — | — | W577 | — | — | Cs-5 | — | — | — | — | — |
Cs-6 | — | — | W389 | — | — | W433 | Cs-6 | — | W113 | — | — | — |
Cs-7 | — | W602 | — | — | — | — | Cs-7 | — | W398 | — | — | — |
— | — | W519 | — | — | — | — | Cs-8 | — | — | W511 | — | — |
Cs-9 | W12 | W427 | W351 | W627 | — | W422 | Cs-9 | W2039 | — | W629 | W322 | W336 |
Cs-10 | — | W698 | — | — | — | — | Cs-10 | — | — | — | — | — |
Cs-11 | W125 | — | — | — | — | — | Cs-11 | — | — | — | — | — |
Cs-12 | — | — | — | — | — | — | — | — | — | — | — | — |
Ca-1 | Ca-1 | Ca-1 | Ca-1 | Ca-1 | Ca-1 | Ca-1 | Ca-1 | Ca-1279 | Ca-1 | Ca-1 | Ca-1 | Ca-1 |
Na-2 | Na-2 | Ca-401 | Ca-284 | Na-402 | — | Ca-277 | Na-2 | Na-1278 | W359 | Ca-403 | W305 | W294 |
Cl-1 | W18 | W885 | — | — | — | W489 | Cl-1 | — | — | W582 | W299 | — |
— | W97 | W503 | W404 | W662 | W665 | W484 | Cl-2 | — | W285 | W518 | W326 | — |
Cl-3 | — | W577 | — | — | — | — | Cl-3 | — | — | — | — | — |
Cl-4 | — | — | — | — | — | — | Cl-4 | — | — | — | — | — |
Cl-5 | — | — | — | — | — | — | Cl-5 | — | — | — | — | — |
— | SO4 | — | W334 | — | W590 | — | Cl-6 | SO4 | — | — | — | W304 |
Cl-7 | — | W492 | — | — | — | — | Cl-7 | — | W384 | — | — | — |
Cl-8 | — | — | — | — | W640 | W463 | Cl-8 | — | W372 | — | — | — |
Turning to the Cl− 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− 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−, 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.
The interactions between proteins and counter-ions may have important influences 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.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra06448h |
This journal is © The Royal Society of Chemistry 2014 |