Conceived and designed the experiments: BH MB. Performed the experiments: MG JAG. Analyzed the data: MG DP BH MB. Contributed reagents/materials/analysis tools: MG DP BH MB. Wrote the paper: MG BH MB. Crystallographic expertise and analysis of the data: DP.
The authors have declared that no competing interests exist.
DNase I requires Ca2+ and Mg2+ for hydrolyzing double-stranded DNA. However, the number and the location of DNase I ion-binding sites remain unclear, as well as the role of these counter-ions. Using molecular dynamics simulations, we show that bovine pancreatic (bp) DNase I contains four ion-binding pockets. Two of them strongly bind Ca2+ while the other two sites coordinate Mg2+. These theoretical results are strongly supported by revisiting crystallographic structures that contain bpDNase I. One Ca2+ stabilizes the functional DNase I structure. The presence of Mg2+ in close vicinity to the catalytic pocket of bpDNase I reinforces the idea of a cation-assisted hydrolytic mechanism. Importantly, Poisson-Boltzmann-type electrostatic potential calculations demonstrate that the divalent cations collectively control the electrostatic fit between bpDNase I and DNA. These results improve our understanding of the essential role of cations in the biological function of bpDNase I. The high degree of conservation of the amino acids involved in the identified cation-binding sites across DNase I and DNase I-like proteins from various species suggests that our findings generally apply to all DNase I-DNA interactions.
DNase I requires Ca2+ and Mg2+ for hydrolyzing double-stranded DNA. Here, we show that bovine pancreatic (bp) DNase I contains four ion-binding pockets. Two of them, previously observed in the crystallographic structure of free bpDNase I, strongly bind Ca2+. The other two sites bind Mg2+ and are described in detail for the first time. One Ca2+ stabilizes the functional DNase I structure. The presence of Mg2+ in close vicinity to the catalytic pocket of bpDNase I reinforces the idea of a cation-assisted hydrolytic mechanism. Poisson-Boltzmann-type electrostatic potential calculations demonstrate that the divalent cations collectively control the electrostatic fit between bpDNase I and DNA. Thus, this work reveals the link between cation binding and the biological function of bpDNase I. The high degree of conservation of the amino acids involved in the identified cation-binding sites across DNase I and DNase I-like proteins from various species suggests that our findings generally apply to all DNase I-DNA interactions.
DNase I is a ubiquitous endonuclease that cleaves the phosphodiester backbone of the DNA double helix in the presence of divalent cations, introducing single-stranded nicks through hydrolysis of the P-O3′-bond and yielding 5′-phosphorylated fragments
DNase I, in particular bpDNase I, has been extensively studied. Its activity at physiological pH is at its highest in presence of both Ca2+ and Mg2+
In addition to its effect on enzymatic activity, the importance of Ca2+ for the structural integrity of DNase I has long been recognized. Calcium cations protect bpDNase I from proteolytic degradation
Ca2+ coordination in sites I and II involves amino acids belonging to loops L1 (Leu198 to Thr211, containing site I) and L2 (Tyr97 to Pro113, containing site II), which are structured by two disulfide bridges, Cys173-Cys209 and Cys101-Cys104, respectively
Little has been published on the number and location of Mg2+-binding sites. It has been proposed that Mg2+ is located near the catalytic pocket and contributes to hydrolysis
Thus, despite extensive biochemical and structural characterization of bpDNase I, the number and the location of cation-binding sites in free bpDNase I have still not been resolved. The location of Ca2+-binding sites I and II has been firmly established through both X-ray structures and biochemical studies of DNase I variants. In contrast, Mg2+-binding sites remain much more hypothetical. In this study, molecular dynamics (MD) simulations in explicit solvent were carried out on bpDNase I, with variation in the compositions of metal ions. We identify four cation-binding sites and demonstrate that both Ca2+ and Mg2+ are crucial for optimizing the electrostatic fit between the enzyme and the negatively charged DNA. Two Mg2+-binding sites located within and very close to the active site of DNase I provide the first tangible support for a cation-assisted hydrolysis process. In sum, these findings establish a direct link between cation binding and the biological function of DNase I.
The nine available high-resolution X-ray structures containing bpDNase I are listed in
Four sites (sites I to IVa,b) in bpDNase I are occupied by cations (red). Sites I, II and III are located in loops L1 and L2 (green), which contain disulfide bridges (yellow). Sites IVa and IVb surround the catalytic histidines (side-chains shown in gray, imidazole nitrogens in blue). Ion-binding pockets are within or near the region that interacts with DNA (orange, bottom). The highlighted DNA binding region was defined by the residues losing their solvent accessibility when complexed with DNA, as described in the
DNase I cation-binding site | |||||||
PDB code | Macromolecular ligand | Divalent ions in buffer | I | II | III | IVa | IVb |
3DNI | none | Ca2+ | Ca2+ | Ca2+ | H2O | - | H2O |
1ATN | actin | Ca2+ | Ca2+ | Ca2+ | Ca2+ | - | - |
2D1K, 2A40, 2A41, 2A3Z | actin | Ca2+/Mg2+ | Ca2+ | - | Mg2+ | - | - |
2A42 | actin | Ca2+/Mg2+ | Ca2+ | - | Mg2+ | - | H2O |
2DNJ, 1DNK | DNA | - | H2O | - | H2O | - | H2O |
Nine high-resolution X-ray structures contain bpDNase I. The enzyme is either free or complexed with actin or DNA. The crystallization buffers contained divalent cations, except for the bpDNase I/DNA complexes. The bpDNase I binding sites (
All of these observations are supported by the analysis of bound crystallographic water molecules (
Overall, these analyses show that at least three sites potentially bind divalent cations in bpDNase I. In addition to the well-known sites I and II, with high affinities for Ca2+, the detection of site III is particularly interesting since it seems to preferentially bind Mg2+. It remains to be verified whether this site can stabilize Mg2+ in actin-free bpDNase I. The existence of a fourth putative site located near the bpDNase I catalytic pocket, possibly occupied by Mg2+, is only supported by the presence of bound water molecules in three structures and thus must be explored
The confirmed or putative cation-binding sites identified above were further investigated using molecular dynamics simulations of bpDNase I carried out in the presence of various types of metal ions in explicit solvent. In all seven simulations, the starting positions of Na+ around the enzyme were determined using a Coulombic potential grid and, importantly, were not initially located in cation-binding sites. The characteristics of the seven simulations are summarized in
Bound ions | ||||||
Name | Site I | Site II | Site III | Site IVa | Site IVb | Cα-RMSDav |
Sim1 | Na+ | Na+ | Na+ | - | Na+ | 1.7 (0.1) |
Sim2 | Ca2+ | Ca2+ | Na+ | Na+ | - | 1.4 (0.1) |
Sim3 | Ca2+ | Ca2+ | Mg2+ | Na+ | - | 1.0 (0.1) |
Sim4 | Ca2+ | Ca2+ | Na+ | Mg2+ | - | 1.2 (0.1) |
Sim5 | Ca2+ | Ca2+ | Mg2+ | Mg2+ | - | 1.5 (0.1) |
Sim6 | Ca2+ | Ca2+ | Mg2+ | - | Mg2+ | 1.5 (0.1) |
Sim7 | Ca2+ | Ca2+ | Mg2+ | Mg2+ | Mg2+ | 1.5 (0.1) |
The bpDNase I simulations were performed with solvent containing Na+, Na+ and Ca2+, or Na+, Ca2+ and Mg2+. The ions bound at sites I, II, III and IVa,b (
Sites I and II in bpDNase I, identical to their X-ray counter-parts, were very strong cation-binding pockets, always occupied by ions throughout the trajectories (
Ca2+ (cyan) at sites I and II are involved in coordination spheres with tricapped trigonal prismatic geometry. The coordination spheres of Mg2+ (green) at sites III, IVa and IVb are octahedral. Cys173 interacts with a water molecule belonging to the coordination sphere of site III. The amino acids involved in the coordination spheres are listed in
Site | AAcs | %tocc (Na+) | %tocc (Ca2+) | %tocc (Mg2+) |
Site I | Asp201 | 92 | 99 | N.A. |
Thr203 | 99 | 99 | N.A. | |
Thr205 | 100 | 100 | N.A. | |
Thr207 | 100 | 100 | N.A. | |
Site II | Asp99 | 95 | 100 | N.A. |
Cys101 | 0 | 100 | N.A. | |
Asp107 | 66 | 97 | N.A. | |
Phe109 | 97 | 100 | N.A. | |
Glu112 | 95 | 72 | N.A. | |
Site III | Asp172 | 68 | N.A. | 100 |
Asp198 | 72 | N.A. | 100 | |
Site IVa | Asp168 | 78 | N.A. | 100 |
Asp212 | 99 | N.A. | 100 | |
His252 | 96 | N.A. | 100 | |
Site IVb | Asn7 | 95 | N.A. | 25 |
Ile8 | 96 | N.A. | 0 | |
Glu39 | 55 | N.A. | 100 |
Amino acids involved in the coordination sphere (AAcs) of the four bpDNase I cation-binding sites (
Site | CN | Na+ | Ca2+ | Mg2+ |
Site I | aa | 5.1 | 5.4 | N.A. |
water | 1.2 | 3.6 | N.A. | |
total | 6.3 | 9.0 | N.A. | |
Site II | aa | 3.9 | 6.6 | N.A. |
water | 2.0 | 2.4 | N.A. | |
total | 5.9 | 9.0 | N.A. | |
Site III | aa | 2.3 | N.A. | 2.0 |
water | 3.5 | N.A. | 4.0 | |
total | 5.8 | N.A. | 6.0 | |
Site IVa | aa | 4.3 | N.A. | 4.3 |
water | 1.6 | N.A. | 1.7 | |
total | 5.9 | N.A. | 6.0 | |
Site IVb | aa | 3.0 | N.A. | 2.0 |
water | 3.0 | N.A. | 4.0 | |
total | 6.0 | N.A. | 6.0 |
The coordination number (CN) of the cations bound to the four bpDNase I sites (
Site III, close to site I (
The existence of a fourth site (site IV) specific to Mg2+ has been suspected
In summary, four cation-binding sites were identified in free bpDNase I. The strong sites I, II and IVa, as well as the two additional sites III and IVb with fewer coordinating protein side-chains, were able to bind divalent cations.
The prediction of cation binding sites by molecular dynamics can potentially be biased by incomplete sampling of ion positions. In MD simulations with Ca2+ and Mg2+, these divalent cations were directly located at the sites where Na+ spontaneously binds. Conversely, Na+ ions were randomly distributed around DNase I, yet artifacts may have been caused by attraction from the closest negatively charged side-chains. In order to make sure that no such artifacts apply in the present case, we used two complementary approaches to systematically explore all possible cation pockets on the entire surface of DNase I. First, ion locations were interactively investigated with the “MyPal” approach that allows steering ions within DNase I electrostatic potential maps using a haptic device
The very high residence times of Mg2+ in MD simulations could be another issue. Indeed, this ion, due to its +2 charge and small radius, could be artificially trapped by DNase I. However, the fact that Na+ binds to sites III and IVa,b over the whole Sim 1–3 trajectories precludes the possibility of a specific bias towards Mg2+ in the present case.
The characteristics of theoretical sites I, II and III perfectly parallel those observed in the crystallographic structures containing DNase I, as mentioned in the previous section. Their existence is thus well attested. In order to obtain equally sound experimental evidence for the existence of site IV, we revisited the X-ray experimental electron density maps of the highest resolution structures 2A40 and 2A42. In both maps, significant densities are either unattributed (2A40) or attributed to a water molecule (2A42) at the location of site IVb. These perfectly match a divalent cation interacting with Glu39 and one (2A40) or five (2A42) water molecules (for details,
Sites I and II are located in loops L1 (Leu195 to Tyr211) and L2 (Tyr97 to Pro113) respectively (
Ten snapshots (red) of L1 (left) and L2 (right) loop structures from Sim1 or Sim7 were superimposed on their counterparts in the 3DNI X-ray structure (green). Site I in L1 and site II in L2 bind either Na+ (dark blue, top panels) or Ca2+ (cyan, bottom panels). L2 contains one disulfide bridge (yellow).
Mg2+ at site III was coordinated to Asp172, protecting the Cys173-Cys209 bridge from reduction. Accordingly, one water molecule involved in the Mg2+ coordination sphere was also interacting with Cys173 (
Individually, cations stabilized the enzyme's structure, either directly (Ca2+ at site II) or indirectly (Mg2+ at sites III and IV).
The proximity between the catalytic site and the double-binding site IV, involving two aspartates, one catalytic histidine and one glutamate, suggests that Mg2+ at sub-sites IVa and IVb may assist in the hydrolysis of the DNA phosphodiester bond. The approximate location of DNA with respect to Site IV is depicted in
A close-up view of the double magnesium binding site IVa and IVb, where DNA from the 1DNK complex was modeled in by superimposition of the protein chains from our simulation and from the crystal structure. Acidic side-chains are shown in red, histidines in blue, magnesium in green. The size of the cations is indicated by a transparent van der Waals sphere to assess their possible contact with DNA. The DNA surface is represented colored by underlying atom type, red for oxygen, orange for phosphorus, blue for nitrogen, white for carbon.
At the present stage, our study cannot reveal further details of the bpDNase I hydrolysis process. However, the discovery of site IV strengthens the hypothesis that Mg2+ ions are directly implicated in the enzymatic function of DNase I.
In addition to these diverse roles, we suspected that ions may interfere with the bpDNase I/DNA interaction. Any favorable interaction between two charged macromolecules requires an electrostatic fit between them. Nucleic acid molecules are negatively charged, mainly owing to their phosphate groups. Their interaction with proteins thus requires positively charged protein surfaces
Poisson-Boltzmann electrostatic potential maps were calculated for bpDNase I structures extracted from simulations Sim3–7, focusing on the region where DNA interacts with the enzyme. According to the 3DNI, 2DNJ and 1DNK structures, this region involves 24 amino acids (listed in
The electrostatic potential of the interface was extremely sensitive to the number of charges contributed by the ions (
Electrostatic potentials (Ep, in kT/e units) of the bpDNase I region involved in the interaction with DNA were calculated on ten snapshots extracted from Sim3 (black), Sim4 (pink), Sim5 (green), Sim6 (blue) and Sim7 (red). Ep values are ordered as a function of the number of charges carried by the ions at sites I, II, III or IVa,b. Error bars represent standard deviations.
Electrostatic potential maps colored from -5 kT/e in red to +5 kT/e in blue for the bpDNase I region involved in the interaction with DNA. The top panel shows the map obtained in the absence of any bound cations, the central panel shows the map for sites I & II occupied by Ca2+ (cyan), and III, IVa and IVb by Mg2+ (green). The bottom panel indicates the location of DNA (magenta) at its binding site on DNase I with respect to the ion binding sites.
The existence of crystallographic DNA/DNase I complexes (2DNJ
Poisson-Boltzmann calculations show that ions bound to DNase I can collectively influence the electrostatic potential of the bpDNase I region involved in the DNA interface, and thus drive the electrostatic fit between the enzyme and the DNA substrate.
The importance of individual amino acids in bpDNase I cation-binding sites can be further assessed by comparing the sequences from DNase I and DNase I-like proteins of various species (from 30 for DNase I to 11 for DNase I-like 2). DNase I-like proteins include three variants. The sequences and their percentages of identity with bpDNase are listed in
The conservation of the bpDNase I cation-binding sites is shown in
Sequences of the human DNase I and DNase I-like 1, 2 and 3 proteins were aligned with the bovine pancreatic DNase I sequence. The alignment highlights the bpDNase I cation-binding sites I, II, III, IVa and IVb. Most of the corresponding amino acids belonging to human enzymes are either identical or similar to those of bpDNase I.
DNase I | DNase I-like 1 | DNase I-like 2 | DNase I-like 3 | ||||||
Site | Residue | I (%) | S (%) | I (%) | S (%) | I (%) | S (%) | I (%) | S (%) |
I | Asp201 | 97 | 94 | 91 | 94 | ||||
Thr203 | 93 | 100 | 91 | 82 | |||||
Thr205 | 47 | Ser: 40 | 0 | Ala: 88 | 0 | Gly: 100 | 0 | polar/charged: 88 | |
Thr207 | 77 | polar: 20 | 94 | 9 | Ser: 82 | 59 | Asn: 4 | ||
Asn208 | 17 | polar: 80 | 19 | His: 75 | 0 | polar/charged: 91 | 12 | polar/charged: 68 | |
II | Asp99 | 97 | 94 | 100 | 82 | ||||
Cys101 | 73 | 0 | gap: 100 | 0 | gap: 100 | 0 | polar: 80 | ||
Asp107 | 97 | 94 | 100 | 100 | |||||
Phe109 | 97 | 100 | 100 | 88 | |||||
Glu112 | 90 | 100 | 100 | 88 | |||||
III | Asp172 | 30 | Gly: 63 | 94 | 91 | 47 | Gly: 41 | ||
Asp198 | 97 | 100 | 91 | 100 | |||||
IVa | Asp168 | 97 | 100 | 91 | 100 | ||||
Asp212 | 100 | 100 | 91 | 100 | |||||
His252 | 97 | 94 | 100 | 100 | |||||
IVb | Asn7 | 97 | 100 | 82 | 100 | ||||
Glu39 | 97 | 100 | 91 | 100 |
The bpDNase I sequence was aligned with 30 non-redundant DNase I, 16 DNase I-like 1, 11 DNase I-like 2 and 17 DNase I-like 3 sequences from different species. I (%) corresponds to the percentage of identity for each of the amino acids involved in each of the four bpDNase I cation binding sites, I, II, III and IVa,b. For relevant cases, S (%) is the percentage of the amino acid or the type of amino acid substituting those of bpDNase I.
Overall, the cation-binding sites in bpDNase I are as well conserved as the residues involved in DNase I/DNA contacts
The aim of this work was to investigate the ability of bpDNase I to bind divalent cations, Ca2+ and Mg2+, and to elucidate their possible roles in bpDNase I function.
In several simulations, we used monovalent sodium ions as a reference state, as they may occupy any of the potential binding sites. It should be noted that NaCl effectively inhibits DNase I activity
Molecular dynamics simulations reveal that four sites are able to bind Ca2+ (sites I and II) or Mg2+ (sites III and IVa and b). The existence of sites I, II and III is demonstrated by the X-ray structures of free (sites I and II) and actin-bound bpDNase I (sites I, II and III). Reexamination of the 2A40 and 2A42 electron density maps validates sub-site IVb. Sub-site IVa is indirectly but firmly confirmed by site-directed mutations
At site II, Ca2+ acts on the folding of the L2 loop and reduces its mobility. At site III, Mg2+ is located very close to the Cys173-Cys209 bridge and protects this essential structural element from reduction. Site IV corresponds to two sub-sites, IVa and IVb, with an ion either coordinating (sub-site IVa) or close (sub-site IVb) to the two histidine residues involved in the DNA cleavage process. The discovery of site IV is a first step towards a comprehensive understanding of the bpDNase I enzymatic mechanism. In addition to these local functions, bound ions collectively modify the electrostatic potential of the bpDNase I region implicated in DNA binding. By introducing positive charges, they compensate for the intrinsic repulsion between DNase I and DNA, both negatively charged. A similar effect may have been achieved by engineering Human DNase I mutants, introducing additional positive charges at the DNA binding domain
Beyond the current investigation of Mg2+ and Ca2+ binding, this study also opens the prospect to address the effects of other divalent metal ions, for instance Mn2+, able to enhance DNase I activity
Our results are consistent with a recent study highlighting how the prediction of ion binding sites may improve our understanding of structure-based protein functions
A summary of all MD simulations is given in
Water molecules and cations were energy-minimized and equilibrated in the NVT ensemble at 100 K for 100 ps, with the protein constrained. The entire system (bpDNase I, water molecules and ions) was then heated from 100 to 300 K in 10 ps by 5 K increments with harmonic restraints of 5.0 kcal mol−1 Å−2 on the solute atoms. The simulations were continued in the NPT ensemble, without a noticeable change in volume. Subsequently, production runs lasting 25 ns were carried out.
The same initial configuration was used for Sim1 and Sim2. This starting point was constructed from the crystal structure of the bpDNase I enzyme at 2 Å resolution (PDB code 3DNI
A Coulombic potential grid was used to determine the initial positions of Na+ ions. Ca2+ locations in Sim3–7 were those observed in 3DNI. In addition to these two 3DNI Ca2+ sites, two strong Na+ binding sites were observed in Sim1 and Sim2. These bound Na+ ions were replaced by Mg2+ in Sim3–7.
In all simulations, the two histidines involved in the catalytic pocket, His134 and His252, were in their neutral form to test a potential cation-binding site under favorable conditions. This was consistent with calculations carried out on 3DNI devoid of cations with the WhatIf program
All radial distribution functions were computed using the g_rdf analysis module of the Gromacs software suite. RDF analysis was used to determine ion–oxygen distributions and coordination numbers for Na+, Ca2+ and Mg2+. These coordination numbers were calculated for all ions bound to bpDNase I in each trajectory. Fixed distances of 3.2 Å for Na+ and Ca2+ and 2.9 Å for Mg2+ were used to define the outer limit of the first solvation shell
Electrostatic potential maps were calculated with the Adaptive Poisson-Boltzmann Solver (APBS)
These calculations focused on the bpDNase I region corresponding to the bpDNase I/DNA interface. The amino acids belonging to this interface were determined by comparing the amino acid accessibilities in free (3DNI
Various combinations of ions were tested, from naked (no ion) to maximally charged DNase I,
The first 500 sequences homologous to bpDNase I were extracted from the Basic Local Alignment Search Tool (BLAST)
Secondary structure elements were identified using the STRIDE method by Heinig and Frishman
Reinterpretation of the 2A40 and 2A42 crystal structures.
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Ion binding sites predicted via the CHED server. This Table is related to
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Thirty non-redundant DNase I sequences from various species. This Table is related to
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Sixteen non-redundant DNase I-like 1 sequences from various species. This Table is related to
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Eleven non-redundant DNase I-like 2 sequences from various species. This Table is related to
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Seventeen non-redundant DNase I-like 3 sequences from various species. This Table is related to
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The authors wish to address their acknowledgments to Dr. Olivier Delalande and Dr. Nicolas Férey for technical assistance and helpful discussions, and to Daniel Parton for proof-reading the manuscript.