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Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
Molecular Dynamics Simulation in Vacuo and in Solution of 5,6
8
[Aib -D-Ala ] Cyclolinopeptide A: a Conformational and Comparative Study a
a
Michele Saviano , Filomena Rossi , Vincenzo a
a
Pavone , Benedetto Di Biasio & Carlo Pedone
a
a
Centro di Studio di Biocristallografia del C.N.R. Dipartimento di Chimica , Università degli Studi di Napoli , “Federico II” via Mezzocannone 4, 80134 , Napoli , Italy Published online: 21 May 2012.
To cite this article: Michele Saviano , Filomena Rossi , Vincenzo Pavone , Benedetto Di Biasio & Carlo Pedone (1992) Molecular Dynamics Simulation in Vacuo and in 5,6
8
Solution of [Aib -D-Ala ] Cyclolinopeptide A: a Conformational and Comparative Study, Journal of Biomolecular Structure and Dynamics, 9:6, 1045-1060, DOI: 10.1080/07391102.1992.10507978 To link to this article: http://dx.doi.org/10.1080/07391102.1992.10507978
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Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 9, Issue Number 6 (1992), @Adenine Press (1992).
Molecular Dynamics Simulation in Vacuo and in Solution of [Aib 5, 6-D-Ala 8] Cyclolinopeptide A: a Conformational and Comparative Study
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Michele Saviano, Filomena Rossi, Vincenzo Pavone, Benedetto Di Blasio and Carlo Pedone Centro di Studio di Biocristallografia del C.N.R. Dipartimento di Chimica Universita degli Studi di Napoli "Federico II" via Mezzocannone 4, 80134 Napoli, Italy Abstract The conformation of a Cyclolinopeptide A analogue, c-(Pro-Pro-Phe-Phe-Aib-Aib-Ile-DAla-Val), has been investigated by means of molecular dynamics simulations, in various molecular environments. The molecular dynamics results are compared with that obtained for Cyclolinopeptide A and a detailed analysis of the different behaviour for the two compounds is reported. A complete analysis of hydrogen bonds is presented.
Introduction Cyclolinopeptide A (CLA), c-(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val), a homodetic nonapeptide, isolated from linseed (1 ), belongs to a class of natural peptides (2) that can protect tissues against lesion caused by cell toxins such as phalloidin (3). In particular it is able to inhibit the uptake of cholate by hepatocytes (4).1t has been postulated (4) that this biological activity is linked to well-defined sequential features in the Pro-Pro-Phe-Phe segment and is shared by other cyclic peptides, i.e., antamanide (2) and by several analogues (2). The various experimental studies on CLA (5) underline that the biological activity is critically dependent upon the sequence and conformation of the cyclopeptide. The conformational characteristics ofCLA, both in the crystalline state and in solution, have been deeply examined (6,7,8). NMR studies, in several solvent media of different polarity (9,10,11,12), reveal the large flexibility of the peptide with a great tendency to exist in several quasi-isoenergetic conformations. It has been suggested (8), besides many explanations (13,14), that the existence of multiple conformers, even at low temperature, is related to several different hydrogen bonds between CLA and solvent molecules. Recently the CLA conformational behaviour has also been investigated using the Molecular Dynamics (MD) methods (15,16). The analysis of the simulation in vacuo and in polar solution (16) shows the possibility of existence of various structures depending upon the molecular environment, with high
1045
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Saviano eta/.
Figure 1: Comparison of CLAIB x-ray structure (continuous lines) with that of CLA (dotted lines).
flexibility of the Pro-Pro-Phe-Phe, and with the capability of the solvent molecules to divert the NH and CO groups from the formatiorr of intramolecular hydrogen bonds. The MD results underline the role of the packing effect in determining the peptide conformation in the crystal ( 16). This behaviour is also favoured by the great flexibility to the cis-trans isomerism of the Xxx-Pro bonds. In chloroform solution at 214K one single conformer exists, whose structure is in agreement with the conformation found in the crystal. In order to decrease the backbone flexibility of the cyclic peptide, a well known molecular tool (17, 18,19,20), the Aib residue, has been introduced. This CLA analog, (Aib 5,6 D-Ala8) henceforth called CLAIB, has recently been characterized in solid state and in solution, using NMR technique (21 ). The solid state results indicate that the x-ray structure ofCLAIB is very close that ofCLA (root mean square deviation for the backbone atoms is of0.16 A) (Figure 1) with the same hydrogen bond pattern
1047
Molecular Dynamics of a Cyclolinopeptide A analog
I AE(t) AE
I
AE(t) AE
I
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I
ol.0
50
50
t(ps)
t(ps)
1'
"'T''
AE(t) AE
o~------------~
0
50
t(ps)
t(ps)
..
1 .-------------
30
t(ps)
0~----------------_J
0
30 t(ps)
Figure 2: Energy versus time course plots as obtained from MD simulations: a) UNI 100; b) UNI300; c) CELVAClOO; d) CELVAC300; e) CELSOL; f) UNISOL. Energy has been reported as a normalized function.
Saviano et a/.
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Table I Root mean square deviation (in A) from x-ray data for the C' atoms of CLAIB, as obtained from the MD simulations. T(K)
RMS
100 300 300 100 300 300 300
0.68 0.79 0.24 0.25 0.49 0.30 0.87
Single molecule (UNIIOO) Single molecule (UNBOO) Single molecule min. (UNBOO) CELL vacuo (CELVAC 100) CELL vacuo (CELVAC300) CELL hydrated (CELSOL) Molecule in solution (UNISOL)
Table II a. Average and relative errors of the dihedral angles in the peptide main chain as obtained from the MD simulation in vacuo at lOOK (UNilOO) and 300K (UNBOO).
cp Residue
T=lOOK
T=300K
T=lOOK
Pro1 Pro 2 Phe3 Phe4 Aib5 Aib 6 Ile7 D-Ala 8 Val9
-82.0± 4.9 -81.0± 6.2 -79.0± 11.1 -117.2±13.3 -52.6± 5.3 -53.2± 5.6 -97.3± 8.5 82.1±11.3 -118.5± 5.1
-83.8± 9.4 -87.8± 9.9 -85.0±23.7 -130.3±20.8 -56.4± 7.2 -58.7± 7.2 -100.3±14.9 95.1±19.1 -121.5± 9.5
144.0± 8.2 -11.2± 14.6 -54.8± 8.0 72.4± 6.5 -47.1± 5.4 -34.0± 6.3 8.1± 12.3 28.1 ± 7.3 84.5± 5.9
"'
(J)
T=300K
T=lOOK
T=300K
143.4± 9.9 -13.2± 12.0 -64.9±15.0 92.2±14.6 -37.6± 8.5 -31.7± 10.4 0.8±22.9 28.7±14.1 88.7± 9.2
-0.3±7.0 -164.0±5.5 -173.5±5.5 -175.5±4.9 174.6±3.9 173.2±3.4 -179.0±4.8 -179.1±5.8 178.8±4.7
-1.7±10.1 -172.6± 11.1 -175.3± 8.3 -173.9± 6.7 -178.5± 5.9 -174.6± 7.1 -178.5± 7.6 -179.6± 8.3 173.0± 9.3
b. Comparison between x-ray data and the best minimized conformation of the MD simulations for a single molecule T = 300K (UNI300)
cp Residue
x-ray
MD
x-ray
Pro 1 Pro2 Phe3 Phe4 Aib5 Aib 6 Ile7 D-Ala 8 Va19
-65.3 -89.9 -95.7 -90.9 -53.7 -54.7 -118.0 77.6 -128.3
-81.4 -72.2 -77.1 -89.5 -59.4 -57.0 -95.4 79.1 -121.5
162.2 -17.3 -43.4 57.8 -40.8 -31.4 16.2 27.2 79.6
"'
(J)
MD
x-ray
MD
160.4 -38.6 -52.7 60.3 -40.6 -32.2 6.9 24.0 82.9
9.0 -175.2 -170.4 -178.8 -173.0 -173.6 172.2 179.4 175.1
2.8 -169.5 -172.0 -174.8 175.9 171.8 -174.5 -176.2 173.0
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Molecular Dynamics of a Cyclolinopeptide A analog
1049
a)
b)
c)
d)
e)
f)
Figure 3: Comparisons of the structure of CLAIB in the solid state with those obtained from MD simulations: a) solid state; b) UNI300; c) CELVAClOO; d) CELVAC300; e) CELSOL; f) UNISOL.
and with a cis-peptide bond between the two proline residues. In solution, the same unique conformation has been proposed (21) to be present even at room temperature in chloroform and in acetonitrile solutions, with a structure very similar to that proposed for CLA (8). The biological activity of CLAlB has shown that this cyclopeptide is almost 40 times less active than CLA Therefore a complete structure characterization of two related compounds, CLA and CLAIB, in the solid state, in solution and in vacuo, using three different techniques, may allow a more reliable structureactivity correlation (22,23).
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Saviano et a/.
Table III a. Average and relative errors of the dihedral angles in the peptide main chain for the four units of the crystallographic cell as obtained from the MD simulation in vacuo at lOOK (CELVAC100).
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Residue
(!)
IJI
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-68.3 ± -82.4 ± -87.7 ± -88.3 ± -52.9 ± -53.8 ± -86.0 ± 84.9 ± -124.7 ±
4.9 5.0 7.7 4.6 5.1 6.1 5.7 7.4 4.8
152.8 ± -25.9 ± -51.3 ± 73.0 ± -44.0 ± -50.6 ± 7.5 ± 39.3 ± 80.3 ±
4.7 11.0 5.2 5.9 5.6 10.0 6.4 6.7 3.4
9.3 ± -171.0 ± 179.9 ± 170.3 ± 178.1 ± 177.2 ± -172.5 ± 170.9 ± 173.1 ±
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Va1 9
-66.9 -80.2 -81.5 -80.3 -55.3 -52.3 -99.5 77.5 -123.2
± ± ± ± ± ± ± ± ±
5.1 4.9 8.2 4.3 5.3 6.7 8.0 4.5 8.5
157.1 ± -33.8 ± -51.3 ± 60.7 ± -45.1 ± -42.7 ± 4.6 ± 33.8 ± 83.7 ±
4.9 11.7 5.7 6.2 5.0 11.1 6.3 6.8 3.8
7.7 ± 4.8 -170.3 ± 3.5 -175.9±3.9 178.8 ± 7.5 170.6 ± 3.9 175.7 ± 3.5 -178.1 ± 4.2 177.9 ± 4.0 177.9 ± 3.9
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Va19
-68.8 ± -79.3 ± -88.8 ± -84.0 ± -48.7 ± -52.5 ± -88.6 ± 76.3 ± -128.5 ±
4.7 5.0 8.1 4.5 5.0 6.9 8.4 4.6 8.2
152.2 ± -26.7 ± -56.5 ± 67.1 ± -36.6 ± -50.4 ± 6.6 ± 40.9 ± 82.2 ±
5.0 10.8 5.9 5.6 5.1 11.2 6.7 6.9 3.3
8.0 ± 5.0 -171.0±3.7 -176.5 ± 4.1 176.7 ± 7.2 177.5 ± 4.0 175.2 ± 3.4 -177.2 ± 3.7 177.3 ± 3.5 178.3 ± 4.3
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-68.9 ± -83.5 ± -87.9 ± -87.6 ± -53.5 ± -52.8 ± -87.2 ± 77.5 ± -125.8 ±
5.2 4.9 8.2 4.2 5.3 6.8 8.2 5.1 8.6
146.7 ± -21.6 ± -54.3 ± 70.3 ± -43.2 ± -45.9 ± 10.2 ± 35.8 ± 75.8 ±
4.5 9.6 5.2 5.2 4.9 10.6 6.5 7.3 4.2
5.6 ± -175.0 ± -178.2 ± 176.4 ± 176.1 ± 177.0 ± -175.0 ± 173.3 ± 176.7 ±
5.0 3.4 3.6 7.7 3.8 3.7 3.6 3.9 3.8
4.6 3.8 4.7 7.3 4.2 3.4 3.8 3.9 4.0
In this work we present an analysis of molecular dynamics simulations for CLAIB in vacuo and in solution, both for an isolated molecule and for a periodic crystal.
Materials and Methods The starting coordinates for all the simulations were those obtained from the single crystal x-ray diffraction (21 ). Energy minimizations and molecular dynamics (MD) simulations were performed with a Personal Iris 4025 Turbo Silicon Graphics workstation, using the
1051
Molecular Dynamics of a Cyclolinopeptide A analog Table III b. Comparison of the dihedral angles in the solid state with those obtained from the average of the four sets of MD angles given in Table Ilia.
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Q>
(J)
'I'
Residue
x-ray
MD
x-ray
MD
x-ray
MD
Pro' Pro2 Phe3 Phe4 Aib5 Aib 6 Ile7 D-Ala 8 Val9
-65.3 -89.9 -95.7 -90.9 -53.7 -54.7 -118.0 77.6 -128.3
-68.2 -81.3 -86.5 -85.0 -52.6 -52.8 -90.3 79.0 -125.4
162.2 -17.3 -43.4 57.8 -40.8 -31.4 16.2 27.2 79.6
152.2 -27.0 -53.4 67.8 -42.2 -47.4 7.2 37.4 80.5
9.0 -175.2 -170.4 -178.8 -173.0 -173.6 172.2 179.4 175.1
7.6 -171.8 -178.5 175.5 175.6 176.3 -175.7 174.8 176.5
DISCOVER program ver.2.7 developed by BIOSYM TECHNOLOGIES with the Consistent Valence Forcefield (CVFF) (24,2S,26) for the solute intra- and intermolecular interaction and for the water-peptide and water-water intermolecular interactions. The equations of motion were solved using the so-called Leapfrog integration algorithm (27) with a time step ofO.S fs. For the simulation in cell (in vacuo and hydrated) and in solution, the long range Coulomb interactions were computed using a non-bond cutoff method (28). In all cases, periodic boundary conditions were applied. MD Simulation in Vacuo
Two types of systems were simulated in vacuo: the first was composed of one CLAIB molecule in a large simulation box; the second system was composed of fourCLAIB molecules in the same crystallographic cell as in the solid state. Energy minimization to eliminate hot spots (29) using conjugate gradient method (30) was performed for one CLAIB molecule using the x-ray structure as an initial conformation. The energy-minimized structure was used as the initial structure for the MD simulations in vacuo at lOOK (hereafter denoted UNilOO) and at 300K (UNBOO). For both simulations the computational conditions were chosen to avoid boundary effects (16,31 ). For the simulations in the crystallographiccell,carriedoutat lOOK(CELVAClOO) and at 300K (CELVAC300), the x-ray structure was used as the starting conformation: four units per cell (Z = 4) were placed in a crystallographic box of29.92X 19.8SX9.90 A3 with the symmetry of the space P2 12 12 1• In each simulation performed with a time step ofO.Sfs, the CLAIB was equilibrated for SOps. After this first step, an additional 60ps of simulation without rescaling was carried out, since energy conservation was observed and the average temperature remained essentially constant around the target value of lOOK (for UNilOO and CELVAClOO) and 300K (for UNI300 and CELVAC300). In Figures 2a, 2b, 2c and 2e we have reported an energy versus time course plot for the initial steps of the various simulations. The analysis ofthese plots shows that a SOps equilibration was adequate in our simulations. Coordinates and velocities for the four simulations were dumped to a disk every 10
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Saviano eta/.
Table IV a. Average and relative errors of the dihedral angles in the peptide main chain for the four units of the crystallographic cell as obtained from the MD simulation in vacuo at 300K (CELVAC300).
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Residue
Ill
(J)
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile7 D-Ala8 Va1 9
-76.8 -81.3 -89.8 -131.1 -54.7 -58.7 -90.6 118.3 -114.2
± ± ± ± ± ± ± ± ±
8.1 7.6 12.5 10.5 8.8 8.5 11.4 15.3 10.0
130.7 ± 9.1 -35.9±11.1 -12.8 ± 10.2 76.1 ± 6.6 -42.0 ± 9.5 -36.3 ± 9.7 23.7 ± 11.4 29.7 ± 10.0 89.6 ± 7.0
7.0 ± 8.9 -171.2 ± 6.7 177.1 ± 6.2 171.5 ± 9.4 178.6 ± 6.2 173.9 ± 5.8 -174.9 ± 6.5 176.9 ± 7.2 164.6 ± 7.3
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-78.3 ± -83.4 ± -90.2 ± -135.6 ± -57.7 ± -56.8 ± -94.8 ± 115.8 ± -116.7 ±
8.6 7.8 11.2 11.0 8.9 9.0 18.8 15.0 11.1
140.5 ± -38.7 ± -14.8 ± 78.4 ± -45.0 ± -42.7 ± 25.9 ± 33.8 ± 87.7 ±
10.2 13.4 11.8 6.2 9.5 10.1 11.0 12.0 7.2
9.7 ± 9.0 -176.6±7.0 -173.7±6.7 176.9 ± 9.5 175.6 ± 6.9 179.9 ± 5.2 -170.3 ± 6.9 175.8 ± 7.6 172.8 ± 7.6
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-75.5 ± -81.6 ± -89.8 ± -137.8 ± -55.9 ± -57.2 ± -88.7 ± 107.4 ± -124.3 ±
8.0 7.7 9.2 10.2 8.7 8.3 17.4 13.6 9.5
137.3 ± -37.7 ± -16.8 ± 69.6 ± -36.9 ± -45.4 ± 23.9 ± 36.5 ± 81.3 ±
10.5 11.8 10.2 6.8 10.0 10.8 11.3 11.4 9.2
8.3 ± -175.0 ± -174.3 ± 173.3 ± 178.5 ± 176.4 ± 173.5 ± 177.4 ± 171.4 ±
9.3 7.4 6.5 7.3 7.2 5.8 6.7 7.7 7.6
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib 6 Ile7 D-Ala8 Val9
-70.5 ± 7.9 -78.5 ± 4.9 -85.8 ± 8.9 -132.6 ± 11.4 -55.5 ± 8.2 -60.7 ± 8.2 -93.8 ± 16.7 108.9 ± 13.1 -111.1 ± 9.8
140.0 ± -31.8 ± -13.8 ± 70.9 ± -44.7 ± -42.4 ± 25.7 ± 31.4 ± 89.6 ±
9.1 9.6 11.4 7.7 9.2 ·9.2 10.2 10.3 9.7
10.1 ± -175.2 ± -173.1 ± 171.2 ± 176.9 ± 177.1 ± -172.5 ± 176.8 ± 166.7 ±
9.1 3.8 7.3 6.4 7.3 6.5 6.2 7.8 8.8
steps during the last 20ps of the simulations. The dumped data were used for the statistical analysis. For UNI 100 and UNI300, energy minimization procedures were applied several times during the equilibration phase of the MD simulations in order to search for conformations "closer to the global minimum" (29). MD Simulations in Solution and in Hydrated Cell
1\vo kinds of systems were simulated: the first was composed of the CLAIB molecule
1053
Molecular Dynamics of a Cyclolinopeptide A analog Table IV b. Comparison of the dihedral angles in the solid state with those obtained from the average of the four sets of MD angles given in Table IVa.
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q,
(I)
\If
Residue
x-ray
MD
x-ray
MD
x-ray
MD
Pro' Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-65.3 -89.9 -95.7 -90.9 -53.7 -54.7 -118.0 77.6 -128.3
-75.3 -81.2 -88.9 -134.3 -55.9 -58.3 -92.0 112.6 -116.6
162.2 -17.3 -43.4 57.8 -40.8 -31.4 16.2 27.2 79.6
137.1 -36.0 -14.5 73.7 -42.1 -41.7 24.8 32.8 87.6
9.0 -175.2 -170.4 -178.8 -173.0 -173.6 172.2 179.4 175.1
8.8 -174.5 -174.5 173.2 178.3 176.8 -172.8 176.7 168.9
and as many water molecules as were needed to fill the simulation box. The latter box was selected such that all peptide atoms were at lest 6 Aaway from the box boundaries (UNISOL). The second system was composed of the same four molecules per cell in the crystallographic box as in the solid state (CELSOL) and as many water molecules as were needed to fill the cell. The two simulations were carried out at a temperature of 300K with a 0.5fs time step. The simulations were performed as follows. For the UNISOL case, as a first step, 520 water molecules confined in an orthorombic box of 27X25X23 N were equilibrated for a few picoseconds. As a second step, the CLAIB molecule, with coordinates from the x-ray data, was placed in the middle of the simulation box with the smallest moment of inertia parallel to the largest dimension ofthe box. For CELSOL, as a first step, 196 water molecules were equilibrated in an orthorombic box of29.92X 19.85X9.90 N. As a second step, the four units per cell were placed in the crystallographic box. Then for both systems, we removed those water molecules with the water atoms within a 1.6 A distance from any peptide atom, the resulting systems were composed of 452 water molecules and 139 peptide atoms for UNISOL and of 17 water molecules and 556 peptide atoms for CELSOL. In the first step, for both systems, a 1ps simulation was performed by keeping the solute fixed at its initial conformation to allow the water molecules to equilibrate in presence of the solute. Finally, both water and peptide atoms were allowed to move. The simulations were carried out for 30ps in the equilibration phase, and for 40ps without velocity rescaling since the average temperature remained constant around 300K (Figures 2e and 2f). Coordinates and velocities for the systems were dumped to a disk every 10 steps during the last 20ps of the two simulations. The dumped data were again used for the statistical analysis.
Results MD Simulation in Vacuo
The two simulations, UNilOO and UNI300, were carried out for an isolated molecule at 1OOK and 300K, respectively, in order to obtain indication on the characteristic of the isolated CLAIB molecule. In both simulations only notable flexibility of the
1054
Saviano eta/.
Table V a. Average and relative errors of the dihedral angles in the peptide main chain for the four units of the crystallographic cell as obtained from the MD simulation in solution at 300K (CELSOL).
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Residue
IV
(J)
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-65.6 -76.8 -79.7 -88.3 -52.4 -52.2 -87.9 83.0 -129.8
± ± ± ± ± ± ± ± ±
8.1 7.6 10.1 8.7 7.4 9.0 10.9 13.1 8.6
153.9 ± -36.8 ± -49.8 ± 74.6 ± -40.3 ± -57.7 ± 7.9 ± 42.0 ± 89.6 ±
7.3 11.4 9.2 8.4 7.5 10.8 11.3 9.9 6.8
7.6 ± -170.5 ± 176.1 ± 163.1 ± 176.6 ± 179.2 ± -174.4 ± 173.2 ± -173.4 ±
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val 9
-57.7 ± -77.2 ± -76.0 ± - 85.0 ± -59.4 ± -57.0 ± -88.5 ± 84.5 ± -125.5 ±
8.2 7.8 10.2 8.3 7.5 9.3 10.2 ll.5 8.4
153.4 ± -40.4 ± -49.3 ± 65.8 ± -40.1± -45.3 ± 9.0 ± 30.5 ± 75.3 ±
7.5 11.6 9.7 8.3 8.0 10.3 11.4 9.5 6.5
13.8 ± 8.3 -170.4 ± 5.3 -176.3±7.2 173.3 ± 7.8 175.4 ± 7.4 178.3 ± 5.8 176.3 ± 6.5 175.4 ± 9.2 -170.3 ± 6.5
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-60.0 -81.1 -80.7 -86.3 -51.9 -58.8 -93.5 83.3 -121.4
± ± ± ± ± ± ± ± ±
7.9 7.8 9.8 8.5 7.9 9.7 10.5 10.9 9.1
153.9 ± -30.1 ± -55.1 ± 62.3 ± -43.4 ± -29.3 ± 16.3 ± 20.6 ± 85.6 ±
7.8 ll.5 9.6 8.3 8.5 9.7 10.9 10.2 7.0
4.3 ± 8.2 -172.4 ± 5.2 -175.2±7.3 165.9 ± 7.6 173.2 ± 7.3 178.4 ± 5.9 176.2 ± 6.6 172.1 ± 9.1 -170.6 ± 6.8
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-57.8 ± -77.9 ± -76.2 ± -100.4 ± -52.1 ± -62.0 ± -93.9 ± 84.3 ± -138.8 ±
8.6 7.7 10.0 9.2 8.0 9.3 9.7 10.5 9.8
157.1 ± -42.8 ± -48.5 ± 77.1 ± -40.5 ± -43.4 ± 8.4 ± 20.6 ± 80.2 ±
7.6 10.9 9.5 8.3 8.6 9.9 10.5 10.3 7.2
5.0 ± -171.7 ± -169.5 ± 178.1 ± 175.8 ± 177.3 ± -170.0 ± 178.8 ± -172.0 ±
8.0 5.4 7.4 7.7 7.3 5.7 6.1 10.3 6.5
8.2 5.3 7.0 7.6 7.1 6.0 6.5 9.3 7.5
cyclopeptide has been observed in the Pro 2-Phe4 region and the root mean square deviations (RMS) from the experimental data of the average structures of the simulations were 0.68 Aat lOOK and 0.79 Aat 300K. The results are summarized in Table I. In Table Ila, we report the conformational statistical analysis for UNIIOO and UNI300, compared with the x-ray data. The cis bond between Pro 1 and Pro2, presents in the x-ray structure, is conserved in both the two simulations. No large deviations, however, exist between the x-ray data and the simulation results for all dihedral angles.
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Molecular Dynamics of a Cyclolinopeptide A analog TableV b. Comparison of the dihedral angles in the solid state with those obtained from the average of the four sets of MD angles given in Table Va.
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Residue
x-ray
MD
x-ray
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib 6 Ile 7 D-Ala8 Va19
-65.3 -89.9 -95.7 -90.9 -53.7 -54.7 -118.0 77.6 -128.3
-60.4 -78.3 -78.1 -90.0 -53.9 -57.5 -90.9 83.8 -128.7
162.2 -17.3 -43.4 57.8 -40.8 -31.4 16.2 27.2 79.6
"'
(J)
MD
x-ray
MD
154.6 -37.5 -50.7 70.0 -41.1 -43.9 10.4 28.4 80.4
9.0 -175.2 -170.4 -178.8 -173.0 -173.6 172.2 179.4 175.1
7.8 -171.2 -174.3 170.1 175.2 178.3 -174.2 174.9 -171.6
Energy minimizations were repeated using as starting point intermediate coordinates of the MD simulation ofUNI300, and many local minima were obtained with almost the same energy. Similarity of the structures minimized along the trajectory was evaluated by comparing the RMS deviation of the ca atoms for each possible pair of structures. The analysis of these minimized structures, using this procedure, reveals the existence of only one conformational family. The most stable structure, reported in Table lib, is similar to the x-ray data, with a low RMS deviation (0.24 A) and is characterized by larger difference only for Pro2-Phe4 segment. In Figure 3b we show the lowest energy structure. In order to study the temperature and cell packing effect on the conformation, two other simulations, CELVAClOO and CELVAC300, were performed at lOOK and 300K in vacuo, using the crystallographic cell as a simulation box (four units per cell). In Tables III and IV the conformational statistical analyses of the two MD simulations are reported. In Tables lila and IVa we report the analysis results for each unit in the simulation box; the results in Table Illb and IVb have been obtained by averaging over the four units, considered as equivalent molecules. It should be noted that the simulations have been performed without symmetry requirements within the simulation box; the space group symmetry was applied only to the periodic boundary conditions. As it is evident from the results in Tables III and IV, the two structures are similar to the x-ray data. The average conformations are shown in Figures 3c and 3d. CELVAClOO shows the lowest RMS deviation (0.25 A) from the x-ray data. The statistical analysis for CELVAC300 (RMS=0.49 A) shows more flexibility around the Pro 2-Phe3 bond. MD Simulation in Solution and in Hydrated Cell
The two simulations were carried out at 300K: in the system UNISOL, one CLAIB molecule was dissolved in a box containing 452 water molecules; in the system CELSOL, four CLAIB molecules are packed as in the crystalline state with 17 water molecules. In Table V and VI, the statistical analyses obtained from the MD simulations are
Saviano et al.
1056
Table VI Average and relative errors of the dihedral angles in the peptide main chain as obtained from the MD simulation in solution at 300K (UNISOL). Residue
-83.7 -88.7 -87.9 -137.9 -53.8 -52.3 -87.2 95.6 -129.1
± 10.1 ± 11.1 ± 24.0 ± 13.7 ± 8.3 ± 9.3 ± 15.0 ± 19.5 ± 8.5
ljl
(i)
141.9 ± 9.1 -20.7 ± 15.3 -61.3 ± 12.4 98.3 ± 9.9 -43.7 ± 8.0 -42.6 ± 11.1 12.0 ± 18.5 35.4 ± 10.8 82.2 ± 9.1
2.6 ± 10.0 -171.0 ± 7.7 177.3 ± 8.1 176.2 ± 7.5 177.1 ± 6.3 175.0 ± 6.1 -177.0 ± 7.0 177.3 ± 9.4 -176.3 ± 8.9
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Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
Figure 4: Stereo view of the average conformation of the MD simulations for a single molecule in solution at T=300K.
reported. For CELSOL the flexibility around the Pro 2-Phe 3 bond, already presents forCELVAC300, is decreased, and the RMS deviation is 0.30 A. The average conformation is presented in Figure 3e. The statistical analysis for the solution simulation (UNISOL) shows an increase in the flexibility for the dihedral angles in the Pro 1Pro2-Phe 3- Phe 4 and Ile7- D-Ala 8 regions with an RMS deviation of0.83 A. In Figure 3f, the averafe conformation is shown. In particular the side chains ofPhe2, Phe3, Ile7 and Val residues result to be oriented in the same direction (Figure 4). Hydrogen Bond Analysis Hydrogen bonding plays an important role in stabilizing the conformation of
Molecular Dynamics of a Cyclolinopeptide A analog
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Table VII The occurrence of intramolecular hydrogen bonds(%) obtained from MD simulations compared with those from x-ray data (21) Donor
NH 9
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Val 4 Phe 5 Aib 7 Ile D-Ala8
Acceptor
co 4
Phe 9 Val Phe 3 4 Phe 5 Aib
CELL vacuum
CELLhydr.
Solution
X-ray
T=lOOK
T=300K
T=300K
T=300K
100 100 100 100 100
100 99 98 40 98
97 98 68 20 80
94 96 99 30 82
86 96 83 15 86
biological molecules and in their mode of action and interaction with specific biological sites. In our analysis of the MD simulations, we define a hydrogen bond when the distance between a hydrogen atom and an acceptor atom is shorter than 2.5 A Table vn presents the occurrence of hydrogen bonds (%) for the various simulations. We define the occurrence of hydrogen bonds as the ratio of the number of the simulation steps when hydrogen bond is present, to the total number of simulation steps. As shown in Table VII, all hydrogen bonds found in the crystalline state are correctly reproduced in the in vacuo simulations ofCLAIB in cell at lOOK (CELVAClOO) and at 300K (CELVAC300). The analysis shows that only the hydrogen bond between the NH group oflle7 and the CO group ofPhe 4 shows a no high percentage of occurrence. Similar results are found in the energy minimized conformations of an isolated molecule (UNilOO and UNI300). In the simulation of the hydrated cell (CELSOL), we observe the same hydrogen bond patterns as in the x-ray structure with a no high occurrence of hydrogen bond 7 4 between Ile NH and CO Phe (30% of occurrence is predicted by the simulation). As shown in Figure 5, the peptide-solvent hydrogen bonding pattern in CELSOL is close to that found in the solid state (21) with two extra peptide solvent hydrogen bonds: between the oxygen of one water molecule and the CO of Pro 1, between Phe 3 NHandO(w). In the solution simulation (UNISOL), the results are similar to that we observe for CELSOL with a decrease of hydrogen bonds occurrence between Ile 7 NH and CO 4 Phe (20% occurrence is predicted by the simulation). As shown in Table VII, we did not observe other intramolecular hydrogen bonds in addition to that found in x-ray and NMR studies for any simulation.
Discussion The analysis ofthe simulations shows that the structure found in the solid state is independent from the molecular environment. These results are in agreement with experimental observations.
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Figure 5: Arrangement of the CLAIB and water molecules after MD simulation at 300K (CELSOL). Only the hydrogen bonds between peptide and solvent, already found in the x-ray structure, are shown as dashed lines.
The MD simulation ofCELVAClOO and CELVAC300 were performed in order to study the packing effect. It is worth noting that the MD results in the crystallographic cell (the system composed of only four CLAIB molecules) show structural characteristic similar to those obtained by x-ray and NMR data in polar and apolar solvents. No substantial differences are found for CELSOL simulation. The same solvent-peptide hydrogen bonds experimentally detected are found with additional hydrogen bonds between CLAIB and water molecules. The solid state conformational study (21) was performed with a crystal grown from methanolwater mixtures. The increase of the Xxx-Pro flexibility can be explained by the tendency of the NH and CO groups to be involved in both intra-molecular and solvent-peptide hydrogen bonds. It should be emphasized that the arrangement of the water molecules found in CELSOL (Figure 3) is quite similar to that found in the solid state study (21). Besides, the water addition in the crystallographic box improves the agreement of the MD results with the x-ray data (Table 1), compared with those obtained in vacuo in cell at the same temperature (CELVAC300). The MD simulation ofUNISOL was performed in order to characterize the behaviour and the conformational characteristics ofCLAIB in a polar solvent. The simulation result at 300K points out that CLAIB is structured in water, in a conformation as the one observed in the solid state and in polar and apolar solvent at room temperature. The presence of water molecules increases the flexibility ofXxx-Pro and oflle7DAla8 regions for the tendency of the carbonyl groups ofPro 1, Pro2, Phe3, Ile7 and
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Molecular Dynamics of a Cyclolinopeptide A analog
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D-Ala8 to be involved in intermolecular hydrogen bonds. The presence of these peptide-water hydrogen bonds can be a possible ex~lanation of the no high percentage 4 ofoccurrence of the hydrogen bond between NH lle and CO Phe (Table VII). It should be noted the low flexibility of the two Aib residues, during the simulations, and that the side chains of Phe2, Phe3, Ile7 and Val9 for CLAIB point toward the same side with the possible identification of a local hydrophobic region (Figure 4). The MD simulations of UNilOO and UNI300 and the subsequent minimizations were performed to clarify the characteristics of one CLAIB molecule in vacuo. The results point out that the x-ray conformation is a minimum energy structure for CLAIB and, on the contrary ofCLA, indicate that the packingofthe CLAIB molecules in the cell doesn't play an important role in stabilizing the peptide conformation in the crystallographic environment This result is in agreement with that obtained by NMR conformational studies (21 ). The solution studies for CLAIB point out to the presence in polar and apolar solvents, even at room temperature, of a structure closely resembling the one from x-ray analysis. The comparison of the MD results for CLAIB and CLA (16) emphasizes the different behaviour of these cyclic peptides and the ability of the two Aib residues to decrease the conformational accessible space for CLAIB. For CLA it was observed (16) the existence of different conformations with almost the same energy and a most stable structure that can be described as a bowl with a concave hydrophilic side, characterized by four carbonyl oxygens pointing inside the ring backbone, and an opposite convex side with predominant hydrophobic properties. The presence of water molecules induces an overall conformational variation similar to that observed in presence of metal ions (32).1t is interesting to point out that the CD and NMR titrations of CLAIB with metal ions didn't show specific interactions between this molecule and cations (21).
Conclusion This work completes the studies on a full characterization of two related compounds CLA and CLAIB. For both molecules the solid state structure and the behaviour in solution are known. A comparison between the characteristics of the two compounds in vacuo and in solution is presented. The MD confirms our expectation that the introduction in cyclic peptide of two Aib residues further reduces the backbone flexibility, stabilizing in solution one conformation, closely resembling its x-ray structure. The greatest difference between CLA and CLAIB is the behaviour of the two cyclic peptides in polar solution. The decrease of biological activity can be correlated to the decrease of flexibility of the CLAIB as compared to the CLA molecule. This result seems to exclude that the conformation found in the solid state for CLA and CLAIB corresponds to the biological active conformer and it suggests that folding of the ring structure, giving rise to a clustering ofhydrophobic groups in defined stereochemical position, could favour this activity. This arrangement could be realized by the presence of metal ions or by polar solvent molecules. It is noteworthy that Ovchinnikov eta/. (33) have suggested for antamanide a direct correlation between the biological activity and complex-stability constants of this natural peptide with metal ions. More recently it was shown that, also in the solid state, the folding of ring structure of cycloctapeptides (34) is strengthened by the interaction with polar solvent (H 20), and decreases going to semipolar or apolar solvents. Further studies on selected peptides are requested to support this hypothesis.
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Acknowledgments This work was supported by CNR grant no CTBCNR 89.5354
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References and Footnotes l. H.P. Kaufmann and A Tobschirbel, Chern. Ber. 92,2805 (1959). 2. T. Wieland,In Chemistry and Biology ofPeptides; Meinhofer, J., Ed; Ann Arbor Science: Ann Arbor, Ml, 377 (1972). 3. K Ziegler, M. Frimmer, H. Kessler, I. Damm, V. Eirmann, S. Koll and J. Zarkoch, Biochem. Biophys. Acta 86, 845 ( 1985). 4. H. Kessler, M. Klein, K Wagner, J.W. Bats, K Ziegler and M. Frimmer, Angew. Chern. Int.25, 997 (1986). 5. H. Kessler, J.W. Bats, C. Griesinger, S. Koll, M. Will and KJ. Wagner, J. Am. Chern. Soc. 110, 1033 (1986). 6. B. DiBlasio, E. Benedetti, V. Pavone, C. Pedone and M. Goodman,Biopolymers 26,2099 (1987). 7. T. Tancredi, G. Zanotti, F. Rossi, E. Benedetti, C. Pedone and P.A Temussi, Biopo/ymers 28, 513 (1989). 8. B. DiBlasio, F. Rossi, E. Benedetti, V. Pavone, C. Pedone, P.A. Temussi, G. Zanotti and T. Tancredi, J. Amer. Chern. Soc. 111, 9089 ( 1989). 9. F. Naider, E. Benedetti and M. Goodman, Proc. Nat/. Acad. Sci. 68, 1195 (1971). 10. AI. Brewster and F.A. Bovery, Proc. Nat/. Acad. Sci. 68, 1199 ( 1971 ). 11. l.Z. Siemion, WA Klis,A Sucharda-Sobczyk and R Obermeier, Rocniki Chern. 51, 1489 (1977). 12. AE. Tonelli, Proc. Nat/. Acad Sci. 68, 1203 (1971). 13. l.L. Karle,J. Karle, T. Wieland, W. Burgermeister, H. Faulstich and B. Witkop,Proc. Nat/.Acad. Sci. 6, 1836 (1973). 14. l.L. Karle, T. Wieland, D. Schermer and H. C. Ottenheym, Proc. Nat/. Acad. Sci. 76, 1532 (1979). 15. M.A. Castiglione Morelli, A Pastore, C. Pedone, PA Temussi, G. Zanotti and T. Tancredi, Int. J. Peptide Protein Res. 37, 81 (1991). 16. M. Saviano, M. Aida and G. Corongiu,Biopolymers 31, 1017 (1991). 17. B.V. Venkataram Prasad and P. Balaram, CRC Crit. Rev. Biochem. 16,307 (1984). 18. B. DiBlasio, V. Pavone, C. Pedone, A Santini, A Bavoso, C. Toniolo, M. Crisma and L. Sartore,J. Chern. Soc. Perkin Trans. 2, 1829 (1990). 19. V. Pavone, B. Di Blasia, A Santini, E. Benedetti, C. Pedone, C. Toniolo and M. Crisma,J. Mol. Bioi. 214, 633 (1990). 20. E. Benedetti, B. Di Blasia, V. Pavone, C. Pedone, A Santini, M. Crisma and C. Toniolo, Book in HonorofG.N Ramachandran, in press. 21. B. Di Blasia, F. Rossi, E. Benedetti, V. Pavone, M. Saviano, C. Pedone, G. Zanotti, T. Tancredi,]. Am. Chern. Soc., in press. 22. H. Kessler,Angew. Chern. Int. Ed. Engl. 21, 512 (1982). 23. H. Kessler, C. Griesinger, J. Lautz, A. Muller, W.F. van Gunsteren and H.J.C. Berendsen, J. Am. Chern. Soc. 110,3393 (1988). 24. S. Lifson, AT. Hagler and P. Dauber,J. Am. Chern. Soc. 101, 5111 (1979). 25. AT. Hagler, S. Lifson and P. Dauber, I. Am. Chern. Soc. 101,5122 (1979). 26. AT. Hagler, P. Dauber and S. Lifson,J. Am. Chern. Soc. 101,5131 (1979). 27. R.W. Hockney, Methods Comput. Phys. 9, 136 (1970). 28. C.L. Brooks III, B. Montgomery Pettitt and M. Karplus, J. Chern. Phys. 83, 5897 (1985). 29. C.L. Brooks Ill, B. Montgomery Pettitt and M. Karplus, in Proteins: A Theoretical Perspective of Dynamics, Structure and Thermoynamics; John Wiley and Sons, NY (1988). 30. W.H. Press, B.P. Flannery, S.A. Tenkdsky and W.T. Vetterling, in Numerical Recipes, The Art of Scientific Computing; Cambridge University Press, Cambridge (1986). 31. G. Corongiu, M. Aida, M.F. Pas and E. Clementi, in MOTECC-91 Modem Techniques in Computational Chemistry; Chapter 21 pp 910-913, E.Clementi Ed ESCOM, Leiden, 1991. 32. T. Tancredi, E. Benedetti, M. Grimaldi, C. Pedone, F. Rossi, M. Saviano, PA Temussi and G. Zanotti, Biopolymers 31,761 (1991). 33. Yu. A Ovchinnikov, V.T. Ivanov L.l. Barsukov, E.l. Melnik, NA Oreshnikova, N.D. Bogolyubova, l.D. Ryabova, AJ. Miroshnikov and V.A. Rimskaya, Experientia 28, 399 ( 1972). 34. T. Ishida, Y. In, M. Doi, M. Inoue, Y. Hamada and T. Shioiri, Biopolymers, in press ( 1991 ).
Date Received: November 12, 1991
Communicated by the Editor R.H. Sarma
Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
Molecular Dynamics Simulation in Vacuo and in Solution of 5,6
8
[Aib -D-Ala ] Cyclolinopeptide A: a Conformational and Comparative Study a
a
Michele Saviano , Filomena Rossi , Vincenzo a
a
Pavone , Benedetto Di Biasio & Carlo Pedone
a
a
Centro di Studio di Biocristallografia del C.N.R. Dipartimento di Chimica , Università degli Studi di Napoli , “Federico II” via Mezzocannone 4, 80134 , Napoli , Italy Published online: 21 May 2012.
To cite this article: Michele Saviano , Filomena Rossi , Vincenzo Pavone , Benedetto Di Biasio & Carlo Pedone (1992) Molecular Dynamics Simulation in Vacuo and in 5,6
8
Solution of [Aib -D-Ala ] Cyclolinopeptide A: a Conformational and Comparative Study, Journal of Biomolecular Structure and Dynamics, 9:6, 1045-1060, DOI: 10.1080/07391102.1992.10507978 To link to this article: http://dx.doi.org/10.1080/07391102.1992.10507978
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Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 9, Issue Number 6 (1992), @Adenine Press (1992).
Molecular Dynamics Simulation in Vacuo and in Solution of [Aib 5, 6-D-Ala 8] Cyclolinopeptide A: a Conformational and Comparative Study
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Michele Saviano, Filomena Rossi, Vincenzo Pavone, Benedetto Di Blasio and Carlo Pedone Centro di Studio di Biocristallografia del C.N.R. Dipartimento di Chimica Universita degli Studi di Napoli "Federico II" via Mezzocannone 4, 80134 Napoli, Italy Abstract The conformation of a Cyclolinopeptide A analogue, c-(Pro-Pro-Phe-Phe-Aib-Aib-Ile-DAla-Val), has been investigated by means of molecular dynamics simulations, in various molecular environments. The molecular dynamics results are compared with that obtained for Cyclolinopeptide A and a detailed analysis of the different behaviour for the two compounds is reported. A complete analysis of hydrogen bonds is presented.
Introduction Cyclolinopeptide A (CLA), c-(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val), a homodetic nonapeptide, isolated from linseed (1 ), belongs to a class of natural peptides (2) that can protect tissues against lesion caused by cell toxins such as phalloidin (3). In particular it is able to inhibit the uptake of cholate by hepatocytes (4).1t has been postulated (4) that this biological activity is linked to well-defined sequential features in the Pro-Pro-Phe-Phe segment and is shared by other cyclic peptides, i.e., antamanide (2) and by several analogues (2). The various experimental studies on CLA (5) underline that the biological activity is critically dependent upon the sequence and conformation of the cyclopeptide. The conformational characteristics ofCLA, both in the crystalline state and in solution, have been deeply examined (6,7,8). NMR studies, in several solvent media of different polarity (9,10,11,12), reveal the large flexibility of the peptide with a great tendency to exist in several quasi-isoenergetic conformations. It has been suggested (8), besides many explanations (13,14), that the existence of multiple conformers, even at low temperature, is related to several different hydrogen bonds between CLA and solvent molecules. Recently the CLA conformational behaviour has also been investigated using the Molecular Dynamics (MD) methods (15,16). The analysis of the simulation in vacuo and in polar solution (16) shows the possibility of existence of various structures depending upon the molecular environment, with high
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Saviano eta/.
Figure 1: Comparison of CLAIB x-ray structure (continuous lines) with that of CLA (dotted lines).
flexibility of the Pro-Pro-Phe-Phe, and with the capability of the solvent molecules to divert the NH and CO groups from the formatiorr of intramolecular hydrogen bonds. The MD results underline the role of the packing effect in determining the peptide conformation in the crystal ( 16). This behaviour is also favoured by the great flexibility to the cis-trans isomerism of the Xxx-Pro bonds. In chloroform solution at 214K one single conformer exists, whose structure is in agreement with the conformation found in the crystal. In order to decrease the backbone flexibility of the cyclic peptide, a well known molecular tool (17, 18,19,20), the Aib residue, has been introduced. This CLA analog, (Aib 5,6 D-Ala8) henceforth called CLAIB, has recently been characterized in solid state and in solution, using NMR technique (21 ). The solid state results indicate that the x-ray structure ofCLAIB is very close that ofCLA (root mean square deviation for the backbone atoms is of0.16 A) (Figure 1) with the same hydrogen bond pattern
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Molecular Dynamics of a Cyclolinopeptide A analog
I AE(t) AE
I
AE(t) AE
I
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I
ol.0
50
50
t(ps)
t(ps)
1'
"'T''
AE(t) AE
o~------------~
0
50
t(ps)
t(ps)
..
1 .-------------
30
t(ps)
0~----------------_J
0
30 t(ps)
Figure 2: Energy versus time course plots as obtained from MD simulations: a) UNI 100; b) UNI300; c) CELVAClOO; d) CELVAC300; e) CELSOL; f) UNISOL. Energy has been reported as a normalized function.
Saviano et a/.
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Table I Root mean square deviation (in A) from x-ray data for the C' atoms of CLAIB, as obtained from the MD simulations. T(K)
RMS
100 300 300 100 300 300 300
0.68 0.79 0.24 0.25 0.49 0.30 0.87
Single molecule (UNIIOO) Single molecule (UNBOO) Single molecule min. (UNBOO) CELL vacuo (CELVAC 100) CELL vacuo (CELVAC300) CELL hydrated (CELSOL) Molecule in solution (UNISOL)
Table II a. Average and relative errors of the dihedral angles in the peptide main chain as obtained from the MD simulation in vacuo at lOOK (UNilOO) and 300K (UNBOO).
cp Residue
T=lOOK
T=300K
T=lOOK
Pro1 Pro 2 Phe3 Phe4 Aib5 Aib 6 Ile7 D-Ala 8 Val9
-82.0± 4.9 -81.0± 6.2 -79.0± 11.1 -117.2±13.3 -52.6± 5.3 -53.2± 5.6 -97.3± 8.5 82.1±11.3 -118.5± 5.1
-83.8± 9.4 -87.8± 9.9 -85.0±23.7 -130.3±20.8 -56.4± 7.2 -58.7± 7.2 -100.3±14.9 95.1±19.1 -121.5± 9.5
144.0± 8.2 -11.2± 14.6 -54.8± 8.0 72.4± 6.5 -47.1± 5.4 -34.0± 6.3 8.1± 12.3 28.1 ± 7.3 84.5± 5.9
"'
(J)
T=300K
T=lOOK
T=300K
143.4± 9.9 -13.2± 12.0 -64.9±15.0 92.2±14.6 -37.6± 8.5 -31.7± 10.4 0.8±22.9 28.7±14.1 88.7± 9.2
-0.3±7.0 -164.0±5.5 -173.5±5.5 -175.5±4.9 174.6±3.9 173.2±3.4 -179.0±4.8 -179.1±5.8 178.8±4.7
-1.7±10.1 -172.6± 11.1 -175.3± 8.3 -173.9± 6.7 -178.5± 5.9 -174.6± 7.1 -178.5± 7.6 -179.6± 8.3 173.0± 9.3
b. Comparison between x-ray data and the best minimized conformation of the MD simulations for a single molecule T = 300K (UNI300)
cp Residue
x-ray
MD
x-ray
Pro 1 Pro2 Phe3 Phe4 Aib5 Aib 6 Ile7 D-Ala 8 Va19
-65.3 -89.9 -95.7 -90.9 -53.7 -54.7 -118.0 77.6 -128.3
-81.4 -72.2 -77.1 -89.5 -59.4 -57.0 -95.4 79.1 -121.5
162.2 -17.3 -43.4 57.8 -40.8 -31.4 16.2 27.2 79.6
"'
(J)
MD
x-ray
MD
160.4 -38.6 -52.7 60.3 -40.6 -32.2 6.9 24.0 82.9
9.0 -175.2 -170.4 -178.8 -173.0 -173.6 172.2 179.4 175.1
2.8 -169.5 -172.0 -174.8 175.9 171.8 -174.5 -176.2 173.0
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Molecular Dynamics of a Cyclolinopeptide A analog
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a)
b)
c)
d)
e)
f)
Figure 3: Comparisons of the structure of CLAIB in the solid state with those obtained from MD simulations: a) solid state; b) UNI300; c) CELVAClOO; d) CELVAC300; e) CELSOL; f) UNISOL.
and with a cis-peptide bond between the two proline residues. In solution, the same unique conformation has been proposed (21) to be present even at room temperature in chloroform and in acetonitrile solutions, with a structure very similar to that proposed for CLA (8). The biological activity of CLAlB has shown that this cyclopeptide is almost 40 times less active than CLA Therefore a complete structure characterization of two related compounds, CLA and CLAIB, in the solid state, in solution and in vacuo, using three different techniques, may allow a more reliable structureactivity correlation (22,23).
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Table III a. Average and relative errors of the dihedral angles in the peptide main chain for the four units of the crystallographic cell as obtained from the MD simulation in vacuo at lOOK (CELVAC100).
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Residue
(!)
IJI
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-68.3 ± -82.4 ± -87.7 ± -88.3 ± -52.9 ± -53.8 ± -86.0 ± 84.9 ± -124.7 ±
4.9 5.0 7.7 4.6 5.1 6.1 5.7 7.4 4.8
152.8 ± -25.9 ± -51.3 ± 73.0 ± -44.0 ± -50.6 ± 7.5 ± 39.3 ± 80.3 ±
4.7 11.0 5.2 5.9 5.6 10.0 6.4 6.7 3.4
9.3 ± -171.0 ± 179.9 ± 170.3 ± 178.1 ± 177.2 ± -172.5 ± 170.9 ± 173.1 ±
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Va1 9
-66.9 -80.2 -81.5 -80.3 -55.3 -52.3 -99.5 77.5 -123.2
± ± ± ± ± ± ± ± ±
5.1 4.9 8.2 4.3 5.3 6.7 8.0 4.5 8.5
157.1 ± -33.8 ± -51.3 ± 60.7 ± -45.1 ± -42.7 ± 4.6 ± 33.8 ± 83.7 ±
4.9 11.7 5.7 6.2 5.0 11.1 6.3 6.8 3.8
7.7 ± 4.8 -170.3 ± 3.5 -175.9±3.9 178.8 ± 7.5 170.6 ± 3.9 175.7 ± 3.5 -178.1 ± 4.2 177.9 ± 4.0 177.9 ± 3.9
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Va19
-68.8 ± -79.3 ± -88.8 ± -84.0 ± -48.7 ± -52.5 ± -88.6 ± 76.3 ± -128.5 ±
4.7 5.0 8.1 4.5 5.0 6.9 8.4 4.6 8.2
152.2 ± -26.7 ± -56.5 ± 67.1 ± -36.6 ± -50.4 ± 6.6 ± 40.9 ± 82.2 ±
5.0 10.8 5.9 5.6 5.1 11.2 6.7 6.9 3.3
8.0 ± 5.0 -171.0±3.7 -176.5 ± 4.1 176.7 ± 7.2 177.5 ± 4.0 175.2 ± 3.4 -177.2 ± 3.7 177.3 ± 3.5 178.3 ± 4.3
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-68.9 ± -83.5 ± -87.9 ± -87.6 ± -53.5 ± -52.8 ± -87.2 ± 77.5 ± -125.8 ±
5.2 4.9 8.2 4.2 5.3 6.8 8.2 5.1 8.6
146.7 ± -21.6 ± -54.3 ± 70.3 ± -43.2 ± -45.9 ± 10.2 ± 35.8 ± 75.8 ±
4.5 9.6 5.2 5.2 4.9 10.6 6.5 7.3 4.2
5.6 ± -175.0 ± -178.2 ± 176.4 ± 176.1 ± 177.0 ± -175.0 ± 173.3 ± 176.7 ±
5.0 3.4 3.6 7.7 3.8 3.7 3.6 3.9 3.8
4.6 3.8 4.7 7.3 4.2 3.4 3.8 3.9 4.0
In this work we present an analysis of molecular dynamics simulations for CLAIB in vacuo and in solution, both for an isolated molecule and for a periodic crystal.
Materials and Methods The starting coordinates for all the simulations were those obtained from the single crystal x-ray diffraction (21 ). Energy minimizations and molecular dynamics (MD) simulations were performed with a Personal Iris 4025 Turbo Silicon Graphics workstation, using the
1051
Molecular Dynamics of a Cyclolinopeptide A analog Table III b. Comparison of the dihedral angles in the solid state with those obtained from the average of the four sets of MD angles given in Table Ilia.
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Q>
(J)
'I'
Residue
x-ray
MD
x-ray
MD
x-ray
MD
Pro' Pro2 Phe3 Phe4 Aib5 Aib 6 Ile7 D-Ala 8 Val9
-65.3 -89.9 -95.7 -90.9 -53.7 -54.7 -118.0 77.6 -128.3
-68.2 -81.3 -86.5 -85.0 -52.6 -52.8 -90.3 79.0 -125.4
162.2 -17.3 -43.4 57.8 -40.8 -31.4 16.2 27.2 79.6
152.2 -27.0 -53.4 67.8 -42.2 -47.4 7.2 37.4 80.5
9.0 -175.2 -170.4 -178.8 -173.0 -173.6 172.2 179.4 175.1
7.6 -171.8 -178.5 175.5 175.6 176.3 -175.7 174.8 176.5
DISCOVER program ver.2.7 developed by BIOSYM TECHNOLOGIES with the Consistent Valence Forcefield (CVFF) (24,2S,26) for the solute intra- and intermolecular interaction and for the water-peptide and water-water intermolecular interactions. The equations of motion were solved using the so-called Leapfrog integration algorithm (27) with a time step ofO.S fs. For the simulation in cell (in vacuo and hydrated) and in solution, the long range Coulomb interactions were computed using a non-bond cutoff method (28). In all cases, periodic boundary conditions were applied. MD Simulation in Vacuo
Two types of systems were simulated in vacuo: the first was composed of one CLAIB molecule in a large simulation box; the second system was composed of fourCLAIB molecules in the same crystallographic cell as in the solid state. Energy minimization to eliminate hot spots (29) using conjugate gradient method (30) was performed for one CLAIB molecule using the x-ray structure as an initial conformation. The energy-minimized structure was used as the initial structure for the MD simulations in vacuo at lOOK (hereafter denoted UNilOO) and at 300K (UNBOO). For both simulations the computational conditions were chosen to avoid boundary effects (16,31 ). For the simulations in the crystallographiccell,carriedoutat lOOK(CELVAClOO) and at 300K (CELVAC300), the x-ray structure was used as the starting conformation: four units per cell (Z = 4) were placed in a crystallographic box of29.92X 19.8SX9.90 A3 with the symmetry of the space P2 12 12 1• In each simulation performed with a time step ofO.Sfs, the CLAIB was equilibrated for SOps. After this first step, an additional 60ps of simulation without rescaling was carried out, since energy conservation was observed and the average temperature remained essentially constant around the target value of lOOK (for UNilOO and CELVAClOO) and 300K (for UNI300 and CELVAC300). In Figures 2a, 2b, 2c and 2e we have reported an energy versus time course plot for the initial steps of the various simulations. The analysis ofthese plots shows that a SOps equilibration was adequate in our simulations. Coordinates and velocities for the four simulations were dumped to a disk every 10
1052
Saviano eta/.
Table IV a. Average and relative errors of the dihedral angles in the peptide main chain for the four units of the crystallographic cell as obtained from the MD simulation in vacuo at 300K (CELVAC300).
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Residue
Ill
(J)
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile7 D-Ala8 Va1 9
-76.8 -81.3 -89.8 -131.1 -54.7 -58.7 -90.6 118.3 -114.2
± ± ± ± ± ± ± ± ±
8.1 7.6 12.5 10.5 8.8 8.5 11.4 15.3 10.0
130.7 ± 9.1 -35.9±11.1 -12.8 ± 10.2 76.1 ± 6.6 -42.0 ± 9.5 -36.3 ± 9.7 23.7 ± 11.4 29.7 ± 10.0 89.6 ± 7.0
7.0 ± 8.9 -171.2 ± 6.7 177.1 ± 6.2 171.5 ± 9.4 178.6 ± 6.2 173.9 ± 5.8 -174.9 ± 6.5 176.9 ± 7.2 164.6 ± 7.3
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-78.3 ± -83.4 ± -90.2 ± -135.6 ± -57.7 ± -56.8 ± -94.8 ± 115.8 ± -116.7 ±
8.6 7.8 11.2 11.0 8.9 9.0 18.8 15.0 11.1
140.5 ± -38.7 ± -14.8 ± 78.4 ± -45.0 ± -42.7 ± 25.9 ± 33.8 ± 87.7 ±
10.2 13.4 11.8 6.2 9.5 10.1 11.0 12.0 7.2
9.7 ± 9.0 -176.6±7.0 -173.7±6.7 176.9 ± 9.5 175.6 ± 6.9 179.9 ± 5.2 -170.3 ± 6.9 175.8 ± 7.6 172.8 ± 7.6
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-75.5 ± -81.6 ± -89.8 ± -137.8 ± -55.9 ± -57.2 ± -88.7 ± 107.4 ± -124.3 ±
8.0 7.7 9.2 10.2 8.7 8.3 17.4 13.6 9.5
137.3 ± -37.7 ± -16.8 ± 69.6 ± -36.9 ± -45.4 ± 23.9 ± 36.5 ± 81.3 ±
10.5 11.8 10.2 6.8 10.0 10.8 11.3 11.4 9.2
8.3 ± -175.0 ± -174.3 ± 173.3 ± 178.5 ± 176.4 ± 173.5 ± 177.4 ± 171.4 ±
9.3 7.4 6.5 7.3 7.2 5.8 6.7 7.7 7.6
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib 6 Ile7 D-Ala8 Val9
-70.5 ± 7.9 -78.5 ± 4.9 -85.8 ± 8.9 -132.6 ± 11.4 -55.5 ± 8.2 -60.7 ± 8.2 -93.8 ± 16.7 108.9 ± 13.1 -111.1 ± 9.8
140.0 ± -31.8 ± -13.8 ± 70.9 ± -44.7 ± -42.4 ± 25.7 ± 31.4 ± 89.6 ±
9.1 9.6 11.4 7.7 9.2 ·9.2 10.2 10.3 9.7
10.1 ± -175.2 ± -173.1 ± 171.2 ± 176.9 ± 177.1 ± -172.5 ± 176.8 ± 166.7 ±
9.1 3.8 7.3 6.4 7.3 6.5 6.2 7.8 8.8
steps during the last 20ps of the simulations. The dumped data were used for the statistical analysis. For UNI 100 and UNI300, energy minimization procedures were applied several times during the equilibration phase of the MD simulations in order to search for conformations "closer to the global minimum" (29). MD Simulations in Solution and in Hydrated Cell
1\vo kinds of systems were simulated: the first was composed of the CLAIB molecule
1053
Molecular Dynamics of a Cyclolinopeptide A analog Table IV b. Comparison of the dihedral angles in the solid state with those obtained from the average of the four sets of MD angles given in Table IVa.
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q,
(I)
\If
Residue
x-ray
MD
x-ray
MD
x-ray
MD
Pro' Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-65.3 -89.9 -95.7 -90.9 -53.7 -54.7 -118.0 77.6 -128.3
-75.3 -81.2 -88.9 -134.3 -55.9 -58.3 -92.0 112.6 -116.6
162.2 -17.3 -43.4 57.8 -40.8 -31.4 16.2 27.2 79.6
137.1 -36.0 -14.5 73.7 -42.1 -41.7 24.8 32.8 87.6
9.0 -175.2 -170.4 -178.8 -173.0 -173.6 172.2 179.4 175.1
8.8 -174.5 -174.5 173.2 178.3 176.8 -172.8 176.7 168.9
and as many water molecules as were needed to fill the simulation box. The latter box was selected such that all peptide atoms were at lest 6 Aaway from the box boundaries (UNISOL). The second system was composed of the same four molecules per cell in the crystallographic box as in the solid state (CELSOL) and as many water molecules as were needed to fill the cell. The two simulations were carried out at a temperature of 300K with a 0.5fs time step. The simulations were performed as follows. For the UNISOL case, as a first step, 520 water molecules confined in an orthorombic box of 27X25X23 N were equilibrated for a few picoseconds. As a second step, the CLAIB molecule, with coordinates from the x-ray data, was placed in the middle of the simulation box with the smallest moment of inertia parallel to the largest dimension ofthe box. For CELSOL, as a first step, 196 water molecules were equilibrated in an orthorombic box of29.92X 19.85X9.90 N. As a second step, the four units per cell were placed in the crystallographic box. Then for both systems, we removed those water molecules with the water atoms within a 1.6 A distance from any peptide atom, the resulting systems were composed of 452 water molecules and 139 peptide atoms for UNISOL and of 17 water molecules and 556 peptide atoms for CELSOL. In the first step, for both systems, a 1ps simulation was performed by keeping the solute fixed at its initial conformation to allow the water molecules to equilibrate in presence of the solute. Finally, both water and peptide atoms were allowed to move. The simulations were carried out for 30ps in the equilibration phase, and for 40ps without velocity rescaling since the average temperature remained constant around 300K (Figures 2e and 2f). Coordinates and velocities for the systems were dumped to a disk every 10 steps during the last 20ps of the two simulations. The dumped data were again used for the statistical analysis.
Results MD Simulation in Vacuo
The two simulations, UNilOO and UNI300, were carried out for an isolated molecule at 1OOK and 300K, respectively, in order to obtain indication on the characteristic of the isolated CLAIB molecule. In both simulations only notable flexibility of the
1054
Saviano eta/.
Table V a. Average and relative errors of the dihedral angles in the peptide main chain for the four units of the crystallographic cell as obtained from the MD simulation in solution at 300K (CELSOL).
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Residue
IV
(J)
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-65.6 -76.8 -79.7 -88.3 -52.4 -52.2 -87.9 83.0 -129.8
± ± ± ± ± ± ± ± ±
8.1 7.6 10.1 8.7 7.4 9.0 10.9 13.1 8.6
153.9 ± -36.8 ± -49.8 ± 74.6 ± -40.3 ± -57.7 ± 7.9 ± 42.0 ± 89.6 ±
7.3 11.4 9.2 8.4 7.5 10.8 11.3 9.9 6.8
7.6 ± -170.5 ± 176.1 ± 163.1 ± 176.6 ± 179.2 ± -174.4 ± 173.2 ± -173.4 ±
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val 9
-57.7 ± -77.2 ± -76.0 ± - 85.0 ± -59.4 ± -57.0 ± -88.5 ± 84.5 ± -125.5 ±
8.2 7.8 10.2 8.3 7.5 9.3 10.2 ll.5 8.4
153.4 ± -40.4 ± -49.3 ± 65.8 ± -40.1± -45.3 ± 9.0 ± 30.5 ± 75.3 ±
7.5 11.6 9.7 8.3 8.0 10.3 11.4 9.5 6.5
13.8 ± 8.3 -170.4 ± 5.3 -176.3±7.2 173.3 ± 7.8 175.4 ± 7.4 178.3 ± 5.8 176.3 ± 6.5 175.4 ± 9.2 -170.3 ± 6.5
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-60.0 -81.1 -80.7 -86.3 -51.9 -58.8 -93.5 83.3 -121.4
± ± ± ± ± ± ± ± ±
7.9 7.8 9.8 8.5 7.9 9.7 10.5 10.9 9.1
153.9 ± -30.1 ± -55.1 ± 62.3 ± -43.4 ± -29.3 ± 16.3 ± 20.6 ± 85.6 ±
7.8 ll.5 9.6 8.3 8.5 9.7 10.9 10.2 7.0
4.3 ± 8.2 -172.4 ± 5.2 -175.2±7.3 165.9 ± 7.6 173.2 ± 7.3 178.4 ± 5.9 176.2 ± 6.6 172.1 ± 9.1 -170.6 ± 6.8
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
-57.8 ± -77.9 ± -76.2 ± -100.4 ± -52.1 ± -62.0 ± -93.9 ± 84.3 ± -138.8 ±
8.6 7.7 10.0 9.2 8.0 9.3 9.7 10.5 9.8
157.1 ± -42.8 ± -48.5 ± 77.1 ± -40.5 ± -43.4 ± 8.4 ± 20.6 ± 80.2 ±
7.6 10.9 9.5 8.3 8.6 9.9 10.5 10.3 7.2
5.0 ± -171.7 ± -169.5 ± 178.1 ± 175.8 ± 177.3 ± -170.0 ± 178.8 ± -172.0 ±
8.0 5.4 7.4 7.7 7.3 5.7 6.1 10.3 6.5
8.2 5.3 7.0 7.6 7.1 6.0 6.5 9.3 7.5
cyclopeptide has been observed in the Pro 2-Phe4 region and the root mean square deviations (RMS) from the experimental data of the average structures of the simulations were 0.68 Aat lOOK and 0.79 Aat 300K. The results are summarized in Table I. In Table Ila, we report the conformational statistical analysis for UNIIOO and UNI300, compared with the x-ray data. The cis bond between Pro 1 and Pro2, presents in the x-ray structure, is conserved in both the two simulations. No large deviations, however, exist between the x-ray data and the simulation results for all dihedral angles.
1055
Molecular Dynamics of a Cyclolinopeptide A analog TableV b. Comparison of the dihedral angles in the solid state with those obtained from the average of the four sets of MD angles given in Table Va.
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Residue
x-ray
MD
x-ray
Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib 6 Ile 7 D-Ala8 Va19
-65.3 -89.9 -95.7 -90.9 -53.7 -54.7 -118.0 77.6 -128.3
-60.4 -78.3 -78.1 -90.0 -53.9 -57.5 -90.9 83.8 -128.7
162.2 -17.3 -43.4 57.8 -40.8 -31.4 16.2 27.2 79.6
"'
(J)
MD
x-ray
MD
154.6 -37.5 -50.7 70.0 -41.1 -43.9 10.4 28.4 80.4
9.0 -175.2 -170.4 -178.8 -173.0 -173.6 172.2 179.4 175.1
7.8 -171.2 -174.3 170.1 175.2 178.3 -174.2 174.9 -171.6
Energy minimizations were repeated using as starting point intermediate coordinates of the MD simulation ofUNI300, and many local minima were obtained with almost the same energy. Similarity of the structures minimized along the trajectory was evaluated by comparing the RMS deviation of the ca atoms for each possible pair of structures. The analysis of these minimized structures, using this procedure, reveals the existence of only one conformational family. The most stable structure, reported in Table lib, is similar to the x-ray data, with a low RMS deviation (0.24 A) and is characterized by larger difference only for Pro2-Phe4 segment. In Figure 3b we show the lowest energy structure. In order to study the temperature and cell packing effect on the conformation, two other simulations, CELVAClOO and CELVAC300, were performed at lOOK and 300K in vacuo, using the crystallographic cell as a simulation box (four units per cell). In Tables III and IV the conformational statistical analyses of the two MD simulations are reported. In Tables lila and IVa we report the analysis results for each unit in the simulation box; the results in Table Illb and IVb have been obtained by averaging over the four units, considered as equivalent molecules. It should be noted that the simulations have been performed without symmetry requirements within the simulation box; the space group symmetry was applied only to the periodic boundary conditions. As it is evident from the results in Tables III and IV, the two structures are similar to the x-ray data. The average conformations are shown in Figures 3c and 3d. CELVAClOO shows the lowest RMS deviation (0.25 A) from the x-ray data. The statistical analysis for CELVAC300 (RMS=0.49 A) shows more flexibility around the Pro 2-Phe3 bond. MD Simulation in Solution and in Hydrated Cell
The two simulations were carried out at 300K: in the system UNISOL, one CLAIB molecule was dissolved in a box containing 452 water molecules; in the system CELSOL, four CLAIB molecules are packed as in the crystalline state with 17 water molecules. In Table V and VI, the statistical analyses obtained from the MD simulations are
Saviano et al.
1056
Table VI Average and relative errors of the dihedral angles in the peptide main chain as obtained from the MD simulation in solution at 300K (UNISOL). Residue
-83.7 -88.7 -87.9 -137.9 -53.8 -52.3 -87.2 95.6 -129.1
± 10.1 ± 11.1 ± 24.0 ± 13.7 ± 8.3 ± 9.3 ± 15.0 ± 19.5 ± 8.5
ljl
(i)
141.9 ± 9.1 -20.7 ± 15.3 -61.3 ± 12.4 98.3 ± 9.9 -43.7 ± 8.0 -42.6 ± 11.1 12.0 ± 18.5 35.4 ± 10.8 82.2 ± 9.1
2.6 ± 10.0 -171.0 ± 7.7 177.3 ± 8.1 176.2 ± 7.5 177.1 ± 6.3 175.0 ± 6.1 -177.0 ± 7.0 177.3 ± 9.4 -176.3 ± 8.9
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Pro 1 Pro2 Phe 3 Phe4 Aib 5 Aib6 Ile 7 D-Ala8 Val9
Figure 4: Stereo view of the average conformation of the MD simulations for a single molecule in solution at T=300K.
reported. For CELSOL the flexibility around the Pro 2-Phe 3 bond, already presents forCELVAC300, is decreased, and the RMS deviation is 0.30 A. The average conformation is presented in Figure 3e. The statistical analysis for the solution simulation (UNISOL) shows an increase in the flexibility for the dihedral angles in the Pro 1Pro2-Phe 3- Phe 4 and Ile7- D-Ala 8 regions with an RMS deviation of0.83 A. In Figure 3f, the averafe conformation is shown. In particular the side chains ofPhe2, Phe3, Ile7 and Val residues result to be oriented in the same direction (Figure 4). Hydrogen Bond Analysis Hydrogen bonding plays an important role in stabilizing the conformation of
Molecular Dynamics of a Cyclolinopeptide A analog
1057
Table VII The occurrence of intramolecular hydrogen bonds(%) obtained from MD simulations compared with those from x-ray data (21) Donor
NH 9
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Val 4 Phe 5 Aib 7 Ile D-Ala8
Acceptor
co 4
Phe 9 Val Phe 3 4 Phe 5 Aib
CELL vacuum
CELLhydr.
Solution
X-ray
T=lOOK
T=300K
T=300K
T=300K
100 100 100 100 100
100 99 98 40 98
97 98 68 20 80
94 96 99 30 82
86 96 83 15 86
biological molecules and in their mode of action and interaction with specific biological sites. In our analysis of the MD simulations, we define a hydrogen bond when the distance between a hydrogen atom and an acceptor atom is shorter than 2.5 A Table vn presents the occurrence of hydrogen bonds (%) for the various simulations. We define the occurrence of hydrogen bonds as the ratio of the number of the simulation steps when hydrogen bond is present, to the total number of simulation steps. As shown in Table VII, all hydrogen bonds found in the crystalline state are correctly reproduced in the in vacuo simulations ofCLAIB in cell at lOOK (CELVAClOO) and at 300K (CELVAC300). The analysis shows that only the hydrogen bond between the NH group oflle7 and the CO group ofPhe 4 shows a no high percentage of occurrence. Similar results are found in the energy minimized conformations of an isolated molecule (UNilOO and UNI300). In the simulation of the hydrated cell (CELSOL), we observe the same hydrogen bond patterns as in the x-ray structure with a no high occurrence of hydrogen bond 7 4 between Ile NH and CO Phe (30% of occurrence is predicted by the simulation). As shown in Figure 5, the peptide-solvent hydrogen bonding pattern in CELSOL is close to that found in the solid state (21) with two extra peptide solvent hydrogen bonds: between the oxygen of one water molecule and the CO of Pro 1, between Phe 3 NHandO(w). In the solution simulation (UNISOL), the results are similar to that we observe for CELSOL with a decrease of hydrogen bonds occurrence between Ile 7 NH and CO 4 Phe (20% occurrence is predicted by the simulation). As shown in Table VII, we did not observe other intramolecular hydrogen bonds in addition to that found in x-ray and NMR studies for any simulation.
Discussion The analysis ofthe simulations shows that the structure found in the solid state is independent from the molecular environment. These results are in agreement with experimental observations.
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Figure 5: Arrangement of the CLAIB and water molecules after MD simulation at 300K (CELSOL). Only the hydrogen bonds between peptide and solvent, already found in the x-ray structure, are shown as dashed lines.
The MD simulation ofCELVAClOO and CELVAC300 were performed in order to study the packing effect. It is worth noting that the MD results in the crystallographic cell (the system composed of only four CLAIB molecules) show structural characteristic similar to those obtained by x-ray and NMR data in polar and apolar solvents. No substantial differences are found for CELSOL simulation. The same solvent-peptide hydrogen bonds experimentally detected are found with additional hydrogen bonds between CLAIB and water molecules. The solid state conformational study (21) was performed with a crystal grown from methanolwater mixtures. The increase of the Xxx-Pro flexibility can be explained by the tendency of the NH and CO groups to be involved in both intra-molecular and solvent-peptide hydrogen bonds. It should be emphasized that the arrangement of the water molecules found in CELSOL (Figure 3) is quite similar to that found in the solid state study (21). Besides, the water addition in the crystallographic box improves the agreement of the MD results with the x-ray data (Table 1), compared with those obtained in vacuo in cell at the same temperature (CELVAC300). The MD simulation ofUNISOL was performed in order to characterize the behaviour and the conformational characteristics ofCLAIB in a polar solvent. The simulation result at 300K points out that CLAIB is structured in water, in a conformation as the one observed in the solid state and in polar and apolar solvent at room temperature. The presence of water molecules increases the flexibility ofXxx-Pro and oflle7DAla8 regions for the tendency of the carbonyl groups ofPro 1, Pro2, Phe3, Ile7 and
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Molecular Dynamics of a Cyclolinopeptide A analog
1059
D-Ala8 to be involved in intermolecular hydrogen bonds. The presence of these peptide-water hydrogen bonds can be a possible ex~lanation of the no high percentage 4 ofoccurrence of the hydrogen bond between NH lle and CO Phe (Table VII). It should be noted the low flexibility of the two Aib residues, during the simulations, and that the side chains of Phe2, Phe3, Ile7 and Val9 for CLAIB point toward the same side with the possible identification of a local hydrophobic region (Figure 4). The MD simulations of UNilOO and UNI300 and the subsequent minimizations were performed to clarify the characteristics of one CLAIB molecule in vacuo. The results point out that the x-ray conformation is a minimum energy structure for CLAIB and, on the contrary ofCLA, indicate that the packingofthe CLAIB molecules in the cell doesn't play an important role in stabilizing the peptide conformation in the crystallographic environment This result is in agreement with that obtained by NMR conformational studies (21 ). The solution studies for CLAIB point out to the presence in polar and apolar solvents, even at room temperature, of a structure closely resembling the one from x-ray analysis. The comparison of the MD results for CLAIB and CLA (16) emphasizes the different behaviour of these cyclic peptides and the ability of the two Aib residues to decrease the conformational accessible space for CLAIB. For CLA it was observed (16) the existence of different conformations with almost the same energy and a most stable structure that can be described as a bowl with a concave hydrophilic side, characterized by four carbonyl oxygens pointing inside the ring backbone, and an opposite convex side with predominant hydrophobic properties. The presence of water molecules induces an overall conformational variation similar to that observed in presence of metal ions (32).1t is interesting to point out that the CD and NMR titrations of CLAIB with metal ions didn't show specific interactions between this molecule and cations (21).
Conclusion This work completes the studies on a full characterization of two related compounds CLA and CLAIB. For both molecules the solid state structure and the behaviour in solution are known. A comparison between the characteristics of the two compounds in vacuo and in solution is presented. The MD confirms our expectation that the introduction in cyclic peptide of two Aib residues further reduces the backbone flexibility, stabilizing in solution one conformation, closely resembling its x-ray structure. The greatest difference between CLA and CLAIB is the behaviour of the two cyclic peptides in polar solution. The decrease of biological activity can be correlated to the decrease of flexibility of the CLAIB as compared to the CLA molecule. This result seems to exclude that the conformation found in the solid state for CLA and CLAIB corresponds to the biological active conformer and it suggests that folding of the ring structure, giving rise to a clustering ofhydrophobic groups in defined stereochemical position, could favour this activity. This arrangement could be realized by the presence of metal ions or by polar solvent molecules. It is noteworthy that Ovchinnikov eta/. (33) have suggested for antamanide a direct correlation between the biological activity and complex-stability constants of this natural peptide with metal ions. More recently it was shown that, also in the solid state, the folding of ring structure of cycloctapeptides (34) is strengthened by the interaction with polar solvent (H 20), and decreases going to semipolar or apolar solvents. Further studies on selected peptides are requested to support this hypothesis.
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Acknowledgments This work was supported by CNR grant no CTBCNR 89.5354
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Date Received: November 12, 1991
Communicated by the Editor R.H. Sarma
