Introduction to Molecular Modeling Lab X - Modeling Proteins

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1 (Rev 12/2/99, Beta Version) Introduction to Molecular Modeling Lab X - Modeling Proteins Table of Contents Introduction...2 Objectives...3 Outline...3 Preparation of the pdb File...4 Using Leap to build the Biotin/Steptavidin Complex...5 xleap...5 Building the biotin residue from pdb in Leap...5 Carnal...13 Sander...13 summary...15 Carnal-II...17 Appendices...21 Appendix Appendix Appendix Appendix Appendix Appendix Appendix xx...24 Appendix xx...25

2 Biotin Streptavidin Introduction The streptavidin/biotin system is of special interest because it has one of the largest free energies of association as of yet observed for noncovalent binding of a

The streptavidin protomer is organized as an 8-stranded beta-barrel.

2 2 Biotin Streptavidin Introduction The streptavidin/biotin system is of special interest because it has one of the largest free energies of association as of yet observed for noncovalent binding of a protein and small ligand in aqueous solution (K ass = ). The complexes are also extremely stable over a wide range of temperature and ph. The streptavidin protomer is organized as an 8-stranded beta-barrel. Pairs of the barrels bind together to form symmetric dimers, pairs of which in turn interdigitate with their dyad axes coincident to form the naturally-occurring tetramer. If you are interested in the Biotin/streptavidin system and would like to know more about the system in general then consult the two references given below. However, it is not necessary to do so to understand this laboratory exercise. Miyamoto S, Kollman PA. What determines the strength of noncovalent association of ligands to proteins in aqueous solution. PNAS, (1993), 90: Miyamoto S, Kollman PA. Absolute and relative binding free energy calculations of the interaction of biotin and its analogs with streptavidin using molecular dynamics free energy perturbation approaches. Proteins-Structure Function and Genetics, (1993) 16:

3 3 Objectives In this tutorial, we massage a PDB file of the tetramer so that it can be understood by Amber, solvate the region of interest (i.e. around one of the four complexed biotins), and run some dynamics keeping the rest frozen. The equilibration takes 2.3 hours per picosecond on moderately fast CPU (Convex 3820). Allowing the whole system to move takes 4 hours/psec (the whole system would have to be solvated, thus still longer time, for this to be useful). The frozen part provides a more realistic electrostatic environment for the part that moves. Thus the main objectives are 1) Modifying Protein PDB files to suit specific needs. 2) Partially hydrating proteins to decrease computational time yet obtain meaningful results. 3) Run molecular dynamics on a protein 4) Interpret the results of the molecular dynamics run. The initial biotin/streptavidin tetramer was prepared by Richard Dixon from the momomer. The monitor was obtained from the Brookhaven database, structure 1stp by P.C. Weber, D.H. Ohlendorf, J.J. Mendolowski, and F.R. Salemme (1992). Outline 1) Prepare the required pdb file 2) xleap a) Build biotin residue template. b) Load 'frcmod' file (extra force field params) for biotin. c) Load the streptavidin-biotin pdb. d) Add 'cap' of water. e) Save top and crd files for dynamics. 3) Carnal Figure out residues in biotin 'cap' region. 4) Sander a) Minimize and run dynamics to equilibrate. b) Check equilibration. c) Run more dynamics.

4 4 5) Carnal Analyze the trajectory. Setup 1) Create a subdirectory in your home directory call biotin mkdir ~/biotin 2) Copy all the files from /usr/people/amberlab11 into the ~/biotin directory cp /usr/people/amberlab4/* ~/biotin/ The files that should be copied are: biotin.pdb btn.frcmod carnal_grp.in carnal_rms.in carnal_dist.in carnal_hbond.in min.in md0.in md1.in process_md.perl Preparation of the pdb File Hydrogen naming conventions in the given.pdb file are wrong - a not uncommon experience. One option is to try to correct all the hydrogen atoms (about 4000). However, it is much quicker to simply delete all the hydrogren atoms and then let xleap add them back when you load up the pdb file. This can be achieved by using 'egrep -v' to exclude lines matching a pattern with H in either of the 1 st or 2 nd columns of the atom name. egrep -v '^...H' given.pdb > x egrep -v '^...H' x > start.pdb where the '^ starts the pattern at the beginning of the line and the.'s are wild-card single characters Note that there are 13. in the first line and 12. in the second.

5 Using Leap to build the Biotin/Steptavidin Complex xleap Building the biotin residue from pdb in Leap The amber force field lacks parameters for biotin.

5 5 Using Leap to build the Biotin/Steptavidin Complex xleap Building the biotin residue from pdb in Leap The amber force field lacks parameters for biotin. Therefore to get Amber to understand biotin will require you to load a coordinate file for biotin, then make bonds between the connected atoms, and then input parameters for each atom of biotin. While this is a bit tedious, we will not have to actually determine what the parameters are, which is really the hard part. Start xleap and load the residue: > BTN = loadpdb bioti n.pdb > edit BTN When you molecule in you will see atoms of diamonds. colored atom type but lack any look at the biotin the Unit editor each of the biotin appear as They will be according to the structure will bonds.

6 6 To add the atoms, select all of the atoms by snapping a box that encloses all of the atoms. Then, in the World editor, type the following command: > bondbydistance BTN If you now view the biotin molecule you will see bonds have been added.

7 Now, select all of the atoms by dragging a box about the structure with the left mouse button, they will turn

Select all atoms by dragging a box with left mouse button and pull down 'Edit selected atoms': Note: Editing the

When it hangs, you lose all of your work.

7 7 Now, select all of the atoms by dragging a box about the structure with the left mouse button, they will turn purple. Select all atoms by dragging a box with left mouse button and pull down 'Edit selected atoms': Note: Editing the table that appears can be frustrating. The program driving it is not very stable and tends to hang. When it hangs, you lose all of your work. If this crashes more than once on you, let me know and I will provide you with what you need to by-pass this step. Also, if it does crash, try to note exactly what you were doing when it crashed. I am trying to collect this information to help the programmers discover the problem and then, hopefully, fix it.

8 8 ust input an atom type, charge, Pert.name and Pert.type for each atom as shown on the next table. T hi s ta bl e lis ts al l th e at o m s p re s e nt in B io ti n. Y o u m

9 9 Note that for most of the atoms the Name/PERT.name and the Type/PERT.type entries are identical. The exceptions are H71, H81, H91 and H92. When you have finished entering the data, pull down 'Operations / Check table' to make sure all is ok, and 'Save and quit' the table.

10 In the Unit Editor, deselect the atoms by holding down the Shift key and clicking in the background (i.e. not on any atom). This is mainly cosmetic, so that the saved state will not have selection on. Finally, save the residue in xleap format at the command line. The.lib file contains a description of each atom - atom name, type, charge, coordinates, and connectivity information. However, it does not contain force field information. > saveoff BTN btn.lib 10 The residue can be loaded in later sessions by > loadoff btn.lib Load the premade 'frcmod' file for biotin. This file is shown in Appendix 1. It contains the force field parameters which were missing from the Amber force field. When this file is loaded, the parameters in this file are merged with the Amber force field. If there is duplication, the.frcmod file parameters take precedence. > fmod = loadamberparams btn.frcmod Load prepared pdb of streptavidin/biotin complex: > stbt = loadpdb start.pdb > edit stbt

11 11 To manipulate the structure, hold down the two right buttons and push forward/back to zoom in/out. The middle button alone rotates, the right button translates, and the space bar recenters the molecule. Add a 'cap' of waters around the site of the 1 st biotin. This is done by estimating a median x, y, z coordinate by eyeballing the coordinates of the BTN at the beginning of start.pdb, which can be viewed by jot start.pdb: > solvatecap stbt WATBOX216 { } 20

12 12 The number of waters in the cap varies slightly, depending on machine; about 265 is to be expected on SGI. Save system in xleap and pdb formats for future reference: > saveoff stbt built.lib > savepdb stbt stbtcap.pdb Save top and prm files for dynamics & perturbation: > saveamberparm stbt stbtcap.top stbtcap.crd

13 13 Carnal For dynamics, we only want the region of interest to move - the rest is there to provide a more lifelike environment. Use a carnal feature to figure out the residues around the biotin molecule that the water cap is on: carnal < carnal_grp.in > carnal_grp.out Appendix 2 contains a listing of the carnal_grp.in. If you take a look at the input file you will see that first the stbtcap.top and stbtcap.crd files are read. Next the file says to find all of the residues that are within 15 A of group 479. Group 479 is the biotin residue. The output then contains the list of groups found with these constraints and it would be a good idea to examine this file. GROUP format is described in Appendix B of the Amber manual. Sander Introduction When the biotin/stretavidin complex was built, the hydrogens were deleted and then xleap added them back. They may have not all been added back in a way that was sterically favorable. In addition, a water cap was subsequently added. This is a pre-equilibrated volume of water and unfavorable steric interactions between the waters and the biotin/streptavidin complex may exist. Therefore, we first need to do a quick molecular mechanics minimization to remove any particularly bad steric interactions. Molecular Mechanics minimization The command line for this is given below. sander -O -i min.in -o min.out -p stbtcap.top -c stbtcap.crd \ -r min.rst & The file called min.in is the control file for the run and it is listed in the Appendix (3). The key parameters in this file are

14 14 IMIN=1, IMIN determines whether the run is a molecular mechanics (=1) or molecular dynamics run (=0). MAXYCY=100, we will let the minimizer run for 100 cycles. NTPR=20 SCEE=1.2. This is the 1,4-VDW scaling term. When using the all_nuc94.dat data base it should be set to this value. NSNB= NTMIN=2 Look at the output from this run (located in min.out). Note that the first part of this file lists what all the input parameters were, files used, date, etc., which can be very helpful once you start to accumulate a lot of minimization runs. After that, for each cycle, is listed various values such as total energy and each of its components. If a run blows up on you, this can help you diagnose where the problem may lie. Finally, there is some information about the length of the run. Molecular Dynamics Next we are going to gradually warm, using 'belly' option to restrict motion to region of interest as determined by carnal, above. (The group defined by carnal is modified in md0.in to include all the waters.) sander -O -i md0.in -o md0.out -p stbtcap.top -c min.rst \ -x md0.crd -r md0.rst & The input file for this run is given in the Appendix 4. Some of the key parameters are: IBELLY=1, NRUN=1, NTT=1, IREST=0 NTX=1, NTB=0 TEMPI=0, TEMPO=300, TAUTP=0.1, DTEMP=2.0, NTP=0 NSTLIM=1000, DT=0.002, NTC=2, NTF=2 IDIEL=0, SCEE=1.2, CUT=999, NSNB=9999, NTPR=20, NTWX=20 This equilibration immediately warms to 300K; this may be ok in this case since a major part of the system is frozen - if this were not the case, too-rapid warming could disrupt the structure. Similarly, if this was a constant pressure simulation in a periodic box of solvent, too-rapid warming could lead to excessive pressure fluctuations. Although no such critical problems can happen here, we still need to see if the system has truly equilibrated. To determine this we need to take a look at the variantion in temperature energy and its components. This

15 15 information is numerically present in the md0.out file and the relevant numbers can be sorted into files for plotting with a perl script called process_md_perl and then the plots can be viewed with xmgr. process_md_perl < md0.out This script will create a bunch of files called summary_xxx where xxx is temperature (TEMP), total energy (ETOT), kinetic energy (KETOT), etc To plot these files use xmgr. Equilibration: Temperature The temperature seems fairly equilibrated, but this does not necessarily mean the system is fully equilibrated, especially because the temperature coupling we use in AMBER forces the temperature to the desired value. (Note: since this setup uses an effectively infinite cutoff, once it is equilibrated the temperature scaling could be turned off; see NTT in the Sander manual.) Equilibration: Energy Looking at the total energy of the system we see a definite trend to lower energy that presumably has not completed. The total energy of the system can be subdivided into its main components, kinetic and potential energies and are shown below.

16 16 Equilibration: Kinetic Energy Component Equilibration: Potential Energy Component Thus after the initial warming period, kinetic energy is relatively constant (it scales with temperature so is not really worth checking, in fact), but the system is gradually relaxing to a lower-energy conformation. Further equilibration is called for. Note: in a periodic system, we would also want to check pressure fluctuations and density. We restart from the saved coordinates of the previous run, using the same conditions but setting the appropriate variables to use the saved final velocities (IREST=1, NTX=5) and letting it run twice as long (NTSLIM=2000) (see Appendix 5 for a listing of the control file md1.in): sander -O -i md1.in -o md1.out -p stbtcap.top -c md0.rst \ -x md1.traj -r md1.rst & Looking at the potential energy for the combined runs leads to the plots shown below. These were generated are for the first run. You should generate these plots following the procedure given above using the perl script and xmgr. Equilibration 2: Potential Energy Component

17 17 The potential energy clearly has still not fully equilibrated. At this point, it appears that the assumption that fast warming was ok could have been wrong, and a gradual warming protocol or use of cartesian restraints in the initial warmup may have been in order (this more conservative approach is recommended in general). On the other hand, 6-10 psec of equilibration is not an excessive requirement. In effect, we have performed a mild form of simulated annealing on the initial structure, and in any case need to know how far the solute has moved from the crystallographic starting position. For this analysis, we turn to carnal. Carnal-II To get a rough measure of how far the structure has drifted from the crystal form during the equilibration, we perform a root-mean-square (RMS) comparison of the solute atoms that were allowed to move, using the first set as a reference. (If this system were not held in place by the stationary atoms, we would want to get the best fit of each coordinate set to the first using carnal's RMS FIT option.) Three types of RMS are measured: the entire moving part of the system; the moving biotin to get a rough idea of the changes that it experiences in its pocket; and the biotin using the FIT option to see how much of its variation is due to internal (vs. orientational) changes (See appendix 6 for a listing of carnal_rms.in). carnal < carnal_rms.in > carnal_rms.out As might be expected, the average RMS decreases in each successive case:0.9, 0.7 and 0.2 Angstroms respectively. The time results: Equilibration: RMS Deviation from Initial Structure

18 18 Equilibration: RMS Deviation of Backbone from Initial Structure Although the equilibration is not quite complete based on the potential energy above, the overall RMS indicates that the structure is not drifting progressively (as virtually guaranteed by the frozen part). The overall RMS of ca. 1 Angstrom is well within the norm for simulations; beyond 2 Angstroms would be alarming. The backbone RMS is also reasonable: NMR-derived structures tend to differ from X-ray by about 0.8 Angstroms. (Different X-ray structures vary by about 0.4 Angstroms.) Turning to Initial the biotin: Equilibration: RMS Deviation of Biotin from Structure Equilibration: Internal RMS Deviation of Biotin

19 19 The first graph indicates a quasi-periodic motion relative to the initial structure that may be interesting to explore once the trajectory has equilibrated. The internal variations are faster and smaller, as one would expect. Since the solute RMS is not drifting appreciably, a progressive change in water structure presumably is the cause of the progressive dropping of potential energy. This stands to reason, since we simply superimposed a sphere cut from a periodic system of pure water onto the solute and trimmed away any waters that overlapped, then warmed the local system rapidly to 300K, with nothing to prevent the sphere of waters from expanding. Therefore one would expect the drop in potential energy to correlate with the waters settling in toward the solute. Measuring the distance between the geometric centers of two groups of atoms, the biotin and the water oxygens, illustrates: carnal < carnal_dist.in > carnal_dist.out resulting in md01.dist. Treating the trajectory as if it were at equilibrium for didactic purposes, we run carnal again to analyse hydrogen bonding between the ligand and its receptor: carnal < carnal_hbond.in > carnal_hbond.out The summary data from the.out file shows that 6 hbonds persist throughout the trajectory ('grep 100 carnal_hbond.out'). They are: # 63 (SER 13 OG )_(SER 13 HG )..(BTN 479 O3 ) % occupied: # 107 (TYR 29 OH )_(TYR 29 HH )..(BTN 479 O3 ) % occupied: # 146 (ASN 35 N )_(ASN 35 H )..(BTN 479 O2 ) % occupied: # 353 (SER 74 OG )_(SER 74 HG )..(BTN 479 O1 ) % occupied: # 118 (BTN 479 N2 )_(BTN 479 HN2 )..(ASP 114 OD2 ) % occupied: # 244 (BTN 479 N1 )_(BTN 479 HN1 )..(SER 31 OG ) % occupied: Thus all of the potential hbonding atoms of the biotin except for the sulfur are 100% engaged with the acceptor. As one might expect, these hbonds are not weak; e.g. the first in detail: 63 (SER 13 OG )_(SER 13 HG )..(BTN 479 O3 ) % occupied: distance avg dev max min N 150

20 angle(deg) avg dev max min N

21 21 Appendices Appendix 1-btn.frcmod file BTNMOD Modified paramaters for Biotin (RWD) MASS SD DH DC DN BOND SD-C HC-DC DC-DH DC-DN DN-DH DC-DC DH-HC DH-H ANGL N -C -N SD-C -N HC-CT-S HC-CT-N DH-HC-CT DH-H -N DH-HC-DH DH-H -DH CT-HC-DC HC-DC-DH HC-DC-DN HC-DC-DC DC-DN-DH DC-DN-DC DC-HC-DC DH-DC-DN DN-DC-DC DC-DC-DH DC-DC-DC DIHE SD-C -N -X X -CT-HC-X X -N -H -X X -DC-HC-X X -DC-DN-X X -DC-DC-X

22 22 IMPR SD-C -N -N X -X -HC-CT X -X -DC-DH X -X -DN-DH NONB SD DH DC DN Appendix 2 carnal_grp.in # CUTRES: list residues within cutoff of group FILES_IN PARM p1 stbtcap.top; STREAM s1 stbtcap.crd; FILES_OUT DECLARE GROUP g1 (RES 479); CUTRES c1 g1 15.0; OUTPUT END Appendix 3 - min.in &cntrl imin=1, maxcyc=100, ntpr=20, scee=1.2, nsnb=999999, ntmin=2, &end Appendix 4 - md0.in cold start belly equil &cntrl IREST = 0, ibelly= 1, NTX = 1, TEMPI = 0.0, NTB = 0, NRUN = 1, NTT = 1, TEMP0 = 298.0, TAUTP =.1, DTEMP = 2.0, NTP = 0, NSTLIM= 1000, DT =.002, NTC = 2, NTF = 2, IDIEL = 0, SCEE = 1.2, CUT =999.0, NSNB = 9999,

23 23 NTPR = 20, NTWX = 20, &end -- belly = residues 15A from BTN res 479 plus all H2O RES 5 17 RES RES RES RES RES RES RES RES RES RES RES RES RES RES RES RES RES RES RES RES RES RES RES END END Appendix 5 - md1.in cold start belly equil &cntrl IREST = 1, ibelly= 1, NTX = 5, TEMPI = 0.0, NTB = 0, NRUN = 1, NTT = 1, TEMP0 = 298.0, TAUTP =.1, DTEMP = 2.0, NTP = 0, NSTLIM= 2000, DT =.002, NTC = 2, NTF = 2, IDIEL = 0, SCEE = 1.2, CUT =999.0, NSNB = 9999, NTPR = 20, NTWX = 20, &end -- belly = residues 15A from BTN res 479 plus all H2O RES 5 17 RES RES RES RES

24 24 RES RES RES RES RES RES RES RES RES RES RES RES RES RES RES RES RES RES RES END END Appendix 6 - carnal_rms.in # RMS deviation of moving part of solute FILES_IN PARM p1 stbtcap.top; STREAM s1 traj/md01.crd; FILES_OUT TABLE t1 md01.rms; DECLARE GROUP g1 (RES 5-17,25,27-44,59-81,89-101,104,105, , 142, ,172, , , , 216,228,230, , ,438, , ,472,479); RMS r1 g1; GROUP g2 ((GROUP g1) & (ATOM NAME C CA N)); RMS r2 g2; GROUP g3 (RES 479); RMS r3 g3; RMS r4 FIT g3; OUTPUT TABLE t1 r1 r2 r3 r4; END Appendix xx - carnal_dist.in # distance between centers of geometry of 'central' biotin and waters FILES_IN PARM p1 stbtcap.top;

25 25 STREAM s1 traj/md01.crd; FILES_OUT TABLE t1 md01.dist; DECLARE GROUP g1 (RES 479); GROUP g2 (ATOM TYPE OW); DIST d1 g1 g2; OUTPUT TABLE t1 d1; END Appendix xx - carnal_hbond.in # HBOND analysis of ligand-receptor interaction FILES_IN PARM p1 stbtcap.top; STREAM s1 traj/md01.crd; FILES_OUT HBOND h1 md01_hb1 TABLE LIST; HBOND h2 md01_hb2 TABLE LIST; DECLARE GROUP g1 (RES 5-17,25,27-44,59-81,89-101,104,105, , 142, ,172, , , , 216,228,230, , ,438, , ,472); GROUP g2 (RES 479); OUTPUT HBOND h2 DONOR g2 ACCEPTOR g1 STATS; HBOND h1 DONOR g1 ACCEPTOR g2 STATS; END

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