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PEPCRE: an interactive program to create, manipulate and display oligopeptides
PEPCRE: an interactive program to create, manipulate and display oligopeptides C. W. v. d. Lieth,* J. Palm,? A. Sundin,“f R. E. Carter? and T. Liljefors University of Lund, Chemical Centre, Molecular Graphics Laboratory for Organic Chemistry, Organic Chemistry 2 and 3, S-221 00 Lund, Sweden *Deutsches Krebsforschungszentrum, Institut fur Biochemie, Im Neuenheimer Feld, Heidelberg, FRG
PEPCRE is an interactive computer graphics program for the rapid construction, manipulation and display of oligopeptides. Essentially any desired conformation of an oligopeptide can be constructed in a simple and straightforward manner. The program provides various display and output possibilities. It is user-friendly and tk written in FORTRAN 77 for use on inexpensive, monochrome graphics terminals. Keywords: oligopeptides; modeling; computer graphics Received 13 May 1987 Accepted 20 May 1987
Oligopeptides are currently attracting a great deal of attention due to their extremely varied physiological effects. An adequate understanding of their biological effects (i.e., at the molecular level) must be based at least in part on knowledge about their available conformational space.’ A great deal of effort has been expended to develop molecular mechanics and molecular dynamics methods to explore the conformational energetics of peptides and proteins.z-s Most of the programs currently available use crystallographic data taken either from the Brookhaven Protein Data Bank (PDB) or the Cambridge Crystallographic Database to input the 3D structure of the desired molecule. Unfortunately, often no X-ray data are available, and only the amino acid sequence and some angles or distances are known. It is therefore of interest to develop programs that can construct peptides from this knowledge alone. The aim of this work was to develop a flexible, interactive computer graphics program for the construction, manipulation and display of oligopeptides. Such a program should allow the user to input easily all known information about the molecule of interest. The system should be able to create output to be used as input for various computations. In a second paper we will describe how the PDB tiles created by PEPCRE, together with some other input information, can be used as an interface for AMBER,3 MUMOD and GRID7 calculations. BACKGROUND Programs for the rapid and stereospecific construction of organic molecules have been developed and extenVolume 5 Number 3 September 1987
sively used at our Molecular Graphics Laboratory during the past six years,8sgprimarily as an input/ouput interface for molecular mechanics calculations. PEPCRE is a continuation and extension of this work to provide for the treatment of molecules of a specific type. The program was developed for oligopeptides up to 26 residues and 300 atoms for use on inexpensive monochrome raster graphics terminals using PLOT-10 software. The program is written in a flexible and modular way so that the molecule size can be easily expanded or contracted, and color coding can be added for more sophisticated terminals in a simple way. BASIC CONCEPT One feature of the model builder developed at our laboratory ‘3’ lets the user maintain full control of the 3D geometry of the desired structure throughout the building process. PEPCRE follows this philosophy: it is 3D from the very beginning and offers the user full control to set all relevant angles of the peptide bonds and the side chains at any desired value and at any time during construction. For building oligopeptides this is accomplished by joining prestored amino acids with the JOIN function of RINGS,’ similar to the way described for the COGS system.” A standard set of the natural amino acids was prepared with MIMIC*,’ and then minimized with AMBER.’ Other amino acids can be built or their structures taken from x-ray data and easily added to the data base. The amino acid sequence can be expanded at any time during construction. The program makes it possible to construct some standard peptide geometries (e.g., a-helix, p-sheet) for a user-defined sequence of amino acids or to individually set the relevant angles for each residue. A simple minimization routine helps to find suitable positions for the side chains. Various output formats are provided to establish an easy connection to other programs. THE MAN- MACHINE
INTERFACE
As pointed out above, the program is designed to be used by non-computer-oriented chemists and therefore should be user-friendly. The entire program is menudriven and relatively simple to use, so that the user needs essentially no further information to start creating
0263-7855/87/030119-07 $03.00 @ 1987 Butterworth Publishers
119
the
PEPCRE MAIN
menus
MEB
1
ABU
SAR 3
2
MLE 4
VAL 5
MLE 6
ALA 7
F\Lf? 8
MLE 3
MLE 10
MVA
11
MENU
1 set ALPHA
HELIX
2 setBETA
SHEET
3 set ANGLES 4 add AMINO ACIDS 5 ROTATE
6
ALL
SAVE
7 UNDO 8 NAMES 9 DISPLAY y-
DISPLAY OPTIONS
I)
SIDE
+-
10 SPECIAL SET. 11 CALC/MINIMIZE
3) PLUTO
on/off
1) CLOSE PEPTIDE 2) OPEN PEPTIDE
on/off
4) STEREO 5) DOTSURFACE 6) ELECTROSTATIC
3) S-S BRIDGE
5) ALPHA ’ 6) BETA I
8) RAMACHANDRAh
7) BETA II
ALL
10)SCALE 11) MOVE 12) NAMES
8) BETA III 9) GAMMA
Figure 1. The PEPCRE
10) LEFT ALPHA 11) GAMMA
menus
peptides. The main menu (Figure 1) contains the functions for construction and setting the geometry. Two submenus for the display and the minimization options and a menu for less often used angle setting functions are also available. PEPCRE includes routines that check the validity of all user input to enable the return of control to the user if an input error is found. All user input is checked for “reasonableness” and data type appropriateness. To enable the user to escape from a self-created “error condition,” an interrupt function was implemented that jumps back to the input of the corresponding (sub)menu. With the UNDO option the user can always come back to his or her previous step of construction. The keyboard is the only input device used. Figure 2 shows the organization of the screen. The peptide is displayed in the middle of the screen. The current amino acid sequence is given at the top and the active menu at the right side of the screen, where a graphic representation of the definition of the important dihedral angles is also shown.
INPUT OPTIONS Direct amino acid sequence The user inputs the amino acid sequence using the normal three-letter code as an identification.” The program looks up the codes in an internal directory, and the residues are joined together and set to a P-sheet geometry. The main chain, together with the sequence information, is displayed on the screen. The user can then set the relevant dihedral angles as described below. 120
4
ADD
5 6 3
ROTATE ALL SAVE SET ANGLES
7 8
UNDO NAMES
9
DISPLAY
AMINO
CF1LC/tlINI
q,
AC
L
6 J 7 &PHI 6 , 3 1 trm = 0 PSI 4 J 1 2 b-an, - 0 CHI 4 3 5 7 Eil - 0
4) set OMEGA
7) HYDROPHOBICITY
9)ROTATE
HLPHA-HELIX BETA-SHEET
SPECIRL SETTINGS
on/off
2) H-ATOM
11
? 2
10
SPECIAL
20
EXIT
SET.
Figure 2. Cyclosporin A. The peptide was constructed with the compressedformat. The torsion angle information was taken from Ref. 12. The amino acid sequence is given at the top. Backbone and side chains are displayed
Compressed input format Often the relevant dihedral angles of the peptide bonds and the side chains can be found in the literature. It is convenient to input these values via a file. PEPCRE reads the file, picks up the corresponding residues and sets the angles to the given values. Table 1 shows the compressed input for Cyclosporin A. The angle information was taken directly from the literature.” Figure 2 shows the resulting graphics display (backbone and side chain) of the cyclic peptide. Analogues can also be constructed quite easily with the compressed input format. The user has to change only the desired amino acid identification names in the tile, and the program will pick up the corresponding residues. This is demonstrated in Figure 3, where four residues have been changed from the original Cyclosporin A. MIMIC input format If the user wishes to construct peptides with special side chains, he or she can make substitutions on an existing peptide with the aid of MIMIC8*9 functions. PEPCRE reads and writes files in MIMIC format. Figure 4 shows such a substituted peptide. Data from the Cambridge Database can also be utilized easily via a program that converts Cambridge data files to MIMIC format. SETTING
PEPTIDE
CONFORMATION
With a stiff amide bond and with rather rigid bond lengths and bond angles, the conformations of natural peptide chains are essentially described by the dihedral angles cpand w” (see also Figure 2). The rotation around Journal of Molecular Graphics
Table 1 Compressed input file for Cyclosporin A 10 1 2 3 4 5 6 7 8 9 10 11
The first line contains information HIS
fWJ 1
SAR 2
MLE 3
TRP 4
PSI PSI PSI PSI PSI PSI PSI PSI PSI PSI PSI
- 86.0 - 121.0 72.0 - 100.0 - 112.0 - 89.0 - 85.0 88.0 - 117.0 - 139.0 - 60.0
MEB PHI = ABUPHI = SAR PHI = MLE PHI = VALPHI = MLEPHI = ALA PHI = ALA PHI = MLE PHI = MLEPHI = MVAPHI =
5
MLE 6
I?LA 7
PHE 8
= = = = = = = = = = =
123.0 CHI 90.0 CHI -127.0 CHI 21.0 CHI 126.0 CHI lOl.OCHI 52.0 CHI 126.0 CHI 101.0 CHI 65.0 CHI 126.0 CHI
= = = = = = = = = = =
- 168.0 - 178.0 0.0 - 52.0 - 174.0 - 178.0 17.0 21.7 - 58.0 - 165.0 - 178.0
OMG OMG OMG OMG OMG OMG OMG OMG OMG OMG OMG
= = = = = = = = = = =
- 175.0 - 176.0 173.0 - 180.0 166.0 - 164.0 180.0 - 168.0 - 7.0 - 167.0 173.0
about cyclic peptides and S-S bridges. MLE 3
TYR 10
MVA 11
P
Table 2 Backbone angles of some standard peptide geometries that are implemented in PEPCRE’3122
Right-handed a helix
- 60.
180.
180.
a prime
- 150.
- 50.
- 60. -90. - 60. 80. - 60. - 60. 80. 60.
- 30. 0. 120. 0. - 30. - 30. - 65. 60.
i+l i+2 PIP i+l i+2 $111” i+l i+2 i+l y turna Left-handed a helix SI”
Figure 3. A changed Cyclosporin A. The amino acid sequence is given at the top. Backbone and side chains are displayed
-60.
0 sheet
a I indicates the user-chosen residue number. The inverse turns are also possible. the torsion angle w is inhibited in natural peptides13 due to resonance effects. The orientation of the side chain is determined by the torsion angle x. All four torsion angles can be set to any value. For some standard geometries (Table 2), special options have been implemented. A given sequence of residues can be set to the desired conformation, which considerably speeds up construction. Figure 5 shows the same amino acid sequence set to a normal right- and left-handed cc-helix conformation. For cyclic peptides the user must set the ring closure. The program creates a bond between the first and the last residue. If the distance is greater than 281, the user gets a warning. S-S bridges between two cysteine residues must also be set by the user. Again, the user gets a warning if the distance between the two S atoms is too long. ENERGY CALCULATIONS
Figure 4. The side chains of a peptide can be substituted by MIMIC options. Backbone, side chains and H-atoms are displayed. The two +-signs indicate the N- and the C-terminal end of the peptide. Lone pairs are indicated with a pseudo bond and two dots Volume 5 Number 3 September 1987
Because the position of the side chains depends heavily on both the backbone conformation and the adjacent amino acids, an option to check all short distances was implemented. To get some ideas about the energies associated with a rotation around a side chain or the backbone, the van der Waals interactions of a userspecified residue can be calculated. Moreover, an option 121
VAL
SER 1
2
FlLFl 3
HIS TRP 4
flSN 5
6
FlSP 7
GLU a
MET 9
GLY 10
TYR 11
LYS 12
ILE 13
VAL TYR fiRG LYS ASP 12 3 4 5 SHORT DISTANCES
ER
Figure 5. Construction of a left-handed and right-handed helix peptide by setting sequences of residues to a “standard” geometry. The amino acid sequence is given at the top. Only the backbone is displayed
that automatically sets the torsional angles of one or all side chains to achieve a minimum of van der Waals’ interactions is quite useful. The CHECK DISTANCE option displays the two atom numbers and a dashed line between them if they are closer than a user-chosen cutoff distance. The corresponding distance is given in A on the left side of the The option MINIMIZE SIDE lets the user screen. set an indicated residue or all residues together at the minimum of the van der Waals interactions using the EVDW routine of MM2.’ A “rigid rotation” around the bond between C(a) and C(p) is done. To speed up this calculation, only the interactions between the indicated side chain and the rest of the molecule are calculated. If only one side chain is minimized, the rotation increment is set to 10”. If all side chains are minimized in one run, the minimization is first done from the N- to the C-terminal end with an angle increment of 30”. In a second step the minimization is done in the opposite direction with an angle increment of lo”. This procedure produces more reasonable results than just one run, because the true minimum of a side chain is dependent on the positions of the adjacent side chains. Figures 6a and 6b show the short distances of a peptide before and after setting the side chains to a minimum of van der Waals interactions. The options RIGID BACKBONE and RIGID SIDE enable a rigid rotation around either of the two important backbone angles Q and Y or the side chain angle X. The van der Waals interactions can be calculated either alone or together with the dipole interactions. The user can set the angles to be rotated about and their rotational increments. The calculated energies are plotted as an energy map - which is equivalent to the so-called Ramachandran mapI (see Figure 7) - or as a potential curve (Figure 8).14 Although some efforts have been made to speed up 122
669917161836363737374462ala28282-
16= 26= 93= 94= 26= 25= 26' 40= 49= 45= 46= 49' 62= 69= 91= 90= 91= 92=
1.99 1.69 1.66 1.13 1.61 1.54 0.64 1.62 1.90 1.09 0.50 1.45 1.62 1.66 1.64 1.40 1.54 1.66
Figure 6a. CHECK DISTANCE option before minimizing the side chains. The atom numbers of the short distances and0 their values are given at the left side of the screen in A. The atom numbers are also given in the molecule display. Backbone, side chains and H-atoms are displayed. The amino acid sequence is given at the top VAL TYR ARG LYS RSP 12 3 4 5 SHORT DISTANCES 37- 46= 1.92 46- 62= 1.66
Figure 6b. CHECK the side chains
this calculation, this to all other options The time required mately as the square
DISTANCE
option after minimizing
part of the program - in contrast - is no longer really interactive. for calculation increases approxiof the number of atoms. Journal of Molecular Graphics
VAL
SER
ALA
ALA
1
2
1
CYS
2
PHE
3
TYR
4
VAL
5
3
SER
6
,.:::;:::... .. . . .
_
PSI
PHI
Figure 7. Ramachandran map. The energies are calculated for the rotation around PHI and PSI of serine. The increment of rotation was 30”. The numbers in the plot indicate the relative energies in Kcallmol calculated for these angles. All relative energies greater than 99 Kcal/mol are set to 99. The isoenergetic lines are drawn for 3, 10, 20, 40, 60 and 80 Kcal/mol. The map shows clearly that the a-helix region (PHI -60, PSI -60) and an extended chain region (PHI -90, PSI + 120) are connected by a “bridge” of low energies. The energy of the conformation of the left-handed a-helix (phi = 60, psi = 60) is only a little bit higher than those of the right-handed, but it is separated by a higher “bridge” from the chain region VAL
HIS ;I
2
TRP
LYS 3
4 XSTART= DELTAX= YSTFIRT’ DELTAY=
0.0 36.0 0.0 67.6
Figure 8. Energy plot of a rigid rotation of 360” around the side chain of tryptophan in a $-sheet conformation of the hexapeptide VAI-HIS-TRP-LYS-PHE-TYR
DISPLAY OPTIONS Because PEPCRE is designed to construct mainly oligopeptides, it uses only relatively simple and interactive display possibilities. The various functions are listed under the display menu in Figure 1. The default is only to display the backbone of a peptide as a simple stick model. If desired, the user can Volume 5 Number 3 September 1987
Figure 9. Dotsurface display for side chains of cysteine, phenylalanine and serine in a cyclic hexapeptide
change the defaults so that the side chains (SIDE on/off) and H-atoms (H-ATOM on/off) are also displayed. This can be done either as a PLUTO Plot,” which is speeded up by a hardware circle generator, or as a simple stick model (PLUTO on/off). The MOVE and SCALE options allow the user to examine any part of the peptide in greater detail. The STEREO option produces a stereo plot with the current settings of the plot defaults, i.e., as a PLUTO plot or in stick mode, with or without side chains/Hatoms. The NAME option displays the three-letter identification codes at the C(a) position of the corresponding amino acid for all residues, for all residues with the same code or for a particular residue number. The dotted van der Waals surfaces of side chains, indicated by the user, can be visualised with the DOTSURFACE option, which uses a recently developed fast algorithm. l6 Figure 9 shows a conformation of a cyclic hexapeptide in which the side chains of cysteine (residue 2), phenylalanine (residue 3) and serine (residue 6) are drawn with a dotsurface. The electrostatic potential is a powerful tool that has provided insights into intermolecular association and molecular propex,ties of peptides and proteins and their interactions with small molecules.” The option ELECTROSTATIC provides for the calculation of the electrostatic potential for any of the dot points and can be visualized with a relative scale.16 The classical formula for the electrostatic potential V at a point F for a given system of point charges q(zJ at points r in a medium of a dielectric constant E is given by:
v=cLi
s(ri - F)
The point charges are taken from AMBER” and stored in the amino acid data base for each atom. The user can choose the dielectric constant (default value 4). Figure 10 shows the electrostatic potential calculated
123
TYR
GLY 1
GLY 2
PHE 3
MET 4
5
Figure 10. Electrostatic potential display for the side chains of thyrosine, phenylalanine and methionine of a met-enkephalin conformation. The two + - signs indicate the N-and the C-terminal end of the peptide. The symbol] energy code is: “= “: r-19.9 to -12.0]; “- “: [-120.0 to -4.01; “.‘I:r-4. to 4.01; , “+ I’: [4.0 to 12.01; I,#>‘: [12.0 to 19.1 RRG
PRO 1
2
LYS
PRO 3
GLN 4
GLN 5
6
PHE
PHE 7
GLY 8
LEU 9
10
PlET 11
Figure 12. Plot of the main chain dihedral angles of the 11 residues of Cyclosporin A (Ramachandran-Plot). The numbers indicate the corresponding residue position. The plot shows clearly that 9 residues fall in the chain region and only the N-methyl glycine (residue 3) and alanine (residue 8) show unusual (‘tforbidden”) angles of each vector indicates the strength of the attractive/ repulsive forces for water molecules. The option RAMACHANDRAN generates a plot of the cpand v angles of the given peptide, which is known as a Ramachandran plot. I3 Figure 12 which is such a plot for Cyclosporin A, clearly shows that 9 of the 11 residues fall in the chain region and only the N-methyl Glycine (residue 3) and Alanine (residue 8) show unusual (“forbidden”) angles. OUTPUT
Figure Il. Indication of residue based hydrophobicityl hydrophilicitti’ of a conformer of Substance P. The hydrophobic (+) and hydrophilic (-) vectors point toward the center of mass of the corresponding side chains, and the length of each vector indicates the strength of the attractive/repulsive forces for water molecules for the side chains of tyrosine, phenylalanine and methionine of an energy minimized conformation of met-enkephalin.” The HYDROPHOBICITY option gives a qualitative overview of the hydrophobic and hydrophilic regions of a peptide. This representation uses the residue-based matching hydrophobicities (OMH) of optimal Eisenberg.” Figure 11 shows the hydrophobicity distribution of a conformer of Substance P. The hydrophobic ( + ) and hydrophilic ( - ) vectors point toward the center of mass of the corresponding side chains, and the length 124
OPTIONS
To be able to calculate conformational energetics or other properties of a constructed oligopeptide, different output formats are provided. These can be used as input for other programs such as AMBER,3 MUMOD and GRID.’ The most often used format for peptides and proteins is that from the Brookhaven Protein Data Bank (PDB format). As shown above, the MIMIC output format enables the user to change a given peptide with MIMIC substitutions and to minimize a given structure with MM2[85].’ The compressed format described above can also be used to save disk space. HARDWARE
AND IMPLEMENTATION
Most of the graphics and some of the construction routines are taken from MIMIC.8,9 The MIMIC format is therefore expanded only with charges for each atom and the peptide chain information. PEPCRE is written in FORTRAN 77 on a VAX 1l/780 and uses PLOT-l& compatible monochrome graphics (VISUAL 500 series) terminals. These terminals also include a selective erase capability and a scrolling region. Color coding can be added by modifying the corresponding display routines. The number of atoms PEPCRE accepts can be set easily. All intrinsic data assignments can be done from a command file. Journal of Molecular Graphics
SUMMARY
AND CONCLUSION
PEPCRE provides a flexible, interactive program for constructing, manipulating and displaying oligopeptides. Conformational information taken from the literature can be input easily, e.g., in compressed format. PEPCRE can be used as a “front-end” for molecular mechanics calculations or for any type of calculation that requires 3D molecular coordinates and connectivity as input. Although PEPCRE was designed mainly as a construction program for oligopeptides, the display options include a quick view of some properties of a peptide such as the molecular shape, electrostatic potentials and residue-based hydrophobicities. Simple hard-sphere mini~zation helps the user find a good starting conformation for more sophisticated calculations.
REFERENCES 1 Kessler, H. Angew. Chem. Int. Ed. Engl., 1982, 21, 512-523 2 Burkert, U. and Allinger, N. L. ~o~ec~iar mechanics. ACS Monograph. 1982, 177 Washington, D.C. The MM2[85] program includes some parameters for peptides. It will be available through QCPE. 3 Weiner, S. J., Kollman, P. A., Nguyen, D. T. and Case, D. A. J. Corn. Chem., 1986,7,23(X252 4 Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan S. and Karplus, M. J. Comp. Chem., 1983,4,187-217 5a Review: McCammon, J. A., Karplus, M. Act. Chem. Res., 1983, 16, 187-193 5b Review: Berendsen, H. C. J. and van Gunsteren, W. F. Molecular dynamics simulations: techniques and approaches, NATO Adv. Study Inst. Series, Ser. Cl 35,475-500
Volume 5 Number 3 September 1987
6 Teleman, 0. and Jiinsson, B. J. Comp. Chem., 1986, 7,58-66 7 Goodford, P. J. J. Med. Chem., 1985,28,849-857 8 Liljefors, T. .i. Mol. Graph., 1983, l,, 111 9 Lieth, C. W. v. d., Carter, R. E., Dolata, D. P. and Liljefors, T. J. Mol. Graph., 1984,2, 117 10 White, D. J. and Pearson, J. E. J. Mol. Graph., 1986, 4,134 11 IUPAC-IUB commission on Bicohemical Nomenclature 1969(70), Abbreviations and symbols for the description of the confo~ation of polypeptide chains. Biochemistry, 1970, 9, 3471-3479 12 Loosli, H. R., Kessler, H., Oschkinat, H., Weber, H. P., Petcher, T. J. and Widmer, A. Helv. Chim. Acta., 1985, 68,682 13aSchulz, G. E., Schirmer, R. H. Principles of Protein Structure. Springer, New York, 1978 13bRamachandran, G. N. and Ramakrishnan, C. Adv. Pror. Chem., 1968,23,283-437 14 Software for calculating the energy contour taken from an interactive carbohydrate minimization program by A. Sundin (to be published) 15 Motherwell, W. D. S. A program for pIotting molecular and crystal structures - PLUTO 79. University Chemical Laboratory, Lensfield Road, Cambridge, England 16 Palm, J. To be published 17 See for example: Weiner, P. K., Langridge, R., Blaney, J. M., Schaeffer, R. and Kollman, P. A. Proc. Natl. Acad. Sci. USA, 1982,79, 3754-3758 18 Singh, U. C. and Kollman, P. A. J. Comp. Chem., 1984,5,129-145 19 Paine, G. H. and Scheraga, H. A., Biopolymers, 1986, 25, 1547-1563 20 Eisenberg, D. Ann. Rev. Biochem., 1984,53,595-623 21 Smith, J.A. and Pease, L.G. CRC Crit. Rev. Biochem., 1980,8,512
125
PEPCRE is an interactive computer graphics program for the rapid construction, manipulation and display of oligopeptides. Essentially any desired conformation of an oligopeptide can be constructed in a simple and straightforward manner. The program provides various display and output possibilities. It is user-friendly and tk written in FORTRAN 77 for use on inexpensive, monochrome graphics terminals. Keywords: oligopeptides; modeling; computer graphics Received 13 May 1987 Accepted 20 May 1987
Oligopeptides are currently attracting a great deal of attention due to their extremely varied physiological effects. An adequate understanding of their biological effects (i.e., at the molecular level) must be based at least in part on knowledge about their available conformational space.’ A great deal of effort has been expended to develop molecular mechanics and molecular dynamics methods to explore the conformational energetics of peptides and proteins.z-s Most of the programs currently available use crystallographic data taken either from the Brookhaven Protein Data Bank (PDB) or the Cambridge Crystallographic Database to input the 3D structure of the desired molecule. Unfortunately, often no X-ray data are available, and only the amino acid sequence and some angles or distances are known. It is therefore of interest to develop programs that can construct peptides from this knowledge alone. The aim of this work was to develop a flexible, interactive computer graphics program for the construction, manipulation and display of oligopeptides. Such a program should allow the user to input easily all known information about the molecule of interest. The system should be able to create output to be used as input for various computations. In a second paper we will describe how the PDB tiles created by PEPCRE, together with some other input information, can be used as an interface for AMBER,3 MUMOD and GRID7 calculations. BACKGROUND Programs for the rapid and stereospecific construction of organic molecules have been developed and extenVolume 5 Number 3 September 1987
sively used at our Molecular Graphics Laboratory during the past six years,8sgprimarily as an input/ouput interface for molecular mechanics calculations. PEPCRE is a continuation and extension of this work to provide for the treatment of molecules of a specific type. The program was developed for oligopeptides up to 26 residues and 300 atoms for use on inexpensive monochrome raster graphics terminals using PLOT-10 software. The program is written in a flexible and modular way so that the molecule size can be easily expanded or contracted, and color coding can be added for more sophisticated terminals in a simple way. BASIC CONCEPT One feature of the model builder developed at our laboratory ‘3’ lets the user maintain full control of the 3D geometry of the desired structure throughout the building process. PEPCRE follows this philosophy: it is 3D from the very beginning and offers the user full control to set all relevant angles of the peptide bonds and the side chains at any desired value and at any time during construction. For building oligopeptides this is accomplished by joining prestored amino acids with the JOIN function of RINGS,’ similar to the way described for the COGS system.” A standard set of the natural amino acids was prepared with MIMIC*,’ and then minimized with AMBER.’ Other amino acids can be built or their structures taken from x-ray data and easily added to the data base. The amino acid sequence can be expanded at any time during construction. The program makes it possible to construct some standard peptide geometries (e.g., a-helix, p-sheet) for a user-defined sequence of amino acids or to individually set the relevant angles for each residue. A simple minimization routine helps to find suitable positions for the side chains. Various output formats are provided to establish an easy connection to other programs. THE MAN- MACHINE
INTERFACE
As pointed out above, the program is designed to be used by non-computer-oriented chemists and therefore should be user-friendly. The entire program is menudriven and relatively simple to use, so that the user needs essentially no further information to start creating
0263-7855/87/030119-07 $03.00 @ 1987 Butterworth Publishers
119
the
PEPCRE MAIN
menus
MEB
1
ABU
SAR 3
2
MLE 4
VAL 5
MLE 6
ALA 7
F\Lf? 8
MLE 3
MLE 10
MVA
11
MENU
1 set ALPHA
HELIX
2 setBETA
SHEET
3 set ANGLES 4 add AMINO ACIDS 5 ROTATE
6
ALL
SAVE
7 UNDO 8 NAMES 9 DISPLAY y-
DISPLAY OPTIONS
I)
SIDE
+-
10 SPECIAL SET. 11 CALC/MINIMIZE
3) PLUTO
on/off
1) CLOSE PEPTIDE 2) OPEN PEPTIDE
on/off
4) STEREO 5) DOTSURFACE 6) ELECTROSTATIC
3) S-S BRIDGE
5) ALPHA ’ 6) BETA I
8) RAMACHANDRAh
7) BETA II
ALL
10)SCALE 11) MOVE 12) NAMES
8) BETA III 9) GAMMA
Figure 1. The PEPCRE
10) LEFT ALPHA 11) GAMMA
menus
peptides. The main menu (Figure 1) contains the functions for construction and setting the geometry. Two submenus for the display and the minimization options and a menu for less often used angle setting functions are also available. PEPCRE includes routines that check the validity of all user input to enable the return of control to the user if an input error is found. All user input is checked for “reasonableness” and data type appropriateness. To enable the user to escape from a self-created “error condition,” an interrupt function was implemented that jumps back to the input of the corresponding (sub)menu. With the UNDO option the user can always come back to his or her previous step of construction. The keyboard is the only input device used. Figure 2 shows the organization of the screen. The peptide is displayed in the middle of the screen. The current amino acid sequence is given at the top and the active menu at the right side of the screen, where a graphic representation of the definition of the important dihedral angles is also shown.
INPUT OPTIONS Direct amino acid sequence The user inputs the amino acid sequence using the normal three-letter code as an identification.” The program looks up the codes in an internal directory, and the residues are joined together and set to a P-sheet geometry. The main chain, together with the sequence information, is displayed on the screen. The user can then set the relevant dihedral angles as described below. 120
4
ADD
5 6 3
ROTATE ALL SAVE SET ANGLES
7 8
UNDO NAMES
9
DISPLAY
AMINO
CF1LC/tlINI
q,
AC
L
6 J 7 &PHI 6 , 3 1 trm = 0 PSI 4 J 1 2 b-an, - 0 CHI 4 3 5 7 Eil - 0
4) set OMEGA
7) HYDROPHOBICITY
9)ROTATE
HLPHA-HELIX BETA-SHEET
SPECIRL SETTINGS
on/off
2) H-ATOM
11
? 2
10
SPECIAL
20
EXIT
SET.
Figure 2. Cyclosporin A. The peptide was constructed with the compressedformat. The torsion angle information was taken from Ref. 12. The amino acid sequence is given at the top. Backbone and side chains are displayed
Compressed input format Often the relevant dihedral angles of the peptide bonds and the side chains can be found in the literature. It is convenient to input these values via a file. PEPCRE reads the file, picks up the corresponding residues and sets the angles to the given values. Table 1 shows the compressed input for Cyclosporin A. The angle information was taken directly from the literature.” Figure 2 shows the resulting graphics display (backbone and side chain) of the cyclic peptide. Analogues can also be constructed quite easily with the compressed input format. The user has to change only the desired amino acid identification names in the tile, and the program will pick up the corresponding residues. This is demonstrated in Figure 3, where four residues have been changed from the original Cyclosporin A. MIMIC input format If the user wishes to construct peptides with special side chains, he or she can make substitutions on an existing peptide with the aid of MIMIC8*9 functions. PEPCRE reads and writes files in MIMIC format. Figure 4 shows such a substituted peptide. Data from the Cambridge Database can also be utilized easily via a program that converts Cambridge data files to MIMIC format. SETTING
PEPTIDE
CONFORMATION
With a stiff amide bond and with rather rigid bond lengths and bond angles, the conformations of natural peptide chains are essentially described by the dihedral angles cpand w” (see also Figure 2). The rotation around Journal of Molecular Graphics
Table 1 Compressed input file for Cyclosporin A 10 1 2 3 4 5 6 7 8 9 10 11
The first line contains information HIS
fWJ 1
SAR 2
MLE 3
TRP 4
PSI PSI PSI PSI PSI PSI PSI PSI PSI PSI PSI
- 86.0 - 121.0 72.0 - 100.0 - 112.0 - 89.0 - 85.0 88.0 - 117.0 - 139.0 - 60.0
MEB PHI = ABUPHI = SAR PHI = MLE PHI = VALPHI = MLEPHI = ALA PHI = ALA PHI = MLE PHI = MLEPHI = MVAPHI =
5
MLE 6
I?LA 7
PHE 8
= = = = = = = = = = =
123.0 CHI 90.0 CHI -127.0 CHI 21.0 CHI 126.0 CHI lOl.OCHI 52.0 CHI 126.0 CHI 101.0 CHI 65.0 CHI 126.0 CHI
= = = = = = = = = = =
- 168.0 - 178.0 0.0 - 52.0 - 174.0 - 178.0 17.0 21.7 - 58.0 - 165.0 - 178.0
OMG OMG OMG OMG OMG OMG OMG OMG OMG OMG OMG
= = = = = = = = = = =
- 175.0 - 176.0 173.0 - 180.0 166.0 - 164.0 180.0 - 168.0 - 7.0 - 167.0 173.0
about cyclic peptides and S-S bridges. MLE 3
TYR 10
MVA 11
P
Table 2 Backbone angles of some standard peptide geometries that are implemented in PEPCRE’3122
Right-handed a helix
- 60.
180.
180.
a prime
- 150.
- 50.
- 60. -90. - 60. 80. - 60. - 60. 80. 60.
- 30. 0. 120. 0. - 30. - 30. - 65. 60.
i+l i+2 PIP i+l i+2 $111” i+l i+2 i+l y turna Left-handed a helix SI”
Figure 3. A changed Cyclosporin A. The amino acid sequence is given at the top. Backbone and side chains are displayed
-60.
0 sheet
a I indicates the user-chosen residue number. The inverse turns are also possible. the torsion angle w is inhibited in natural peptides13 due to resonance effects. The orientation of the side chain is determined by the torsion angle x. All four torsion angles can be set to any value. For some standard geometries (Table 2), special options have been implemented. A given sequence of residues can be set to the desired conformation, which considerably speeds up construction. Figure 5 shows the same amino acid sequence set to a normal right- and left-handed cc-helix conformation. For cyclic peptides the user must set the ring closure. The program creates a bond between the first and the last residue. If the distance is greater than 281, the user gets a warning. S-S bridges between two cysteine residues must also be set by the user. Again, the user gets a warning if the distance between the two S atoms is too long. ENERGY CALCULATIONS
Figure 4. The side chains of a peptide can be substituted by MIMIC options. Backbone, side chains and H-atoms are displayed. The two +-signs indicate the N- and the C-terminal end of the peptide. Lone pairs are indicated with a pseudo bond and two dots Volume 5 Number 3 September 1987
Because the position of the side chains depends heavily on both the backbone conformation and the adjacent amino acids, an option to check all short distances was implemented. To get some ideas about the energies associated with a rotation around a side chain or the backbone, the van der Waals interactions of a userspecified residue can be calculated. Moreover, an option 121
VAL
SER 1
2
FlLFl 3
HIS TRP 4
flSN 5
6
FlSP 7
GLU a
MET 9
GLY 10
TYR 11
LYS 12
ILE 13
VAL TYR fiRG LYS ASP 12 3 4 5 SHORT DISTANCES
ER
Figure 5. Construction of a left-handed and right-handed helix peptide by setting sequences of residues to a “standard” geometry. The amino acid sequence is given at the top. Only the backbone is displayed
that automatically sets the torsional angles of one or all side chains to achieve a minimum of van der Waals’ interactions is quite useful. The CHECK DISTANCE option displays the two atom numbers and a dashed line between them if they are closer than a user-chosen cutoff distance. The corresponding distance is given in A on the left side of the The option MINIMIZE SIDE lets the user screen. set an indicated residue or all residues together at the minimum of the van der Waals interactions using the EVDW routine of MM2.’ A “rigid rotation” around the bond between C(a) and C(p) is done. To speed up this calculation, only the interactions between the indicated side chain and the rest of the molecule are calculated. If only one side chain is minimized, the rotation increment is set to 10”. If all side chains are minimized in one run, the minimization is first done from the N- to the C-terminal end with an angle increment of 30”. In a second step the minimization is done in the opposite direction with an angle increment of lo”. This procedure produces more reasonable results than just one run, because the true minimum of a side chain is dependent on the positions of the adjacent side chains. Figures 6a and 6b show the short distances of a peptide before and after setting the side chains to a minimum of van der Waals interactions. The options RIGID BACKBONE and RIGID SIDE enable a rigid rotation around either of the two important backbone angles Q and Y or the side chain angle X. The van der Waals interactions can be calculated either alone or together with the dipole interactions. The user can set the angles to be rotated about and their rotational increments. The calculated energies are plotted as an energy map - which is equivalent to the so-called Ramachandran mapI (see Figure 7) - or as a potential curve (Figure 8).14 Although some efforts have been made to speed up 122
669917161836363737374462ala28282-
16= 26= 93= 94= 26= 25= 26' 40= 49= 45= 46= 49' 62= 69= 91= 90= 91= 92=
1.99 1.69 1.66 1.13 1.61 1.54 0.64 1.62 1.90 1.09 0.50 1.45 1.62 1.66 1.64 1.40 1.54 1.66
Figure 6a. CHECK DISTANCE option before minimizing the side chains. The atom numbers of the short distances and0 their values are given at the left side of the screen in A. The atom numbers are also given in the molecule display. Backbone, side chains and H-atoms are displayed. The amino acid sequence is given at the top VAL TYR ARG LYS RSP 12 3 4 5 SHORT DISTANCES 37- 46= 1.92 46- 62= 1.66
Figure 6b. CHECK the side chains
this calculation, this to all other options The time required mately as the square
DISTANCE
option after minimizing
part of the program - in contrast - is no longer really interactive. for calculation increases approxiof the number of atoms. Journal of Molecular Graphics
VAL
SER
ALA
ALA
1
2
1
CYS
2
PHE
3
TYR
4
VAL
5
3
SER
6
,.:::;:::... .. . . .
_
PSI
PHI
Figure 7. Ramachandran map. The energies are calculated for the rotation around PHI and PSI of serine. The increment of rotation was 30”. The numbers in the plot indicate the relative energies in Kcallmol calculated for these angles. All relative energies greater than 99 Kcal/mol are set to 99. The isoenergetic lines are drawn for 3, 10, 20, 40, 60 and 80 Kcal/mol. The map shows clearly that the a-helix region (PHI -60, PSI -60) and an extended chain region (PHI -90, PSI + 120) are connected by a “bridge” of low energies. The energy of the conformation of the left-handed a-helix (phi = 60, psi = 60) is only a little bit higher than those of the right-handed, but it is separated by a higher “bridge” from the chain region VAL
HIS ;I
2
TRP
LYS 3
4 XSTART= DELTAX= YSTFIRT’ DELTAY=
0.0 36.0 0.0 67.6
Figure 8. Energy plot of a rigid rotation of 360” around the side chain of tryptophan in a $-sheet conformation of the hexapeptide VAI-HIS-TRP-LYS-PHE-TYR
DISPLAY OPTIONS Because PEPCRE is designed to construct mainly oligopeptides, it uses only relatively simple and interactive display possibilities. The various functions are listed under the display menu in Figure 1. The default is only to display the backbone of a peptide as a simple stick model. If desired, the user can Volume 5 Number 3 September 1987
Figure 9. Dotsurface display for side chains of cysteine, phenylalanine and serine in a cyclic hexapeptide
change the defaults so that the side chains (SIDE on/off) and H-atoms (H-ATOM on/off) are also displayed. This can be done either as a PLUTO Plot,” which is speeded up by a hardware circle generator, or as a simple stick model (PLUTO on/off). The MOVE and SCALE options allow the user to examine any part of the peptide in greater detail. The STEREO option produces a stereo plot with the current settings of the plot defaults, i.e., as a PLUTO plot or in stick mode, with or without side chains/Hatoms. The NAME option displays the three-letter identification codes at the C(a) position of the corresponding amino acid for all residues, for all residues with the same code or for a particular residue number. The dotted van der Waals surfaces of side chains, indicated by the user, can be visualised with the DOTSURFACE option, which uses a recently developed fast algorithm. l6 Figure 9 shows a conformation of a cyclic hexapeptide in which the side chains of cysteine (residue 2), phenylalanine (residue 3) and serine (residue 6) are drawn with a dotsurface. The electrostatic potential is a powerful tool that has provided insights into intermolecular association and molecular propex,ties of peptides and proteins and their interactions with small molecules.” The option ELECTROSTATIC provides for the calculation of the electrostatic potential for any of the dot points and can be visualized with a relative scale.16 The classical formula for the electrostatic potential V at a point F for a given system of point charges q(zJ at points r in a medium of a dielectric constant E is given by:
v=cLi
s(ri - F)
The point charges are taken from AMBER” and stored in the amino acid data base for each atom. The user can choose the dielectric constant (default value 4). Figure 10 shows the electrostatic potential calculated
123
TYR
GLY 1
GLY 2
PHE 3
MET 4
5
Figure 10. Electrostatic potential display for the side chains of thyrosine, phenylalanine and methionine of a met-enkephalin conformation. The two + - signs indicate the N-and the C-terminal end of the peptide. The symbol] energy code is: “= “: r-19.9 to -12.0]; “- “: [-120.0 to -4.01; “.‘I:r-4. to 4.01; , “+ I’: [4.0 to 12.01; I,#>‘: [12.0 to 19.1 RRG
PRO 1
2
LYS
PRO 3
GLN 4
GLN 5
6
PHE
PHE 7
GLY 8
LEU 9
10
PlET 11
Figure 12. Plot of the main chain dihedral angles of the 11 residues of Cyclosporin A (Ramachandran-Plot). The numbers indicate the corresponding residue position. The plot shows clearly that 9 residues fall in the chain region and only the N-methyl glycine (residue 3) and alanine (residue 8) show unusual (‘tforbidden”) angles of each vector indicates the strength of the attractive/ repulsive forces for water molecules. The option RAMACHANDRAN generates a plot of the cpand v angles of the given peptide, which is known as a Ramachandran plot. I3 Figure 12 which is such a plot for Cyclosporin A, clearly shows that 9 of the 11 residues fall in the chain region and only the N-methyl Glycine (residue 3) and Alanine (residue 8) show unusual (“forbidden”) angles. OUTPUT
Figure Il. Indication of residue based hydrophobicityl hydrophilicitti’ of a conformer of Substance P. The hydrophobic (+) and hydrophilic (-) vectors point toward the center of mass of the corresponding side chains, and the length of each vector indicates the strength of the attractive/repulsive forces for water molecules for the side chains of tyrosine, phenylalanine and methionine of an energy minimized conformation of met-enkephalin.” The HYDROPHOBICITY option gives a qualitative overview of the hydrophobic and hydrophilic regions of a peptide. This representation uses the residue-based matching hydrophobicities (OMH) of optimal Eisenberg.” Figure 11 shows the hydrophobicity distribution of a conformer of Substance P. The hydrophobic ( + ) and hydrophilic ( - ) vectors point toward the center of mass of the corresponding side chains, and the length 124
OPTIONS
To be able to calculate conformational energetics or other properties of a constructed oligopeptide, different output formats are provided. These can be used as input for other programs such as AMBER,3 MUMOD and GRID.’ The most often used format for peptides and proteins is that from the Brookhaven Protein Data Bank (PDB format). As shown above, the MIMIC output format enables the user to change a given peptide with MIMIC substitutions and to minimize a given structure with MM2[85].’ The compressed format described above can also be used to save disk space. HARDWARE
AND IMPLEMENTATION
Most of the graphics and some of the construction routines are taken from MIMIC.8,9 The MIMIC format is therefore expanded only with charges for each atom and the peptide chain information. PEPCRE is written in FORTRAN 77 on a VAX 1l/780 and uses PLOT-l& compatible monochrome graphics (VISUAL 500 series) terminals. These terminals also include a selective erase capability and a scrolling region. Color coding can be added by modifying the corresponding display routines. The number of atoms PEPCRE accepts can be set easily. All intrinsic data assignments can be done from a command file. Journal of Molecular Graphics
SUMMARY
AND CONCLUSION
PEPCRE provides a flexible, interactive program for constructing, manipulating and displaying oligopeptides. Conformational information taken from the literature can be input easily, e.g., in compressed format. PEPCRE can be used as a “front-end” for molecular mechanics calculations or for any type of calculation that requires 3D molecular coordinates and connectivity as input. Although PEPCRE was designed mainly as a construction program for oligopeptides, the display options include a quick view of some properties of a peptide such as the molecular shape, electrostatic potentials and residue-based hydrophobicities. Simple hard-sphere mini~zation helps the user find a good starting conformation for more sophisticated calculations.
REFERENCES 1 Kessler, H. Angew. Chem. Int. Ed. Engl., 1982, 21, 512-523 2 Burkert, U. and Allinger, N. L. ~o~ec~iar mechanics. ACS Monograph. 1982, 177 Washington, D.C. The MM2[85] program includes some parameters for peptides. It will be available through QCPE. 3 Weiner, S. J., Kollman, P. A., Nguyen, D. T. and Case, D. A. J. Corn. Chem., 1986,7,23(X252 4 Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan S. and Karplus, M. J. Comp. Chem., 1983,4,187-217 5a Review: McCammon, J. A., Karplus, M. Act. Chem. Res., 1983, 16, 187-193 5b Review: Berendsen, H. C. J. and van Gunsteren, W. F. Molecular dynamics simulations: techniques and approaches, NATO Adv. Study Inst. Series, Ser. Cl 35,475-500
Volume 5 Number 3 September 1987
6 Teleman, 0. and Jiinsson, B. J. Comp. Chem., 1986, 7,58-66 7 Goodford, P. J. J. Med. Chem., 1985,28,849-857 8 Liljefors, T. .i. Mol. Graph., 1983, l,, 111 9 Lieth, C. W. v. d., Carter, R. E., Dolata, D. P. and Liljefors, T. J. Mol. Graph., 1984,2, 117 10 White, D. J. and Pearson, J. E. J. Mol. Graph., 1986, 4,134 11 IUPAC-IUB commission on Bicohemical Nomenclature 1969(70), Abbreviations and symbols for the description of the confo~ation of polypeptide chains. Biochemistry, 1970, 9, 3471-3479 12 Loosli, H. R., Kessler, H., Oschkinat, H., Weber, H. P., Petcher, T. J. and Widmer, A. Helv. Chim. Acta., 1985, 68,682 13aSchulz, G. E., Schirmer, R. H. Principles of Protein Structure. Springer, New York, 1978 13bRamachandran, G. N. and Ramakrishnan, C. Adv. Pror. Chem., 1968,23,283-437 14 Software for calculating the energy contour taken from an interactive carbohydrate minimization program by A. Sundin (to be published) 15 Motherwell, W. D. S. A program for pIotting molecular and crystal structures - PLUTO 79. University Chemical Laboratory, Lensfield Road, Cambridge, England 16 Palm, J. To be published 17 See for example: Weiner, P. K., Langridge, R., Blaney, J. M., Schaeffer, R. and Kollman, P. A. Proc. Natl. Acad. Sci. USA, 1982,79, 3754-3758 18 Singh, U. C. and Kollman, P. A. J. Comp. Chem., 1984,5,129-145 19 Paine, G. H. and Scheraga, H. A., Biopolymers, 1986, 25, 1547-1563 20 Eisenberg, D. Ann. Rev. Biochem., 1984,53,595-623 21 Smith, J.A. and Pease, L.G. CRC Crit. Rev. Biochem., 1980,8,512
125