An important aim of modeling is to contribute to understanding of the function of the modeled protein. Inspection of the 4mdhA template structure revealed that loop 93-100, one of the functionally most important part of the enzyme, is more disordered than the rest of the protein. The long active site loop appears to be flexible in the absence of a ligand and could not be seen well in the diffraction map. The unreliability of the template coordinates and the inability of MODELLER to model long insertions is why this loop was poorly modeled in TvLDH, as indicated by PROSAII (Figure 4). Since we are interested in understanding differences in specificity between two similar proteins, we need to build precise and accurate models. Therefore, we need to search for another template malate dehydrogenase structure, which may have a lower overall sequence similarity to TvLDH, but a better resolved active site loop. The old and new templates can then be used together to get a model of TvLDH. The active site loop tends to be more defined if the structure is solved together with its physiological ligand and a co-factor. The model based on a template with ligands bound is also expected to be more relevant for the purposes of our study of enzymatic specificity, especially if we also build the model with the ligands.
1emd, a malate dehydrogenase from E. coli was identified in PDB. While the 1emd sequence shares only 32% sequence identity with TvLDH, the active site loop and its environment are more conserved. The loop in the 1emd structure is well resolved. Moreover, 1emd was solved in the presence of a citrate substrate analog and the NADH cofactor. The new alignment in the PAP format is shown below (file `TvLDH-4mdh.pap').
_aln.pos 10 20 30 40 50 60 1emd_ed -------------------------------------------------------------------- 4mdhA -SEPIRVLVTGAAGQIAYSLLYSIGNGSVFGKDQPIILVLLDITPMMGVLDGVLMELQDCALPLLKDV TvLDH MSEAAHVLITGAAGQIGYILSHWIASGELYG-DRQVYLHLLDIPPAMNRLTALTMELEDCAFPHLAGF _aln.p 70 80 90 100 110 120 130 1emd_ed ------------------SAGVRRKPGMDRSDLFNV--------------NAGI-------------- 4mdhA IATDKEEIAFKDLDVAILVGSM--------------PRRDGMERKDLLKANVKIFKCQGAALDKYAKK TvLDH VATTDPKAAFKDIDCAFLVASMPLKPGQVRADLISS--------------NSVIFKNTGEYLSKWAKP _aln.pos 140 150 160 170 180 190 200 1emd_ed -------------------------------------------------------------------- 4mdhA SVKVIVVGNPANTNCLTASKSAPSIPKENFSCLTRLDHNRAKAQIALKLGVTSDDVKNVIIWGNHSST TvLDH SVKVLVIGNPDNTNCEIAMLHAKNLKPENFSSLSMLDQNRAYYEVASKLGVDVKDVHDIIVWGNHGES _aln.pos 210 220 230 240 250 260 270 1emd_ed -------------------------------------------------------------------- 4mdhA QYPDVNHAKVKLQAKEVGVYEAVKDDSWLKGEFITTVQQRGAAVIKARKLSSAMSAAKAICDHVRDIW TvLDH MVADLTQATFTKEGKTQKVVDVLDHD-YVFDTFFKKIGHRAWDILEHRGFTSAASPTKAAIQHMKAWL _aln.pos 280 290 300 310 320 330 340 1emd_ed -------------------------------------------------------------------- 4mdhA FGTPEGEFVSMGIISD-GNSYGVPDDLLYSFPVTIK-DKTWKIVEGLPINDFSREKMDLTAKELAEEK TvLDH FGTAPGEVLSMGIPVPEGNPYGIKPGVVFSFPCNVDKEGKIHVVEGFKVNDWLREKLDFTEKDLFHEK _aln.pos 350 360 370 380 390 400 1emd_ed ----------VKNLVQQVAKTCPKACIGIITNPVNTTVAIAAEVLKKAGVYDKNKLFGVTTLDIIRSN 4mdhA ETAFEFLSSA---------------------------------------------------------- TvLDH EIALNHLAQ----------------------------------------------------------- _aln.p 410 420 430 440 450 460 470 1emd_ed TFVAELKGKQPGEVEVPVIGGHSGVTILPLLSQVPGVSFTEQEVADLTKRIQNAGTEVVEAKAGGGSA 4mdhA -------------------------------------------------------------------- TvLDH -------------------------------------------------------------------- _aln.pos 480 490 500 510 520 530 540 1emd_ed TLSMGQAAARFGLSLVRALQGEQGVVECAYVEGDGQYARFFSQPLLLGKNGVEERKSIGTLSAFEQNA 4mdhA -------------------------------------------------------------------- TvLDH -------------------------------------------------------------------- _aln.pos 550 560 1emd_ed LEGMLDTLKKDIALGQEFVNK/-.. 4mdhA ---------------------/.-- TvLDH ---------------------/..-
The modified alignment refers to an edited 1emd structure (see below), 1emd_ed, as a second template. The alignment corresponds to a model that is based on 1emd_ed in its active site loop and on 4mdhA in the rest of the fold. Four residues on both sides of the active site loop are aligned with both templates to ensure that the loop has a good orientation relative to the rest of the model.
The modeling script below has several changes with respect to `model-single.top'. First, the name of the alignment file assigned to ALNFILE is updated. Next, the variable KNOWNS is redefined to include both templates. Another change is an addition of the `SET HETATM_IO = ON' command to allow reading of the non-standard pyruvate and NADH residues from the input PDB files. The script is shown next (file `model-multiple-hetero.top').
INCLUDE
SET ALNFILE = 'TvLDH-4mdh-1emd_ed.ali'
SET KNOWNS = '4mdhA' '1emd_ed'
SET SEQUENCE = 'TvLDH'
SET STARTING_MODEL = 1
SET ENDING_MODEL = 5
SET HETATM_IO = ON
CALL ROUTINE = 'model'
SUBROUTINE ROUTINE = 'special_restraints'
ADD_RESTRAINT ATOM_IDS = 'NH1:161' 'O1A:335', ;
RESTRAINT_PARAMETERS = 2 1 1 22 2 2 0 3.5 0.1
ADD_RESTRAINT ATOM_IDS = 'NH2:161' 'O1B:335', ;
RESTRAINT_PARAMETERS = 2 1 1 22 2 2 0 3.5 0.1
ADD_RESTRAINT ATOM_IDS = 'NE2:186' 'O2:335', ;
RESTRAINT_PARAMETERS = 2 1 1 22 2 2 0 3.5 0.1
RETURN
END_SUBROUTINE
A ligand can be included in a model in two ways by MODELLER. The
first case corresponds to the ligand that is not present in the
template structure, but is defined in the MODELLER residue topology
library. Such ligands include water molecules, metal ions,
nucleotides, heme groups, and many other ligands (see FAQ 18 in the
MODELLER manual). This situation is not explored further here. The
second case corresponds to the ligand that is already present in the
template structure. We can assume either that the ligand interacts
similarly with the target and the template, in which case we can rely
on MODELLER to extract and satisfy distance restraints
automatically, or that the relative orientation is not necessarily
conserved, in which case the user needs to supply restraints on the
relative orientation of the ligand and the target (the conformation of
the ligand is assumed to be rigid). The two cases are illustrated by
the NADH cofactor and pyruvate modeling, respectively. Both NADH and
cofactor are indicated by the `.' characters at the end of each
sequence in the alignment file above (the `/' character indicates a
chain break). In general, the `.' character in MODELLER indicates
an arbitrary generic residue called a ``block'' residue (for details
see the MODELLER manual [72]). The 1emd structure file
contains a citrate substrate analog. To obtain a model with pyruvate,
the physiological substrate of TvLDH, we convert the citrate analog in
1emd into pyruvate by deleting the
group, thus
obtaining the 1emd_ed template file. A major advantage of using the
`.' characters is that it is not necessary to define the residue
topology.
To obtain the restraints on pyruvate, we first superpose the structures of several LDH and MDH enzymes solved with ligands. Such a comparison allows to identify absolutely conserved electrostatic interactions involving catalytic residues Arg 161 and His 186 on one hand, and the oxo groups of the lactate and malate ligands on the other hand. The modeling script can now be expanded by appending a routine that specifies the user defined distance restraints between the conserved atoms of the active site residues and their substrate.
The ADD_RESTRAINT command has two arguments. ATOM_IDS
defines the restrained atoms, by specifying their atom types and the
residue numbers as listed in the model coordinate file.
RESTRAINT_PARAMETERS defines the restraints, by specifying
the mathematical form (e.g., harmonic, cosine, cubic spline), modality,
the type of the restrained feature (e.g.,
distance, angle, dihedral angle), the number of atoms in the
restraint, and the restraint parameters. In this case, a harmonic
upper bound restraint of
is imposed on the distances
between the specified pairs of atoms. A trick is used to prevent
MODELLER from automatically calculating distance restraints on the
pyruvate-TvLDH complex; the ligand in the 1emd_ed template is moved
beyond the upper bound on the ligand-protein distance restraints
(i.e., 10).
The new script produces a model with a significantly improved PROSAII profile (Figure 4). The predicted error in the 90-100 active site loop is much less and practically resolved in the loop region 220-250. The overall Z-score is improved from -10.7 to -11.7, which compares well with the template Z-score of -12.7. With this favorable evaluation, we gain confidence in the final model. The model was used for interpreting site-directed mutagenesis experiments aimed at elucidating the determinants of enzyme specificity in this class of enzymes [73].