Current addresses:

(1) Bioinformatics Unit, Research Institute of Molecular Pathology, Dr. Bohrg.7, 1030 Wien, Austria.

(2) European Media Laboratory, Villa Bosch, Schloss-Wolfsbrunnenweg 33, D-69118 Heidelberg and European Molecular Biology Laboratory Heidelberg, Meyerhofstr.1,D-69117 Heidelberg.

(3) Molecular Design and Informatics, N.V. Organon, Molenstraat 110, PO Box 20, 5340 BH Oss, The Netherlands.

Keywords: cytochrome P450, molecular dynamics simulation, protein-ligand binding, protein dynamics, molecular animation, random expulsion molecular dynamics, interactive molecular graphics visualisation

Content:

• Abbreviations
• Introduction
• Methods:
  • 1. The REMD approach
  • 2. How to use Molsurfer
  • 3. How to look at the animations
• Results
  • 1. Substrate exit pathway classification in P450cam and P450BM-3
  • 2. Application of the REMD method to P450BM-3
  • 3. Substrate exit pathways obtained in P450cam
    • 3.a Pathway class 1
    • 3.b Pathway class 2
    • 3.c Pathway class 3
  • 4. The role of salt-links in substrate exit/access
  • 5. Overview of expulsion trajectories
• Conclusions
• Acknowledgments

Abbreviations

Fig.1: The inner active site surface and the outer molecular surface computed by the GRASP program ⁴ using a probe radius of 1.4Å are shown. They are not connected to each other and therefore, no substrate access or product egress channel can be seen.

In the following paragraph, the colors used for residues or secondary structure elements in the text correspond to the color code used in fig.2.

Examination of the crystal structure ^{2, 5} prompted speculation about a possible entry channel located between the B' helix and the F/G loop (see Fig. 2, below). This is supported by the following data: (i) Site-directed mutagenesis and stopped flow kinetic measurements that show that Tyr29 (N-terminus) and Phe193 (F/G loop) have an important influence on substrate access ⁶. (ii) The crystal structure of P450cam with a large inhibitor bound that shows that this inhibitor which is larger than the natural substrate occupies part of the putative access channel. In this structure the sidechains of residues Phe87, Tyr96 and Phe193 are displaced with respect to the sidechains in the camphor bound structure ¹.   Recently, the structure of an (artifically large) ruthenium sensitizer-linked substrate was solved and showed the ligand extending along and opening up the putative access channel ⁷ . (iii) In the crystal structures of two structurally similar proteins of the P450 family, cytochrome P450BM-3 ⁸ and nitric oxide reductase ⁹, in which this channel is open.

Fig.2: Overview of specific secondary structure elements and residues in P450cam.

Adjacent to the proposed substrate entrance channel, there is a ring of four salt links formed by the residues, Asp251, Lys178, Arg186, and Asp182. Experiments indicate that substrate access to the active site of P450cam is modulated by electrostatic interactions involving residue Asp251 ¹⁰. This salt-link tetrad and a further salt-link between Asp97 and Lys197 are amongst the most electrostatically stable salt bridges in P450cam ¹¹. The combined action of the salt-link tetrad and the salt-link Asp97-Lys197 could be important for the tethering of the F/G helix+loop to the I helix and the B' helix in order to keep the substrate access channel closed. Such a tethering mechanism might be important for sufficient desolvation of the buried active site upon substrate binding and consequently for catalysis.

In order to investigate possible ligand exit channels from the active site, a novel method, random expulsion molecular dynamics, was developed ¹². During molecular dynamics simulations, a randomly oriented additional force is imposed on the ligand in addition to the standard molecular dynamics force field. In a set of over 20 simulations of ligand egress from P450cam, three classes of exit pathways were observed. One of these pathways is the proposed substrate access channel. By applying steered molecular dynamics and adiabatic mapping techniques, this channel was found to be the most probable pathway ¹³. Here, we present animations of representative trajectories and a new interactive trajectory visualization tool.These facilitate investigation of the transient conformational changes involved in ligand exit during the simulations. For interactive visualization, we have adapted our MolSurfer software (see also Methods) to enable investigation of the trace of the center of mass of the ligand along its exit pathway. MolSurfer was originally designed to aid visualization of macromolecular interfaces by linking 2D-projections of the properties of the interfaces to the 3D-structures. The 3D-structures are displayed with the Java macromolecular viewer, WebMol ¹⁴. In this paper, the ligand trajectory is displayed by a tube through the protein structure (in the WebMol window). Navigation of the tube is coupled to a second window (the MolSurfer window) which permits simultaneous display of trajectory properties such as the simulation time in ps, the residues closest to the ligand trajectory, and computed and crystallographic thermal factors.

Methods

The force constants of the expulsion trajectories shown in the present paper range from 500-1000 kJ/mol*nm. Concomitantly, values of r_min between 0.001 and 0.016Å together with N of 10-80 have been chosen.

Initial cartesian coordinates of P450cam with camphor bound and P450BM-3 with palmitoleic acid bound were taken from the Brookhaven protein data bank (entries 2cpp and 1fag, respectively).

Simulations were performed with the ARGOS MD program ¹⁵ using the CHARMm 22 ¹⁶ all atom force field. Parametrization, set up and simulation details are described elsewhere ^{11, 12}.

2. How to use MolSurfer for interactive display of the ligand exit trajectories

• The WebMol window shows the crystal structure of cytochrome P450cam in an all atom representation and a tube representing the center of mass trace of camphor along its exit pathway. Features of 3D viewing as stereo, colouring, selecting etc. can be changed in the WebMol window. Although camphor has an approximate diameter of 6.5Å, a radius of 0.5Å was chosen for the tube for clarity during visualisation. The color of the tube can be changed with the MolSurfer window: change color-button. The right mouse button scales the size of the protein, while the middle one performs x,y translations and the left mouse button performs rotations around the x and y axes of the image.
• The MolSurfer window can be used to browse along the the trace of the ligand trajectory.  (The first bar represents pathway 1, the second pathway 2a, the third pathway 2c, the fourth pathway 3, cf   Substrate exit pathways obtained in P450cam .) When a certain time step along the 1D trace of the ligand trajectory is chosen, a third window (the WebMol: Focus window) pops up in which a focus radius can be chosen. (If you want to go back to view the total protein, click the "ResetSlab" button in the WebMol window.)  The following quantities can  be monitored via the "View Menu" in the MolSurfer window:

1.

Time (ps): During expulsion, frames were saved at intervals of 0.2ps. For each frame, properties 2 and 3 were determined.

2.

Closest residue is the number of the residue closest to every point of the expulsion pathway of camphor. The closest distance between any protein atom and any camphor atom is used as a criterion.

3.

Distance (nm) is the shortest distance between any protein atom and any camphor atom.

4.

Computed B-factor (Ų) is the temperature factor of the closest residue (as described under 2). The temperature factor is determined from the last 20ps of expulsion.

5.

X-ray B-factor (Ų) is the temperature factor of the closest residue (as described under 2) taken from the crystal structure.

If you want to switch between the representation of all pathways at once and only one single pathway,  click on "File", "Open"  in the MolSurfer window and choose the representation you are interested in. Use menu View to switch between the properties to be displayed on the 2D map.

The colour of the tube can be changed with the MeshColor button in the MolSurfer window. To adjust the range for displaying properties on 2D map, click left/right mouse button on the color bar.

Use menu File--Quit when done.

3. How to look at the animations

Due to the high compression and the non-standard size of our mpeg movies, some mpeg players (in particular those commonly installed in the internet explorer) cannot play our movies. Look at the quicktime movies instead. It is important to note that some of the quicktime movies are lower quality as they have been obtained by conversion from mpeg.

In P450BM-3, a pathway similar to pw2a in P450cam was found together with a further pathway subclass 2d ¹², for which no animation is shown in the present paper. Animations can be obtained when clicking on the left icons which show the center of mass traces of the ligand along a single representive trajectory for each pathway class.

2. Application of the REMD method to P450BM-3

Animation 1 (3.3Mb):  Pathway 2a for exit of PAM from P450BM-3 (traj#1). The red line shows the center of mass of the ligand. Secondary structure elements that undergo significant conformational changes during ligand exit are coloured. The heme, PAM and Arg-47 are shown in ball-and-stick representation. In the animation (for mpeg format, click on the image to left to view, for quicktime format click below image), PAM is shown in yellow, Arg47 is shown in stick representation in light blue and the heme ring in light grey.

Click here for Animation 1 in quicktime format (5.8 Mb)

The location of the expulsion pathways observed with the REMD method (applied on the closed substrate-bound crystal structure) matches with the location of the open channel seen in the substrate-free crystal structure. Furthermore, the rms deviations for each residue from the crystal structure averaged over the expulsion phase are in good agreement with the rms differences between substrate-free and substrate-bound crystal structure, cf fig.5 in ref ¹².

3. Substrate exit pathways obtained in P450cam

3.a Pathway class 1

Exit MECHANISM:
The ligand has to pass a region with little flexibility (heme ring), before the exit occurs at a region with high flexibility (C' helix) predominantly populated by small aliphatic amino acids (Val, Ala and Leu). Upon ligand exit the heme plane is transiently forced into a strained conformation that deviates from planarity. Of all pathways, it shows the largest backbone deformation upon ligand egress (local C rms fluctuations of 2.4-3.9Å in the C' helix).

Animation 2 (4.4Mb):  Pathway 1 for exit of CAM from P450cam (traj#2). The red line shows the center of mass trace of the ligand. In the animation (for mpeg format, click on the image on the left to view; for quicktime format click below image), the backbone of cytochrome P450cam is shown as a ribbon, CAM is shown in yellow and the heme ring in white.

Click here for Animation 2 in quicktime format (13.2 Mb)

Start MolSurfer representation
(by clicking on the picture)

3.b Pathway class 2

Pw2 consists of 3 different pathway subclasses 2a-2c in P450cam, the endpoints of which lie up to 15Å apart from each other on the protein surface. This class shows the lowest global rms deviations (1.2-1.7Å) and the smallest C rms fluctuations (0.9-1.8Å).

Key features of the mechanism: Small backbone fluctuations are accompanied by rotation of the aromatic sidechains of residues Tyr29, Phe87, Tyr96, Phe98, Phe193 and Tyr201. The aromatic sidechains act like doors that are opened via transitions of the sidechain torsions of  and . Thus, small fluctuations (below 2.4Å) suffice to allow CAM (with a diameter of approximately 6.5Å) to exit. Pw2c, additionally, requires the perturbation of the salt-links between D97 and K197.

Animation 3a (4.4Mb):  Pathway 2a for exit of CAM from P450cam (traj#3) . The red line shows the center of mass trace of the ligand. In the animation (mpeg format, click on the image to the left to view click; for quicktime format click below image), the backbone of cytochrome P450cam is shown as a ribbon, CAM is shown in yellow, Phe 87 in white, Phe 193 in light blue and Tyr96 in green.

Click here for Animation 3a in quicktime format (14.3Mb)

Animation 3b (11.7Mb):  Pathway 2a for exit of CAM from P450cam (traj#4) . In the animation (mpeg format, click on the image to the left to view; for quicktime format click below image), of this alternative trajectory of pw2a, Phe 29 (shown in red) and Phe 193 (shown in blue) are displaced upon exit of CAM (shown in yellow).

Click here for Animation 3b in quicktime format (9.5Mb)

Animation 3c (18.0Mb):  Pathway 2b for exit of CAM from P450cam (traj#5). In the animation (mpeg format, click on the image to the left to view; for quicktime format click below image), CAM is shown in yellow. Pathway 2b is located between the B' helix and the and  sheets. This pathway was only observed once and with a higher expulsion force constant. Therefore, it is considered to be a lower probability pathway. From the animation one can see that CAM is blocked for about 30ps at the same position in proximity to the  and  sheets before it succeeds in exiting the protein. Click here for Animation 3c in quicktime format (14.7Mb)

Animation 3d (2.4Mb):  Pathway 2c for exit of CAM from P450cam (traj#6). In the animation (mpeg format, click on the image to the left to view; for quicktime format click below image), CAM is shown in yellow. Exit involves the displacement of the sidechains of residues Phe98 and Tyr201 (shown in light blue) and perturbation of the salt-link beween Asp97 and Lys197 (colored atomwise).

Click here for Animation 3d in quicktime format (2.0Mb)

Start MolSurfer representation
(by clicking on the picture)

3.c Pathway class 3

Exit MECHANISM:
Ligand exit along this pathway involves the displacement of backbone and sidechains of aliphatic residues, e.g. L200, V177 and V247.

Animation 4(12.4Mb):  Pathway 3 for exit of CAM from P450cam (traj#7) . The red line shows the center of mass trace of the ligand. In the animation (mpeg format, click on the image to the left to view; for quicktime format click below image), the backbone of cytochrome P450cam is shown as a ribbon and CAM is shown in yellow. CAM tries to exit via pw2a, but then it proceeds to the region below the F and G helices. Finally, ligand egress is observed close to the E/F loop.

Click here for Animation 4 in quicktime format (20.2Mb)

Start MolSurfer representation
(by clicking on the picture)

4. The role of salt-links in substrate exit/access

Perturbation of the salt-links Asp251-Arg186, Asp251-Lys178 or Asp97-Lys197 has been observed in nearly all REMD trajectories of all pathways.  On the other hand, we have found in steered molecular dynamics simulations ¹³ that the energy barrier for ligand exit is not affected by neutralising the salt-links. Most probably, therefore, the salt-links affect the slow frequency dynamics of P450cam which are of importance for the opening of the substrate access channel on a much slower time scale than our expulsion trajectories.

  Animation 5.a (7.2Mb):   Pw2a for exit of CAL from P450cam (traj#8). In the animation (mpeg format, click on the image on the left to view; for quicktime format click below image), the backbone of cytochrome P450cam is shown as a ribbon, CAM is shown in yellow and the heme ring in white. The residues Asp251, Arg186 and Lys178 forming salt-links are colored by atom. Phe87 is colored in white and Phe29 in red. Expulsion of CAL along pw2a and the concomitant perturbation of salt-links Asp251-Arg186 and Asp251-Lys178 is shown. Hydrogen bonds formed by the salt-links are displayed by continuous lines to make the conformational changes more visible. At the end of the animation, the salt-link between Asp251-Lys178 (left bond), although still shown as a bond, is broken, while the salt-link between Asp251-Arg186 (right bond) is reformed again, as the side- chain of Asp251 has rotated around by approx. 180^o. The perturbations of the salt-links are triggered by the,rotations of the aromatic residues Phe87 and Phe29 which cause a structural relaxation in their proximity.

Click here for Animation 5a in quicktime format (5.9Mb)

  Animation 5.b (2.4Mb):   Pw2c for exit of CAM from P450cam (traj#6). In the animation (mpeg format, click on the image on the left to view; for quicktime format click below image),the backbone of cytochrome P450cam is shown as a ribbon, CAM is shown in yellow and the heme ring in white. The residues Phe98 and Tyr201 are shown in light blue. The perturbation of salt-link Asp97-Lys197 along pw2c is shown. The salt-link is transiently broken (with a distance between  N and OD1 of 4.5Å.) This perturbation is triggered by the conformational changes of the aromatic residues Phe98 and Tyr201 upon substrate exit.

Click here for Animation 5b in quicktime format (2.0Mb)

5. Overview of expulsion trajectories

protein traj # pw # rmin/ N k/ expulsion traj. +           route length1/ ligand     Å   (kJ/mol nm) (residue #) ps                 P450BM-3               PAM  1 2a 0.008 40 1000 B' helix, F/G ~10                 P450cam               CAM  2 1  0.004 20 1000 C and H helices ~30 - `` -  3 2a 0.002 10 800 F/G loop, ~20             B' helix (via 87)   - `` -  4 2a 0.016 80 600 - `` - (via 29, 193) ~50 - `` -  5 2b 0.005 10 1000 B/B' loop, , ~60 - `` -  6 2c 0.008 40 700 B', I and G helices ~10             (via 98, 201)   - `` -  7 3 0.001 10 800 E/F loop ~50 CAL  8 2a 0.008 40 700 F/G loop, ~70             B' helix (via 193)

Table2   List of all expulsion trajectories in P450cam for which a MolSurfer representation is shown. The pathway location, the pathway classification and the REMD parameters are given. For definition of r_min, N and k,  cf. Methods.

protein	traj#	pw#	rmin/	N	k/	expulsion	traj.
+						route	length1/
ligand			Å		(kJ/mol nm)	(residue#)	ps
P450cam
CAM	9	1	0.02	100	600	C' helix, G/H loop	15.8
	3	2a	0.002	10	800	F/G loop,	11.2
						B' helix (via 87)
	6	2c	0.008	40	700	B', I and G helices	7.4
						(via 98, 201)
	7	3	0.001	10	800	E/F loop	~50
							only last 25.2ps  shown

Conclusions

Acknowledgments