3. Results and Discussion
3.2. Molecular dynamic simulations of the CTLD of perlucin and MBP-‐A
3.2.4. Association of calcium ions to residues of the CTLD of perlucin and MBP-‐A
and Gready (Zelensky & Gready [2003]) – is investigated and not any further details of atomic/residue positions or orientations.
3.2.4. Association of calcium ions to residues of the CTLD of perlucin and
Fig.
3.2.13. Calcium ion positions in several CTLDs. In every part of the figure the large red beads are the calcium ions and the small red beads are oxygen atoms (on amino acids) within a distance of 3 Å to a calcium ion. The residues to which the aforementioned oxygen atoms belong to are represented as well. The calcium ion positions are named according to the common nomenclature (e.g. Zelensky & Gready [2005]) for CTLDs Ca-‐1, Ca-‐2, Ca-‐3 and Ca-‐4. In brackets the number of amino acid oxygen atoms within a distance of 3 Å to the corresponding calcium ion is given. The left image shows the ASGR (1DV8, chain A) with its three calcium ions from the crystal structure. In the middle the CTLD of MBP-‐A (1KWV, chain A) is shown and on the right hand side the perlucin model with four calcium ions is shown. The latter structure was taken from one MD simulation series after the minimization step (run09, no MD performed). It has to be pointed out that the oxygen of Asn115 of perlucin is not within a 3 Å distance. But this is the case for the two exemplary CTLDs shown in A) (1DV8, Asn264) and B) (1KWV, Asn205). Since these Asn residues are part of the conserved WND motif it might be a shortcoming of the perlucin model. (Visualization software acknowledgements given in Fig. 3.2.12.)
In Fig. 3.2.13. the number of oxygen atoms within a 3 Å distance of every calcium ion is given as further information in brackets after the ion labels. Note that the Ca-‐2 in perlucin (Fig. 3.2.13.C is the CTLD structure of perlucin after the minimization step of one exemplary MD simulation) has indeed six coordinated oxygen atoms but only from four residues. The distance between the sidechain oxygen of Asn115 and Ca-‐2 is about 5 Å. This is contrary to at least the crystal structures (no MD or minimization performed) of ASGR and MBP-‐A shown in Fig. 3.2.13.A and 3.2.13.B. In both cases the oxygen of the Asn residue is within 3 Å distance of Ca-‐2. Since these residues are part of the well-‐conserved WND motif of CTLDs (see e.g. Drickamer [1993], Zelensky &
Gready [2005]) it is assumed that in the case of perlucin the initial orientation of Asn115 might be incorrect. In the perlucin model used for all MD simulations with calcium ions the initial distance between the oxygen of Asn115 and Ca-‐2 is 3.47 Å.
Every MD simulation containing Ca2+ ions was analysed with “ptraj”. For every protein oxygen or nitrogen (nitrogen atoms were included due to their negative partial charge) the frames were counted in which those atoms had a distance to a calcium ion less or equal to 3 Å. This resulted in occupancy values (relative number of frames) for every
“bond” between an oxygen atom and a calcium ion. Note that the sole criterion for a
“bond” is a distance between oxygen and calcium of ≤ 3 Å. Occupancies (in the aforementioned sense) below 5% were not reported to avoid a possible “spillage” of the “ptraj” output. Since nitrogen atoms never appeared in the results list the occupancy of a nitrogen-‐calcium ion distance of 3 Å or less had to be below 5%. For every “bond” that occurred the occupancy values were averaged over the MD simulations performed with the same initial structure. If the average occupancy was greater or equal to 75% the oxygen-‐calcium interaction was assumed to be sufficiently stable. The residue that contributes the oxygen to the interaction with calcium is marked in Fig. 3.2.14. with the identifier of the calcium ion which is involved in the interaction. Note that the time dependency of the interaction between the oxygen atoms and the calcium ions was not investigated here explicitly.
Fig. 3.2.14. summarises those residues – in perlucin and MBP-‐A – that have at least one oxygen within a distance of 3 Å to a Ca2+ in at least 75% of the trajectory frames averaged over the simulations of one MD series.
A) perlucin ------ number | 1 10 20 30 40 50 60 70 80 90 100 110 120 130 PERLUCIN | GCPLGFHQNRRSCYWFSTIKSSFAEAAGYCRYLESHLAIISNKDEDSFIRGYATRLGEAFNYWLGASDLNIEGRWLWEGQRRMNYTNWSPGQPDNAGGIEHCLELRRDLGNYLWNDYQCQKPSHFICEKER w/ 4 calcium | 4 4 1 & 21 + 2 4 w/ 2 calcium | 4 4 2 2 2 4 ------ B) MBP-A ------ number | 1 10 20 30 40 50 60 70 80 90 100 110 118 1KWV chain A | GKKSGKKFFVTNHERMPFSKVKALCSELRGTVAIPRNAEENKAIQEVAKTSAFLGITDEVTEGQFMYVTGGRLTYSNWKKDEPNDHGSGEDCVTIVDNGLWNDISCQASHTAVCEFPA
w/ 3 calcium | (1) & 2 21 +& 2 w/ 1 calcium | 2 2 2 2
--- "+" - Ca-1 and Ca-2 "&" - Ca-1 and Ca-3 Fig. 3.2.14. Summary of the residues of perlucin and the CTLD of MBP-‐A that have at least one oxygen atom with a distance ≤ 3 Å to a calcium ion in at least 75% of the trajectory – averaged over every simulation of a MD simulation series. In both parts of the figure the first line contains the residue numbering (in the case of MBP-‐A this is not the PDB numbering). The next line contains the amino acid sequence. The following two lines contain the condensed results of the MD simulation series. For perlucin this includes the six simulations performed with the structure with four calcium ions (run09) and the three simulations with two calcium ions (run21). In the case of the CTLD of MBP-‐A this includes three MD simulations each for the structure with three (run07) and one (run10) calcium ion. The numbers 1, 2 and 4 denote the calcium ions Ca-‐1, Ca-‐2 and Ca-‐4. Since it is possible that one oxygen or several oxygens of one residue have a short distance to two different calcium ions following characters are introduced. A “+” marks a residue that contributes oxygen atoms to Ca-‐1 and Ca-‐2 whereas “+” identifies residues that contributes oxygen atoms to Ca-‐1 and Ca-‐3. Concerning the value in brackets: the Oδ1 of Asp58 had on average only in 73% of all frames a distance to Ca-‐1 not exceeding 3 Å.
Concerning perlucin Ca-‐4 maintains a close distance to the Glu residues of α2 (Glu45) and β5 (Glu128) in both MD simulation series. The oxygen of Asn115 (in perlucin) does not return to a distance less than 3 Å to Ca-‐2 (remember the 5% occupancy threshold:
at least such a close distance in maximal 250 frames might be possible). Interestingly the average occupation of the 3 Å distance between the corresponding Asn (Oδ1 oxygen) in the WND motif of MBP-‐A (Asn205 [PDB numbering] corresponds to Asn102 [simulation numbering]) and Ca-‐2 is quite low as well: 26% (run10 with one calcium ion) and 53% (run07 with three calcium ions). Nonetheless this is in accordance with MD simulations (100 𝑝𝑝𝑝𝑝 lenght) of MBP-‐A carried out by Harte and Bajorath (Harte Jr.
& Bajorath [1994]) who found that Oδ1 of Asn205 increased its distance to Ca-‐2.
Concerning Gln92 of perlucin it is not flagged in Fig. 3.2.12. to maintain a short distance to Ca-‐2. The occupancies were: 68% (run09 with four calcium ions) and less than 5%
(run21 with two calcium ions). In contrast Glu82 of MBP-‐A keeps a closer distance to Ca-‐2. In the MD simulation series of perlucin with four calcium ions (run09) in total five oxygen atoms of the protein were on average in at least 75% of the simulations frames within 3 Å distance to Ca-‐2. In case of the perlucin simulations with two calcium ions the latter statement is true for four oxygen atoms. For the MD simulations of the CTLD of MBP-‐A one could find six oxygen atoms in close proximity to Ca-‐2.
Concerning the perlucin MD simulations that incorporated Ca-‐3 it can be stated that it remained in four simulations within 3 Å of Glu72 (Oε1 oxygen), during one simulation it did not stay within 3 Å distance to any of the proteins oxygen or nitrogen atoms and in another simulation it shifted its position from Glu72 to Asp68. Since during most of the simulation time only one close contact to an oxygen atom was maintained Ca-‐3 seems not to be stable associated to the molecule. The Ca-‐3 in the MBP-‐A simulation series remained on average in at least 75% of the frames within a 3 Å distance to three oxygen atoms from the residues Glu62 and Asp91 and therefore seems to be more stable bound to the protein structure.
In the perlucin simulation series the Ca-‐1 was on average in at least 75% of the frames within a 3 Å distance to five oxygen atoms from four residues. For MBP-‐A there are as well five oxygen atoms from four residues in close distance to Ca-‐1.
These results give first hints that perlucin might be able to bind calcium ions at different sites. But in light of the discussion concerning the Lennard-‐Jones parameter of the calcium ion (see section 3.2.1.) inappropriate ion parameters or simulation
artefacts cannot be ruled out. Ion parameters specifically adjusted to the interactions with oxygen atoms of protein structures might give more realistic results.
Unfortunately Joung and Cheatham optimized only the parameters of monovalent ions (Joung & Cheatham III. [2008]) but they discussed the importance of careful derived ion parameters (see section “Issues and Artifacts in Simulations with Common Ion Potentials”). Two examples from this section are anomalous crystallisation behaviour below experimental saturation salt concentrations and unexpected instability of certain DNA conformations. Additionally the authors stressed that the water model, the non-‐bonded interaction combining rules and the Ewald treatment can have an influence on the suitability of the ion parameters.
Nonetheless – as it will be discussed in the next section – the calcium ions might contribute to the stability of the long loop region.
Beyond the scope of this thesis is a possible pH dependency of the calcium ion association to the perlucin structure. But as it can be seen from Fig. 3.2.14. His101 is close to the hypothetical binding site of Ca-‐1 and Ca-‐3. MBP-‐A has an Asp91 at the corresponding position, which is negatively charged in the physiological pH regime (pKa ≈ 3.7, Thurlkill et al. [2006]). Since His has a pKa value in the physiological range (pKa ≈ 6.5, Thurlkill et al. [2006]) one could speculate about a pH dependency of the calcium binding. Note however that Thurlkill et al. derived the pKa values for pentapeptides and that the actual pH value of residues depends on the local environment that is the actual protein fold. This can be inferred for example from the theory underlying the computational pKa-‐shift predictor presented by Li et al. (Li et al.
[2005]).
3.2.5. Atomic positional fluctuations of residues and RMSd values of the